NEURODEGENERATIVE DISEASES - LABORATORY AND CLINICAL RESEARCH
DROSOPHILA MELANOGASTER MODELS
OF MOTOR NEURON DISEASE
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NEURODEGENERATIVE DISEASES -
LABORATORY AND CLINICAL RESEARCH
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NEURODEGENERATIVE DISEASES - LABORATORY AND CLINICAL RESEARCH
DROSOPHILA MELANOGASTER MODELS
OF MOTOR NEURON DISEASE
RUBEN J. CAUCHI
EDITOR
New York
Copyright © 2013 by Nova Science Publishers, Inc.
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CONTENTS
Preface vii
Chapter 1 Genetics of Motor Neuron Disorders: From Gene Diversity to
Common Cellular Conspirators in Selective Neuronal Killing 1 Rebecca K. Sheean and Bradley J. Turner
Chapter 2 A Secreted Ligand for Growth Cone Receptors, VAP Mediates the
Cellular Pathological Defects in ALS 35 Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda
Chapter 3 Flies in Motion: What Drosophila Can Tell Us about Amyotrophic
Lateral Sclerosis 57 Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu
Chapter 4 Maintaining Long Supply Lines: Axon Degeneration and the
Function of Hereditary Spastic Paraplegia Genes in Drosophila 85 Belgin Yalçın and Cahir J. O’Kane
Chapter 5 Drosophila as a Model for CMT Peripheral Neuropathy: Mutations
in tRNA Synthetases as an Example 121 Georg Steffes and Erik Storkebaum
Chapter 6 Lessons from Drosophila in Neurodegeneration: Mechanisms
of Toxicity and Therapeutic Targets in Spinal and Bulbar
Muscular Atrophy 147 Adrienne M. Wang
Chapter 7 Spinal Muscular Atrophy: Insights from the Fruit Fly 171 Stuart J. Grice, Kavita Praveen, A. Gregory Matera and
Ji-Long Liu
Chapter 8 Genetic Screens in Drosophila and Their Application in Motor
Neuron Disease Models 185 Liya E. Jose, Patrik Verstreken and Sven Vilain
Index 211
PREFACE
A RESPONSIBLE CHOICE OF MODEL ORGANISM
Motor neuron diseases (MNDs) are the most catastrophic of neurodegenerative disorders
in that cognitive function is spared yet motor neuron degeneration translates into progressive
muscle weakness and paralysis that propel the afflicted patient to eventual death.
Neurodegenerative disorders constitute one of the major challenges of modern medicine in
view of the current lack of effective therapies.
The fruit fly, Drosophila melanogaster, has a distinguished history as an important model
organism capable of shaping our fundamental understanding of life including neuromuscular
development and physiology. Through the efforts of the Drosophila and human genome
projects, more then a decade ago, we learned that the genetic makeup of the fruit fly is
remarkably similar to humans. In this respect, it’s no surprise that the vast majority of all
known human disease genes have a similar fly counterpart that is devoid of the genetic
complexity so typical of the human gene structure and families.
Compared to vertebrate models, the fly is small, has a rapid lifecycle and gives rise to a
large numbers of offspring. Importantly, at a molecular and physiological level, the basic
principles of neuromuscular function are amazing conserved. Combine this with the presence
of numerous genetic tools developed over the last century allowing genes and the proteins
they encode to be manipulated swiftly to decipher their in vivo function and you have a
superb genetic animal model organism of disease. This publication singles out the past and
recent accomplishments of Drosophila in modelling MNDs with particular emphasis on the
emerging molecular pathways underpinning these diseases.
The volume opens with Chapter One where Rebecca K. Sheean and Bradley J. Turner
(University of Melbourne, Australia) introduce us to the features of the two groups of neurons
that are primarily affected in MND, namely upper motor neurons (also known as giant
pyramidal cells or Betz cells) and lower motor neurons (also known as anterior horn cells in
the spinal cord). MNDs are categorised according to which group of motor neurons is
affected and in this context, the authors give us the core clinical and pathological features of
upper MNDs including primary lateral sclerosis (PLS) and hereditary spastic paraplegia
(HSP); lower MNDs including progressive muscular atrophy (PMA), progressive bulbar
palsy (PBP), spinal muscular atrophy (SMA), spinobulbar muscular atrophy (SBMA), axonal
Charcot-Marie-Tooth disease, which significantly overlaps with distal hereditary motor
Ruben J. Cauchi viii
neuropathy (dHMN) and lethal contracture syndrome (LCCS); and amyotrophic lateral
sclerosis (ALS), in which there is combined upper and lower motor neuron loss.
Chapter One also includes a detailed account of the multitude of genes linked to motor
neuron degeneration. Interestingly, most of these genes encode ubiquitously expressed and
fundamental proteins that, when defective, lead to selective motor neuron injury. Based on
this rich genetic evidence captured in the past decades as well as seminal contributions by
mammalian models, the authors eloquently highlight the key cellular pathways that are
disrupted in both motor neurons and their neighbouring cells including mitochondrial
function, intracellular membrane trafficking, axonal transport, cytoskeletal dynamics, RNA
processing, proteostasis, myelination and lipid metabolism. Importantly, various mentioned
genes and the proteins they encode will be subject to deeper investigation in the chapters that
follow.
In Chapter Two, Hiroshi Tsuda et al. (McGill University, Canada) kick-start a series of
accounts that underline the contribution of Drosophila to our understanding of the
pathophysiology of motor neuron degeneration. The authors focus on ALS (also known as
Lou Gehrig’s disease), a mercilessly fatal neurodegenerative disease, which was first
described by the founder of modern neurology, the French neurologist Jean-Martin Charcot.
ALS occurs in hereditary or sporadic forms and since both these forms share several features,
insights into the mechanisms through which gene mutations have a negative impact on motor
neuron physiology can potentially lead to novel therapeutic approaches that are effective in
both forms of the disease.
Identification of the first ALS-linked gene, superoxide dismutase 1 (SOD1), 20 years ago
spurred a successful hunt for additional causative genes among which was the VAMP
associated protein B (VAPB). VAPB, an endoplasmic reticulum (ER) transmembrane protein,
is conserved in Drosophila and numerous other species, including the N-terminal major
sperm protein (MSP) domain, which harbours the dominantly-inherited missense mutation
(proline 56 to serine) that confers motor neuron dysfunction. Human VAPB and its fly
orthologue are functionally interchangeable with regards to their influence on the architecture
and electrophysiological properties of Drosophila neuromuscular junctions (NMJs).
Furthermore, Drosophila studies were pivotal to reveal the VAPB MSP domain cleaved from
the full length protein is secreted in a cell type-specific fashion and acts as a diffusible
hormone. Importantly, VAPB-MSP was identified as a ligand for Ephrine (Eph), Roundabout
(Robo) and leukocyte-antigen related (LAR) family receptors, which were originally
identified as mediators of axon growth cone guidance cues during nervous system
development.
The downstream effects of VAPB signalling are still a work in progress though studies in
the fly implicate a function in the maintenance of muscle mitochondria. The authors conclude
this compelling narrative by delving into the cellular defects associated with mutant VAPB,
which can potentially shed light on the pathophysiology of ALS. Based on several lines of
evidence, the ALS mutation is thought to cause two different types of defects: failed secretion
of mutant VAPB (loss-of-function) and accumulation of mutant VAPB as ubiquitinated ER
inclusions that lead to ER stress (gain-of-function), a common defect observed in the
pathology of both familial and sporadic forms of ALS.
The subject of Chapter Three remains ALS though Daniela C. Zarnescu and colleagues
(University of Arizona, USA) focus on TAR DNA binding protein (TDP-43) and fused in
sarcoma (FUS), two RNA binding proteins that not only associate with ubiquitinated
Preface ix
intracellular inclusions but also act as causative agents of disease in view of the recent
discovery of MND-linked mutations in the respective gene. The identification of these
proteins in addition to the involvement of additional RNA binding proteins such as Senataxin
and Angiogenin as well as RNA itself (C90RF72 noncoding expanded repeats) in ALS,
prompted an ‘earth-shaking’ shift in our thinking about the pathophysiology of ALS whereby
RNA metabolism is presently seen as a central disrupted pathway in the presence of disease.
The authors discuss the contribution of the fruit fly to our understanding of ALS through
a review of the studies on TDP-43 and FUS. At a sequence level, both these proteins share
several domains including those that enable RNA binding and nucleocytoplasmic shuttling.
They also have a similar job description and in this regard, they have been implicated in
several RNA processing steps including transcriptional regulation, mRNA splicing, miRNA
processing as well as mRNA transport and local translation. Drosophila was vital for
demonstrating a link between FUS and TDP-43, whereby through genetic interaction
approaches it was shown that FUS acts downstream of TDP-43. A flurry of studies have tried
to unravel the function of these two proteins through loss-of-function of the orthologous gene
or overexpression of either wild-type or mutant human protein in an otherwise wild-type
background (the Drosophila orthologue of the disease gene is intact). Interestingly, in the
case of TDP-43, overexpression studies tended to produce similar ALS-like phenotypes to
those observed on loss-of-function which raise the question of whether protein
overexpression mirrors the loss-of-function condition. The authors also discuss the genetic
interactions reported in the fly models of TDP-43 and FUS which highlight the cellular
pathways that are functionally important in ALS including protein folding, proteasome-
mediated degradation, apoptosis and microtubule organisation. Differences do exist between
fly models of ALS and human pathology such as the absence of ubiquitinated inclusions,
which are a hallmark feature of the disease. However, the fly model could be telling us that
cytoplasmic aggregates are not a prerequisite for motor neuron degeneration and most
probably these pathological inclusions are a consequence rather than a cause of motor neuron
injury. Rest assured that you will hear about further twists in this riveting story in the years to
come.
It is worth noting that whereas Drosophila was heavily exploited to make great strides in
deciphering the biology of ALS-linked VAMPB, TDP-43 and FUS, the same cannot be said
of SOD1. In this regard, only a few scattered reports exist in the literature. Particularly,
Watson et al. (J Biol Chem 2008) report that expression of wild-type or disease-linked
mutants of human SOD1 selectively in motor neurons induced progressive climbing defect
which were accompanied by defective neural circuit electrophysiology, focal accumulation of
the human SOD1 protein and stress response in glia surrounding motor neurons. The utility of
Drosophila to model SOD1 pathophysiology might have been overtaken by the mutant SOD1
mouse model of ALS. However, since therapeutic success in the mouse model has not
translated into effective therapy for human ALS in clinical trials (Benatar Neurobiol Dis
2007; Aggarwal & Cudkowicz Neurotherapeutics 2008), the use of Drosophila for high-
throughput screening to identify pharmacological and genetic modifiers of disease phenotype
might eventually come of age.
The length of motor axons, which can be up to 105 times longer than that of cell bodies,
presents great challenges to the subcellular trafficking machinery of motor neurons.
Impairment of the mechanisms that maintain axonal function can lead to axon degeneration
diseases, particularly in the distal regions of the axons that lie furthest from the cell body.
Ruben J. Cauchi x
Chapter Four deals with one such group of diseases, namely hereditary spastic paraplegias
(HSPs), which are characterised by progressive spasticity and weakness in lower extremities,
caused by progressive distal axonopathy mostly in the longest upper corticospinal motor
neurons. The large number of genetic loci identified as causative explains the phenotypic
heterogeneity of HSPs although the gene products point at an unexpectedly limited range of
disease mechanisms, including endoplasmic reticulum organisation and function, axonal
microtubule-based transport and endosomal trafficking and signalling, mitochondrial function
as well as the interactions of axons with the myelin sheath.
Most but not all causative human genes have orthologues in Drosophila. In view of the
powerful genetic tools for generation of specific mutant or transgenic flies, as well as the
myriad of analytic tools for understanding the cellular roles of these gene products in neurons,
particularly in axons and synapses, Drosophila offers a compelling system to study HSP-
linked genes as well as the consequences on mutation. Belgin Yalçın and Cahir J. O’Kane
(University of Cambridge, UK) review the major contributions from flies so far including the
dissection of the roles of several HSP proteins in ER organisation, transport of specific
cargoes in axons and in pathways including bone morphogenetic protein (BMP) signalling.
As additional HSP-linked proteins are identified, the fly model offers a great opportunity to
understand their cellular roles and ultimately provide plausible mechanisms for these
diseases.
Charcot-Marie-Tooth (CMT) disease is characterised by the degeneration of peripheral
motor and sensory neurons, leading to progressive muscle weakness and wasting, and sensory
loss. The disease is clinically heterogeneous although electrophysiological and pathological
criteria allow the distinction between demyelinating, axonal and intermediate forms of CMT.
Since more than 30 genes have been causally associated with CMT to date, the disease is also
genetically heterogeneous. These genes encode proteins with often very different molecular
functions suggesting that peripheral motor and sensory neuropathy can result from
impairment of multiple molecular pathways including myelination and myelin maintenance,
axonal transport, mitochondrial dynamics, endosomal trafficking, axon-Schawann cell
interaction, transcriptional regulation and protein chaperone activity. The exact molecular
underpinnings of the peripheral motor and sensory neuropathy are still poorly understand, and
there is no effective drug treatment available. Chapter Five concentrates on the use of
Drosophila as a genetic organism to model CMT in view of the possibility of studying the
effect of CMT-associated mutant proteins on motor and sensory neurons in their
physiological context as well as the suitability of this model system to perform genetic
screens. The organisational principles of the nervous system as well as basic
neurophysiological principles including but not limited to conduction of action potentials,
signal transmission through release of neurotransmitters and the synaptic vesicle cycle, are
remarkably conserved between flies and humans. The development and anatomy of the
Drosophila neuromuscular system is beautifully described in this section.
Mutations in the genes encoding tyrosyl-tRNA synthetase (YARS), glycyl-tRNA
synthetase (GARS), alanyl-tRNA synthetase (AARS) and possibly lysyl-tRNA synthetase
(KARS) and histidyl-tRNA synthetase (HARS) give rise to axonal and intermediate forms of
CMT. Such enzymes ligate amino acids to their cognate tRNA and therefore catalyse an
important step in protein synthesis. Aimed at illustrating the usefulness of the fly as a model
for CMT, Georg Steffes and Erik Storkebaum (Max Planck Institute for Molecular
Biomedicine, Germany) highlight the features of the Drosophila model of CMT associated
Preface xi
with mutations in YARS, which aminoacylates tyrosyl-tRNA with tyrosine. The authors
underline a series of experiments, the results of which suggest unexpectedly that loss of
aminoacylation activity per se is not necessary to cause peripheral motor and sensory
neuropathy, although the possibility that altered subcellular localization of aminoacylation-
active mutants could lead to defects in local protein synthesis and terminal axonal
degeneration cannot be excluded at the present moment. Current evidence suggests that the
disease may be caused by a gain-of-toxic function mechanism, the molecular nature of which
remains elusive. The future use of Drosophila CMT models in genetic screens for disease-
modifying genes may be of great value to unravel the molecular mechanisms of disease, and
to identify possible therapeutic targets.
Chapter Six focuses on spinal and bulbar muscular atrophy (SBMA) or Kennedy’s
disease, a progressive X-linked motor neuron disorder arising from the build-up of toxic
aggregates due to an abnormal expansion of the glutamine tract in the androgen receptor (AR)
gene as well as loss of the endogenous function of the AR. SBMA forms part of an ensemble
of neurodegenerative disorders referred to as polyglutamine (polyQ) diseases which, although
affecting different neuronal subtypes, share several features including an earlier disease onset
and a more acute disease progression, the longer the glutamine expansion. Interestingly,
polyQ-expanded AR is necessary to induce motor impairment, hence suggesting a gain-of-
function by the pathogenic AR in motor neurons and in this regard, patients with loss-of-
function mutations in the AR gene only show androgen insensitivity.
Fly models of SBMA were key to establish that toxicity is dependent on glutamine length
as well as the ligand-dependent activation of the AR, hence, flies that ectopically express the
human AR with either a non-toxic glutamine tract or with an expanded glutamine tract in the
absence of dihydrotestosterone exhibit no neurodegeneration or motor defects. Activation
gives the mutant AR unrestrained access to the nucleus where it is thought to alter numerous
processes. In this context, restricting the mutant AR to the cytosol by genetic manipulation
strategies in the fly was shown to abolish toxicity. Furthermore, Drosophila studies, including
genetic screens for modifiers of polyQ-induced eye degeneration (‘rough-eye phenotype’),
revealed several cellular pathways such as gene expression, axonal trafficking and
mitochondrial physiology that are affected by the disease. Considering that a myriad of
cellular mechanisms sustain a negative impact from a polyQ pathology and highlighting
evidence from SBMA model organisms, Adrienne M. Wang (University of Washington,
USA) makes the case for the stimulation of the cell’s innate protein quality control pathways
as one of the best therapeutic approaches aimed at clearing the mutant protein upstream of its
toxic effects.
Chapter Seven addresses spinal muscular atrophy (SMA) which is the most common
autosomal recessive disorder in the population following cystic fibrosis. The causative gene is
the survival of motor neuron 1 (SMN1), where its homozygous loss in patients with SMA
leads to a situation of low SMN levels resulting from a partially-functional duplicate gene,
SMN2. SMN2 copy number is inversely correlated with disease severity and in this regard,
SMA is usually classified into three types. SMN forms a multimeric complex that participates
in the cytoplasmic phase of spliceosomal Uridine-rich small nuclear ribonucleoprotein
biogenesis. Stuart J. Grice and colleagues (University of Oxford, UK and University of North
Carolina, USA) review the present fly models of SMA by giving a thorough description of
the Smn mutant and transgenic flies that were generated so far. Importantly, the authors
highlight the developmental defects observed in Drosophila SMA models including alteration
Ruben J. Cauchi xii
in P body organisation and nuclear architecture in the Smn knockout germline; growth
defects, stem cell defects, abnormal neuromuscular junction morphology and reduced motor
function at the larval stage; and, flight defects as well as muscle atrophy in the adult stage.
Importantly, this chapter discusses the recent thrilling findings in Drosophila, which shed
light on the selective vulnerability of motor systems. Interestingly, such studies introduce a
concept that was first observed in ALS, specifically, the possibility that a subpopulation of
neurons might be prone to degeneration as a result of alterations in the function of neuronal
circuits that impinge onto these neurons.
The final chapter (Chapter Eight) of this collection revolves around the application of
Drosophila in the conduction of genetic screens aimed at identifying novel genes that cause
motor neuron degeneration or finding modifiers – enhancement or suppression – of the
phenotype resulting from disruption of MND causative genes. Both approaches hold promise
to decipher the molecular mechanisms underpinning both normal physiology and
pathophysiology. Patrik Verstreken and colleagues (KU Leuven, Belgium) first highlight the
key features of the screen phenotype as well as discussing some of the phenotypes that are
amenable to genetic screens such as lifespan, behaviour (crawling of larvae during the larval
stage and climbing or flight during the adult stage), retinal morphology, electroretinogram
(ERG) recordings and NMJ architecture.
Through the use of engaging diagrams, the authors also discuss different screening
strategies including classic genetic screens using either Ethyl Methane Sulphonate (EMS)-
based or transposable element (TE)-based mutagenesis; clonal genetic screens, where
homozygous tissue is generated in an otherwise heterozygous animal, hence allowing the
investigator to assess the phenotypes of genes required for organismal viability; dominant
genetic screens, which aim at identifying dominant modifiers of a phenotype; and, UAS/Gal4-
based screens, whereby the use of the potent UAS/Gal4 system enables researchers to
manipulate gene expression through either overexpression or RNAi-mediated knockdown in a
spatially- and temporally-controlled manner. This account also includes examples of genetic
screens that were successful in fly models of MND including SMA, SBMA and ALS.
Furthermore, the plethora of genetic tools presently available are adequately described, and
exploiting their use will undoubtedly help us to further understand the molecular mechanisms
giving rise to MNDs. Importantly, the ability to conduct high-throughput genetic screens with
relative ease augurs well for the future application of high-throughput pharmacological
screens aimed at identifying novel therapeutics, which can be considered as the final frontier
in MND research.
Whilst editing this assemblage of stimulating works, I confess that I have learned several
interesting things even though I have been in the ‘business’ for quite some time. In this
regard, I am definitely convinced that this timely collection will be welcomed not only by
Drosophilists and MND aficionados alike but also by newcomers to the field. Whilst thanking
wholeheartedly the authors for their expert contribution, I would like to invite the reader to
enjoy this volume, an inspiring look at the indispensability of the fruit fly, and of model
organisms in general, to neuroscience research.
Ruben J. Cauchi
Dept. of Physiology & Biochemistry,
Faculty of Medicine & Surgery,
University of Malta, MALTA G.C.
January 2013
Preface xiii
The author’s laboratory acknowledges the support of research grants from the University
of Malta and Malta’s National Research & Innovation Programme.
In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 1
GENETICS OF MOTOR NEURON DISORDERS:
FROM GENE DIVERSITY
TO COMMON CELLULAR CONSPIRATORS
IN SELECTIVE NEURONAL KILLING
Rebecca K. Sheean and Bradley J. Turner Florey Institute of Neuroscience and Mental Health, University of Melbourne,
Parkville, Victoria, Australia
ABSTRACT
Motor neuron disorders (MNDs) are a spectrum of progressive and incapacitating
neurological diseases leading to motor impairment, disability and in most cases death.
The clinical phenotype of MNDs depends on the relative degeneration of (a) upper and
lower motor neurons and (b) their proximal cell bodies and distal axons. Despite limited
progress in the search for effective therapies, there has been recent and explosive
discovery of dozens of causative genes in MNDs. This arsenal of genetic evidence has
illuminated key cellular pathways disrupted in motor neurons, axons and supporting
ensheathing glial cells, providing fresh insights into determinants of neuronal
susceptibility and loss in MNDs. Strikingly, most of these genes encode ubiquitously
expressed and fundamental proteins that, when defective, provoke selective motor neuron
killing. In this chapter, we provide an introduction to the core clinical and pathological
features of upper motor neuron disorders (primary lateral sclerosis and hereditary spastic
paraplegia); lower motor neuron disorders (progressive muscular atrophy, progressive
bulbar palsy, spinal muscular atrophy, spinal bulbar muscular atrophy, axonal Charcot-
Marie-Tooth disease which significantly overlaps with distal hereditary motor
neuropathy, and lethal congenital contracture syndrome); and finally amyotrophic lateral
sclerosis with combined upper and lower motor neuron loss. We provide a systematic
summary of MND causative genes and functionally group them according to six
dominant pathogenic themes established and emerging in MNDs: (1) oxidative stress and
Correspondence: Bradley Turner, Florey Institute of Neuroscience and Mental Health, Kenneth Myer Building,
University of Melbourne, Parkville, Victoria 3010, Australia. Tel: +61 3 90356521, Fax: +61 3 93470446,
Email: [email protected].
Rebecca K. Sheean and Bradley J. Turner 2
mitochondrial dysfunction, (2) dysregulated intracellular membrane trafficking, (3)
abnormal axonal transport and cytoskeleton, (4) defective RNA processing, (5) impaired
proteostasis, and (6) defective myelination and lipid metabolism. We also highlight the
seminal contribution of transgenic, knockout and mutant mice in elucidating MND gene
function and testing disease hypotheses and therapeutic interventions, which are
complemented by other model systems such as Drosophila.
Keywords: Amyotrophic lateral sclerosis; Charcot-Marie-Tooth disease; hereditary spastic
paraplegia, spinal muscular atrophy; spinal bulbar muscular atrophy
ABBREVIATIONS
AD, autosomal dominant
ALS, amyotrophic lateral sclerosis
AR, autosomal recessive
CMT, Charcot-Marie-Tooth disease
CST, corticospinal tract
dHMN, distal hereditary motor neuropathy
DI, dominant intermediate
FALS, familial amyotrophic lateral sclerosis
FTD, frontotemporal dementia
FTDP, frontotemporal dementia-Parkinsonism
HNA, hereditary neuralgic amyotrophy
HSP, hereditary spastic paraplegia
LCCS, lethal congenital contracture syndrome
LMN, lower motor neuron
PBP, progressive bulbar palsy
PLS, primary lateral sclerosis
PMA, progressive muscular atrophy
RI, recessive intermediate
SALS, sporadic amyotrophic lateral sclerosis
SBMA, spinal bulbar muscular atrophy
SMA, spinal muscular atrophy
SPG, spastic paraplegia
X-linked D, X-linked dominant
X-linked R, X-linked recessive
INTRODUCTION
Motor neuron disorders (MNDs) are a collection of clinically and pathologically
heterogeneous neurological diseases characterised by progressive and selective dysfunction
and degeneration of motor neurons. They strike adults, children and infants, occurring in
sporadic and hereditary forms, causing progressive motor disability and in most cases
premature death. The nature and progression of clinical symptoms depends on the relative
Genetics of Motor Neuron Disorders 3
involvement of two groups of neurons primarily affected, upper motor neurons (UMNs) and
lower motor neurons (LMNs). UMNs, also known as giant pyramidal cells or Betz cells, with
diameters up to 100 m, are located in laminar sheets of layer V of the primary motor cortex
(Brodmann area 4) and project long axons to the brainstem and spinal cord through pathways
such as the corticobulbar and corticospinal tracts (CST) (Ravits and La Spada, 2009). These
descending pathways synapse directly, or in most cases indirectly via interneurons, with
LMNs in humans. LMNs, also called anterior horn cells in the spinal cord, with diameters
ranging 35-100 m, are stacked into columns in motor nuclei in the tegmentum of the
brainstem and ventral horns of the spinal cord and send axons to innervate skeletal muscles
(Ravits and La Spada, 2009).
Table 1. Motor neuron disorders classification and clinical features
MN
involvement
Disease Onset Duration Prevalence Clinical features
UMN HSP Childhood Non-fatal 2-5/100,000 Progressive lower limb
spasticity
PLS 50 years 20 years 1/100,000 Slowly progressive limb
and bulbar spasticity
LMN PMA 45-65 years 5-10 years 1/100,000 Slowly progressive
weakness and atrophy
PBP 50-70 years 1-3 years 5-6/100,000 Progressive bulbar
weakness
SMA 6 months 2 years 1/6,000 Symmetrical weakness
and atrophy
SBMA 30-60 years Non-fatal 1-2/100,000 Limb and bulbar
weakness and atrophy,
gynecomastia
CMT/dHMN
Adolescence Non-fatal 1/2,500 Distal lower limb
weakness and
atrophy
LCCS Foetal 10-weeks 1/25,000 Severe muscle atrophy,
foetal hydrops and
akinesia
UMN and
LMN
ALS 45-65 years 2-3 years 7/100,000 Rapidly progressive
spasticity, weakness,
atrophy and paralysis
Abbreviations: ALS, amyotrophic lateral sclerosis; CMT, Charcot-Marie-Tooth disease; dHMN, distal
hereditary motor neuropathy; HSP, hereditary spastic paraplegia; LCCS, lethal congenital
contracture syndrome; LMN, lower motor neuron; PBP, progressive bulbar palsy; PLS, primary
lateral sclerosis; PMA, progressive muscular atrophy; SBMA, spinal bulbar muscular atrophy;
SMA, spinal muscular atrophy.
Rebecca K. Sheean and Bradley J. Turner 4
LMNs are divided into three functional classes: -motor neurons which innervate skeletal
muscle fibres and drive contraction and voluntary movement, -motor neurons which
innervate muscle spindles and sense motor control, and -motor neurons which innervate
both muscle fibres and spindles (Kanning et al., 2010). -motor neurons which are the most
abundant are further classed into fast-fatiguable, fast fatigue-resistant and slow subtypes,
reflecting the contractile properties of their target muscle fibres (Kanning et al., 2010).
Interestingly, fast-fatiguable -motor neurons with large somas and large diameter fast
conducting axons are most vulnerable in MNDs (Pun et al., 2006). MNDs are classified
according to the nerve cells affected whether UMN, LMN or both and the resulting clinical
signs (Table 1). UMN damage is manifested by spasticity and pathological reflexes such as
Babinski and Hoffman signs (Talbot, 2009).
These symptoms arise primarily from the loss of descending motor control and inhibition
of spinal cord reflexes. In contrast, LMN degeneration results in muscle weakness, atrophy,
paralysis and fasciculation caused by chronic muscle denervation (Talbot, 2009). Pure UMN
disorders are hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS). Pure
LMN involvement occurs in progressive muscular atrophy (PMA), progressive bulbar palsy
(PBP), spinal muscular atrophy (SMA), spinal bulbar muscular atrophy (SBMA), Charcot-
Marie-Tooth (CMT) disease which occurs in demyelinating (CMT1 and CMT4) or axonal
forms (CMT2), the latter significantly overlapping with distal hereditary motor neuropathy
(dHMN), and lethal congenital contracture syndrome (LCCS). Finally, amyotrophic lateral
sclerosis (ALS) is distinguished by both UMN and LMN degeneration. ALS, PLS, PMA and
PBP represent the spectrum of motor neuron diseases which are defined by adult-onset and
progressive symptoms that are uniformly fatal and pathologically distinct from SMA, SBMA,
CMT and LCCS, the key differences being the presence of neuronal ubiquitinated inclusions
commonly containing TAR DNA binding protein 43 (TDP-43) and extensive gliosis. In this
chapter, we will refer to all the diseases listed in Table 1 as MNDs which encompasses motor
neuron diseases.
1. AMYOTROPHIC LATERAL SCLEROSIS
ALS, also known as Lou Gehrig's disease in North America or MND in Commonwealth
nations, is the most common motor neuron disease accounting for 85% of cases (Talbot,
2002). It is characterised by a combination of muscle weakness, wasting and spasticity with
rapid progression to respiratory paralysis and death usually within 2-3 years from diagnosis
(Talbot, 2009). Approximately one-third of patients present with lower-limb onset symptoms,
one-third with upper-limb symptoms, and one-third with bulbar-onset (Talbot, 2009). In cases
of limb-onset ALS, symptoms often appear focal and asymmetrical and spread outwardly,
laterally and ascendingly, suggesting dissemination of pathology (Ravits and La Spada,
2009). Although ALS involves death of both UMNs and LMNs, there is evidence for a dying-
back axonopathy preceding cell body loss, at least for LMNs (Fischer et al., 2004). Motor
neuron loss is also associated with cytoplasmic accumulation of misfolded proteins, notably
TDP-43 in over 90% of ALS cases (Neumann et al., 2006) and superoxide dismutase 1
(SOD1) in approximately 50% of ALS cases examined (Bosco et al., 2010).
Genetics of Motor Neuron Disorders 5
Table 2. Familial ALS genes and cellular functions
Type
Inheritance Onset Gene Protein Cellular function Reference
ALS1 AD Adult SOD1 Superoxide
dismutase 1
Oxidative stress (Rosen et
al., 1993)
ALS2 AR Juvenile ALS2 ALS2/alsin Endosomal
trafficking
(Hadano et
al., 2001;
Yang et al.,
2001)
ALS4 AD Juvenile SEXT Senataxin RNA processing (Chen et al.,
2004)
ALS5 AR Juvenile SPG11 Spatacsin Endosomal
trafficking
(Stevanin et
al., 2007)
ALS6 AD Adult FUS Fused in sarcoma RNA processing (Kwiatkows
ki et al.,
2009; Vance
et al., 2009)
ALS8 AD Adult VAPB Vesicle-associated
protein B
ER and Golgi
trafficking
(Nishimura
et al., 2004)
ALS9 AD Adult ANG Angiogenin RNA processing (Greenway
et al., 2006)
ALS10 AD Adult TARDBP TAR DNA
binding protein 43
RNA processing (Sreedharan
et al., 2008)
ALS11 AD Adult FIG4 Factor induced
gene 4
Endosomal
trafficking
(Chow et al.,
2009)
ALS12 AD Adult OPTN Optineurin Golgi and
endosomal
trafficking
(Maruyama
et al., 2010)
ALS13 AD Adult ATXN2 Ataxin-2 RNA processing (Elden et al.,
2010)
ALS14 AD Adult VCP Valosin-
containing protein
Ubiquitin-
proteasome
degradation
(Johnson et
al., 2010)
ALS15 X-linked D Adult UBQLN2 Ubiquilin 2 Ubiquitin-
proteasome
degradation
(Deng et al.,
2011)
ALS16 AD Adult SIGMAR1 non-opioid
intracellular
receptor 1
ER trafficking (Al-Saif et
al., 2011)
ALS-
FTD2
AD Adult C9ORF72 C9ORF72 RNA processing (DeJesus-
Hernandez
et al., 2011;
Renton et
al., 2011)
ALS-
FTD3
AD Adult CHMP2B Charged
multivesicular
body protein 2B
Endosomal
trafficking
(Parkinson
et al., 2006)
ALS-
FTDP
AD Adult MAPT Microtubule-
associated protein
tau
Cytoskeleton (Hutton et
al., 1998)
Rebecca K. Sheean and Bradley J. Turner 6
Table 2. (Continued)
Type
Inheritance Onset Gene Protein Cellular function Reference
ALS AD Adult DCTN1 Dynactin 1 Axonal transport (Puls et al.,
2003)
ALS
AD Adult DAO D-amino acid
oxidase
Translation (Mitchell et
al., 2010)
ALS AD Adult SQSTM1 Sequestosome 1 Ubiquitin-
proteasome
degradation
(Fecto et al.,
2011)
ALS
AD Adult PFN1 Profilin 1 Cytoskeleton (Wu et al.,
2012)
Abbreviations: ALS, amyotrophic lateral sclerosis; AD, autosomal dominant; AR, autosomal recessive;
FTD, frontotemporal dementia; FTDP, frontotemporal dementia-Parkinsonism; X-linked D, X-
linked dominant.
The finding of TDP-43 pathology in affected neurons in frontotemporal dementia (FTD)
and co-occurrence of ALS-FTD strongly suggests that ALS represents one extreme of a
clinical spectrum with a similar underlying pathological process, the other extreme being
FTD (Neumann et al., 2006). PLS and PMA (or Duchenne-Aran disease) are rare variants of
motor neuron disease which account for 1-2% and 4% of cases, respectively (Talbot, 2009).
Both are slowly progressive disorders involving predominantly spastic paresis in PLS and
muscle wasting in PMA. PBP is also regarded as distinct from bulbar-onset ALS due to
weakness that rapidly generalises to limbs (Talbot, 2009). While PLS, PMA and PBP are
considered subtypes of motor neuron disease, they may also be phenotypic manifestations of
ALS. It is well established that 5-10% of ALS cases are inherited. To date, 25 loci and 20
genes have been identified in familial ALS (FALS) involving predominantly autosomal
dominant inheritance (Table 2). Most of these genes encode proteins involved either in
intracellular membrane and organelle trafficking or RNA processing and regulation. They are
summarised here according to function and cellular pathways implicated in ALS.
1.1. Common Pathogenic Mechanisms in ALS
1.1.1. Oxidative Stress and Mitochondrial Dysfunction
Mutations in SOD1 were first identified in FALS (Rosen et al., 1993) and over 160
mutations have been reported to date (Al-Chalabi et al., 2012). SOD1 mutations account for
20% of FALS and some sporadic ALS (SALS) cases. SOD1 is a ubiquitously expressed
enzyme that binds copper and zinc ions and degrades superoxide anions produced by normal
mitochondrial metabolism. There are two main proposals for how mutant SOD1 causes motor
neuron damage in ALS. Firstly, mutations induce misfolding of SOD1 leading to abnormal
catalysis and copper-mediated chemistry, producing reactive oxygen species that damage
motor neurons (Wiedau-Pazos et al., 1996). Secondly, mutant SOD1 misfolding promotes
formation of protein aggregates that are injurious to motor neurons (Bruijn et al., 1998).
Irrespective of the primary mechanism of toxicity, tissue markers of oxidative damage and
mitochondrial dysfunction feature prominently in spinal cords of transgenic mice expressing
Genetics of Motor Neuron Disorders 7
ALS-linked SOD1 mutations (Turner and Talbot, 2008), suggesting that oxidative stress
contributes to motor neuron degeneration in ALS.
1.1.2. Dysregulated Intracellular Membrane Trafficking
A number of causative genes have been identified in ALS which regulate endosomal
trafficking. In 2001, two groups identified mutations in ALS2 encoding the protein alsin
(Hadano et al., 2010; Yang et al., 2001). Alsin is a guanine nucleotide exchange factor for
Rab5 which is localised to early endosomes and involved in endocytosis and endocytic
trafficking. Mutations in alsin commonly ablate its vacuolar protein sorting 9 domain required
for Rab5 interaction. ALS2 null mice show only mild subclinical motor dysfunction, however
aberrant endosome and vesicle trafficking was observed, as well as a decrease in Rab5-
dependent endosome fusion and accumulation of early endosomes (Devon et al., 2006;
Hadano et al., 2006). Mutations in FIG4 encoding factor induced gene 4 have also been
identified in ALS (Chow et al., 2009). FIG4 regulates the abundance of phosphatidylinositol
3,5-bisphosphate, a signalling lipid located on late endosome membranes, and retrograde
trafficking of endosomes from the trans-Golgi network. Mutations in charged multivesicular
body protein 2B (CHMP2B) in FTD have been reported (Skibinski et al., 2005) and more
recently have been identified in ALS patients (Parkinson et al., 2006). CHMP2B is a
component of the endosomal sorting complex required for transport (ESCRT) which sorts
cargoes to form multivesicular bodies. Mutant CHMP2B expression in transgenic mice
triggers progressive axonal degeneration and accumulation of neuronal inclusions containing
p62, TDP-43 and CHMP2B (Ghazi-Noori et al., 2012). At least 4 genes linked to ALS are
involved in ER-Golgi trafficking. SIGMAR1 encodes an ER chaperone that is mutated in ALS
(Al-Saif et al., 2011), in addition to mutations in OPTN encoding optineurin, a key regulator
of membrane trafficking, exocytosis and Golgi-lysosome trafficking (Maruyama et al., 2010).
Mutations have also been identified in SPG11, encoding spatacsin, which have been linked to
ALS with juvenile onset (Orlacchio et al., 2010) and are discussed further in the HSP section
of this chapter. Finally, VAPB which encodes vesicle-associated protein B involved in ER-
Golgi trafficking is mutated in ALS (Nishimura et al., 2004).
1.1.3. Abnormal Axonal Transport and Cytoskeleton
Three genes associated with axonal transport are associated with ALS. A mutation in the
gene encoding the axonal transport protein, dynactin (DCTN1), which is important for
retrograde axonal transport, was identified in ALS patients, linking disruption of axonal
transport to ALS pathology (Puls et al., 2003). Mutations in DCTN1 are predicted to induce
its misfolding, reducing interaction of the dynactin complex with microtubules, thus slowing
axonal transport. Microtubule-associated protein tau (MAPT) has been linked to a number of
neurodegenerative diseases such as Alzheimer’s disease. Tau-positive inclusions have also
been reported in frontotemporal dementia and parkinsonism linked to chromosome 17
(FTDP), a disease that closely resembles ALS-parkinsonism-dementia complex, and
mutations in MAPT have been identified in FTDP patients (Hutton et al., 1998). These
mutations result in an imbalance in the ratio of tau isoforms and are hypothesized to
destabilise microtubules, leading to impaired axonal transport. Most recently, mutations in
profilin 1 (PFN1) were identified in ALS (Wu et al., 2012). Profilin 1 promotes actin
polymerisation and formation of microfilaments which are vital for cytoskeletal function.
Rebecca K. Sheean and Bradley J. Turner 8
Mutant PFN1 impairs axonal outgrowth and promotes formation of ubiquitinated aggregates
containing TDP-43 (Wu et al., 2012).
1.1.4. Defective RNA Processing
The discovery that TDP-43 is the major component of ubiquitinated inclusions in ALS
(Neumann et al., 2006) drew attention to the contribution of RNA processing defects in ALS.
Links between RNA metabolism and ALS had been formed prior to this, through discovery of
mutations in genes encoding senataxin (SETX) and angiogenin (ANG) (Chen et al., 2004;
Greenway et al., 2006). Mutations in senataxin cause juvenile onset ALS through a
mechanism that may disrupt its DNA/RNA helicase activity involved in transcription (Chen
et al., 2004). Angiogenin is involved in rRNA transcription and regulates expression of
neurotrophic factors such as vascular endothelial growth factor (VEGF). Mutations in ANG
cause aberrant ribosomal transcription, reduced VEGF expression and subsequently, a
reduction in cell repair and proliferation (Greenway et al., 2006). Spurred on by the discovery
of TDP-43 pathology in ALS (Neumann et al., 2006), mutations in TARDBP were soon
identified in ALS (Sreedharan et al., 2008). TDP-43 contains two RNA recognition sites,
nuclear import and localisation sequences and glycine-rich region, with majority of the
known mutations located within the glycine-rich region. TDP-43 is predicted to have a
number of RNA processing functions including regulation of gene expression, transcription
and splicing and microRNA processing. Mutations can result in both a hyper-phosphorylated,
high molecular weight product (45 kDa) and a C-terminal fragment (25 kDa) and cause the
mislocalisation of TDP-43 from the nucleus to cytoplasm (Neumann et al., 2006). TDP-43
depletion from mouse brain causes widespread and abnormal transcription and RNA splicing
defects affecting multiple gene targets (Polymenidou et al., 2011; Tollervey et al., 2011). A
number of these targets have also been identified and include the RNA/DNA binding protein,
fused in sarcoma (FUS) which is also mutated in ALS (Kwiatkowski et al., 2009; Vance et
al., 2009). FUS is a nucleoprotein containing an RNA binding motif, RGG-rich repeat,
transcriptional activation and zinc-ring finger domains which are important for RNA
processing (Vance et al., 2009). A number of similarities exist between TDP-43 and FUS,
with both proteins involved in transcriptional regulation and processing of mRNA and
microRNA. Mutations in FUS also result in its nuclear exclusion which leads to formation of
cytoplasmic FUS-containing stress granules, likely to cause a number of RNA processing
defects (Kwiatkowski et al., 2009). In addition to these DNA/RNA binding proteins,
mutations in the gene encoding D-amino acid oxidase (DAO) were reported in ALS (Mitchell
et al., 2010). DAO is responsible for catabolism of D-amino acids including D-serine and
mutations promote excess D-serine levels which may result in over-activation of NMDA
receptors leading to excitotoxicity. Non-coding gene expansions in Ataxin 2 (Elden et al.,
2010) and very recently C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2011) have
also been identified in ALS. Ataxin 2 contains a polyglutamine (polyQ) repeat and expansion
in this region can cause spinocerebellar ataxia type 2 (SCA2) (Lorenzetti et al., 1997).
However, intermediate length expansions (27-33 repeats) are associated with ALS which
promote ataxin 2 interaction with TDP-43 to modify its toxicity (Elden et al., 2010). More
recently, two groups identified an expansion in the non-coding GGGGCC hexanucleotide
repeat within C9ORF72 (DeJesus-Hernandez et al., 2011; Renton et al., 2011). This
expansion was shown to cause both ALS and FTD phenotypes and accounts for ~40% of
FALS, which is the most common genetic cause identified to date (Renton et al., 2011).
Genetics of Motor Neuron Disorders 9
Although the function of C9ORF72 is currently unknown, the expansion results in a loss of
one of the splice variants of C9ORF72 which causes the formation of RNA foci in the nucleus
(DeJesus-Hernandez et al., 2011). Like most disease-associated repeat expansions, the
C9ORF72 expansion is considered to create an accumulation of toxic mRNA and lead to
disturbances in transcription and other important RNA processes.
1.1.5. Impaired proteostasis
Proteostasis refers to protein homeostasis which is the sum of biogenesis, folding,
trafficking and degradation of intracellular and extracellular proteins. Mutations in valosin-
containing protein (VCP) which is required for maturation of autophagosomes were reported
in ALS, linking dysfunction of autophagy to motor neuron loss (Johnson et al., 2010).
Sequestosome 1 (SQSTM1), also known as p62, is another key regulator of protein
degradation, playing a dual role in both proteasome and autophagic degradation. SQSTM1 is
a ubiquitin binding protein that is found in neuronal inclusions in ALS patients (Mizuno et al.,
2006), and thus was an obvious candidate gene for ALS. This was confirmed by identification
of SQSTM1 mutations in ALS, which were predicted to cause a toxic gain-of-function,
increasing protein-protein interactions and causing deregulation of autophagic signalling
cascades (Fecto et al., 2011). Finally, mutations in the ubiquitin-like protein, ubiquilin 2
(UBQLN2) cause ALS, with UBQLN2 localised to protein inclusions of spinal cords in both
FALS and SALS patients (Deng et al., 2011). UBQLN2 is implicated in regulation of
ubiquitin-dependent protein degradation, with mutations impairing protein clearance and
enhancing inclusion formation.
2. HEREDITARY SPASTIC PARAPLEGIA
HSP, also known as familial spastic paraplegia or Strumpell-Lorrain disease, refers to a
large group of inherited disorders characterised by progressive muscle stiffness, contraction
and weakness predominantly in lower limbs, while upper limbs are largely spared
(Blackstone, 2012).
Table 3. HSP genes and cellular functions
Type Inheritance Gene Protein Cellular function(s) Reference
SPG1
X-linked R L1CAM L1 cell adhesion molecule Cytoskeleton (Jouet et al.,
1994)
SPG2
X-linked R PLP1 Myelin proteolipid protein 1 Myelination (Saugier-Veber
et al., 1994)
SPG3A AD ATL1 Atlastin 1 ER trafficking (Zhao et al.,
2001)
SPG4
AD SPAST Spastin ER and endosome
trafficking,
cytoskeleton
(Hazan et al.,
1999)
SPG5A AR
CYP7B1 Cytochrome P450 family 7
subfamily b polypeptide 1
Myelination, lipid
metabolism
(Tsaousidou et
al., 2008)
SPG6
AD NIPA1 Non-imprinted in Prader-
Willi/Angleman syndrome
region protein 1
Endosomal trafficking (Rainier et al.,
2003)
Rebecca K. Sheean and Bradley J. Turner 10
Table 3. (Continued)
Type Inheritance Gene Protein Cellular function(s) Reference
SPG7 AR SPG7 Paraplegin Mitochondrial
function
(Casari et al.,
1998)
SPG8 AD KIAA0196 Strumpellin Endosomal
trafficking,
cytoskeleton
(Valdmanis et
al., 2007)
SPG10 AD KIF5A Kinesin family member 5A
Axonal transport,
cytoskeleton
(Reid et al.,
2002)
SPG11 AR SPG11 Spatacsin
Endosomal trafficking (Stevanin et al.,
2007)
SPG12
AD RTN2 Reticulon 2 ER trafficking (Montenegro et
al., 2012)
SPG13
AD HSPD1 Heat shock protein 60 Mitochondrial
function
(Hansen et al.,
2002)
SPG15 AR ZFYVE26 Spastizin Endosomal trafficking (Hanein et al.,
2008)
SPG17
AD BSCL2 Seipin ER trafficking, lipid
metabolism
(Windpassinger
et al., 2004)
SPG18
AR ERLIN2 Erlin 2 ER and membrane
trafficking, ubiquitin-
proteasome system
(Alazami et al.,
2011)
SPG20
AR SPG20 Spartin Endosomal
trafficking, lipid
metabolism
(Patel et al.,
2002)
SPG21 AR SPG21 Maspardin Endosomal trafficking (Simpson et al.,
2003)
SPG22 X-linked R SLC16A2 Solute carrier family 16
member 2
Membrane trafficking
(Bohan et al.,
2004)
SPG31
AD REEP1 Receptor-expression
enhancing protein 1
ER trafficking,
cytoskeleton
(Zuchner et al.,
2006)
SPG39
AR PNPLA6 Neuropathy target esterase Lipid metabolism (Rainier et al.,
2008)
SPG35
AR FA2H Fatty acid 2-hydroxylase Myelination, lipid
metabolism
(Dick et al.,
2010)
SPG42
AD SLC33A1 Acetyl-CoA transporter Lipid metabolism (Lin et al.,
2008)
SPG44
AR GJC2 Connexin 47 Myelination (Orthmann-
Murphy et al.,
2009)
SPG47
AR AP4B1 Adaptor-related protein
complex 4,beta 1 subunit
Endosomal trafficking (Bauer et al.,
2012)
SPG48
AR AP5Z1 Adaptor-related protein
complex 5, zeta 1 subunit
Endosomal trafficking (Słabicki et al.,
2010)
SPG50
AR AP4M1 Adaptor-related protein
complex 4, mu 1 subunit
Endosomal trafficking (Verkerk et al.,
2009)
SPG51
AR AP4E1 Adaptor-related protein
complex 4, epsilon 1 subunit
Endosomal trafficking (Moreno-De-
Luca et al.,
2011)
SPG52
AR AP4S1 Adaptor-related protein
complex 4, sigma 1 subunit
Endosomal trafficking (Abou Jamra et
al., 2011)
Abbreviations: HSP, hereditary spastic paraplegia; AD, autosomal dominant; AR, autosomal recessive;
SPG, spastic paraplegia; X-linked R, X-linked recessive.
Genetics of Motor Neuron Disorders 11
HSP occurs in pure spastic or complicated forms, the latter involving neurological
symptoms that may include cerebellar ataxia, dementia, mental retardation and sensory
deficits (Harding, 1983). The core pathological feature of HSP is the selective degeneration of
the longest CST axons which innervate the lower extremities. HSP affects distal axons and
there is little neuronal death, thus conforming to a distal axonopathy, and most patients have
normal lifespans (Blackstone, 2012). HSPs are the most genetically diverse of MNDs with
nearly 50 distinct loci and 30 genes identified to date, involving X-linked recessive,
autosomal dominant or autosomal recessive inheritance (Table 3). Most of these genes encode
proteins involved in intracellular membrane trafficking, cytoskeletal function, organelle
shaping and lipid metabolism in axons. We group and discuss them below according to
function and cellular pathways implicated in HSP pathogenesis.
2.1. Common Pathogenic Mechanisms in HSP
2.1.1. Oxidative Stress and Mitochondrial Dysfunction
Paraplegin and heat shock protein 60 (HSP60) are mitochondrial matrix proteases and
chaperones, respectively, involved in regulation of mitochondrial protein quality control
(Casari et al., 1998; Hansen et al., 2002). HSP patients with paraplegin mutations show
abnormal mitochondrial morphology and energetic defects in skeletal muscle (Casari et al.,
1998), while paraplegin knockout mice develop gait abnormalities preceded by distal
degeneration of central and peripheral axons, accumulation of mitochondria in axonal
swellings and slowing of retrograde axonal transport (Ferreirinha et al., 2004). The phenotype
of HSP60 deficient mice although masked by embryonic lethality, was attributed to
mitochondrial dysfunction early in development (Christensen et al., 2010).
2.1.2. Dysregulated Intracellular Membrane Trafficking
The majority of HSP genes encode proteins involved in endosomal trafficking, such as
NIPA1, strumpellin, spatacsin, spastizin, spartin and maspardin. NIPA1 is an integral
membrane protein localised to endosomes involved in receptor signalling coupled to axonal
growth and mutations have been implicated in abnormal bone morphogenic protein (BMP)
signaling (Rainier et al., 2003). Strumpellin is an endosomal protein containing spectrin
repeats which interact with ankyrin, suggesting roles in stabilising the cell membrane-
cytoskeletal network and vesicle trafficking (Valdmanis et al., 2007). Spatacsin and spastizin
are found in the ER and endosomes with putative roles in endocytic trafficking, particularly
of inositol 1,4,5-trisphosphate receptors (IP3Rs) required for calcium signalling in neurons
(Hanein et al., 2008; Stevanin et al., 2007). Mutations in spartin which contains microtubule
and ESCRT-III interacting domains were predicted to disrupt neuronal endocytic trafficking
in HSP (Patel et al., 2002). Gene deletion of spartin results in progressive locomotor deficits
in adult mice and abnormal axonal branching of cortical neurons without evidence for LMN
loss (Renvoise et al., 2012). Lastly, maspardin which distributes to endosomes has putative
roles in vesicle trafficking between endocytic and trans-Golgi compartments (Simpson et al.,
2003). Maspardin deficient mice produce a similar phenotype to spartin knockouts, revealing
progressive hindlimb weakness and excessive branching of cortical neuron axons (Soderblom
et al., 2010), collectively implicating these two HSP genes as negative regulators of UMN
Rebecca K. Sheean and Bradley J. Turner 12
axon outgrowth. More recently, mutations in subunits of multiple adaptor protein complexes,
AP4 and AP5, involved in endocytic transport of vesicles, were found in HSP families
(Abou Jamra et al., 2011; Bauer et al., 2012; Moreno-De-Luca et al., 2011; Słabicki et al.,
2010; Verkerk et al., 2009). Interestingly, AP4 trafficks AMPA receptors to the cell surface
and HSP mutations were predicted to perturb glutamate-mediated signalling in neurons,
eliciting excitotoxicity (Verkerk et al., 2009). In mice deficient for AP4B, AMPA receptors
were mistrafficked to autophagosomes which accumulate in neurons and animals show motor
impairment (Matsuda et al., 2008). At least six HSP genes encoding atlastin 1, REEP1,
reticulon 2, seipen and erlin 2, are involved in ER formation, morphogenesis and associated
trafficking pathways. Atlastin 1 is an ER-localised GTPase belonging to the dynamin-related
superfamily of large GTPases and mutations are predicted to disrupt ER morphology and
axon outgrowth (Zhao et al., 2001b). REEP1, which interacts with atlastin 1, is involved in
receptor trafficking through the ER, binds microtubules and may play a role in maintaining
the ER network (Zuchner et al., 2006). Mice deficient for REEP1 develop adult-onset gait
abnormalities consistent with spasticity and axonal degeneration in the spinal cord (Beetz,
2010). Mutations in the prototypical ER shaping protein reticulon 2 were recently identified
in HSP, implicating abnormal ER morphogenesis in axonal degeneration (Montenegro et al.,
2012). BSCL2 or seipin is an ER integral membrane protein and HSP mutations disrupt its N-
linked glycosylation site leading to its aggregation in motor neurons (Windpassinger et al.,
2004). Transgenic mice expressing mutant seipin develop spastic motor deficits with
degeneration of peripheral axons and evidence of seipin-positive inclusions, ER stress and
impaired retrograde axonal transport (Yagi et al., 2011). Erlin 2 associates with lipid rafts and
is involved in ER-associated degradation of IP3Rs, implicating dysregulation of calcium
signalling in this form of HSP (Alazami et al., 2011). Finally, SLC16A2 which encodes a
monocarboxylic acid transporter at the plasma membrane may be linked to HSP (Bohan and
Azizi, 2004).
2.1.3. Abnormal Axonal Transport and Cytoskeleton
Spastin, L1CAM and KIF5A are key proteins involved in axon development, dynamics
and maintenance. Mutations in spastin, which account for 40% of autosomal-dominant HSP,
were predicted to disrupt its ATPase and microtubule binding domains required for
microtubule severing and cytoskeletal remodeling (Hazan et al., 1999). This was confirmed in
spastin deficient mice which develop progressive axonopathy with disruption of the
cytoskeleton and motor deficits, in the absence of neuronal loss (Tarrade et al., 2006).
L1CAM is a cell surface glycoprotein and adhesion molecule required for neuronal migration
and axon growth during development. Loss-of-function mutations in L1CAM identified in
HSP were proposed to abolish axonal pathfinding (Jouet et al., 1994). This was supported by
L1CAM knockout mice which show reduced length of CST and locomotor deficits (Dahme et
al., 1997). Lastly, mutations in the anterograde motor protein KIF5A underlie a form of HSP,
providing direct evidence of axonal transport defects leading to neurodegeneration (Reid et
al., 2002). Postnatal deletion of KIF5A in neurons provoked sensory neuron loss,
accumulation of neurofilament subunits in cell bodies and hindlimb paralysis (Xia et al.,
2003).
Genetics of Motor Neuron Disorders 13
Table 4. Axonal CMT disease genes and cellular functions
Type Inheritance Gene Protein Cellular function(s) Reference
CMT2A1 AD KIF1B Kinesin family member 1B
Axonal transport (Zhao et al.,
2001)
CMT2A2 AD MFN2
Mitofusin 2 Mitochondrial
function
(Zuchner et al.,
2004)
CMT2B AD RAB7A Ras-associated protein 7
Endosomal
trafficking
(Verhoeven et
al., 2003)
CMT2B1
AR LMNA Lamin A Cytoskeleton (De Sandre-
Giovannoli et
al., 2002)
CMT2B2
AR MED25 Mediator complex subunit 25 Transcription,
myelination
(Leal et al.,
2009)
CMT2C AD TRPV4
Transient receptor potential
cation channel, subfamily v,
member 4
Membrane
trafficking
(Auer-
Grumbach et
al., 2010)
CMT2D
AD GARS Glycyl-tRNA synthetase Translation (Antonellis et
al., 2003)
CMT2E
AD NEFL Neurofilament light chain Cytoskeleton (Mersiyanova
et al., 2000)
CMT2F AD HSPB1 Heat shock protein 27
Protein degradation (Evgrafov et
al., 2004)
CMT2H/K AD GDAP1 Ganglioside-induced
differentiation-associated
protein 1
Mitochondrial
function
(Baxter et al.,
2002; Cuesta et
al., 2002)
CMT2I/J AD MPZ Myelin protein zero
Myelination (De Jonghe et
al., 1999)
CMT2L AD HSPB8 Heat shock protein 22
Protein degradation (Irobi et al.,
2004)
CMT2M AD DNM2 Dynamin 2
Membrane
trafficking
(Zuchner et al.,
2005)
CMT2N AD AARS Alanyl-tRNA synthetase
Translation (Latour et al.,
2010)
CMT2O AD DYNC1H1 Dynein cytoplasmic heavy
chain 1
Axonal transport
(Weedon et al.,
2011)
CMT2P AR LRSAM1 Leucine-rich repeat and sterile
alpha motif containing 1
Protein degradation,
endosomal
trafficking
(Guernsey et
al., 2010)
CMT2Q
AD HARS Histidyl-tRNA synthetase Translation (Vester et al.,
2012)
CMTDIB AD DNM2 Dynamin 2
Membrane
trafficking
(Zuchner et al.,
2005)
CMTDIC AD YARS Tyrosyl-tRNA synthetase
Translation (Jordanova et
al., 2006)
CMTDID
AD MPZ Myelin protein zero Myelination (Mastaglia et
al., 1999)
CMTDIE
AD INF2 Inverted formin 2 Cytoskeleton (Boyer et al.,
2011)
CMTRIA
AR GDAP1 Ganglioside-induced
differentiation-associated
protein 1
Mitochondrial
function
(Nelis et al.,
2002)
CMTRIB
AR KARS Lysyl-tRNA synthetase Translation (McLaughlin et
al., 2010)
HNA
AD SEPT9 Septin 9 Cytoskeleton (Kuhlenbaumer
et al., 2005)
Abbreviations: ALS, AD, autosomal dominant; AR, autosomal recessive; CMT, Charcot-Marie-Tooth
disease; DI, dominant intermediate; HNA, hereditary neuralgic amyotrophy; RI, recessive
intermediate.
Rebecca K. Sheean and Bradley J. Turner 14
2.1.4. Defective Myelination and Lipid Metabolism
At least 6 HSP genes are linked to myelination and metabolism of lipids and sterols in
axons. Interestingly, 3 of these encoding proteolipid protein 1 (PLP1), connexin 47 and fatty
acid 2-hydroxylase, are expressed by oligodendrocytes, implicating non-cell autonomous
axonal degeneration in HSP. PLP1 is the major structural protein of CNS myelin and HSP
mutations were proposed to disrupt oligodendrocyte maturation and myelination (Saugier-
Veber et al., 1994). In mice either lacking PLP1 or harbouring spontaneous mutations in
PLP1 called jimpy and rumpshaker, oligodendrocyte development was normal, although there
was evidence for dysmyelination at late age (Klugmann et al., 1997; Schneider et al., 1992).
Connexin 47, together with connexin 43, are the main gap junction proteins expressed by
oligodendrocytes essential for proper maintenance of myelin and connexin 47 mutations
linked to HSP disrupt function of these heterotypic channels (Orthmann-Murphy et al., 2009).
Mice with deficiency of connexin 47 are viable, however combined deletion with connexin 32
triggers premature death resulting from severe demyelination and axonal loss (Menichella et
al., 2003). Lastly, mutations in fatty acid 2-hydroxylase which synthesises myelin
galactolipids cause a form of HSP (Dick et al., 2010) and mice lacking this gene develop CNS
demyelination and axonal degeneration (Potter et al., 2011).
CYP7B1 encodes an enzyme essential for cholesterol catabolism in the brain that is
mutated in HSP, providing direct evidence for defective cholesterol metabolism driving motor
neuron degeneration (Tsaousidou et al., 2008). Although CYP7B1 knockout mice appear
normal probably due to functional compensation by CYP7A1, inactivation of liver X receptor
which regulates CYB7B1 results in lipid accumulation and degeneration of motor neurons
in adult mice (Andersson et al., 2005). Next, neuropathy target esterase is an ER protein that
modifies phospholipids that is mutated in HSP, suggesting that altered neuronal membrane
composition can trigger axonal loss (Rainier et al., 2003). Finally, mutations in the acetyl-
CoA transporter necessary for ganglioside and glycoprotein formation in the Golgi apparatus
underlie one form of HSP, suggesting links between acetyl-CoA metabolism and axon
outgrowth and maintenance (Lin et al., 2008).
3. CHARCOT-MARIE-TOOTH DISEASE
CMT disease, also known as hereditary motor and sensory neuropathy, is one of the most
common inherited neurological diseases and peripheral neuropathy (Bucci et al., 2012). It is
characterised by slowly progressive weakness and wasting in distal muscles, commonly
affecting the foot, leading to the classical CMT phenotype of high arch and claw toe. Patients
may also present with mild distal sensory loss, hyporeflexia and skeletal deformity, although
lifespan is not generally affected (Patzko and Shy, 2011). CMT results from selective and
distal degeneration of peripheral nerves. Clinically, CMT disease is divided into two main
types according to nerve conduction velocities (NCVs) of the motor median nerve. CMT1
with NCVs below 38 m/s account for 30% of patients and CMT2 with NCVs above 38 m/s,
which is considered normal, account for 40% of patients (Bucci et al., 2012). This distinction
is useful when classifying CMT disease according to genetics (Table 4). CMT1, or
demyelinating CMT, is associated with 15 genes primarily expressed in Schwann cells,
causing peripheral demyelination and secondary axonal loss. CMT2, or axonal CMT, is
Genetics of Motor Neuron Disorders 15
presently linked to over 20 genes expressed in neurons causing primary defects in axons
without demyelination (Bucci et al., 2012). Furthermore, some forms of CMT2 show genetic
and pathological overlap with dHMNs which are predominantly motor neuropathies with
little sensory deficits (Rossor et al., 2012).
In this chapter, we will discuss only CMT2 genes since the focus model organism of this
volume Drosophila lack clear orthologues of myelin genes and Schwann cells. Most CMT2
genes encode proteins involved in mitochondrial morphogenesis, translation machinery and
organelle, vesicle and protein transport in axons as listed below according to pathogenic
themes.
3.1. Common Pathogenic Mechanisms in CMT2
3.1.1. Oxidative Stress and Mitochondrial Dysfunction
MFN2 is a mitochondrial GTPase required for mitochondrial fusion which coupled to
fission controls morphology, function and motility of mitochondria in axons (Zuchner et al.,
2004). Mutations in MFN2 linked to CMT2 are predicted to disrupt mitochondrial fusion-
fission balance in peripheral nerves (Zuchner et al., 2004). Postnatal gene deletion of MFN2
in the cerebellum causes mitochondrial pathology, electron transport chain defects and loss of
Purkinje cells (Chen et al., 2007), consistent with a role of MFN2 in neurodegeneration.
GDAP1 is implicated in mitochondrial fission and dynamics in axons and mutant forms are
linked to both autosomal dominant (Baxter et al., 2002; Cuesta et al., 2002) and recessive-
intermediate forms of CMT (Nelis et al., 2002).
3.1.2. Dysregulated Intracellular Membrane Trafficking
At least 4 CMT2 genes are associated with membrane and protein trafficking events in
axons. Rab7A which mediates trafficking between late endosomes and lysosomes is mutated
in CMT2 (Verhoeven et al., 2003). Endocytic Rab proteins are implicated in neurite
outgrowth and polarised sorting of vesicles in neurons which may account for axonal
pathology (Verhoeven et al., 2003). LMNA which encodes lamin A/C proteins essential for
maintaining nuclear envelope architecture and transport is linked to a form of CMT2 (De
Sandre-Giovannoli et al., 2002). Interestingly, mice deficient for LMNA develop locomotor
deficits and peripheral axonopathy, consistent with CMT (De Sandre-Giovannoli et al., 2002;
Sullivan et al., 1999). Dynamin 2 is a large GTPase involved in receptor-mediated
endocytosis and trafficking from the plasma membrane which is linked to CMT (Zuchner et
al., 2005). Depletion of dynamin 2 in neurons impaired clathrin-mediated endocytosis and
myelination in mice (Sidiropoulos et al., 2012), highlighting the role of endocytic dysfunction
in axons as a determinant of CMT2. Finally, mutations in the calcium channel TRPV4 which
affect its cell surface trafficking and assembly occur in CMT2 (Auer-Grumbach et al., 2010),
again linking dysregulation of calcium signalling cascades to motor neuron degeneration.
3.1.3. Abnormal Axonal Transport and Cytoskeleton
A primary role for axon transport defects in CMT2 is underscored by discovery of
mutations in 5 genes, KIF1B, DYNC1H1, NEFL, INF2 and SEPT9. K1F1B belongs to the
same superfamily of motor proteins as KIF5A and is involved in synaptic vesicle transport
Rebecca K. Sheean and Bradley J. Turner 16
(Zhao et al., 2001a). Mutations in KIF1B associated with CMT2 were shown to abolish its
ATPase activity necessary for vesicle motility and mice partially deficient for K1F1B develop
progressive weakness, motor deficits and impaired axonal transport (Zhao et al., 2001a).
Conversely, DYN1CH1 which encodes the retrograde motor protein cytoplasmic dynein
heavy chain 1 is also mutated in CMT2 (Weedon et al., 2011). This finding was corroborated
by previous studies in two lines of ENU mutant mice, legs at odd angles and cramping 1,
which develop progressive motor deficits and axonal loss due to independent DYNCH1
mutations (Hafezparast et al., 2003). NEFL which encodes the light chain of neurofilament
protein fundamental to axonal structure, organisation and transport is mutated in CMT2
(Mersiyanova et al., 2000) and transgenic mutant NEFL mice show paralysis with selective
spinal motor neuron loss and abnormal accumulation of neurofilaments (Lee et al., 1994).
INF2 which is involved in remodelling actin filaments and microtubules is also defective in a
form of CMT and expression of INF2 variants disrupts the neuronal cytoskeleton (Boyer et
al., 2011). Lastly, SEPT9, which is implicated in filament formation with actin and tubulin is
mutated in hereditary neuralgic amyotrophy (HNA) which shows clinical overlap with CMT2
(Kuhlenbaumer et al., 2005). SEPT9 knockout mice die in utero, revealing evidence for
abnormal cell morphology, cell adhesion and mitotic spindle formation (Fuchtbauer et al.,
2011).
3.1.4. Defective Myelination and Lipid Metabolism
Interestingly, two genes linked to peripheral myelin and expressed in Schwann cells have
been linked to axonal CMT. MED25 which encodes a transcriptional activator implicated in
regulation of myelin genes is mutant in CMT2 (Leal et al., 2009). Mutations in MPZ
predicted to disrupt adhesion and compaction of myelin were reported in CMT patients with
normal NCVs (De Jonghe et al., 1999; Mastaglia et al., 1999). MPZ mutations are
paradoxically linked to CMT1, suggesting that this gene contributes to both demyelinating
and axonal forms of CMT.
3.1.5. Impaired proteostasis
Eight genes associated with translation and protein degradation have been causally linked
to CMT2 forms. HSPB1 and HSPB8 encode heat shock protein chaperones with roles in cell
stress caused by protein misfolding, stabilisation of cytoskeleton and axonal transport.
Mutations in HSPB1 and HSB8 linked to CMT2 promote their aggregation neurons (Evgrafov
et al., 2004; Irobi et al., 2004). Neuronal expression of mutant HSPB1 in transgenic mice
provokes late-onset motor deficits, slowing of retrograde axonal transport and distal axon loss
(d'Ydewalle et al., 2011). LRSAM1 is a predicted E3 type ubiquitin ligase implicated in
trafficking of polyubiquitinated proteins and endosomal vesicle trafficking that is mutated in
CMT (Guernsey et al., 2010). One of the most intriguing discoveries in CMT2, and indeed
biology, was the identification of mutations in aminoacyl tRNA synthetases which charge
tRNAs with their cognate amino acids. Mutations in at least 5 aminoacyl tRNA synthetases
have been reported to date, involving GARS (Antonellis et al., 2003), YARS (Jordanova et
al., 2006), KARS (McLaughlin et al., 2010), AARS (Latour et al., 2010) and most recently
HARS (Vester et al., 2012). How mutations in such ubiquitously expressed and fundamental
enzymes cause peripheral neuropathy is unclear, however proposals include translation
deficiency, defective aminoacylation leading to mis-incorporation of amino acids promoting
protein misfolding and abnormal distribution of tRNA synthetases in axons and terminals
Genetics of Motor Neuron Disorders 17
(Latour et al., 2010). Insights into the pathogenesis of mutant aminoacyl tRNA synthetases in
CMT2 were gained from multiple lines of ENU mutant GARS mice which developed
locomotor deficits with peripheral denervation and neuropathy in the absence of reduced
aminoacylation activity (Achilli et al., 2009; Seburn et al., 2006), suggesting a novel toxic
gain-of-function mechanism.
Table 5. SMA and other MND genes and cellular functions
Disease/
Type
Inheritance Gene Protein Cellular function Reference
PLS
AR ALS2 ALS2/Alsin Endosomal
trafficking
(Yang et al.,
2001)
SMA AR SMN1 Survival motor neuron
RNA processing,
axonal transport
(Lefebvre et
al., 1995)
SBMA X-linked R
NR3C4 Androgen receptor Transcription (La Spada et
al., 1991)
SMAX2
X-linked R UBE1 Ubiquitin-like modifier
activating enzyme 1
Protein degradation (Ramser et al.,
2008)
SMAX3
X-linked R ATP7A Menkes copper ATPase Membrane
trafficking
(Kennerson et
al., 2010)
HMN2A AD HSPB8 Heat shock protein 22 Protein degradation (Irobi et al.,
2004)
HMN2B
AD HSPB1 Heat shock protein 27 Protein degradation (Evgrafov et
al., 2004)
HMN2C
AD HSPB3 Heat shock protein 27-like
protein
Protein degradation (Kolb et al.,
2010)
HMN4 AR PLEKHG5 Pleckstrin homology domain
containing, family G member 5
Membrane
trafficking
(Maystadt et
al., 2007)
HMN5A AD GARS
Glycyl-tRNA synthetase Translation (Antonellis et
al., 2003)
HMN5B AD REEP1 Receptor expression-enhancing
protein 1
ER trafficking,
cytoskeleton
(Beetz et al.,
2012)
HMN5C
AD BSCL2 Seipin ER trafficking, lipid
metabolism
(Windpassinger
et al., 2004)
HMN6 AR IGHMBP2
Immunoglobulin helicase -
binding protein 2
RNA processing (Grohmann et
al., 2001)
HMN7B AD DCTN1 Dynactin subunit 1 Axonal transport
(Puls et al.,
2003)
LCCS1
AR GLE1 GLE1 RNA export mediator
homolog
RNA processing (Nousiainen et
al., 2008)
LCCS2
AR ERBB3 V-ERB-B2 avian erythroblastic
leukaemia viral oncogene
homolog 3
Membrane
trafficking
(Narkis et al.,
2007b)
LCCS3
AR PIP5K1C phosphatidylinositol-4-
phosphate 5-kinase, type I,
Endosomal
trafficking
(Narkis et al.,
2007a)
Abbreviations: AD, autosomal dominant; AR, autosomal recessive; dHMN, distal hereditary motor
neuropathy; LCCS, lethal congenital contracture syndrome; PLS, primary lateral sclerosis; SBMA,
spinal bulbar muscular atrophy; SMA, spinal muscular atrophy; SMAX, X-linked spinal muscular
atrophy; X-linked R, X-linked recessive.
Rebecca K. Sheean and Bradley J. Turner 18
5. SPINAL MUSCULAR ATROPHIES AND OTHER MOTOR
NEURON DISORDERS
Spinal muscular atrophies refer to a group of inherited disorders characterised by
selective anterior horn cell death, leading to progressive muscle wasting. They are classified
according to the type of muscles affected, whether proximal or distal, and mode of inheritance
(Table 5). Autosomal recessive proximal SMA, or simply known as SMA, accounts for 95%
of cases and is the most common genetic cause of infant death (Burghes and Beattie, 2009).
SMA results from inactivating mutations in the SMN1 gene and retention of SMN2, which is
polymorphic in copy number, and determines the clinical severity of SMA, which ranges
from infant mortality by 2 years of age (SMA type 1), intermediate (SMA type 2), juvenile
(SMA type 3) and adult-onset forms (SMA type 4) with normal lifespans and mild weakness
(Lefebvre et al., 1995). X-linked SMA shows clinical overlap with SMA Type I, although it is
linked to mutations in 3 distinct genes (Table 5). Next, distal SMA which clinically and
genetically overlaps with dHMN or CMT2, is characterised by muscle weakness in the
extremities with minor sensory abnormalities (Rossor et al., 2012). Around ten genes are
associated with distal SMA or dHMN at present (Table 5). SBMA, also called Kennedy's
disease, is an X-linked recessive disorder characterised by slowly progressive weakness,
atrophy and fasciculations of bulbar, facial and limb muscles (Katsuno et al., 2012). It
predominantly affects males with onset usually in adolescence or middle adult life and is
generally not fatal (Katsuno et al., 2012). SBMA results from pathogenic expansion of a
trinucleotide repeat CAG encoding polyQ in the first exon of the AR gene and was the first
so-called polyQ disease discovered, followed by Huntington's disease (La Spada et al., 1991).
Patients also present with gynecomastia resulting from androgen insensitivity. Lastly, LCCS
is a rare autosomal recessive condition characterised by severe muscle atrophy, anterior horn
cell loss and total immobility of the foetus detected at 13 weeks pregnancy, leading to
premature death by 32 weeks gestation (Nousiainen et al., 2008). Three genes have been
linked to LCCS (Table 5). Most genes associated with SMA and these other MNDs encode
ubiquitous proteins involved in transcription, RNA processing and translation which is
striking given the selective pattern of anterior horn cell death. There are also familiar themes
such as membrane trafficking and axonal transport linked to genes underlying these MNDs as
discussed below.
5.1. Common Pathogenic Mechanisms in SMA and Other MNDs
5.1.1. Dysregulated Intracellular Membrane Trafficking
ATP7A is a copper transporter ATPase required for copper efflux from cells and more
importantly, copper transport across the blood-brain barrier. Mutations in ATP7A occur in
dHMN, implicating defective copper trafficking and deficiency of copper-dependent enzymes
in motor neuropathy (Kennerson et al., 2010). PLEKHG5 encodes an intracellular protein
with a pleckstrin homology domain involved in cell signalling and cytoskeletal function that
is mutant in dHMN (Maystadt et al., 2007). Impaired NFB signalling is linked to axonal
degeneration in this form of motor neuropathy (Maystadt et al., 2007). Seipin and REEP1 are
Genetics of Motor Neuron Disorders 19
also mutated in dHMN (Beetz et al., 2012; Windpassinger et al., 2004), again reinforcing the
importance of ER trafficking abnormalities in triggering axonopathy.
5.1.2. Abnormal Axonal Transport and Cytoskeleton
A mutation in the p150 subunit of dynactin, part of the retrograde motor in neurons, was
reported in dHMN and ALS (Puls et al., 2003), and together with dynein mutations in CMT,
provide a strong mechanistic link between abnormal axonal transport and neurodegeneration.
5.1.3. Defective RNA Processing
SMN1 encodes survival motor neuron (SMN) which interacts with many proteins,
including Gemins 2-8, to form the mega-Dalton SMN complex which functions in assembly
of small nuclear ribonucleoproteins involved in pre-mRNA splicing (Burghes and Beattie,
2009). There are two main proposals to account for how SMN deficiency causes SMA.
Firstly, SMN reduction may lead to abnormal pre-mRNA splicing to the detriment of motor
neurons, which is supported by findings of altered small nuclear RNA stoichiometry and
defective mRNA processing in spinal cords of SMN deficient mice (Baumer et al., 2009;
Zhang et al., 2008), although this occurred late in disease. Secondly, SMN may be important
for axonal transport of mRNAs required for distal translation in motor neurons and
maintenance of terminals, as evidenced by peripheral denervation in SMA model mice (Le et
al., 2005). IGHMBP2 encodes a DNA/RNA binding protein with helicase activity that is
mutated in SMA with respiratory distress type 1 or dHMN (Grohmann et al., 2001).
Interestingly, the spontaneous mouse mutant neuromuscular degeneration or nmd, which
develops limb and respiratory weakness, results from an IGHMBP2 mutation (Cox et al.,
1998).
The androgen receptor (AR) is a nuclear receptor which mediates the effects of
androgens, testosterone and dihydrotestosterone, by binding androgen response elements in
target genes (Katsuno et al., 2012). Interestingly, AR is highly expressed by anterior horn
cells where it is also required for trophic factor signalling (Katsuno et al., 2012). Transgenic
mice expressing an expanded AR develop progressive and fatal muscle weakness and nuclear
polyglutamine inclusions characteristic of SBMA (Adachi et al., 2001). Two main
mechanisms have been proposed to link pathogenic expanded AR to motor neuron death.
Firstly, the loss of normal AR signalling contributes to neurodegeneration, presumably by
disrupting transcriptional activities linked to neuronal survival. Secondly, expanded AR
acquires properties that are toxic to motor neurons, such as nuclear accumulation of
inclusions, leading to transcriptional dysregulation (Katsuno et al., 2012). GLE1 linked to
LCCS encodes a protein involved in mRNA export from the nucleus to cytoplasm in cells and
mutations are predicted to disrupt gene expression required for early development and
maturation of anterior horn cells (Nousiainen et al., 2008). Interestingly, LCCS mutations in
ERBB3 and PIP5K1C encoding regulators of the phosphatidyl inositol pathway, PIPK and
HER3, are linked to nuclear mRNA export, again pointing towards defective mRNA transport
in promoting motor neuron degeneration (Narkis et al., 2007a; Narkis et al., 2007b).
5.1.4. Impaired Proteostasis
Five genes associated with intracellular protein homeostasis have been linked to SMA or
dHMN. The first UBE1 encodes an E1 ubiquitin activating enzyme that catalyses the first step
Rebecca K. Sheean and Bradley J. Turner 20
in protein ubiquitination and mutations were found in X-linked SMA, implicating defective
protein degradation (Ramser et al., 2008). HSPB1 and HSPB8 mutations were also reported
in dHMN (Evgrafov et al., 2004; Irobi et al., 2004). A third heat shock protein HSPB3 was
recently implicated in dHMN (Kolb et al., 2010), strengthening the case for defective protein
chaperone activity in causing motor neuropathies. Lastly, GARS mutations were also linked
to dHMN (Antonellis et al., 2003), in addition to CMT.
CONCLUSION
Nearly 100 genes have been linked to susceptibility and degeneration of upper and motor
neurons and their distal axons in MNDs to date. Despite the clinical, pathological and genetic
diversity of MNDs, this accumulation and wealth of genetic evidence has highlighted key
cellular pathways and molecular biology disturbed in all forms of MNDs, leading to proposal
of six dominant and unifying pathogenic themes: (1) oxidative stress and mitochondrial
defects; (2) dysregulation of intracellular vesicle, membrane and organelle traffic; (3)
abnormalities in axonal transport and cytoskeletal architecture; (4) dysmyelination and
impaired lipid metabolism in axons; (5) defective splicing and transport of RNA species; and
(6) abnormal proteostasis. These pathways offer important insights into pathogenesis of both
inherited and sporadic forms of MNDs and selection of urgently needed therapeutic targets.
ACKNOWLEDGMENTS
This work was supported by an Australian NHMRC Project Grant 1008910, Mick
Rodger MND Research Grant from MND Research Institute of Australia and Bethlehem
Griffiths Research Foundation Grant. The Florey Institute of Neuroscience and Mental Health
acknowledges the strong support from the Victorian Government and in particular the funding
from the Operational Infrastructure Scheme.
ABOUT THE AUTHORS
Bradley J. Turner is a Research Fellow at the Florey Institute of Neuroscience and
Mental Health at the University of Melbourne, Australia. His research interests include
functional modelling of genes linked to amyotrophic lateral sclerosis (ALS), spinal muscular
atrophy (SMA) and distal hereditary motor neuropathy (dHMN), and investigating the
molecular pathogenesis of ALS in mouse and rat models. His work is focused on the role of
exosomes in the processing, secretion and propagation of misfolded proteins linked to
neurodegeneration, autophagy dysregulation in neuronal death and therapeutic application of
survival motor neuron protein for broad motor neuron injury and disorders.
Rebecca K. Sheean is a Postdoctoral Researcher at the Florey Institute of Neuroscience
and Mental Health at the University of Melbourne, Australia. Her research interests include
investigating the cellular and molecular mechanisms underlying motor neuron degeneration in
Genetics of Motor Neuron Disorders 21
ALS, focusing on the role of the endosome-lysosome system and interactions between glial
cells and the immune system in ALS pathogenesis.
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 2
A SECRETED LIGAND FOR GROWTH CONE
RECEPTORS, VAP MEDIATES THE CELLULAR
PATHOLOGICAL DEFECTS IN ALS
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda* Department of Neurology and Neurosurgery,
Montreal Neurological Institute, McGill University, Montreal, Canada
ABSTRACT
Drosophila studies have contributed greatly to our understanding of the biological
pathways that might be relevant to the pathogenesis of amyotrophic lateral sclerosis
(ALS). ALS is a progressive neurodegenerative disorder characterized by selective death
of motor neurons. Human VAMP associated protein B (VAPB) is the causative gene of a
familial form of ALS, ALS8. Human VAPB, Drosophila VAP33, and C. elegans VPR-1
are homologous type II transmembrane proteins containing a highly conserved major
sperm protein (MSP) domain in their N-terminal region. A point mutation (P56S) in the
human VAPB MSP domain causes ALS8. Drosophila studies revealed that VAP is
secreted in a cell type-specific fashion and acts as a diffusible hormone. VAP is a ligand
for Ephrin (Eph) and Roundabout (Robo), two receptors originally identified as mediators
of growth cone guidance cues. The ALS mutation causes two different types of defects:
mutant VAP fails to be secreted (loss-of-function) and accumulates as ubiquitinated
inclusions in the endoplasmic reticulum (ER), resulting in ER stress (gain-of-function),
which is a common defect observed in the pathology of familial and sporadic forms of
ALS. Thus, Drosophila studies have revealed the cellular defects associated with mutant
VAP and have helped us understand key pathological features of ALS.
Keywords: Neurodegeneration, ER stress, Mitochondria, Eph, Robo, LAR
* Correspondence: [email protected].
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 36
ABBREVIATIONS
ALS Amyotrophic lateral sclerosis
ER endoplasmic reticulum
LAR Leukocyte common antigen-related
MSP Major sperm protein
Robo Roundabout
SOD1 superoxide dismutase 1
VAP VAMP associated protein
INTRODUCTION
Neurodegenerative disorders constitute one of the major challenges of modern medicine.
Although these diseases are relatively common and highly debilitating, the mechanisms
responsible for their pathologies are poorly understood and there are currently no effective
therapies. Therefore, it is crucial to reveal genetic factors involved in the various pathways
affected, as well as to provide potential therapeutic drug targets.
Powerful genetic techniques available for use in Drosophila have provided new insights
into various aspects of the disease and guided many research efforts in vertebrate
neuroscience. Importantly, Drosophila studies have successfully uncovered several novel
signalling pathways which may be relevant to the pathology of many malignant neurological
diseases, including ALS. The research activities in Drosophila have enabled us to analyse the
resulting cellular phenotypes and uncover the molecular pathways underlying many
neurological diseases.
STUDIES ON MUTATIONS ASSOCIATED WITH FAMILIAL FORMS OF
ALS ENABLE US TO UNDERSTAND THE CORE PATHOLOGY OF ALS
A French neurologist, Jean-Martin Charcot, first noted the characteristics of ALS in 1874
and named the fatal syndrome based on what he found. Damage to upper motor neurons,
which begin at the top of the brain, results in muscle weakness, stiffness, and augmented
reflexes. Lower motor neurons start at the base of the brain and spinal cord; injury to these
neurons causes muscle atrophy, twitching, weakness, and reduced reflexes. Despite over
135 years of vigorous investigations following the discovery of the disease, ALS, also known
as Lou Gehrig’s disease, remains a mercilessly fatal disorder that affects two to eight per
100,000 persons in the world (Kiernan et al., 2011). Unfortunately, there is no primary
therapy for this disease and its pathogenesis is poorly understood, with most persons affected
dying within 3-5 years of diagnosis of the fatal syndrome.
Both environmental and genetic factors have been attributed to the pathology of ALS;
familial cases account for approximately 10% of all instances of the disease. Since both
sporadic and familial forms affect the same types of neurons with comparable disease
hallmarks, they most likely share a similar pathogenesis (Pasinelli and Brown, 2006). Thus,
understanding the mechanisms by which these mutations cause the pathology in ALS may
A Secreted Ligand for Growth Cone Receptors … 37
lead to novel therapeutic strategies of both familial and sporadic forms of the disease. A
landmark discovery reported in 1993 initiated the molecular era of ALS research: the
identification of mutations in the superoxide dismutase 1 (SOD1) gene in a familial form of
ALS (Rosen et al., 1993). Importantly, mice expressing the mutant SOD1 recapitulated the
motor defects observed in ALS patients (Wong et al., 1995). Use of these mice has been
crucial in characterizing the mechanisms of familial and sporadic ALS. On the basis of these
studies, mutant SOD1 has been postulated to be involved in many key aspects of the disease:
(1) accumulation of ubiquitinated SOD1 contributes to ALS cellular toxicity (Bruijn et al.,
1998), (2) synaptic glutamate receptors are aberrantly clustered and cause excitotoxicity
(Bottino et al., 2002; Boyce et al., 2005), (3) motor neuron aberrations alone (cell-
autonomous defects) are insufficient to cause the disease and other cells such as microglia are
involved (cell non-autonomous defect) (Boyle et al., 2006; Brooks, 1994), (4) mitochondrial
morphology and function is impaired (Brostrom et al., 1995; Vielhaber et al., 1999), and (5)
the ER stress response is triggered (Nishitoh et al., 2008; Saxena et al., 2009). The effects of
mutant SOD1 in ALS pathogenesis do not seem to be associated with its wild-type function
and as of yet, there is no unifying hypothesis to explain these observations.
Mutations were subsequently identified in other familial forms of ALS (Table 1),
including TAR DNA-binding protein (TDP)-43 (Sreedharan et al., 2008), fused in sarcoma
(FUS) (Kwiatkowski et al., 2009), valosin-containing protein (VCP) (Johnson et al., 2010),
C9ORF72 (Kudin et al., 1999; Strempel et al., 1999), and VAPB (Nishimura et al., 2004).
Importantly, the proposed functions of these genes are distinct. For example, TDP-43 and
FUS have been implicated in RNA regulation (Brunner et al., 1994; Doering et al., 1999),
while a variety of roles have been proposed for VCP, notably in ER-associated degradation
(Leon and McKearin, 1999; Patel et al., 1998), autophagy, and the ubiquitin-proteasome
system (Cantor et al., 2009). Given that no obvious link between the functions of these
proteins has been established, it is crucial to identify the pathological defects caused by these
mutant proteins implicated in both sporadic and familial ALS to determine the core defects in
ALS. To this end, VAP (ALS8) has been intensively researched in order to elucidate the core
pathology of ALS. Indeed, studies have shown that defects in pathways associated with VAP
lead to key pathological features implicated in ALS (Chai et al., 2008; Chen et al., 2010;
Ratnaparkhi et al., 2008; Tsuda et al., 2008). Thus, uncovering the functions and pathways in
which VAP is operating will provide clues to develop strategies to delay the course of both
the familial and sporadic forms of this disease.
DOMINANTLY INHERITED MISSENSE MUTATIONS IN VAP
ARE ASSOCIATED WITH A FAMILIAL FORM OF ALS
A dominantly inherited proline 56 to serine (P56S) missense mutation in the VAPB gene
was first identified in a large Brazilian family with a slowly progressive and late-onset,
atypical form of ALS, ALS8 (Nishimura et al., 2004) (Figure 1). Interestingly, individuals
carrying the same mutation have been described with three distinct conditions: a late-onset
slowly progressing form of spinal muscular atrophy, an atypical slowly progressing form of
ALS, and a typical severe and rapidly progressing ALS. In a branch of the same large family,
the P56S mutation has been shown to cause a lower motor neuron disorder accompanied by
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 38
autonomic involvement and dyslipidaemia (Marques et al., 2006). The mutated proline is
present in a stretch of amino acids that is very highly conserved in species such as yeast,
worms, flies and humans (Figure 1).
Table 1. Genes identified in familial ALS
Mutations in several genes, including the SOD1, TDP-43, FUS, C9orf72, and VAPB genes, cause
familial ALS (Chow et al., 2009; DeJesus-Hernandez et al., 2011; Greenway et al., 2006;
Maruyama et al., 2010; Renton et al., 2011; Topp et al., 2004; Yang et al., 2001).
VAP consists of three domains, MSP domain (MSP), coiled coil domain (CC) and a transmembrane
domain (TM). MSP domain is named from the high similarity with Major Sperm Protein in C.
elegans. Conserved Proline is mutated to Serine in patients associated with ALS8. Note that VAP
and MSP have no signal sequence.
Figure 1. A graphical overview of VAP and MSP proteins.
A Secreted Ligand for Growth Cone Receptors … 39
The same mutation was identified in Germany (Kirches et al., 1999) as well as in a
patient of Japanese descent (Millecamps et al., 2010). Additionally, a recent study found a
new mutation (T46I) in a British ALS patient that leads to similar molecular effects as the
P56S alteration (Chen et al., 2010). Human genetic research on ALS8 has raised three
important questions: (1) What is the function of the wild-type VAP protein, (2) what are the
effects of pathological mutations of this protein, and (3) how does VAP or its associated
pathways contribute to the pathogenesis of sporadic or other familial forms of ALS.
VAP IS AN ER TRANSMEMBRANE PROTEIN CONTAINING
A MSP DOMAIN
VAPB is closely related to VAPA, which has been shown to associate with the
cytoplasmic face of the ER (Soussan et al. 1999; Skehel et al. 2000; Kaiser, 2005) or to tight
junctions as a type II transmembrane protein (Lapierre et al., 1999). The human VAPB
(hereafter named hVAP) protein is about 30kDa and has homologs in numerous other species,
including C. elegans (F33D11.11 or VPR-1), Drosophila (DVAP33-A, hereafter named
dVAP) (Pennetta et al., 2002) and yeast (Suppressor of Choline Sensitivity, Scs2p)
(Kagiwada and Zen, 2003). VAPs contain an amino (N)-terminal domain of about 125
residues called the major sperm protein (MSP) domain, which is conserved among all VAP
family members (Figure 1) (Weir et al. 1998; Nishimura et al. 1999). The MSP domain was
named for its similarity to nematode MSPs, the most abundant proteins in their sperm
(Bottino et al., 2002). The central region of the protein contains an amphipathic helical
structure and is predicted to form a coiled-coil protein-protein interaction motif. The
hydrophobic carboxyl (C)-terminus acts as a membrane anchor to the ER (Kaiser et al., 2005;
Skehel et al., 2000; Soussan et al., 1999). In studies done with Aplysia, VAP was implicated
in synaptic transmission through its interaction with VAMP (Skehel et al., 2000). However, in
other organisms, VAP is not likely to function in synaptic transmission (Chai et al., 2008).
Indeed, VAP proteins have rather been implicated in other biological functions, including
glucose transport trafficking, expression of phospholipid biosynthetic genes, ER-Golgi and
intra-Golgi transport, neurite extension and calcium homeostasis (Foster et al., 2012; Peretti
et al., 2008) (De Vos et al., 2012; Matsuzaki et al., 2011). Yeast homologs are involved in
phosphatidylinositol-4-phosphate (PIP) synthesis and ceramide transport. Strains lacking
SCS2p, a VAP homolog, are unable to activate the inositol-1-phosphate synthase (INO1)
gene. These strains cannot grow in temperatures above 34C due to the absence of inositol
(Brickner and Walter, 2004; Kagiwada and Zen, 2003). Overexpression of hVAP in HeLa
cells affects the structural integrity of the ER through interaction with Nir (N-terminal
domain-interacting receptor) proteins. These interactions are mediated through FFAT (two
phenylalanines [FF] in an acidic tract) domains, which are present in Nir proteins (Amarilio et
al., 2005). VAPs also interact with a lipid-binding protein, oxysterol-binding protein (OSBP),
ceramide transfer protein (CERT) and phosphatidylinositol-four-phosphate adaptor-protein-2
(FAPP-2), all of which contain a FFAT motif (Amarilio et al., 2005; Kaiser et al., 2005;
Loewen and Levine, 2005; Mikitova and Levine, 2012). Therefore, VAPs are also thought to
play a role in the metabolism and transport of lipids (Jansen et al., 2011; Perry and Ridgway,
2006). Importantly, hVAP and dVAP are functionally interchangeable in fly assays. To
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 40
further define the role of VAPs in animals, Pennetta et al. characterized the function of dVAP
in flies (Pennetta et al., 2002). Pennetta and colleagues showed that dVAP regulates the
division of boutons at the synaptic terminals at the neuromuscular junctions (NMJs). Loss of
dVAP causes a severe decrease in the number, as well as an increase in the size, of boutons at
the NMJs while presynaptic overexpression of dVAP induces an increase in the number of
boutons and a decrease in their size. Loss of dVAP also causes a disruption of the presynaptic
microtubule architecture, whereas gain of dVAP enhances it. The loss of function of dVAP
has also been shown to lead to an increase in size of miniature excitatory junction potentials
(mEJPs), as well as an increased clustering of postsynaptic glutamate receptors, whereas
presynaptic overexpression of the wild-type dVAP decreases the size of the mEJPs (Chai et
al., 2008). Of note, overexpression of hVAP shows the similar phenotypes as overexpression
of dVAP, and expression of hVAP can rescue the defects associated with loss of dVAP in
flies (Chai et al., 2008), indicating that the function of VAP might be evolutionarily
conserved in Drosophila and humans. The similarity in their structures also suggests a
common function between MSP and VAP. The C. elegans MSP proteins and VAP MSP
domains both fold into evolutionarily conserved immunoglobulin-type seven-stranded beta
sandwiches (Baker et al., 2002; Kaiser et al., 2005), though MSP does not contain a coiled-
coil motif nor a transmembrane domain (Ward et al., 1988). MSP function is required for
fertilization in C. elegans (Kim et al., 2013). Indeed, MSPs are abundantly expressed in
sperm and have an intracellular cytoskeletal function, which depend on their ability to
polymerize in the absence of actin or myosin (Bottino et al., 2002). MSP also has an
important extracellular signalling function during fertilization (Miller et al., 2001). MSP is
secreted from the sperm cytosol into the reproductive tract by an unconventional process
(Kosinski et al., 2005) and extracellular MSP binds to the VAB-1 Eph (Ephrin) receptor and
other, yet to be identified receptors, on the surfaces of unfertilized oocytes and the
surrounding ovarian sheath cells (Corrigan et al., 2005; Govindan et al., 2006; Miller et al.,
2003). Secreted MSP induces oocyte maturation, an essential process that prepares oocytes
for fertilization and embryogenesis, as well as ovarian sheath cell contraction (Corrigan et al.,
2005; Miller et al., 2003). Somatic cAMP signalling in the gonadal sheath cells functions
downstream of MSP signalling and coordinates oocyte growth and meiotic maturation via
soma-germline gap-junction communication (Govindan et al., 2009). Similar molecular
functions to that of MSP were not characterized for VAP proteins.
VAP FUNCTIONS IN NOVEL SIGNALLING PATHWAYS
The presence of the MSP domain in its sequence suggests that VAP might share
signalling functions with MSP sperm-specific proteins. In agreement with this notion,
microinjection of worm, fly, or human VAP MSP domains to the MSP-deficient gonad of C.
elegans rescues the gonadal phenotype (Tsuda et al., 2008). VAP carrying the P56S ALS
mutation mimics the wild-type protein in this assay, demonstrating that the mutant protein
still retains functional signalling properties. This further suggests that the defects caused by
the ALS mutation are primarily a result of aberrant trafficking or localization. This chapter
strives to explain how studies on VAP in Drosophila have revealed that pathways associated
with MSP may be relevant to the pathology of ALS.
A Secreted Ligand for Growth Cone Receptors … 41
CLEAVAGE OF VAP RELEASES MSP DOMAIN-CONTAINING
FRAGMENTS THAT ARE SECRETED
An important feature associated with ALS is that the disease may have a cell non-
autonomous component (Boyle et al., 2006; Brooks, 1994). Complementary in vivo and in
vitro studies have suggested a possible role for trophic factors in ALS (Acsadi et al., 2002;
Kaspar et al., 2003). Tsuda et al. and Han et al. showed that VAP MSP domain cleaved from
full length protein can be secreted in flies and worms (Corey et al., 2012).
VAP exists as an endoplasmic reticulum (ER)-localized type II membrane protein and modulates
diverse pathways including ER homeostasis and ceramide and sphingolipid metabolism. VAP is
cleaved by an unknown protease, releasing MSP domain-containing fragments that are secreted.
The resulting VAP fragments bind to Eph and Robo/LAR receptors. VAP/Eph signalling could act
in an autocrine way to modulate neuronal activity, for example, via glutamate receptors (GluRs).
VAP/Robo/LAR signalling is required for mitochondria fission. The proline 56 to serine (P56S)
mutation (P58S in Drosophila) in VAPB results in aggregation of the mutant (red) and wild-type
(green) VAP protein in the ER, which leads to an upregulation of the ER stress. The mutation
causes loss of VAP function including Eph and Robo/LAR receptor signalling and probably other
ER-associated activities of VAP.
Figure 2. Model of ALS pathology caused by ALS8 (P56S) mutant VAP.
Consistently, the VAP MSP domain is also present in human blood serum, and its
presence was also confirmed in a large survey of serum proteins identified by mass
spectrometry (Omenn et al., 2005). VAP lacks an N-terminal signal sequence and, while still
uncharacterized, VAP MSP secretion likely involves an unconventional mechanism similar to
that of MSP in C. elegans (Kosinski et al., 2005). Significantly, the secreted VAP MSP acts
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 42
as a trophic factor via receptors including Eph, Roundabout (Robo) and leukocyte-antigen
related (LAR) in flies and worms, the details of which will be described in this chapter
(Figure 2).
VAP ACTS AS A LIGAND FOR EPH RECEPTOR
AND MEDIATES EPH SIGNALLING
MSP binds to multiple receptors, including the C. elegans VAB-1 Eph-related receptor
protein tyrosine kinase. (Miller et al., 2003). The Eph are an evolutionarily conserved class of
receptor protein-tyrosine kinases that bind to membrane-attached ligands called ephrins
(Palmer and Klein, 2003; Pasquale, 2005). Ephrins inhibit oocyte maturation in the absence of
sperm, and MSP functions to antagonize this inhibitory circuit (Govindan et al., 2006; Miller
et al., 2003; Whitten and Miller, 2007). MSP induces activation of the MAP kinase and
Ca2+
/calmodulin-dependent protein kinase II cascades (Corrigan et al., 2005; Miller et al.,
2001) as well as reorganization of the oocyte microtubule cytoskeleton (Govindan et al.,
2006). Intriguingly, Tsuda et al. have shown that secreted VAP MSP domains also bind to
Eph on the extracellular surface of cells (Tsuda et al., 2008). The genetic and biochemical
binding data shows that VAP acts as a ligand and modulates Eph receptor signalling
pathways. In flies and worms, mutants lacking VAP show phenotypes that overlap with those
observed in Eph mutants (Tsuda et al., 2008). In addition, loss-of-function of the Eph receptor
suppresses the muscle phenotypes induced by overexpression of wild-type dVAP in flies. The
similarity between the phenotypes of VAP mutants and Eph receptor mutants suggests that
VAP MSP functions as an agonist to activate signalling upon binding Eph. In contrast, VAP
functions to antagonize ephrin signalling in chemosensory neuron migration of the C.
elegans, just as MSP antagonizes ephrin signalling during oocyte maturation (Miller et al.,
2003). Biochemical competition assay results are consistent with MSP domains competing
with ephrin for Eph binding (Tsuda et al., 2008). Taken together, the relationship between
MSP and ephrin ligands in Eph signalling may depend on the developmental and cellular
context, as previously observed for ephrins and Eph in mammals (Mawrin et al., 2004).
The agonist effects of VAP MSP on Eph receptors contribute to glutamate excitotoxicity,
which likely plays a role in the pathogenesis of ALS (Bruijn et al., 2004; Rothstein et al.,
1990). Three lines of evidence suggest that VAP MSP domains might regulate glutamate
receptor signalling.
First, Eph receptors directly associate with NMDA-subtype glutamate receptors and
regulate clustering in cultured neurons (reviewed in Dalva et al., 2000; Palmer and Klein,
2003; Takasu et al., 2002). Triple EphB knock-out mice lacking EphB1-3 exhibit homeostatic
upregulation of NMDA receptor surface expression and loss of proper targeting to synaptic
sites (Nolt et al., 2011).
Second, loss of dVAP function in flies is associated with increased glutamate receptor
clustering and increased mEJPs at the NMJs (Chai et al., 2008).
Finally, MSP and VAB-1 Eph regulate NMDA receptor function during worm oocyte
maturation (Corrigan et al., 2005). These observations suggest that defective VAPB-P56S
signalling in ALS patients might alter the susceptibility of motor neurons to potential
pathogenic mechanisms, such as glutamate excitotoxicity.
A Secreted Ligand for Growth Cone Receptors … 43
Alternatively, the antagonist effects of VAP MSP on Eph receptors contribute to the ALS
pathology. In vertebrates, multiple ephrins and Ephs, including EphA4 and A7, are expressed
throughout the adult nervous system and skeletal muscle (Iwamasa et al., 1999; Kullander et
al., 2003; Lai et al., 2001; Martone et al., 1997).
Genetic as well as pharmacological inhibition of EphA4 signalling rescues the mutant
SOD1 phenotype in zebrafish and increases survival in mouse and rat models of ALS (Van
Hoecke et al., 2012).
In humans with ALS, EPHA4 expression inversely correlates with disease onset and
survival, and loss-of-function mutations in EPHA4 are associated with long survival (Van
Hoecke et al., 2012).
Although the mechanism through which deletion of EphA4 suppresses motor neuron
degeneration is not yet understood, MSP VAP may play a role in motor neuron survival or
muscle function through interactions with Eph in the pathogenesis of ALS. Whether the VAP
ligand/Eph receptor interaction leads to positive or negative regulation of downstream
signalling cascades, and whether the effects of VAP/Eph signalling act in an autocrine or
paracrine fashion, or both, will be addressed in the future.
VAP MSP MEDIATES ROBO AND LAR RECEPTORS AND FUNCTIONS
IN THE MAINTENANCE OF THE MITOCHONDRIA IN MUSCLE
MSP domains bind to Eph and other evolutionarily conserved receptors that function
together to regulate a variety of developmental processes (Miller et al., 2003). Accordingly, it
was shown that the cleaved VAP fragment, VAP-MSP, interacts with LAR and Robo family
receptors on the muscle cell surface, which ultimately modulate actin organization and
changes mitochondrial morphology and function (Han et al., 2012). Mitochondria are
remarkably dynamic organelles that migrate, divide and fuse. Since mitochondria cannot
replicate de novo, fission and fusion allow the mixing of metabolites and mitochondrial DNA,
the proliferation and distribution of mitochondria and cellular adaptation to changing energy
demands (Knott et al., 2008). As individual mitochondria are subject to injury and
dysfunction, it is likely that mitochondrial fusion serves as a protective mechanism.
Significantly, Han et al. showed that neuronal VAP is a critical regulator of muscle
mitochondria in C. elegans and Drosophila. Adult C. elegans vap mutants exhibit an
imbalance of mitochondrial fission and fusion in muscle and these aberrant mitochondrial
networks have low transmembrane potential and respiration. Abnormal mitochondrial shapes
and/or electron transport activity have been reported in motor neurons and skeletal muscle of
ALS patients (Crugnola et al., 2010; Wiedemann et al., 2002). Indeed, ALS patients with a
SOD1 mutation show a muscle mitochondrial oxidative defect (Corti et al., 2009).
Cytochrome c oxidase (COX)-negative fibres is a common histochemical finding in skeletal
muscle from patients with sporadic ALS, suggesting the muscle mitochondrial respiratory
chain dysfunction (Crugnola et al., 2010). Therefore, defects in muscle mitochondria may
contribute as a primary cause in ALS pathogenesis.
Importantly, non-autonomous signalling by VAP and sperm-derived MSPs regulates
mitochondria in striated muscles and oocytes in C. elegans. Han et al. observed that the
defects in muscle mitochondria of the vap null mutant can be rescued by expression of VAP
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 44
in neurons, but not in the muscles themselves (Han et al., 2012). In motor neurons, secreted
MSPs signal through Eph (Tsuda et al., 2008), but in muscle, loss of Eph does not alter
mitochondrial phenotypes (Boyle et al., 2006). Thus, Han et al. searched for other receptors
mediating MSP signalling in muscle cells. Using genome-wide microarrays and subsequent
functional analysis, Han et al. identified two additional MSP receptors, the Robo and LAR
receptors. Like Eph receptors, Robo, and LAR-like receptors are called growth cone guidance
receptors because of their established roles in regulating the actin cytoskeleton during nervous
system development. However, these receptors are often expressed after guidance decisions
are made, particularly in the adult central nervous system and muscles (Longo et al., 1993;
Zabolotny et al., 2001; Zhang and Goldstein, 1991).
VAP or MSP signals to mitochondria through Robo and LAR receptor pathways, which
controls actin remodelling (Han et al., 2012). Both LAR and Robo mutants have altered
muscle mitochondrial morphology and function in C. elegans. Interestingly, both VAP and
Robo antagonize LAR signalling in muscle mitochondria. Other groups have demonstrated
genetic interactions and shared signalling pathways between LAR and Robo in neurons. For
example, Robo and LAR have opposing functions during midline axon guidance in
Drosophila, suggesting some type of inhibitory crosstalk between these two receptors (Sun).
The data presented by Han et al. in muscle provides additional evidence of crosstalk between
LAR and Robo. They propose the intriguing hypothesis that Robo facilitates VAP and MSP
binding to LAR or Robo/LAR complexes, down-regulating LAR signalling. Similarly, Robo
functions as the stepwise refinement in growth cone guidance (Stein and Tessier-Lavigne,
2001). Growth cones undergo two mutually reinforcing changes upon midline crossing: loss
of response to a midline attractant, and up-regulation of response to a midline repellent, that
helps to expel them from the midline and move them on to the next leg of their trajectory.
Slit, another Robo ligand is a midline repellent. Upregulation of Slit causes loss of response
to the chemoattractant, netrin. This silencing effect of Slit on netrin attraction is mediated by
a direct physical interaction of Slit receptor Robo with the netrin receptor DCC.
In C. elegans, VAP/Robo/Lar signalling antagonizes Arp2/3 activity to position
mitochondria at I-bands in the muscle (Han et al., 2012), actin-enriched sites containing
structures analogous to focal adhesions (Lecroisey et al., 2007). CLR-1 Lar and Arp2/3
complex activity are required for maintenance or elongation of mitochondrial tubule length
along I-bands, a process that may facilitate mitochondrial fusion. In C. elegans vap mutants,
mitochondria mislocalise to the muscle belly along with ectopic actin filaments. Lar or
Arp2/3 loss suppresses the vap mutant mitochondrial position, morphology, and
transmembrane potential defects. These results reveal a critical role for the Arp2/3 complex in
modulating multiple aspects of mitochondrial biology. The actin cytoskeleton has been shown
to regulate mitochondrial position in cultured neurons and yeast (Boldogh and Pon, 2006;
Pathak et al., 2010). The regulation of actin and microtubule effectors downstream of
VAP/LAR and Robo signalling raises intriguing questions regarding the complexity and
specificity of the regulatory process that supports normal mitochondrial structure and function
in muscle cells. This novel signalling pathway mediated by secreted VAP might be important
for the pathogenesis of ALS. Consistently, analysis of single nucleotide polymorphisms
(SNPs) in genome-wide association studies suggested that common variants in Robo and Eph
are associated with sporadic ALS (Lesnick et al., 2008).
A Secreted Ligand for Growth Cone Receptors … 45
ALS MUTATION CAUSES TWO DIFFERENT TYPES OF DEFECTS:
A FAILURE OF VAP SECRETION (LOSS-OF-FUNCTION) AND
ER STRESS (GAIN-OF-FUNCTION OR TOXIC DEFECTS)
Establishing the relevance of VAP signalling to the pathology of ALS will rest on
defining the cellular activities of VAP that are critical for the normal function and
maintenance of motor neurons, and characterizing how each is affected by the P56S mutation.
Significantly, Tsuda et al. demonstrated that the P56S mutation causes a failure of
secretion of VAP MSP protein, resulting in defects in VAP/receptor signalling. Disrupted
secretion of the VAPB MSP domain could mediate the defects of cellular non-autonomous
signalling from glia, endothelia, or muscle cells to the motor neurons described in ALS
patients and in transgenic animal models expressing mutant SOD1 (Pasinelli and Brown,
2006). Tsuda et al. demonstrated that the P56S VAP accumulates as inclusions in the ER.
These inclusions recruit wild-type VAP protein, which causes a dominant negative defect
(Chai et al., 2008; Ratnaparkhi et al., 2008; Tsuda et al., 2008). Interestingly, Ratnaparki et al.
showed that the mutant VAP interferes with BMP signalling at the synapse, suggesting a
possible link between BMP signalling and the ALS pathology.
Accumulated P56S VAP shows key characteristics associated with ALS. First, P56S
dVAP protein induces ubiquitinated inclusions, as observed in ALS patients and mutant
SOD1 mice (Tu et al., 1996). These intracellular ubiquitinated inclusions are a hallmark of
ALS (Wiedemann et al., 2002). Second, the protein inclusions are associated with the ER and
appear to be electron-dense expansions of the ER (Tsuda et al., 2008). Third, several key ER
proteins were found to be associated with these inclusions, including the chaperones Boca
(Culi and Mann, 2003) and PDI (Wilkinson and Gilbert, 2004). Finally, the P56S dVAP
induces ER stress. This data shows at least three important parallels with ALS and SOD1
mouse models: cytoplasmic inclusions, ubiquitination, and ER stress. Consistently, viral-
mediated expression of the ALS mutant human VAPB leads to an ER stress response that
contributes to the selective death of primarily cultured motor neurons (Langou et al., 2010).
The ER stress is typically induced as a response to ER associated stress to refold proteins
(Schroder and Kaufman, 2005). ER stress is initially defensive by up-regulating specific ER
stress-regulated genes and inhibiting general protein translation. If the ER stress is too severe
or prolonged, it can induce cell death and apoptosis (Schroder and Kaufman, 2005). It is
therefore possible that a slow and protracted accumulation of ER protein aggregates
eventually leads to cellular damage and neuronal death, in agreement with recent observations
that overexpression of P56S dVAP causes neuronal death in flies (Chai et al., 2008).
Recent evidence indicates that ER stress primarily contributes to the ALS pathogenesis
(Walker and Atkin, 2011). Activation of ER stress-regulated genes is one of the earliest
events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant
SOD1 (Atkin et al., 2006; Mori et al., 2011). Genetic manipulation of ER stress in several
different SOD1 mouse models was shown to alter disease onset and progression, implicating
an active role for ER stress in disease mechanisms (Mori et al., 2011). ER stress also occurs
in spinal cord tissues of human sporadic ALS patients (Atkin et al., 2008; Sasaki, 2010), and
recent evidence suggests that the perturbation of the ER could occur in ALS cases associated
with TDP-43 and FUS (Farg et al., 2012; Suzuki and Matsuoka, 2012). Together, these
Amina Moustaqim-Barrette, Mario Maira and Hiroshi Tsuda 46
findings implicate ER stress as a potential upstream mechanism involved in both familial and
sporadic forms of ALS.
Based on our data and the published literature, we would like to propose the following
model for the pathogenesis of ALS8, an autosomal dominant disease (Figure 2). The P56S
hVAP protein accumulates in the ER of cells, while the wild-type protein is functional.
However, with time, the aggregates become more prominent, P56S hVAP protein becomes
ubiquitinated, and functional wild-type proteins become trapped in the inclusions. These
protein inclusions initiate ER stress and defects in lipid metabolism that eventually affect cell
viability and lead to a decrease in secretion of the MSP domain. The MSP domain binds to
receptors including Ephrin, Robo and LAR receptors through which it mediates at least some
of its actions. The mutant protein therefore causes two very different types of defects: first,
accumulation in the ER creates the ER stress, which may be toxic to the cells, and could
account for the cell autonomous component. Second, reduced secretion of VAP MSP, which
may function as an autocrine or paracrine signal, exacerbates the problem. Both defects may
synergize to produce the key features of ALS pathology.
Hence, the collective evidence suggests the model that impaired function of VAP causes
core pathological defects in ALS. This model is also supported by other reports showing that
sporadic ALS patients exhibit reduced VAP protein (Teuling et al., 2007) or mRNA levels
(Anagnostou et al., 2008), suggesting that loss of VAP contributes to the pathology of
sporadic ALS.
CONCLUSION
Over the last century, research activities in Drosophila have had a huge impact on our
understanding of vertebrate neuroscience. In particular, the wealth of powerful research tools
available with this model organism has driven important discoveries into the mechanisms
underlying neurodegenerative diseases. In this chapter, we provide evidence that dissecting
pathways in which a disease gene is operating in fruit flies is a productive approach to
understanding the mechanisms leading to the pathology of ALS. The recent Drosophila
studies into the normal function of VAP have provided invaluable novel insights into the
mechanism of the ALS pathogenesis, while analysis of the cellular defects associated with
mutant VAP has helped us understand key pathological features implicated in this disorder.
Future studies aiming at uncovering the functions and pathways in which VAP is operating
will help develop strategies and identify targets for therapeutic intervention, in order to
prevent or delay the course of familial and sporadic ALS.
ABOUT THE AUTHORS
Amina Moustaqim-Barrette is an undergraduate student studying Neuroscience and
Philosophy at McGill University, Montreal, Canada. Upon joining the Tsuda lab at the
Montreal Neurological Institute in early 2012, her main research initiatives have revolved
around the VAP protein and its implication in ALS.
A Secreted Ligand for Growth Cone Receptors … 47
Mario Maira is a research associate scientist at the Montreal Neurological Institute.
After obtaining is PhD in biochemistry, he studied developmental neurobiology at UCSF and
cell signalling at McGill University as a post-doctoral fellow. Then he joined the private
sector to investigate potential drug candidates for treatment of neurodegenerative diseases. He
is currently working in the Tsuda Lab at McGill University where his research is centered
around the molecular mechanisms relevant to the ALS.
Hiroshi Tsuda is an Assistant Professor in the Department of Neurology and
Neurosurgery at the McGill University. He obtained his MD from Kobe University, Japan
and his board certification in neurology. Then he decided to work on the basic mechanisms of
neurodegeneration and obtained his PhD from Kyoto University. He carried out his Postdoc
studies on learning in fruit flies in the Hugo Bellen lab at the Baylor College of Medicine. His
own laboratory in the Montreal Neurological Institute at the McGill University is focused on
understanding neurodegenerative diseases including ALS through the use of the fly and
mouse model.
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 3
FLIES IN MOTION: WHAT DROSOPHILA CAN TELL
US ABOUT AMYOTROPHIC LATERAL SCLEROSIS
Andrés A. Morera, Alyssa Coyne
and Daniela C. Zarnescu
Department of Molecular and Cellular Biology,
Department of Neuroscience and Department of Neurology,
University of Arizona, Tucson AZ, US
ABSTRACT
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with
a prevalence of 1 in 30,000 individuals. Generally diagnosed between 40 and 70 years of
age, ALS is accompanied by progressive loss of motor neuron function and a life
expectancy of 2-5 years. Although age is considered to be the highest risk factor for ALS,
it is clear that the disease has a genetic basis and may also be influenced by
environmental factors. Familial ALS (fALS) affects 10% of patients and has been linked
to several loci, the most common of which is C9ORF72, a gene of unknown function. The remaining 90% of ALS cases are sporadic (sALS) and remain poorly understood,
although some loci have been linked to both fALS and sALS. In recent years a dramatic
shift in our thinking about ALS has been catalysed by findings that the RNA binding
proteins TAR DNA binding protein 43 (TDP-43) and fused in sarcoma/translocated in
liposarcoma (FUS/TLS) constitute markers of pathology and when mutated, neural
degeneration occurs in human patients. Studies in a wide range of model systems
including worms, flies, zebrafish and rodents support the notion that alterations in these
RNA binding proteins and RNA metabolism cause motor neuron disease. Together with
the recent discovery of GGGGCC repeat expansions in C9ORF72, these studies led to the
hypothesis that ALS is a disease of RNA dysregulation. Here we will review the
contributions of the fruit fly Drosophila melanogaster to our understanding of ALS with
a focus on TDP-43, FUS/TLS and RNA dysregulation as a disease mechanism.
Corresponding author: Daniela C Zarnescu, PhD, Associate Professor, Molecular and Cellular Biology.
Neuroscience, Neurology, 1007 E Lowell St, Life Sciences South 552, University of Arizona, Tucson AZ
85721, email: [email protected].
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 58
Keywords: Drosophila, ALS, motor neuron disease, TDP-43, FUS, RNA binding proteins
INTRODUCTION
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig’s disease, is a
neurodegenerative disease with an incidence of 2 in 100,000 people (Roman, 1996). Without
a clear diagnostic test, patients are identified mainly by ruling out a host of other ALS-like
disorders. Although examples of young-adult onset have been reported, most patients are
diagnosed in their forties or later in life (Cleveland and Rothstein, 2001; Gouveia and de
Carvalho, 2007; Logroscino et al., 2005). Those affected by this devastating disease suffer
progressive muscle atrophy due to degeneration of upper and lower motor neurons, leading to
paralysis and death within 2-5 years of diagnosis. Interestingly, about 20% of all ALS
patients also exhibit FrontoTemporal Lobar Degeneration (FTLD), a related
neurodegenerative disorder with overlap at the pathology level (Banks et al., 2008). The
clinical presentation of ALS is heterogeneous, which is in part, a reflection of the complex
genetic and environmental factors linked to this disease. At this time there is no cure for ALS
and the only available treatment, Riluzole, is at best palliative (Miller et al., 2012).
Familial ALS (fALS) affects approximately 10% of patients and has been linked to
several genes, including the recently discovered C9ORF72, which is thought to be
responsible for ~37.6% of fALS cases (DeJesus-Hernandez et al., 2011; Majounie et al.,
2012; Renton et al., 2011). The remaining 90% of ALS cases are sporadic (sALS) and remain
poorly understood, although some loci have been linked to both fALS and sALS (Alexander
et al., 2002). A plethora of studies have focused on superoxide dismutase 1 (SOD1) the first
locus to be identified as causative of ALS (Rosen et al., 1993). SOD1, an enzyme responsible
for converting superoxide radicals (by-products of oxidative phosphorylation in the
mitochondria) into hydrogen peroxide and molecular oxygen, plays an important role in
preventing cellular damage during times of oxidative stress. SOD1 mutations are found in
~20% of all fALS and ~1% of sALS cases, while altered expression of wild-type SOD1 has
been found in a significant fraction of sporadic cases (reviewed in Andersen, 2006).
Interestingly, ALS has been linked to several different genes encoding proteins of
sometimes seemingly unrelated or even unknown functions, as is the case with the recently
identified C9ORF72 locus. In addition to C9ORF72 and SOD1, these include alsin, senataxin,
VAMP/synaptobrevin-associated protein B (VAPB), P150 dynactin, angiogenin, fused in
sarcoma/translocated in liposarcoma (FUS/TLS, referred herein as FUS), and TAR DNA
binding protein 43 (TDP-43) (Beleza-Meireles and Al-Chalabi, 2009; Lagier-Tourenne and
Cleveland, 2009). Based on the known functions of these loci and extensive pathological
studies in autopsy samples and model organisms, ALS appears to be the result of defects in
several cellular processes including oxidative stress, intracellular transport, RNA metabolism
and apoptosis (reviewed in Beleza-Meireles and Al-Chalabi, 2009; Lagier-Tourenne and
Cleveland, 2009). How these diverse processes converge on a common path to motor neuron
disease remains an open question. A combination of molecular and genetic approaches, both
in vitro and in vivo, using a host of model systems is being employed to elucidate the
mechanisms by which individual gene mutations and other, non-genetic factors lead to ALS
(Joyce et al., 2011; McDonald et al., 2011; Tovar et al., 2009).
Flies in Motion 59
In recent years a dramatic shift in our thinking about ALS has been catalysed by findings
that the RNA binding proteins TDP-43 and FUS constitute markers of pathology and when
mutated, neural degeneration occurs in human patients (Colombrita et al., 2011; Kabashi et
al., 2008; Kwiatkowski et al., 2009; Sreedharan et al., 2008; Vance et al., 2009). Studies in
models including yeast, worms, flies, zebrafish and mouse support the notion that alterations
in these RNA binding proteins and RNA metabolism cause motor neuron disease (Couthouis
et al., 2011; Estes et al., 2011; Kabashi et al., 2011; Lanson et al., 2011; Li et al., 2010;
Liachko et al., 2010; Lu et al., 2009; Ramesh et al., 2010; Wegorzewska et al., 2009).
Together with the recent discovery of GGGGCC repeat expansions in the noncoding region
of C9ORF72, these studies support the emerging hypothesis that ALS is a disease of RNA
dysregulation (DeJesus-Hernandez et al., 2011; Lagier-Tourenne and Cleveland, 2009;
Renton et al., 2011).
A plethora of genetic and clinical studies indicate that ALS is a complex disease with a
clear genetic basis although epigenetics and environmental factors are also thought to play a
role (Ahmed and Wicklund, 2011; Horner et al., 2008). Genome Wide Association Studies
(GWAS) have contributed to the identification of several loci linked to ALS and have
provided insights into the genetic complexity of the disease (reviewed in Valdmanis et al.,
2009). Elegant genetic studies in yeast and flies identified functional interactions between
TDP-43 and ataxin 2, which led the way to the discovery that ataxin 2 is a susceptibility
factor for ALS in human patients (Elden et al., 2010). These findings underscore the
importance of studies in model systems, which can provide critical insights into the
mechanisms by which individual gene mutations and mutation combinations contribute to
ALS. Furthermore, model systems can help elucidate the complexity of gene networks and
cellular pathways involved in disease that could be later validated in patients. It is only
through a concerted collaboration between basic scientists, human geneticists and clinicians
that we can expect major progress in ALS research and therapeutics in the coming years.
ALS IN MODEL ORGANISMS: YEAST TO MICE
Some of the early models of ALS based on SOD1 were developed in mice (Gurney,
1997) and rats (Howland et al., 2002; Nagai et al., 2001). These transgenic rodent models
expressing mutant human SOD1 recapitulate many of the disease features seen in human ALS
patients, including muscle atrophy, upper and lower motor neuron degeneration, disease-
associated cytoplasmic inclusions in neurons, and higher mortality (Gurney et al., 1996).
Other organisms in which SOD1-based ALS has been modelled include dog (Awano et al.,
2009), zebrafish (Ramesh et al., 2010), fruit fly (Watson et al., 2008), worm (Gidalevitz et al.,
2009; Wang et al., 2009) and yeast (Nishida et al., 1994). These different models have distinct
advantages as well as limitations that need to be carefully considered.
Recently, models of ALS based on TDP-43 and FUS have also been generated in model
systems ranging from yeast to worms to flies, fish and rodents (Couthouis et al., 2011; Estes
et al., 2011; Kabashi et al., 2011; Laird et al., 2010; Lanson et al., 2011; Li et al., 2010;
Liachko et al., 2010; Lu et al., 2009; Wegorzewska et al., 2009). Although some
discrepancies exist between these models, overall they recapitulate several aspects of ALS
including neurodegeneration, TDP-43 aggregation, locomotor dysfunction and reduced
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 60
survival. Here we will review the contributions of the fruit fly Drosophila melanogaster to
our understanding of ALS with a focus on TDP-43, FUS and RNA dysregulation as a disease
mechanism.
Current Hypotheses for ALS
Similar to other neurodegenerative diseases, including Alzheimer’s disease and
Parkinson’s disease, ALS pathology is accompanied by ubiquitin-positive cytoplasmic
aggregates within neurons, with shapes ranging from round to fusiform (Neumann, 2009).
This led to the hypothesis that ALS is a proteinopathy and suggested that protein misfolding,
possibly as a result of cellular stress, is a potential toxic mechanism.
As with other neurodegenerative disorders, the significance of protein aggregates remains
unclear and evidence exists to support both a toxic as well as a protective role for these
abnormal intracellular structures (Young, 2009). Importantly, age, which is the highest risk
factor for ALS, correlates with reduced proteostasis and increased potential for protein
misfolding and aggregation.
This model is supported by several findings including the propensity of TDP-43 and FUS
to aggregate in vitro (Johnson et al., 2009; Sun et al., 2011) and their association with
cytoplasmic puncta in cell models (Liu-Yesucevitz et al., 2010; McDonald et al., 2011). In
addition, TDP-43 and FUS harbour prion domains that are involved in aggregation and thus
are likely to be relevant to the pathology of ALS (Gitler and Shorter, 2011).
Oxidative stress has offered a particularly attractive model for SOD1 cases where toxicity
has been proposed to stem from free radical accumulation. This mechanism however, remains
uncertain due to lack of consistent evidence for oxidative damage (reviewed in Rothstein,
2009). Defects in axonal transport, which are typically found in neural degeneration were also
linked to ALS via mutations in dynactin, a subunit of the motor protein dynein (Munch et al.,
2004).
Additional hypotheses include an imbalance in trophic factors such as VEGF, which is
thought to be neuroprotective (Dodge et al., 2010; Kulshreshtha et al., 2011; Shimazawa et
al., 2010; Tovar-y-Romo and Tapia, 2012; Wang et al., 2007) and synaptic hyperexcitability,
likely due to an inability of glial cells to buffer excess glutamate within the synaptic cleft
(Foran and Trotti, 2009; Vucic and Kiernan, 2009).
RNA Dysregulation in ALS
Although the proteinopathy hypothesis, primarily driven by the overwhelming
pathological evidence for cytoplasmic aggregates, has dominated the field, recent gene
discovery studies point to the involvement of RNA binding proteins including TDP-43 and
FUS, both of which associate with intracellular inclusions and also act as causative agents of
disease (Kabashi et al., 2008; Kwiatkowski et al., 2009; Sreedharan et al., 2008; Vance et al.,
2009). TDP-43 was found to be a major component of cytoplasmic inclusions in motor
neurons of non-SOD1 fALS and sALS cases (Neumann et al., 2006).
Notably, TDP-43and FUS positive inclusions have been found in a wider spectrum of
neurodegenerative disorders including FrontoTemporal Lobar Degeneration and Alzheimer’s
Flies in Motion 61
disease (reviewed in Baloh, 2012; Blair et al., 2010; Gitler and Shorter, 2011). Extensive
mutation analyses in ALS patients showed that TDP-43 and FUS are linked to 4-5% of fALS
and 2% of sALS cases across ethnicities (reviewed in Colombrita et al., 2011; Lagier-
Tourenne and Cleveland, 2009).
Although at this time we do not fully understand how TDP-43 and FUS function in motor
neurons or the surrounding glial cells and how mutations in these RNA binding proteins lead
to motor neuron disease, new details are emerging on their connection to various aspects of
RNA regulation including RNA splicing, export, stability and translation (reviewed in Lagier-
Tourenne and Cleveland, 2009).
The discovery of TDP-43 and FUS together with the involvement of additional RNA
binding proteins (senataxin, angiogenin) and RNA itself (C9ORF72 noncoding expanded
repeats) in ALS, has led to a repositioning of hypotheses now centered on RNA-based
mechanisms. Notably, this newly emerging hypothesis and the protein aggregation model are
not necessarily mutually exclusive and may be interconnected at the molecular level by
multiprotein/RNA complexes known as RNA granules that have been proposed to act as
precursors of protein aggregates (Dewey et al., 2011; Parker et al., 2012).
TAR DNA Binding Protein (TDP-43)
The RNA binding protein TDP-43 has first captured the attention of the ALS field due to
its identification as a component of cytoplasmic inclusions in neurons and the surrounding
glia (Maekawa et al., 2009; Neumann et al., 2006; Tan et al., 2007). Originally identified as a
transcriptional repressor of HIV-1, TDP-43 consists of two RNA recognition motifs (RRM1,
2) and a glycine-rich domain within the C-terminus (Figure 1, Ou et al., 1995). Its cellular
functions are just beginning to be understood and include in addition to transcriptional
repression, pre-mRNA splicing, miRNA biogenesis and apoptosis (Figure 2, reviewed in
Banks et al., 2008). In vitro assays and RNA sequencing approaches have shown that TDP-43
binds with high affinity UG-rich sequences and regulates splicing of numerous mRNA targets
(Ayala et al., 2011; Buratti and Baralle, 2001; Polymenidou et al., 2011; Sephton et al., 2011;
Tollervey et al., 2011). TDP-43 is ubiquitously expressed and co-localizes with Survival of
Motor Neuron (SMN) and Gemin proteins in the nucleus. In hippocampal neurons, TDP-43
associates with cytoplasmic RNA granules and co-localizes with Fragile X mental retardation
protein (FMRP), Staufen and HuD in an activity-dependent manner, suggesting that TDP-43
may regulate synaptic plasticity in vivo by regulating synaptic mRNAs (Fallini et al., 2012;
Wang et al., 2008). Recently, individual TDP-43 mutations have been shown to differentially
regulate stress granule formation in various cell lines subjected to cellular stress (Dewey et
al., 2011; Liu-Yesucevitz et al., 2010; McDonald et al., 2011). RNA granules play critical
roles in post-transcriptional gene expression and are comprised of large RNA/protein
complexes that can be visualized as cytoplasmic puncta. Among these, stress granules
represent foci of RNA storage that form in response to cellular stress and correlate with
translation inhibition (Anderson and Kedersha, 2009).
The majority of TDP-43 mutations found in ALS patients represent aminoacid
substitutions that are thought to increase TDP-43 phosphorylation and caspase cleavage of the
C-terminus followed by proteasome-mediated degradation (Kabashi et al., 2008; Rutherford
et al., 2008; Sreedharan et al., 2008; Xu et al., 2010). It has been proposed that overwhelming
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 62
the proteasome degradation machinery may lead to the accumulation of TDP-43 C-terminal
fragments in the cytoplasm, which have been shown to be toxic through a gain of function
mechanism (Zhang et al., 2009). More work is needed to elucidate the mechanisms by which
these individual alterations in TDP-43 lead to motor neuron disease and degeneration.
Interestingly, proteomic analyses of the TDP-43 complex indicate that its protein partners,
which are enriched in components of the splicing and translational machinery do not differ
between wild-type and A315T or M337V variants (Freibaum et al., 2010). In keeping with
this observation, several different TDP-43 variants (i.e., wild-type, A382T, Q331K and
M337V) exhibit comparable binding affinity for specific mRNA targets (Colombrita et al.,
2012). Using biochemical purification approaches coupled with microarray analyses or RNA
sequencing, several RNAs associated with TDP-43 have been identified (Colombrita et al.,
2012; Polymenidou et al., 2011; Sephton et al., 2011; Tollervey et al., 2011). These include
numerous splicing targets containing (TG)n sequences, with a clear preference for RNAs with
long introns (Polymenidou et al., 2011; Tollervey et al., 2011). Given its presence in
cytoplasmic inclusions and the identification of several mutations linked to ALS, TDP-43 has
emerged as a common denominator for the majority of clinical cases known to date (Banks et
al., 2008; Neumann, 2009) and this suggests that TDP-43’s involvement in motor neuron
disease extends beyond its role in pre-mRNA splicing. The identification of TDP-43-
associated RNA targets linked to disease as well as synaptic function and neuronal
development position TDP-43 as a player not only in specific aspects of RNA regulation but
also in the various cellular processes in which its targets are involved. For example, knock-
out (KO) mice lacking TDP-43 exhibit alterations in fat metabolism (Chiang et al., 2010)
while the Drosophila homolog of TDP-43, TBPH has been identified in a genome wide RNAi
screen for genes involved in neuronal development (Sepp et al., 2008). It will be interesting to
see what specific targets mediate these developmental and physiological roles of TDP-43
outside of its involvement in neural degeneration.
Fused in Sarcoma (FUS)
Analysis of brain and spinal cord tissues from patients with ALS has led to the discovery
of the presence of FUS mutations resulting in cytoplasmic inclusions in neurons and glial
cells (Kwiatkowski et al., 2009; Vance et al., 2009). FUS, initially discovered for its role in
human sarcoma, is part of the TET/FET family of proteins that also includes Ewing’s
Sarcoma protein EWSR1 and TATA-binding protein associated factor TAF15 (reviewed in
Gitler and Shorter, 2011). At the structural level, FUS contains an N-terminal domain with a
QGSY region, a glycine-rich region, and RRM domain, several RGG repeats, and a zinc
finger motif in the C-terminus (Figure 1). The majority of the missense mutations associated
with ALS are located in the nuclear localization signal (NLS) region of the C-terminus
(Kwiatkowski et al., 2009; Vance et al., 2009). Although the full function(s) of FUS are still
not fully understood, several reports demonstrate its involvement in transcription, pre-mRNA
splicing, and local translation at the synapse (Figure 2 and reviewed in Colombrita et al.,
2011; Lagier-Tourenne and Cleveland, 2009). In hippocampal neurons, FUS has been shown
to be involved in mRNA transport to dendritic spines and to control spine morphology,
presumably by regulating local protein synthesis of specific targets (Fujii et al., 2005; Fujii
and Takumi, 2005).
Flies in Motion 63
Figure 1. TDP-43 and FUS are nucleocytoplasmic shuttling proteins containing RNA binding domains.
Mutations in the NLS domain impair the protein’s ability to localize to the nucleus and as
a result, FUS is targeted to the cytosol, consistent with the discovery of cytoplasmic
inclusions observed in post-mortem ALS samples. NLS mutations have also been shown to
promote the co-localization of FUS with stress granules in the cytoplasm of cells including
neurons and glial cells (Bosco et al., 2010; Gal et al., 2011). Recently, biochemical
purification and gene profiling approaches have identified CAGGACAGCCAG motifs as
FUS-specific targets (Colombrita et al., 2012; Lagier-Tourenne et al., 2012).
TDP-43 and FUS – Shared and Distinct Features
At the sequence level, both TDP-43 and FUS contain RNA binding domains and harbour
NLS and NES (nuclear export signal) domains that permit nucleocytoplasmic shuttling
(Figure 1). At the functional level, both TDP-43 and FUS have been implicated in similar
RNA processing steps including transcription, splicing, association with the miRNA
machinery as well as mRNA transport, transcript stabilization and local translation (Figure 2
and reviewed in Colombrita et al., 2011; Lagier-Tourenne and Cleveland, 2009). In addition,
both TDP-43 and FUS have been shown to associate with RNA stress granules. A major
difference between the two proteins is that while both wild-type and mutant TDP-43 variants
associate with stress granules, in the case of FUS, only the mutant forms co-localize with
stress granule markers (Bosco et al., 2010; Liu-Yesucevitz et al., 2010; McDonald et al.,
2011). In addition to differences in direct binding sequences their targets are also mostly
distinct with the exception of 45 transcripts that are down-regulated in the absence of either
TDP-43 or FUS (Lagier-Tourenne et al., 2012). Immunoprecipitation experiments indicate
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 64
that TDP-43 and FUS associate in a protein complex in whole brains or HEK cells (Freibaum
et al., 2010; Kim et al., 2010; Sephton et al., 2011) but not in motoneuronal NSC-34 cells
(Colombrita et al., 2012). Given their participation in similar RNA processing steps and their
involvement in ALS, these findings are somewhat surprising and suggest cell-type and
context specific interactions between TDP-43 and FUS.
In addition to an NLS and an NES, TDP-43 contains two RNA Recognition Motifs,
RRM1 and RRM2 and a C-terminus glycine-rich region, which harbours the majority of
disease linked mutations as well as a prion domain (Gitler and Shorter, 2011). FUS contains a
Q/G/S/Y rich region followed by a glycine-rich region, which also harbours a prion domain
(Gitler and Shorter, 2011). The C-terminus half of FUS contains an RNA recognition motif
(RRM), an RGG rich region harbouring a Zinc finger motif as well as a second prion domain
in addition to an NES and NLS (Gitler and Shorter, 2011).
Disease linked mutations in FUS cluster within the NLS. Domains defined according to
http://www.uniprot.org and predicted nuclear export signals defined according to
http://www.cbs.dtu.dk/services/NetNES.
Indeed, with the exception of one report of FUS positive inclusions in a patient
harbouring the G298S mutation within TDP-43, pathological inclusions contain either one or
the other but not both (Davidson et al., 2012; Deng et al., 2010). While it is possible that
interactions between TDP-43 and FUS are context dependent, these findings raise questions
about the functional relationship between the two proteins and about where in the pathway
that leads to neuronal dysfunction they may intersect. Using genetic interaction approaches,
experiments in Drosophila have shown that the FUS homolog cabeza (caz) and the TDP-43
homolog, TBPH may function in a common pathway with caz acting downstream of TBPH
(Wang et al., 2011).
Figure 2. Both TDP-43 and FUS are implicated in similar steps of RNA processing.
Flies in Motion 65
A. Regulation of transcription. TDP-43 has been demonstrated to bind TG-rich promoter
regions of target genes, inhibiting transcription, as in the case of mouse acrv1 and SP-10
genes (Abhyankar et al., 2007). FUS/TLS is also involved in transcriptional regulation,
having been shown to be recruited to the promoter region of cyclin D1 by non-coding RNAs
induced by DNA damage (Wang, 2008; Lalmansingh et al., 2011). Also, evidence of
association of FUS to the TFIID complex implicates it in general transcriptional regulation
(Bertolotti et al., 1998). B. pre-mRNA splicing. Recent studies demonstrated that TDP-43
binds to numerous pre-mRNA splicing targets containing (UG)n sequences, with a clear bias
for RNAs with long introns, and likely participates in splicing of these targets (Ayala et al.,
2011; Buratti and Baralle, 2001; Polymenidou et al., 2011; Sephton et al., 2011; Tollervey et
al., 2011). There is also strong evidence indicating that FUS participates in pre-mRNA
splicing as it has been found to be part of the spliceosome (Hartmuth et al., 2002; Zhou et al.,
2002). C. miRNA processing. TDP-43 and FUS/TLS are thought to be involved in miRNA
processing, as both bind to Drosha (Gregory et al., 2004), and TDP-43 interacts with the
Dicer complex (Kawahara and Mieda-Sato, 2012). D. mRNA transport. Both TDP-43 and
FUS proteins are shown to transport mRNA to dendritic spines in hippocampal neurons,
suggesting that they may also be involved in transporting target mRNAs throughout the cell
(Fujii et al., 2005; Fujii and Takumi, 2005). E. Stress granules. FUS and TDP-43 have also
been shown to localize to stress granules, which are associated with repression of translation
(Bosco et al., 2010; Colombrita et al., 2009; Liu-Yesucevitz et al., 2010; McDonald et al.,
2011). F. Regulation of local translation. Through regulation of mRNA transport and stress
granule formation, FUS and TDP-43 may also regulate local translation (Liu-Yesucevitz et
al., 2011; Wang et al., 2008).
Modelling ALS in Drosophila
In recent years Drosophila melanogaster has proven to be a powerful model system for
studying the basic biology of human disease genes as well as elucidating the mechanisms of
disease. Notably, about 75% of the known human disease genes have homologs in
Drosophila (Reiter et al., 2001). Although obvious differences exist between flies and
humans, the conservation of cellular and developmental signalling pathways lends a clear
advantage for employing Drosophila to elucidate the basic biology of genes and to generate
models of human disease. A recent example of success lies with modelling the most common
form of inherited mental retardation, Fragile X syndrome, which led to the identification of
novel neuroanatomical and functional phenotypes as well as interacting genes and small
molecules with therapeutic potential (Chang et al., 2008; Jin et al., 2004; McBride et al.,
2005; Wan et al., 2000; Zarnescu et al., 2005). The Drosophila model offers an unparalleled
set of genetic tools that allow for tissue specific and temporally controlled expression of
genes of interest; these could be wild-type or mutant alleles linked to human disease, or RNAi
constructs for gene knock-down (Brand and Perrimon, 1993; McGuire et al., 2003; Roman et
al., 2001). In addition, clonal analysis approaches allow for loss- and gain-of-function as well
as lineage studies (Lee and Luo, 1999; Pignoni and Zipursky, 1997; Xu and Rubin, 1993).
The use of these sophisticated genetic tools has helped uncover novel components and
functions of the EGFR/Ras, Notch and other signalling pathways, which in turn has advanced
our knowledge of human biology and mechanisms of disease (Doroquez and Rebay, 2006; Hu
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 66
and Li, 2010). Recently, Drosophila has proven its usefulness as a tool for drug discovery
(reviewed in Pandey and Nichols, 2011). Together with the relatively low costs involved and
a short generation time (10-12 days at 25oC), Drosophila offers powerful tools for gene and
drug discovery as well as elucidating the pathophysiological mechanisms underlying various
diseases in humans.
TDP-43 and FUS in Drosophila
There are two TDP-43 orthologues in the fly genome, TBPH and CG7804 (Estes et al.,
2011). Interestingly, CG7804 lacks the entire C-terminus domain that harbours the majority
of missense mutations in TDP-43 and is thought to play a major role in disease (Pesiridis et
al., 2009). Although at this time it is not clear what the precise function or evolutionary
significance of CG7804 is, it shares 41.5% amino acid identity to TBPH and 34.5% to TDP-
43 (Estes et al., 2011). Interestingly, loss-of-function of TBPH is sufficient to cause motor
neuron and ALS-like phenotypes (Feiguin et al., 2009). FUS has a single orthologue in
Drosophila, namely caz, which shares 53% aminoacid identity with its human counterpart
(Wang et al., 2011).
TDP-43 Based Models of ALS in Flies
Initial studies focused on elucidating the requirement of TBPH in motor neurons using
loss-of-function approaches. Hypomorphic and null alleles of TBPH exhibit smaller larval
neuromuscular junctions (NMJs), adult climbing defects and reduced longevity. Importantly,
expressing human TDP-43 in motor neurons only rescues these phenotypes indicating that
TBPH and TDP-43 are functionally conserved (Feiguin et al., 2009). Several groups took a
reverse translational approach, whereby either wild-type or mutant human TDP-43 were
overexpressed in various types of neurons including photoreceptors, motor, dendritic
arborisation (da) or mushroom body neurons (Elden et al., 2010; Estes et al., 2011; Hanson et
al., 2010; Lanson et al., 2011; Li et al., 2010; Lu et al., 2009; Miguel et al., 2011; Ritson et
al., 2010; Voigt et al., 2010; Wang et al., 2011). These experiments showed that TDP-43
overexpression is neurotoxic and leads to neuronal degeneration, locomotor dysfunction and
reduced survival, all of which are remarkably similar to the human disease. Notably,
mutations in TDP-43 that affect the RRM1 domain and its RNA binding ability exhibit milder
phenotypes, suggesting that RNA binding is required for toxicity (Voigt et al., 2010).
An important question in the field concerns the role of the C-terminal domain of TDP-43
in disease. It has been proposed that the missense mutations found in human patients, the
majority of which reside in the C-terminus, promote caspase cleavage, which in turn leads to
accumulation of C-terminal fragments in aggregates. While experiments in mammalian cells
have provided support to this model, with the exception of one report (Gregory et al., 2012),
all other in vivo studies using C-terminus overexpression did not produce any obvious
phenotypes (Estes et al., 2011; Li et al., 2010; Voigt et al., 2010; Yang et al., 2010). It will be
interesting to see whether this is a fly-specific response or perhaps, a reflection of the
increased ability of the whole organism to handle excess C-terminal fragments compared to
cultured cells.
Flies in Motion 67
TBPH/TDP-43 Localization and Toxicity: Loss of Nuclear Function or Gain
of Cytoplasmic Function?
Endogenous TBPH appears to localize primarily to the nucleus, while both nuclear,
perinuclear and some cytoplasmic puncta containing TBPH have been observed upon
overexpression (Estes et al., 2011; Hazelett et al., 2012; Lin et al., 2011). This is not unlike
studies of human TDP-43, which appears restricted to the nucleus when expressed at
endogenous levels but associates with cytoplasmic granules upon overexpression in
mammalian neurons (McDonald et al., 2011; Xu et al., 2010). In keeping with this
observation, upon overexpression in Drosophila motor neurons, TDP-43 is primarily
restricted to the nucleus although some reports of axonal TDP-43 also exist (Estes et al.,
2011; Li et al., 2010; Lu et al., 2009). These discrepancies could, in part, be explained by
differences in levels of expression between various published models. Interestingly, when
expressed in the developing neuroepithelium, TDP-43 associates with cytoplasmic puncta
within axons, which may be due to a differential response of photoreceptor neurons compared
to motor neurons (Estes et al., 2011).
A major question remains whether toxicity is conferred by TDP-43 in the nucleus,
cytoplasm or both. In other words, is toxicity due to a loss of nuclear function or a gain of
cytoplasmic function? This issue has been elegantly addressed in flies by expressing TDP-43
mutants that lack either the NLS or the NES (Miguel et al., 2011; Ritson et al., 2010). These
experiments showed that cytoplasmic TDP-43 is sufficient to generate neurotoxic phenotypes
and that in motor neurons, cytoplasmic TDP-43 (NLS- TDP-43) is more toxic than nuclear
TDP-43 (NES- TDP-43).Together, these data suggest that TDP-43 expression is toxic both
in the nucleus and the cytoplasm and support a gain of cytoplasmic function model (Miguel et
al., 2011; Ritson et al., 2010). Notably, these as well as several studies using disease-linked
variants found that cytoplasmic puncta are not a prerequisite for toxicity (Estes et al., 2011;
Hanson et al., 2010; Miguel et al., 2011). Genetic interaction experiments between human
TDP-43 and Drosophila TBPH result in an enhancement of the ALS-like phenotypes, which
provides further support to the model that TDP-43 overexpression mimics a loss-of-function
(Estes et al., 2011). In contrast, a recent transcriptome analysis using TBPH loss-of-function
and overexpression showed that these two conditions affect gene expression in very different
ways (Hazelett et al., 2012). Surprisingly, although phenotypically these conditions are rather
similar, at the molecular level there were only 57 transcripts commonly changed between
TBPH loss-of-function and overexpression. Collectively, these localization, phenotypic and
genetic interaction studies coupled with bioinformatics suggest that the overexpression of
TDP-43 variants (wild-type or mutant) shares both loss- and gain-of-function features.
Elucidating what aspects of TDP-43 function are impacted in these models that recapitulate
the pathology remarkably well, will provide much needed insights into the pathophysiology
of ALS.
TDP-43 Cytoplasmic Puncta: RNA Stress Granules or Protein Aggregates?
While in mammalian models TDP-43 has been clearly associated with RNA stress
granules, it remains to be seen what types of RNA granules if any, contain TDP-43 in flies
(McDonald et al., 2011). The role of RNA stress granules is to pull specific mRNAs from the
translational pool under less favourable environmental conditions and protect them from
degradation for some period of time (Anderson and Kedersha, 2009). At the same time,
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 68
biochemical and pathological studies indicate that TDP-43 associates with insoluble
aggregates (Voigt et al., 2010). Could these two cellular structures be connected? While it is
tempting to speculate that RNA stress granules may be precursors of protein aggregates, it is
possible that these two structures represent distinct entities (Dewey et al., 2012). More work
is needed to address this important aspect of ALS pathophysiology.
Reduced Survival and Locomotor Dysfunction
A hallmark of ALS is a progressive reduction in locomotor function and reduced
survival. Importantly, both loss-of-function and overexpression approaches in the Drosophila
model show that this feature is well recapitulated. Removal of TBPH from motor neurons
specifically using RNAi results in larval and adult locomotor dysfunction (Estes et al., 2011;
Feiguin et al., 2009; Li et al., 2010; Voigt et al., 2010). Interestingly, overexpression of TDP-
43 variants in motor neurons produces similar effects with TBPH loss, which lends further
support to the notion that phenotypically, TDP-43 overexpression resembles a loss-of-
function condition. Notably, some differences between wild-type and mutant TDP-43 variants
have begun to emerge. For example, while one study found that mutant TDP-43 exerts more
severe phenotypes compared to wild-type (Guo et al., 2011), others reported that wild-type
TDP-43 is more toxic than mutant variants when expressed at comparable levels (Estes et al.,
2011; Lu et al., 2009; Voigt et al., 2010). Thus while all TDP-43 variants exhibit various
levels of toxicity in flies, phenotypic nuances among different alleles may provide useful
insights into the mechanisms of disease.
Neuroanatomical Phenotypes
Another major feature of ALS pathology involves neuroanatomical defects accompanied
by denervation at the NMJ and more recently, evidence for axonal growth abnormalities
(Fallini et al., 2012). Loss of TBPH results in under grown NMJs characterized by fewer
synaptic boutons (Feiguin et al., 2009). As with the locomotor function studies,
overexpression of either wild-type or mutant human TDP-43 also leads to smaller NMJs,
consistent with a loss-of-function mechanism (Estes et al., 2011; Godena et al., 2011; Li et
al., 2010). At least one report finds that wild-type TDP-43 overexpression generates bigger
NMJs while mutant TDP-43 has no effect, suggesting a gain of function mechanism (Wang et
al., 2011). Despite some differences in the net effect of TDP-43 on NMJ growth, which could
be explained in part by genetic background effects, these findings indicate that TBPH/TDP-
43 modulate the morphology of the larval NMJ synapse. A notable difference between
Drosophila and mammalian models is the lack of obvious denervation and motor neuron
degeneration at least at the larval stage when these experiments were performed. Future
studies involving adult NMJs as well as physiological recordings from the synaptic terminals
are likely to provide additional insights into the precise role of TDP-43 and FUS in the
morphology and function of neuromuscular synapses.
Drosophila Models of ALS Based on FUS
Several recent studies have sought to determine the role of FUS and the mechanisms
underlying FUS pathology in ALS using flies as a model (Chen et al., 2011; Lanson et al.,
Flies in Motion 69
2011; Miguel et al., 2012; Sasayama et al., 2012; Wang et al., 2011; Xia et al., 2012). Loss-
of-function and overexpression approaches have shown that caz/FUS plays a role in both
locomotion and the growth of the NMJ synaptic terminal (Chen et al., 2011; Lanson et al.,
2011; Sasayama et al., 2012; Wang et al., 2011; Xia et al., 2012). caz is ubiquitously
expressed and loss-of-function approaches using either RNAi knock-down or classical
deletions result in lethality, which supports the notion that Caz, like TBPH is required for the
survival of the organism (Sasayama et al., 2012). Overexpression of wild-type and disease
variants of human FUS in the Drosophila eye leads to a progressive degenerative phenotype
accompanied by a loss of ommatidia organization. In motor neurons, FUS overexpression
leads to architectural defects at the NMJ synapse, locomotor dysfunction and reduced
survival, which resemble essential features of ALS pathology (Lanson et al., 2011; Sasayama
et al., 2012; Wang et al., 2011; Xia et al., 2012).
FUS Subcellular Localization and Toxicity
Recent studies aimed at examining the role of FUS mutations in ALS have shown that
wild-type FUS is primarily nuclear while mutant FUS localizes to the cytoplasm, which is
consistent with the inclusions found in disease pathology (Kwiatkowski et al., 2009; Lanson
et al., 2011; Miguel et al., 2012; Vance et al., 2009). Its cytoplasmic localization is required
for toxicity as disease mutants lacking the NES ( FUS) exhibit reduced ALS-like
phenotypes (Lanson et al., 2011). Evidence also exists that the nuclear localization of FUS is
critical for toxicity (Xia et al., 2012). As with TBPH, caz loss-of-function results in locomotor
impairment, suggesting a loss-of-function mechanism (Sasayama et al., 2012). Thus while the
FUS overexpression phenotypes are consistent with a gain of toxicity, it remains unclear
whether the human disease is caused by a loss of nuclear function, gain of cytoplasmic
toxicity or both.
Locomotor Phenotypes and Lifespan Effects
Neuronal specific knock-down of caz using RNAi results in impaired locomotor activity
albeit it has no apparent effect on lifespan (Sasayama et al., 2012). On the other hand,
complete loss of caz function using a null allele leads to severe locomotor defects affecting
both walking and flying as well as decreased lifespan (Wang et al., 2011). Importantly,
neuronal specific expression of caz in a null mutant background rescued most but not all
locomotor phenotypes, suggesting that control of locomotor function is partly intrinsic to
motor neurons. Furthermore, human wild-type FUS overexpression in caz mutant neurons
rescued locomotion defects but mutant FUS linked to fALS did not (Wang et al., 2011).
When overexpressed in wild-type motor neurons, all FUS variants (wild-type and mutants)
led to severe locomotor dysfunction in adult flies (Xia et al., 2012).
Neuroanatomical Defects
Loss-of-function studies show that Caz is required for proper morphology at the larval
NMJ (Sasayama et al., 2012; Wang et al., 2011). Although the structure of the synaptic
boutons themselves appears unaltered, loss of caz leads to undergrown synapses, containing
fewer synaptic boutons (Sasayama et al., 2012). As with TDP-43, FUS overexpression in
motor neurons is neurotoxic. While some report a disorganization of motor neurons and a
reduced NMJ area accompanied by a decrease in the number of small and large axonal
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 70
boutons (Xia et al., 2012), others report an increase in the number of synaptic boutons or no
change when FUS is overexpressed (Lanson et al., 2011; Wang et al., 2011). As with TDP-
43, despite some differences between various studies, it is clear that FUS is required to
regulate the architecture of the NMJ synapse.
Pathways and Networks
One of the strongest assets of Drosophila as a model for human disease is the power of
the genetic toolbox. The ability to sort through biochemically identified candidate targets and
partners to determine functional interactions in vivo represents a major strength of the fly
model. While full genome genetic and drug screens have yet to be published, several genetic
interactions have been reported in the fly models of TDP-43 and FUS. Importantly, TDP-43
and FUS exhibit genetic interactions with each other, suggesting that at least some functional
partners are common (Lanson et al., 2011; Wang et al., 2011).
In keeping with the RNA dysregulation hypothesis, TAF15, an RNA binding protein with
a domain structure similar to that of FUS has recently been reported to co-localize with TDP-
43 in pathological inclusions and to mimic several ALS features with the TDP-43 fly model
(Couthouis et al., 2011). It would not be surprising for several other RNA binding proteins to
exhibit similar interactions with TDP-43 and FUS. Identifying which of the known protein
partners can modify TDP-43 or FUS neurotoxicity may be critical for the development of
future therapeutic strategies.
Additional genetic interactions reported for TDP-43 include the HSP70 chaperone, the
caspase inhibitor P35, which suppress photoreceptor neurodegeneration as well as a dominant
negative construct of the small proteasome subunit, which enhances TDP-43 phenotypes
(Estes et al., 2011). These findings provide further evidence that the TDP-43 Drosophila
model recapitulates not only pathological features but also exhibits in vivo interactions with
well-established pathways including protein folding, apoptosis and proteasome mediated-
degradation. Similar to TDP-43, the neurotoxicity induced by FUS in the retina is alleviated
by overexpression of the HSP70 (HSPA1L) chaperone that has also been shown to mitigate
polyglutamine-mediated neurodegeneration (Miguel et al., 2012; Warrick et al., 1999).
In recent years, the discovery of novel genetic and environmental factors for
neurodegenerative disorders have led to the emergence of multi-hit models for
neurodegeneration, similar to Weinberg’s breakthrough cancer models (Hanahan and
Weinberg, 2000). Thus perhaps it is not surprising to discover that mutations in ataxin 2
accompany TDP-43 mutations in patients, making ataxin 2 a risk factor for ALS (Elden et al.,
2010). Similarly, the Type 1, inositol 1,4,5 Triphosphate (IP3) Receptor, ITPR1, a ER
resident, IP3 gated Ca2+
channel, which has been previously linked to cerebellar ataxia,
modulates TDP-43’s nucleocytoplasmic shuttling and its clearance by autophagy (Kim et al.,
2012; van de Leemput et al., 2007). Another example providing support to the multi-hit
model is Valosin Containing Protein (VCP), an ATP-ase involved in segregating
ubiquitinated substrates from large protein complexes and linked to inclusion body myopathy
associated with Paget’s disease of bone and frontotemporal dementia, which interacts
genetically with TBPH (Ritson et al., 2010). This functional interaction was identified in an
unbiased screen for VCP modifiers and suggests that VCP toxicity is mediated by TDP-43, as
evidenced by the redistribution of TDP-43 from the nucleus to the cytoplasm, which is
Flies in Motion 71
consistent with inclusions containing TDP-43 in inclusion body myopathy associated with
Paget’s disease of bone and frontotemporal dementia patients.
Another emerging theme is that TDP-43 and FUS may regulate the stability of the
microtubule network and possibly affect the transport processes dependent on the microtubule
cytoskeleton, which is one of the current ALS hypotheses. For example, HDAC6, a histone
deacetylase implicated in transcriptional control as well as acetylation of tubulin has been
shown to be a target of TDP-43 (Polymenidou et al., 2011). In human cells, TDP-43 and FUS
compete for binding HDAC6 mRNA, which supports the notion that they act in a common
pathway to regulate HDAC6 controlled processes (Kim et al., 2010). At the larval NMJ, the
microtubule associated protein MAP1B/Futsch, which like HDAC6 acts to stabilize
microtubules, is reduced in TBPH mutants, providing a possible explanation for the observed
undergrowth of the synaptic terminals (Feiguin et al., 2009; Godena et al., 2011).
Recently, RNA sequencing approaches using loss-of-function and overexpression
conditions identified significant changes in transcripts linked to the Wnt and BMP pathways,
suggesting that TBPH/TDP-43 may regulate the output of these pathways in the nervous
system (Hazelett et al., 2012).
CONCLUSION: CHALLENGES AND OPPORTUNITIES
The past years since the first reports of TDP-43 mutations in fALS and sALS patients
(Kabashi et al., 2008; Sreedharan et al., 2008; Van Deerlin et al., 2008) have seen an
avalanche of studies aimed at elucidating the basic biology of TDP-43 and the underlying
mechanisms of disease. The discovery of FUS followed shortly (Kwiatkowski et al., 2009;
Vance et al., 2009), which prompted a repositioning of ideas in the field and led to the
emergence of the RNA dysregulation hypothesis (Lagier-Tourenne and Cleveland, 2009).
Does the fly provide a good model for ALS and for studying these new hypotheses? The
sceptics remain concerned about the gulf between the fly model and humans. Eyebrows are
still raised upon discussions of ALS (or other human disease) modelling in the tiny fruit fly.
The evidence however stands to demonstrate that there are remarkable similarities between
the fly phenotypes and ALS pathology. These include among others, locomotor dysfunction
and reduced lifespan, which are, after all, the main problem with this devastating disease.
There are of course caveats that we need to be mindful of, including the overexpression
paradigms used by most models and the differences in glial biology, especially when
considering that glia seems to play a major role in motor neuron disease (Howland et al.,
2002). However, when one considers the strengths of the fly model, they weigh a lot heavier
than the caveats. For example, the ability to express or knock-down genes in a tissue, cell-
type and even temporal-specific manner, allows us to address why various types of neurons
are differentially affected in neurodegenerative disorders. Furthermore, the genetic toolbox
offers multiple strategies for unbiased genetic and drug screens.
Given the high degree of conservation between flies and humans, these screens provide a
relatively high-throughput means of discovering genes and compounds with therapeutic
promise in humans. Most surely, we will see data coming from such endeavours in the
coming years.
Andrés A. Morera, Alyssa Coyne and Daniela C. Zarnescu 72
So, what have we learned from the fruit fly models? First, loss-of-function studies
demonstrate the requirement of TDP-43 and FUS during development as well as proper
neuronal function afterwards. Thus, there may be a previously unappreciated developmental
component for this neurodegenerative disease. Second, we learned that fALS and sALS
mutations in TDP-43 and FUS affect motor neurons intrinsically, in ways that mimic their
effects in humans, i.e., their function and survival are compromised. Furthermore, genetic
interactions in the fly confirmed that suspected cellular pathways are functionally important
in ALS. These include protein folding, proteasome-mediated degradation, apoptosis and
microtubule organization.
A major difference between fly models of ALS and human pathology remains,
specifically, according to most studies, the absence of ubiquitinated inclusions, which
represent a hallmark of the disease. While the reason and significance for this apparent
discrepancy remains to be seen, it is worth noting that the fly model unequivocally shows that
cytoplasmic aggregates are not a prerequisite of motor neuron disease. This suggests that
pathological inclusions are more likely to be a consequence rather than a cause for motor
neuron dysfunction and death. In addition, this finding shifts the focus from cytoplasmic
aggregates to other aspects of disease pathophysiology, including neuroanatomical and
synaptic function defects.
How far can the fly model ”fly”? Even the most passionate Drosophila geneticists will
probably agree that while the fly provides a rapid and efficient evaluation of the pathways
relevant to disease, it is just the first step to developing strategies with therapeutic potential in
humans. Perhaps a most reasonable approach is to learn as much as possible about the basic
biology of ALS from the fly and other genetically amenable models ranging from yeast to
zebrafish, then validate these findings in mammalian models, which have been the golden
standard for advancing therapies to humans.
A similar logic could be applied to drug screening: compounds identified as
neuroprotective in the fly should be subjected to further testing and validation in rodent
models. However, given the limited success of translating findings from mouse to humans at
least when it comes to the SOD1 model and taking into the account the lack of therapies for
ALS patients who face a rapid loss of motor function and death within 2-5 years of diagnosis,
one has to ask: should we take the tiny fruit fly a little more seriously?
ACKNOWLEDGMENTS
We are grateful to the Jim Himelic foundation as well as the Himelic family and friends
for providing seed funding and inspiration for modelling ALS in Drosophila. We also thank
the Muscular Dystrophy Association (MDA173230) and NIH (1R21NS078429-01A1) for
financial support and Drs. K. Scherer, H. Horak, B. Coull and D. Labiner (UA, Neurology) as
well as members of the Zarnescu laboratory for helpful discussions.
Flies in Motion 73
ABOUT THE AUTHORS
Andrés Morera earned his BS in Biotechnology at Indiana University. He is presently a
PhD student in Biochemistry and Molecular & Cellular Biology (BMCB) at University of
Arizona. His research is focused on the involvement of the TOR pathway and autophagy in
neurodegeneration.
Alyssa Coyne obtained her BS in Biology at Springfield College, MA. Alyssa is
currently pursing a PhD in Neuroscience at the University of Arizona where her research
interests lie in the area of protein homeostasis during neurodevelopment and
neurodegeneration.
Daniela C Zarnescu obtained her BS in Physics at University of Bucharest, Romania.
Following her PhD degree in Biochemistry and Molecular Biology at Penn State, Daniela
trained as a postdoctoral fellow in Molecular Genetics at Emory University School of
Medicine. She is currently Associate Professor of Molecular and Cellular Biology,
Neuroscience and Neurology at University of Arizona. Daniela’s current research interests lie
in the broad area of gene expression with a focus on the role of RNA binding proteins during
development and in disease
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 4
MAINTAINING LONG SUPPLY LINES:
AXON DEGENERATION AND THE FUNCTION
OF HEREDITARY SPASTIC PARAPLEGIA GENES
IN DROSOPHILA
Belgin Yalçın and Cahir J. O’Kane* Department of Genetics, University of Cambridge, UK
ABSTRACT
The length of motor system axons presents great challenges to the subcellular
trafficking machinery of neurons. Impairment of the mechanisms that maintain axonal
function can lead to axon degeneration diseases, particularly in the distal regions of axons
that lie furthest from the cell body. Hereditary spastic paraplegias (HSPs) are one such
group of diseases – these share a common feature of degeneration of distal upper motor
axons, sometimes with a spectrum of additional mainly neurological symptoms. Their
phenotypic heterogeneity is reflected in their genetic causes - some 50 genetic loci have
been identified as causative, and over 25 of these have been cloned. These spastic
paraplegia gene (SPG) products do however point at an unexpectedly limited range of
disease mechanisms, including endoplasmic reticulum (ER) organization and function,
axonal microtubule-based transport, and endosomal traffic and signalling. Most but not
all causative human genes have orthologues in Drosophila. Despite the shorter lifespan
and short axons compared to humans, Drosophila offers a powerful system to study both
the cellular functions of many of these genes, and what goes wrong when they are
mutated. This stems both from the powerful genetic tools for generation of specific
mutant or transgenic flies, as well as the powerful analytic tools for understanding the
cellular roles of these gene products in neurons, particularly in axons and synapses.
Major contributions from flies so far have included dissection of the roles of several SPG
proteins in ER organization, transport of specific cargoes in axons, and in pathways
including bone morphogenetic protein (BMP) signalling. As additional SPG proteins are
* Correspondence should be addressed to: Cahir J O’Kane, Department of Genetics, University of Cambridge,
Downing Street, Cambridge, CB2 3EH, United Kingdom. Tel: +44-1223-333177; Fax: +44-1223-333992;
Email: [email protected].
Belgin Yalçın and Cahir J. O’Kane 86
identified, Drosophila offers a great opportunity to understand their cellular roles, and
ultimately providing plausible mechanisms for these diseases.
Keywords: Axon degeneration; axon transport; endoplasmic reticulum
INTRODUCTION
Maintaining the functionality of motor axons, whose length can be up to 105 times longer
than that of cell bodies, presents great challenges for the trafficking machinery of motor
neurons. Impairment of this machinery can therefore lead to degeneration of axons,
particularly in the distal regions that lie furthest from the cell body. Hereditary spastic
paraplegias (HSPs) are a diverse group of genetic disorders that show this effect. They are
characterized by progressive spasticity and weakness in lower extremities, caused by
progressive distal axonopathy mostly in the longest “upper” corticospinal motor neurons.
HSPs are conventionally classed as either pure (uncomplicated) or complex (complicated)
depending on whether there are additional neurological symptoms such as dementia,
intellectual impairment, epilepsy or amyotrophy. However, it is becoming clearer that this
division is simplistic, and that HSPs show a spectrum of additional symptoms. The diversity
of HSP-associated symptoms and causative genes suggests that impairments in a range of
cellular processes can result in axonal degeneration.
Many cases of HSP are clearly inherited. Over 50 causative spastic paraplegia genes
(SPGs) have now been mapped, that can give rise to either dominant or recessive forms of the
disease, and about 25 of these have been cloned (Table 1). This suggests great heterogeneity
in the causes of HSP, and in the cellular processes necessary for integrity of longer axons.
However, a closer look suggests a more limited range of cellular processes that are affected –
in particular endoplasmic reticulum (ER) organization and function, microtubule (MT)
trafficking, endosomal trafficking and signalling, mitochondrial function, and interactions of
axons with the myelin sheath.
The axons affected in HSP can be around a meter long in humans, enormous compared to
their cell body diameters of a few tens of µm. Maintenance of distal axons requires transport
of most organelles and proteins from the cell body, transport of other components such as
lipids, and other communication with the cell body. These are problems both of fundamental
interest, and highly relevant to HSP disease mechanisms. Understanding the cellular roles and
mutant phenotypes of SPG proteins is a route to addressing these problems, and for this the
fruit fly Drosophila is an excellent model. Flies have orthologues of most (but not all) SPGs
(Table 2), they have motor axons that house most of the axon transport and cell signalling
machinery of human neurons (albeit much shorter than human neurons), and sophisticated
genetic tools to generate mutant or transgenic genotypes, more rapidly than in mice. Here we
review the contributions of Drosophila to understanding the cellular roles of SPG proteins,
and the processes that may be affected when their functions are lost or altered. We focus
mainly on the many SPG genes with functions in organization or trafficking of the neuronal
endomembrane system, where Drosophila has been most valuable to understand the roles of
these genes in axonal or synapse function.
Table 1. Cloned HSP genes with predicted domains and protein functions. Predictions were performed using the SMART prediction
program (http://smart.embl-heidelberg.de/)
Category Human
Protein
Predicted Domain Protein
length
(AAs)
Protein Function Main References
Intracellular
trafficking
(ER shaping)
SPG3A
atlastin
558 Member of dynamin GTPase
superfamily, localized to ER three-way
junctions
Zhao et al., 2001; Hu et
al., 2009; Orso et al.,
2009; Lee et al., 2009
Intracellular
trafficking
(ER shaping)
SPG4
spastin
616 AAA protein, predominantly ER
localized, binds and severs
microtubules
Fonknechten et al., 2000;
Evans et al., 2005; Roll-
Mecak and Vale, 2005;
Trotta et al., 2004; Du et
al., 2010
Intracellular
trafficking
(ER shaping)
SPG12
reticulon 2
545 ER shaping protein, generating ER
curvature
Shibata et al., 2008;
Montenegro et al., 2012;
O’Sullivan et al., 2012
Intracellular
trafficking
(ER shaping)
SPG31
REEP 1
208
ER shaping protein, generating ER
curvature, might localize to
mitochondria
Zuchner et al., 2006; Park
et al., 2010
Table 1. (Continued)
Category Human
Protein
Predicted Domain Protein
length
(AAs)
Protein Function Main References
Intracellular
trafficking
(axonal
transport)
SPG10
KIF5A
1032 Member of kinesin family, microtubule
motor protein in intracellular transport
Reid et al., 2002; Ebbing
et al., 2008
Intracellular
trafficking
(axonal
transport)
SPG30
KIF1A
1791
Member of kinesin family, microtubule
motor protein in intracellular transport
Klebe et al., 2012
Intracellular
trafficking
(endosomal
trafficking)
SPG6
NIPA1
329 Endosomal trafficking, BMP signalling Wang et al., 2007
Intracellular
trafficking
(endosomal
trafficking)
SPG8
strumpellin
1159 Subunit of WASH complex, involved
in microtubule dynamics
Valdmanis et al., 2007;
Clemen et al., 2010;
Harbour et al., 2010
Intracellular
trafficking
(endosomal
trafficking)
SPG11
spatacsin
2443 Protein might be involved in cell
growth, cell cycle or transcriptional
regulation
Stevanin et al., 2007;
Murmu et al., 2011
Intracellular
trafficking
(endosomal
trafficking)
ALS2
alsin
1657 Localized to early endosomes, mediates
endosome fusion via Rab5 GEF activity
Kunita et al., 2004; Otomo
et al., 2011
Category Human
Protein
Predicted Domain Protein
length
(AAs)
Protein Function Main References
Intracellular
trafficking
(endosomal
trafficking)
SPG15
spastizin
2539 Protein might be involved in axonal
transport and membrane trafficking
Hanein et al., 2008;
Murmu et al., 2011
Intracellular
trafficking
(endosomal
trafficking)
SPG21
maspardin
308 Protein might function in protein
transport and sorting of endosomes
Simpson et al., 2003
Intracellular
trafficking
(endosomal
trafficking)
SPG33
Protrudin
(ZFYVE27)
416 Interacts with spastin, localizes to
endosomes
Mannan et al., 2006;
Zhang et al., 2012
Intracellular
trafficking
(endosomal
trafficking)
SPG42
SLC33A1
549 Localizes to ER membrane, acetyl-CoA
transporter
Lin et al., 2008; Jonas et
al., 2010
mitochondrial SPG7
paraplegin
795 Mitochondrial AAA protease, protein
folding, and proteolysis
Nolden et al., 2005;
Mancuso et al., 2012
mitochondrial SPG13
HSP60
573 Mitochondrial chaperonin Hansen et al., 2002, 2008
Lipid
metabolism
SPG5A
CYP7B1
506 Localizes to ER, hydroxylation
reactions of some steroids and
oxysterols
Tsaousidou et al., 2008;
Rose et al., 2001
Lipid
metabolism
SPG17
seipin
462 Integral ER membrane protein, lipid
droplet biogenesis
Patel et al., 2001;
Windpassinger et al.,
2004; Ito et al., 2008
Table 1. (Continued)
Category Human
Protein
Predicted Domain Protein
length
(AAs)
Protein Function Main References
Lipid
metabolism
SPG18
ERLIN2
339 Mediates ER degradation (ERAD)
pathway
Alazami et al., 2011
Lipid
metabolism
SPG20
spartin
666 Multifunctional protein, binds ESCRT-
III complex involved in endosomal
transport. Binds to Eps15, involved in
lipid droplet maintenance
Ciccarelli et al., 2003;
Bakowska et al., 2005; Lu
et al., 2006
Lipid
metabolism
SPG35
FA2H
372 Hydroxylation of myelin lipids
Dick et al., 2010; Eckhardt
et al., 2005
Lipid
metabolism
SPG39 NTE
1375 Membrane lipid homeostasis Kienesberger et al., 2008;
Rainier et al., 2008
Cell adhesion SPG1
L1CAM
1257 Immunoglobulin-related neuronal cell
adhesion molecule
Jouet et al., 1994; De
Angelis et al., 2002
Category Human
Protein
Predicted Domain Protein
length
(AAs)
Protein Function Main References
Myelination SPG2 PLP1
277 Predominant myelin protein present in
the central nervous system
Grossi et al., 2011; Zappia
et al., 2011
Abbreviations:
AAA: ATPases associated with a variety of cellular activities
Abhydrolase_1: alpha/beta hydrolase fold
cNMP: Cyclic nucleotide-monophosphate binding domain
FHA: Forkhead associated domain
FN3: Fibronectin type 3 domain
FYVE: zinc finger domain
GBP: Guanylate-binding proteins (a family of GTPases)
IG: Immunoglobulin
IGc2: Immunoglobulin C-2 Type
KISc: kinesin motor catalytic domain, ATPase
MIT: Microtubule Interacting and Trafficking molecule domain
MORN: Possible plasma membrane-binding motif in junctophilins, PIP-5-kinases and protein kinases.
PH: Pleckstrin homology domain.
PHB: Prohibitin homologues
PLP: Myelin proteolipid protein (PLP or lipophilin)
RhoGEF: Rho guanine exchange factor (GEF)
TCP1/cpn60: chaperonin containing domain
TM: transmembrane domain; note that SMART usually scores intramembrane loops as TM domains.
VPS9: vacuolar sorting protein 9-like GEF
Table 2. Cloned HSP genes and their Drosophila homologues. Homology search was performed using BLAST
(Basic Local Alignment Search Tool)
Category Gene Inheritance Pure/
complex
Gene product Drosophila
homologue
AA identity of longest
homology, BLASTP
E value
Intracellular trafficking
(ER shaping)
SPG3A Autosomal
Dominant
Pure atlastin CG6668
atlastin
57%, 0.0
Intracellular trafficking
(ER shaping)
SPG4 Autosomal
Dominant
Pure spastin CG5977
spastin
52%, 5e-160
Intracellular trafficking
(ER shaping)
SPG12 Autosomal
Dominant
Pure reticulon 2 CG33113
Reticulon-like 1
40%, 2e-48
Intracellular trafficking
(ER shaping)
SPG31 Autosomal
Dominant
Pure REEP 1 CG42678
REEP A
63%, 2e-57
Intracellular trafficking
(axonal transport)
SPG10 Autosomal
Dominant
Complex KIF5A CG7765
Khc
59%, 0.0
Intracellular trafficking
(axonal transport)
SPG30 Autosomal
Recessive
Complex KIF1A CG8566
unc-104
55%, 0.0
Intracellular trafficking
(endosomal trafficking)
SPG6 Autosomal
Dominant
Pure NIPA1 CG12292
spichthyin
40%, 4e-67
Intracellular trafficking
(endosomal trafficking)
SPG8 Autosomal
Dominant
Pure strumpellin CG12272
strumpellin
44%, 0.0
Intracellular trafficking
(endosomal trafficking)
SPG11 Autosomal
Recessive
Complex spatacsin CG13531
spatacsin
27%, 3e-33
Intracellular trafficking
(endosomal trafficking)
ALS2 Autosomal
Recessive
Complex alsin CG7158
alsin
25%, 5e-53
Intracellular trafficking
(endosomal trafficking)
SPG15 Autosomal
Recessive
Complex spastizin CG5270
spastizin
36%, 4e-39
Category Gene Inheritance Pure/
complex
Gene product Drosophila
homologue
AA identity of longest
homology, BLASTP
E value
Intracellular trafficking
(endosomal trafficking)
SPG21 Autosomal
Recessive
Complex maspardin No
Intracellular trafficking
(endosomal trafficking)
SPG33 Autosomal
Dominant
Pure Protrudin
(ZFYVE27)
No
Intracellular trafficking
(endosomal trafficking)
SPG42 Autosomal
Dominant
Pure SLC33A1 CG9706
SLC33A1
52%, 4e-172
mitochondrial SPG7 Autosomal
Recessive
Complex paraplegin CG2658
paraplegin
58%, 0.0
mitochondrial SPG13 Autosomal
Dominant
Pure HSP60 CG12101
HSP60
74%, 0.0
Lipid metabolism SPG5A Autosomal
Recessive
Pure CYP7B1 many homologues
Lipid metabolism SPG17 Autosomal
Dominant
Complex seipin CG9904
seipin
40%, 7e-59
Lipid metabolism SPG18 Autosomal
Recessive
Complex ERLIN2 No
Lipid metabolism SPG20 Autosomal
Recessive
Complex spartin CG12001
spartin
28%, 7e-36
Lipid metabolism SPG35 Autosomal
Recessive
Complex FA2H CG30502
FA2H
35%, 7e-66
Lipid metabolism SPG39 Autosomal
Recessive
Complex NTE CG2212
Swisscheese
44%, 0.0
Cell adhesion SPG1 X-linked Complex L1CAM CG1634
neuroglian
29%, 2e-146
Myelination SPG2 X-linked Complex PLP1 CG7540
PLP1
24%, 8e-10
Belgin Yalçın and Cahir J. O’Kane 94
A ROLE FOR MIS-SHAPING OF ENDOPLASMIC RETICULUM IN HSP?
Most cases of autosomal dominant HSP are caused by mutations affecting four proteins
that shape ER morphology – spastin, atlastin-1, REEP1 and reticulon-2 – suggesting that
defects in ER morphology could be one cellular cause of HSPs. These proteins share a
common feature of one or more intramembrane hairpin-loop domains that insert into the
cytosolic face of the ER membrane (Voeltz et al., 2006; Shibata et al., 2008; Zürek et al.,
2011), increasing its surface area and thus inducing membrane curvature, as in ER tubules or
the edges of sheet ER (Hu et al., 2009; Shibata et al., 2008). Before considering how this
process might relate to axonal function, let us first look at the relevant SPG products.
Spastin (SPG4)
Mutations in spastin are the most common cause of autosomal dominant HSP, normally
pure (Hazan et al., 1999). Spastin is a MT-severing AAA (ATPase associated with various
cellular activities) protein. Many missense, nonsense and splice site point mutations, deletions
and insertions have been observed in patients (Fonknechten et al., 2000), suggesting either a
haploinsufficient and/or dominant-negative disease mechanism, although there is a suggestion
of dominant gain-of-function toxicity caused by mutations in the intramembrane hairpin
domain (Solowska et al., 2010).
The most abundant spastin isoforms are a longer M1 isoform of 616 amino acid residues,
and a shorter M87 isoform translated from a downstream start codon, that lacks the N-
terminal 86 residues of M1 (Claudiani et al., 2005; Mancuso et al., 2008). Both forms contain
a MIT domain that interacts with the late endosomal ESCRT-III component CHMP1B (Reid
et al., 2005), an MT-binding domain, and a C-terminal AAA domain responsible for hexamer
formation and MT-severing activity (Beetz et al., 2004; Claudiani et al., 2005; Roll-Mecak et
al., 2008). The M1 variant contains a single intramembrane hairpin, which inserts in ER
membrane, and mediates interactions with atlastin-1 and reticulon-1 (Sanderson et al., 2006;
Mannan et al., 2006; Evans et al., 2006; Connell et al., 2009). In contrast, the M87 isoform is
abundant in endosomes, probably due to interaction of its MIT domain with the ESCRT-III
component CHMP1B.
It is not yet clear which molecular function(s) and population(s) of spastin are most
relevant in HSP. Both M1 and M87 are found in spinal cord (Claudiani et al., 2005; Salinas et
al., 2005; Solowska et al., 2008); and missense mutations are found throughout the coding
region, including the M1-specific region (Shoukier et al., 2009; McCorquodale et al., 2011).
While the MT-severing activity of spastin suggests a link to MT-based axonal transport, this
could be required on the ER, endosomes, or elsewhere. Other functions of spastin could also
be relevant to SPG4 pathology. First, spastin (like three other HSP proteins spartin, NIPA1,
and atlastin-1) appears to antagonize BMP signalling (Tsang et al., 2009), although a
mechanism for this is lacking. Second, spastin is also found in the cytokinetic midbody
during cell division due to its interaction with centrosome protein NA14 (Errico et al., 2004)
and ESCRT-III (Yang et al., 2008), where it is involved in MT abscission (Connell et al.,
2009).
Maintaining Long Supply Lines 95
Drosophila has one spastin orthologue (CG5977), with one known transcript that encodes
an M1-like protein with a likely intramembrane hairpin. Homozygous spastin mutant flies are
predominantly lethal, and adult escapers have severely disrupted motor function, as do human
patients (Sherwood et al., 2004). Spastin is abundant in axons and synaptic areas (Trotta et al.,
2004). RNAi knockdown results in smaller synaptic area and increased MTs at the
neuromuscular junction (NMJ), whereas overexpression lowers synaptic strength and synaptic
MTs (Trotta et al., 2004). In contrast to knockdown larvae, spastin null mutant larvae exhibit
fewer MTs at the NMJ, and additional satellite boutons (Sherwood et al., 2004). Exogenous
expression of full-length human spastin rescues spastin null phenotypes (Du et al., 2010),
showing phylogenetic conservation of at least some spastin functions, but it is not known
whether rescue is mediated by the M1 and/or M87 variants. Taken together, analysis of
spastin mutant phenotypes in flies suggest possible mechanisms of pathology involving
dysfunction of the neuronal MT cytoskeleton, although it would be simplistic to regard these
Drosophila genotypes as replicas of the human disease.
Drosophila phenotypes also illuminate mutant spastin functions. For example, expression
of a pathogenic ATPase domain mutant has dominant-negative effects and is not simply non-
functional (Orso et al., 2005). Co-expression of different spastin variants shows that as in
human patients, amino acid substitutions in the N-terminal M1-specific domain exacerbate
phenotypes caused by mutations in the ATPase domain (Du et al., 2010), arguing that
impairment of ER-specific spastin function is important for spastin mutant phenotypes in
both flies and humans.
Drosophila genetic screens have also revealed cellular processes that interact with
spastin. Spastin loss-of-function phenotypes are strongly suppressed by loss of the actin
regulatory kinase Pak3 (Ozdowski et al., 2011), suggesting that many effects of spastin loss
act via Pak3-dependent processes. Pak family kinases might therefore be potential therapeutic
targets in SPG4 patients, although this will require further investigation in mammals. A link
with another neurological condition has also been found in Drosophila; heterozygous loss of
spastin dominantly suppresses phenotypes of Drosophila FMRP (Fragile X mental retardation
protein) overexpression (Yao et al., 2011), and so FMRP may act upstream of or in parallel to
spastin.
Atlastin (SPG3A)
Mutations in atlastin-1 cause autosomal dominant usually pure HSP (Zhao et al., 2001). It
is the second most common HSP, generally caused by missense mutations (Zhao et al., 2001).
Humans have three atlastin paralogues: atlastin-1 is predominantly expressed in the CNS
(Zhu et al., 2003) and atlastin-2 and atlastin-3 are widely expressed (Rismanchi et al., 2008).
Atlastins belong to the dynamin GTPase superfamily, and contain a C-terminal
intramembrane loop (Zhu et al., 2003; Rismanchi et al., 2008). Knockdown, or expression of
mutant forms, suggests that atlastin-1 is important for the reticular network of tubular ER,
whereas atlastin-2 and atlastin-3 regulate Golgi morphology (Namekawa et al., 2007;
Rismanchi et al., 2008). Atlastin-1 interacts with spastin, REEP and reticulon family proteins
via their intramembrane loop regions (Evans et al., 2006; Sanderson et al., 2006; Hu et al.,
2009; Park et al., 2010), suggesting that these proteins participate in a common
pathomechanism of HSP.
Belgin Yalçın and Cahir J. O’Kane 96
Drosophila has a single atlastin orthologue (CG6668), which has been central to our
understanding of atlastin function. It localizes to ER membranes, and overexpression causes
ER expansion, while loss of its function causes ER network fragmentation. Moreover atlastin
enhances the in vitro fusion of liposomes. The GTPase domain is required to form trans-
oligomeric complexes, promote membrane fusion in vitro, and form a normal reticular
network in vivo (Orso et al., 2009). The fusion function of the yeast atlastin, Sey1p, is
antagonized by another ER hairpin protein lunapark/lnp-1 (Chen et al., 2012), hinting at a
machinery that regulates ER fusion. Drosophila has a lunapark homolog (CG8735), but there
is no information on its function.
Drosophila atlastin loss-of-function and overexpression phenotypes also show many
similarities to those of spastin, perhaps resulting from their interaction. Loss of atlastin causes
accumulation of stable MTs in muscles whereas its overexpression results in the opposite;
loss of atlastin also causes additional satellite boutons at the NMJ (Lee et al., 2009).
Reticulon 2 (SPG12)
Mutations in reticulon 2 (RTN2) cause autosomal dominant pure HSP (Montenegro et al.,
2012). Mammals have four reticulons, RTN1-4 (Shibata et al., 2008). They are predominantly
localized in the ER (van de Velde et al., 1994; Grandpre et al., 2000; Di Sano et al., 2003).
RTN4 has also been characterized as Nogo, an inhibitor of axon regeneration (reviewed by
Zörner and Schwab, 2010), although its in vivo role in regeneration appears subtle (Lee et al.,
2009).
Reticulons contain two hydrophobic hairpins inserted in the ER membrane that mediate
formation of reticulon oligomers and heteromeric complexes with other hairpin loop proteins
(Shibata et al., 2008). They are localized mainly in curved regions of ER (Voeltz et al., 2006;
Kiseleva et al., 2007; Shibata et al., 2010). Overexpression of reticulons results in long
unbranched and mostly bundled ER tubules in both mammalian and yeast cells (Voeltz et al.,
2006). Deletion of all yeast reticulons disrupts peripheral tubular ER in stress conditions, but
otherwise leaves it largely intact, implying that they are not alone responsible for forming it
(Voeltz et al., 2006).
There are two Drosophila reticulons, reticulon-like 1 (Rtnl1) and reticulon-like 2 (Rtnl2).
Sequence analysis suggests that a single ancestral reticulon was duplicated independently in
fly and mammalian lineages, giving two paralogues in Drosophila and four in mammals.
Rtnl1 is widely expressed and is evolving relatively slowly, suggesting that it is the main
functional orthologue of all mammalian reticulons. Rtnl2 is evolving faster and so is less
functionally constrained, and its expression is restricted mainly to fat body and testis,
suggesting a more limited or specialized function (O’Sullivan et al., 2012). Rtnl1 deletion has
no overt effects on viability (Wakefield and Tear, 2006). However, Rtnl1 knockdown causes
expansion of ER sheets in epidermal cells and induces ER stress in epithelial and neuronal
cells. It also disrupts smooth ER and MTs in longer distal motor axons (O’Sullivan et al.,
2012). This finding is significant for HSP, providing a possible model for how ER-shaping
proteins may be essential for distal axon function – impaired ER organization might affect
physiological functions such as calcium signalling preferentially in these axonal regions.
Maintaining Long Supply Lines 97
REEP1 (SPG31)
REEP1 (receptor expression enhancing protein) mutations are the third most common
cause of autosomal dominant HSP, (Züchner et al., 2006). There are six REEP genes in
humans, REEPs 1-6, which all have two hairpin-loop domains in ER membrane, that enable
at least REEP1 to interact with atlastin-1 and spastin (Voeltz et al., 2006; Hu et al., 2008;
Park et al., 2010).
REEPs 1-4 have an MT-binding domain on their C-terminal cytoplasmic region, unlike
REEPs 5-6 (Park et al., 2010), and may therefore tether ER tubules with MTs (Park et al.,
2010). At least one HSP-causing mutation of REEP1 encodes a truncated protein that cannot
bind MTs and causes ER disruption (Park et al., 2010). REEP1 is predominantly localized to
tubular ER but it may also be mitochondrial (Saito et al., 2004; Behrens et al., 2006; Züchner
et al., 2006).
Drosophila has six REEP genes. Using the same criteria as with the reticulons, CG42678
(which we designate ReepA) appears to be the main functional orthologue of mammalian
REEPs 1-4, and CG8331 (which we designate ReepB) appears to be the main functional
orthologue of mammalian REEPs 5-6. Since Drosophila has two REEP proteins that
presumably do the job of six mammalian REEPs, it offers a system to analyse REEP function
with less redundancy than in mammalian models. However, there is no published information
on their roles in ER or axonal function in Drosophila.
Hairpin-Loop ER-Shaping Proteins: Conclusions
What is the relevance of ER hairpin protein function to axon degeneration? Axons have
extensive stretches of smooth ER tubules (Terasaki et al., 1991; Droz et al., 1975), and the
effects of Rtnl1 knockdown in Drosophila axons (O’Sullivan et al., 2012) hint at a role for
SPG proteins in its integrity. How could impairment of axonal ER lead to degeneration? The
continuous tubular organization of smooth ER suggests the model of a “neuron within a
neuron”, which can carry signals along axons and dendrites independently of action
potentials, and faster than MT-based transport (Berridge, 2002). Disruption of this network
could impair processes such as calcium or BMP signalling, that could lead to degeneration
preferentially in distal axons, which may be more dependent on continuity of this network.
Drosophila offers an exciting model to test some of these ideas. Spastin and atlastin
mutants clearly have synaptic defects, and loss of reticulon leads to ER defects in distal axons
that have not yet been ultrastructurally defined. Axonal ER is also a fascinating compartment
in its own right, and Drosophila offers many tools to investigate its role in neuronal function
and dysfunction.
Belgin Yalçın and Cahir J. O’Kane 98
HSP MUTATIONS AFFECT ADDITIONAL ER PROTEINS
Seipin (SPG17)
Mutations in BSCL2, which encodes seipin, cause an autosomal dominant complex HSP
that is known as Silver syndrome. Loss-of-function seipin mutations cause recessive
Berardinelli-Seip congenital lipodystrophy, characterized by loss or absence of body fat and
mental retardation, but without abnormalities in motor neurons (Agarwal and Garg, 2003,
2004; Fu et al., 2004).
Seipin is a homo-oligomer of about nine subunits located in ER membrane, mostly at
junctions with lipid droplets (Windpassinger et al., 2004; Szymanski et al., 2007; Fei et al.,
2008; Binns et al., 2010). It has cytosolic N- and C-termini, and two transmembrane domains
in the ER membrane (Lundin et al., 2006). Unlike the hairpin proteins above, seipin has a
glycosylated loop in the ER lumen. Dominant HSP results from mutations in the ER lumenal
domain, which cause an unfolded protein response (UPR) (Ito et al., 2008; Yagi et al., 2011).
Drosophila seipin loss-of-function mutants also show reduced lipid storage in the fat
body, and ectopic lipid droplets in the non-adipose salivary gland (Tian et al., 2011).
However, there is as yet no published work on HSP-related gain-of-function effects in
Drosophila.
Neuropathy Target Esterase (SPG39)
Mutations in neuropathy target esterase (NTE) cause autosomal recessive complex HSP
(Rainier et al. 2008; 2011). Modification of NTE by organophosphorus (OP) compounds also
causes neuropathy (Johnson and Glynn, 1995). NTE is a 150-kDa ER membrane protein, with
a short lumenal N-terminus, a transmembrane domain, and a large cytosolic C-terminus
(Akassoglou et al., 2004; Chang et al., 2010).
NTE has phospholipase and lysophospholipase activity in its cytosolic domain (Lush et
al., 1998; van Tienhoven et al., 2002; Quistad et al., 2003), and hence regulates membrane
phospholipid content (Akassoglou et al., 2004). It hydrolyses lysophosphatidylcholine (LPC),
protecting cell membranes from LPC accumulation (Zaccheo et al., 2004; Vose at al., 2008),
and degrades ER-associated phosphatidylcholine (Zaccheo et al., 2004). Deletion of NTE in
mouse brain causes neurodegeneration, with disrupted ER and vacuolation in nerve cell
bodies, and abnormal reticular aggregates (Akassoglou et al., 2004). While neuropathy or
neurodegeneration could be due to impaired phospholipid metabolism, the finding of
abnormal ER (albeit not axonal) could also be relevant to HSP.
The Drosophila NTE orthologue is swiss cheese, named after the extensive vacuolation
in mutant nervous systems, as both glia and neurons undergo cell death (Kretzschmar et al.,
1997). Swiss Cheese protein also localizes to ER, and mutants show elevated levels of
phosphatidylcholine (Mühlig-Versen et al., 2005). Whether axonal ER is affected is
unknown.
Maintaining Long Supply Lines 99
CYP7B1 (SPG5A)
Mutations in CYP7B1, which encodes a cytochrome P450, can cause either autosomal
recessive pure HSP with variable age of onset (Tsaousidou et al., 2008; Schule et al., 2009),
or liver failure (Stiles et al., 2009). CYP7B1 is a widely expressed 506-amino-acid enzyme,
thought to be localized in ER, that catalyzes 6- and 7-hydroxylation of some steroids and
oxysterols including cholesterol, and the variety of disease phenotypes may result from the
range of substrates that the enzyme can metabolize (reviewed by Stiles et al., 2009).
Gene duplication has given rise to many CYP7B1 orthologues in Drosophila
(www.flybase.org). This may be due to selection for the ability to metabolize numerous
substrates, perhaps ones encountered in the wild. However without better knowledge of which
substrates are most relevant to HSP, it is not clear which Drosophila CYP7B1 orthologues are
useful for modelling SPG5A.
FA2H (SPG35)
Mutations affecting fatty acid 2-hydroxylase (FA2H), cause autosomal recessive complex
HSP, with additional symptoms including leukodystrophy (myelin sheath defects), and
neurodegeneration with brain iron accumulation (Alderson et al., 2009; Dick et al., 2010).
FA2H is a membrane-bound ER enzyme (Eckhardt et al., 2005). There are four putative
transmembrane domains of human FA2H and the N- and C-terminals, including a cytochrome
b5 domain and an ER retention signal, are on the cytoplasmic side of the membrane (Haak et
al., 1997; Mitchell and Martin, 1997). FA2H catalyses 2-hydroxylation of sphingolipids and
straight chain fatty acids (Zöller et al., 2008; Maldonado et al., 2008). Sphingolipids with 2-
hydroxy fatty acids are abundant in myelin (Norton and Cammer, 1984), and FA2H is also
abundant in the oligodendrocytes that form the myelin sheath – so disease mechanisms may
involve myelin dysfunction rather than impaired neuronal ER function. Drosophila has a
single FA2H orthologue, which is a sphingolipid-specific fatty acid hydroxylase (Carvalho et
al., 2010), but there is little knowledge of its role in neuronal or glial function.
SLC33A1 (SPG42)
A missense mutation affecting the acetyl-CoA transporter SLC33A1 segregates with
autosomal dominant pure HSP in a large Chinese pedigree (Lin et al., 2008). SLC33A1 is
localized in ER membrane, where it is important for lumenal acetylation of ER proteins and
for cell viability (Jonas et al., 2010). SLC33A1 is induced by the ER unfolded protein
response. In its absence, failure to acetylate the lumenal domain of Atg9A leads to induction
of autophagy, and potentially to autophagy-mediated cell death (Pehar et al., 2012). Loss-of-
function mutations cause a human syndrome with cataracts, hearing loss and severe
developmental delay (Huppke et al., 2012), and SLC33A1 inhibition in zebrafish inhibited
axon outgrowth (Lin et al., 2008). The dominant HSP allele is predicted to disrupt the second
transmembrane domain, and potentially reverse the topology of all downstream domains (Lin
et al., 2008), and could therefore lead to either 50% loss-of-function, or to dominant gain-of-
Belgin Yalçın and Cahir J. O’Kane 100
function via a misfolded protein. SLC33A1 has a Drosophila homologue, CG9706. However,
no in-depth characterization of this gene has been published.
MICROTUBULE MOTOR PROTEINS
Two types of HSP are caused by mutations in different kinesin heavy chains: KIF5A
(SPG10), and KIF1A (SPG30). Kinesins are plus-end-directed MT motors that carry a variety
of cargoes such as synaptic vesicles and mitochondria in an anterograde direction in axons
(Hirokawa, 1998; Goldstein and Yang, 2000; Barkus et al., 2008). They are multi-subunit
complexes that include two identical heavy chains (kinesin heavy chains or KHCs) and two
identical light chains.
KIF5A (SPG10)
Mutations in KIF5A cause autosomal dominant complex HSP (Reid et al., 2002). This is
a component of kinesin-I. Mammals have three kinesin-I heavy chains, KIF5A, KIF5B and
KIF5C. KIF5A is expressed in all neurons and it is localized in the cytoplasm of cell body,
dendrites and axons (Kanai et al., 2000). Experiments with primary motor neuron cultures of
knockout mice suggest that KIF5A is required for neuronal survival and outgrowth, and
transport of mitochondria (Karle et al., 2012); other functions include anterograde
transportation of secretory vesicles to growth cones (Burgo et al., 2012).
KHCs contain three domains: a globular N-terminal motor domain, a -helical coiled-
coil stalk domain that mediates dimerization, and a C-terminal globular tail with roles in
light-chain and cargo binding (Wickstead and Gull, 2006; Lo Giudice et al., 2006). Mutations
in both the motor and stalk domains of KIF5A are found in HSP patients, and can cause
impaired axonal transport (Ebbing et al., 2008). Since the motor protein is made of two
chains, one non-functional chain can lead to non-functional kinesin.
Drosophila Khc (CG7765) is the single orthologue of human KIF5A, KIF5B and KIF5C.
Homozygous mutant larvae show impaired growth rate, and severe loss of muscle activity in
the posterior region, a striking similarity to human HSP. Adults also exhibit impaired sensory
and motor activity (Saxton et al., 1991) and defects in anterograde and retrograde axonal
transport (Hurd and Saxton, 1996). Drosophila has been useful in identifying additional
components of Khc-dependent axonal transport such as the mitochondrial adaptor Milton,
Unc-76 and Unc-51 kinase (Stowers et al., 2002; Gindhardt et al., 2003; Toda et al., 2008),
potential cargoes such as mitochondria and synaptic vesicles (Stowers et al., 2002; Hurd and
Saxton, 1996; Toda et al., 2008), and additional neuronal roles of Khc such as transport of
some cargoes to dendrites (Henthorn et al., 2011), that may also be relevant to the complex
disease phenotypes of SPG10.
Maintaining Long Supply Lines 101
KIF1A (SPG30)
Mutations in KIF1A, from the kinesin-3 subfamily, cause autosomal recessive complex
HSP (Erlich et al., 2011; Klebe et al., 2012). Its C-terminus has a lipid-binding Pleckstrin
homology (PH) domain, which is required for vesicle transport (Klopfenstein and Vale,
2004). KIF1A is the primary motor for fast axonal transport of neuropeptide-containing
dense-core vesicles, into axons and dendrites to pre- and postsynaptic sites (Scalettar, 2006;
Lo et al., 2011).
The Drosophila KIF1A orthologue is unc-104 (also known as imac). Like Khc it also has
a central role in axon transport, but appears to transport different cargoes. Whereas synaptic
vesicles accumulate in axons in khc mutants rather than the synapse (Hurd and Saxton, 1996;
Gindhardt et al., 2003), in unc-104 mutants, they fail to enter axons (Pack-Chung et al.,
2007), implying that these kinesins have complementary roles in synaptic vesicle transport. In
contrast to Khc, unc-104 has little role in axonal transport of mitochondria (Barkus et al.,
2008).
Therefore the KIF5/Khc and KIF1A/unc-104 kinesins transport different axonal cargoes
(impairment of which might lead to distal axon degeneration by different mechanisms), or
might play complementary roles in transport of the same cargoes that are essential to prevent
degeneration. Interestingly in Caenorhabditis elegans, unc-104 mutations redistribute the
smooth ER protein lunapark/Lnp-1 (Chen et al., 2012) from axons to cell bodies (Ghila et al.,
2008), hinting that SPG30 pathology might also involve axonal ER.
ENDOSOMAL SPG PROTEINS
Many SPG proteins are localized on components of the endosomal-lysosomal
endomembrane system. The significance of this for the disease is not clear, but may represent
roles for these proteins in intracellular signalling processes that are required for axonal
maintenance.
NIPA1 (SPG6)
Mutations in NIPA1 (non-imprinted in Prader-Willi/Angelman) cause autosomal
dominant HSP. It is a membrane protein with seven to nine transmembrane domains (Chai et
al., 2003; Lefevre, et al., 2004) and is predominantly localized in the early endosomal
pathway and peripheral surface (Goytain et al., 2007).
All known mutations are missense mutations at a limited number of amino acid residues
in diverse populations, which tentatively suggests a dominant gain-of-function mechanism.
These mutations could be affecting trafficking of NIPA1, and trapping of misfolded NIPA1 in
the ER could induce ER stress, giving the disease a gain-of-function feature (Goytain et al.,
2007; Beetz et al., 2008; Zhao et al., 2008). NIPA1 is also suggested to be a Mg2+
transporter
(Goytain et al., 2007). This role is potentially important since Mg2+
is the second most
abundant cation in the cell, but its relationship to HSP is unclear.
Belgin Yalçın and Cahir J. O’Kane 102
Spict (Spichthyin) is the Drosophila NIPA1 orthologue, and it is predominantly localized
on early endosomes. It is involved in the inhibition of BMP signalling, so it mediates the
growth of NMJ pre-synaptically. Spict can redistribute BMP receptors from the plasma
membrane to endosomes, and inhibit BMP signalling (Wang et al., 2007), properties shared
by NIPA1 in mammalian cells (Tsang et al., 2009). Loss of BMP signalling impairs axonal
transport (Aberle et al., 2002; Wang et al., 2007; Ellis et al., 2010); although the mechanisms
are not clear, this is a possible route by which SPG6 mutations could lead to spastic
paraplegia.
Three endosomal HSP proteins, spastin, spartin and NIPA1, and the ER protein atlastin-1,
are inhibitors of BMP signalling (Tsang et al., 2009; Fassier et al., 2010; Zhao and Hedera,
2012), although there is no indication of the mechanisms for spastin and spartin. However,
this shared involvement suggests abnormal BMP signalling as a possible mechanism for at
least some forms of HSP, a model that requires further investigation.
Strumpellin (SPG8)
Strumpellin mutations cause autosomal dominant pure HSP (Valdmanis et al., 2007).
Strumpellin is a subunit of the WASH complex, which regulates actin dynamics (Jia et al.,
2010). It interacts with the retromer complex, which traffics cargo from endosomes to Golgi,
and it promotes formation of endosomal tubules (Harbour et al., 2010). It is also localized to
ER (Clemen et al., 2010), and it is interesting to speculate whether it could promote extension
of ER tubules. Strumpellin has a Drosophila homologue (CG12272) but little is known about
its function.
Spartin (SPG20)
A frameshift mutation in spartin causes a syndromic autosomal recessive complex HSP
that is also known as Troyer Syndrome. Endogenous spartin is found in a cytoplasmic pool
that can be recruited to endosomes, lipid droplets, the cytokinesis midbody, and mitochondria
(Robay et al., 2006; Bakowska et al., 2007; Eastman et al., 2009; Edwards et al., 2009;
Renvoisé et al., 2010; Joshi and Bakowska, 2010), and it can regulate properties or trafficking
of some of these organelles.
Spartin has an N-terminal MIT domain that interacts with ESCRT-III (Ciccarelli et al.,
2003), and like spastin M87 this can explain its localization to endosomes and to the
midbody. At endosomes it functions in EGF receptor traffic and degradation (Bakowska et
al., 2007), and perhaps also in BMP receptor signalling (Tsang et al., 2009; Renvoisé et al.,
2012). It can also recruit ubiquitin ligases AIP4 and AIP5, and PKC- at lipid droplets
(Eastman et al., 2009; Edwards et al., 2009; Hooper et al., 2010; Urbanczyk and Enz, 2011),
and thus regulate properties including their subcellular distribution. Spartin is multiply
monoubiquitinated (Bakowska et al., 2007); although this appears to be constitutive, it could
allow spartin to act as an adaptor to recruit ubiquitin-binding proteins such as Eps15
(Bakowska et al., 2005). The multiple roles and localizations of spartin may contribute to the
Maintaining Long Supply Lines 103
syndromic phenotype of its loss, but their relationship to HSP is still unknown. Spartin has a
single Drosophila orthologue, CG12001, but little is known of its function.
Alsin (ALS2)
Mutations in alsin can cause complex infantile-onset ascending hereditary spastic
paraplegia (IAHSP), as well as early-onset forms of ALS or primary lateral sclerosis (PLS)
(Hadano et al., 2001; Yang et al., 2001; Eymard-Pierre et al., 2002; Panzeri et al., 2006).
Alsin has three predicted guanine exchange factor (GEF) domains: an N-terminal RCC
domain characteristic of Ran-GEFs, Dbl-homology and PH domains characteristic of Rho-
GEFs, and a C-terminal Vps9 domain characteristic of Rab5-GEFs. Rab5-GEF activity and
effects on endosome traffic have been demonstrated (e.g. Otomo et al., 2003; Topp et al.,
2004; Kunita et al., 2004; Devon et al., 2006; Deng et al., 2007; Kunita et al., 2007; Lai et al.,
2009), although a reported activity as a Rac1-GEF (Topp et al., 2004) might be accounted for
by being an effector of activated Rac1 (Kunita et al., 2007). The role of alsin in regulating
endosomal traffic is suggestive of a role in regulating one or more signalling pathways that
are relevant to axon degeneration (e.g. BMP?), but to date there is no information on this.
Drosophila has a single alsin orthologue, CG7158, but there is little information on its
function.
AUTOSOMAL RECESSIVE HSP WITH THIN CORPUS CALLOSUM, AND
THE AP5 ADAPTOR COMPLEX
Three autosomal recessive HSPs can be caused by mutations affecting proteins that are
members of the same multiprotein complex: SPG11 (spatacsin), SPG15 (spastizin) and
SPG48 (Stevanin et al., 2007; Hanein et al., 2008; Słabicki et al., 2010). At least SPG11 and
SPG15 have very similar complex phenotypes, HSP with thin corpus callosum (HSP-TCC;
the corpus callosum is the main tract linking the two brain hemispheres), and frequent
cognitive impairment. While this multiprotein complex was originally suggested to have
DNA repair function (Słabicki et al., 2010), it now appears to be a novel adaptor/coat protein
complex, AP-5 (Hirst et al., 2011).
Spatacsin (SPG11)
Spatacsin mutations are the most common cause of HSP-TCC (Stevanin et al., 2007), and
some spatacsin mutations are also causative for juvenile-onset ALS (Orlacchio et al., 2010)
or Parkinson’s Disease (Stevanin et al., 2008). Spatacsin is a large protein of some 2400
amino acid residues. Although it lacks obvious known domains, a sensitive homology search
suggests similarity to clathrin heavy chain (Hirst et al., 2011). It appears localized to vesicles,
ER, mitochondria and MTs (Murmu et al., 2011). It has a single Drosophila orthologue,
CG13531, about which little is known.
Belgin Yalçın and Cahir J. O’Kane 104
Spastizin (SPG15)
Mutations in spastizin cause a broadly similar HSP-TCC as spatacsin mutations.
Spastizin is also a large protein of some 2500 residues, and contains a C2H2 zinc finger, a
FYVE domain predicted to bind to endosomal membrane, and a possible leucine zipper
(Hanein et al., 2008). Spastizin partially co-localizes with spatacsin and both are found in the
same multi-protein complex (Hirst et al., 2011; Murmu et al., 2011). It has a single
Drosophila orthologue, CG5270, about which little is known.
SPG48
The multi-protein complex that contains spastizin and spatacsin was recently identified as
another adaptor complex, AP-5, for budding of a late endosomal compartment that is not yet
well defined (Hirst et al., 2011). Mutations in a large subunit of this adaptor, AP5-, are
found in a family with late-onset autosomal recessive SPG48 (Słabicki et al., 2010; Hirst et
al., 2011). Although this is a phylogenetically ancient gene, it appears to have been lost from
the protosomes, including Drosophila and most invertebrate phyla, together with other AP5
subunits (Hirst et al., 2011).
It is at first sight puzzling why AP5 should have been lost from the Drosophila lineage,
yet spatacsin and spastizin retained. Spatacsin and spastizin must therefore have roles in
addition to those in the AP-5 complex, that have been retained in Drosophila. If spatacsin is
indeed a distant homolog of clathrin, then it could function (aided somehow by spastizin) as a
coat protein for a number of adaptor complexes and not only AP-5, analogously to clathrin. If
HSP is caused by impaired AP-5 function, the roles of spatacsin and spastizin in Drosophila
may not be immediately relevant to HSP pathology, but may well be relevant to other disease
phenotypes of spatacsin and spastizin mutations, which appear to be more severe than those
of the single published AP5- mutation (Słabicki et al., 2010).
OTHER ENDOMEMBRANE SPG PROTEINS NOT FOUND
IN DROSOPHILA
In addition to SPG48, some other HSPs are caused by mutations affecting endomembrane
proteins that have no Drosophila orthologue.
Adaptor Complex AP-4 (SPG47)
Mutations affecting three components of the AP-4 adaptor complex give rise to a
complex condition that includes severe intellectual disability and spastic paraplegia (Abou
Jamra et al., 2010); one of these loci encoding the beta subunit, AP4B1, is designated SPG47.
AP-4 is a low-abundance adaptor associated with a subset of Golgi and other vesicles (Hirst
et al., 1999), which is also required for normal dendritic localization of AMPA receptor
complexes (Matsuda et al., 2008). AP-4 has a wide phylogenetic distribution that includes
Maintaining Long Supply Lines 105
vertebrates, sponges, and many plants, but appears to have been lost from protostomes
(including Drosophila).
Maspardin (SPG21)
SPG21 is an autosomal recessive complex HSP, caused by mutations in maspardin
(Simpson et al., 2003), a largely Golgi-associated protein that is a predicted hydrolase
(Zeitlmann et al., 2001). Maspardin is found in a wide range of animals including some
insects, but has been lost recently from the Drosophila lineage.
Erlin-2 (SPG18)
SPG18 is an autosomal recessive complex HSP, caused by mutations in erlin-2 (Alazami
et al., 2011), a putative hairpin-loop protein found in detergent-insoluble domains of the ER
(Browman et al., 2006). It has orthologues in a wide range of animals including some insects,
but has been lost from the Drosophila lineage.
MITOCHONDRIAL PROTEINS
Mitochondria are transported to distal axons via fast anterograde transport using kinesin
motors, principally the Khc1/KIF5 family (Hollenback, 1996; Tanaka et al., 1998; Kanai et
al., 2000). HSP could conceivably result from a limited energy supply in distal axons, and
indeed at least two HSPs are caused by mutations in mitochondrial proteins.
Paraplegin (SPG7)
Mutations in paraplegin cause an autosomal recessive complex HSP. Paraplegin is an
ATP-dependent m-AAA protease in the inner membrane of mitochondria (Hanson and
Whiteheart, 2005; Rugarli and Langer, 2006), involved in degradation of misfolded proteins,
cleavage of mitochondrial target sequences (Nolden et al., 2005), and maturation of
mitochondrial enzymes (Koppen et al., 2009). Drosophila CG2658 is the orthologue of
human paraplegin, but has not yet been studied in depth.
Heat Shock Protein 60 (SPG13)
Mutations in heat shock protein 60 (HSP60) cause autosomal dominant pure HSP
(Hansen et al., 2002) and autosomal recessive hypomyelinating leukodystrophy (Magen et al.,
2008). Hsp60 forms a large multisubunit complex with its co-chaperonin Hsp10 in the
mitochondrial matrix (Hansen et al., 2002), where it mediates protein folding, prevents
misfolding, and helps to clear misfolded and damaged non-functional proteins (Hansen et al.,
Belgin Yalçın and Cahir J. O’Kane 106
2008). Hsp60 has one Drosophila orthologue; although it is well studied (www.flybase.org),
little is known of its functions in motor neuron degeneration.
INTERACTIONS OF NEURONS WITH SUBSTRATES OR
NEIGHBORING CELLS
L1CAM (SPG1)
Loss-of-function mutations in L1CAM lead to X-linked hydrocephalus, MASA (Mental
retardation, Aphasia, Shuffling gait and Adducted thumbs) syndrome or complex recessive
HSP (Jouet et al., 1994). L1CAM is a single pass transmembrane protein with six Ig-like
domains and five fibronectin repeats extracellularly, and a short cytoplasmic tail (De Angelis
et al., 2002). Drosophila neuroglian is an orthologue of four human paralogues: neural cell
adhesion molecule, neurofascin, L1CAM, and neural cell adhesion molecule L1-like protein.
The structure of neuroglian is similar to L1CAM, but it also contains ankyrin and ezrin-
radixin-moesin (ERM) binding motifs in the cytoplasmic region (Davis and Bennnett, 1994;
Dickson et al., 2002; Cheng et al., 2005).
Drosophila neuroglian is involved in neurite growth, axon guidance, and sensory-neuron
migration (Bieber et al., 1989; Davis et al., 1994; Hall and Bieber, 1997; Dickson et al., 2002;
Islam et al., 2004; Cheng et al., 2005; Kristiansen et al., 2005; Godenschwege et al., 2006;
Chen and Hing, 2008). Its roles include axon fasciculation (Goossens et al., 2011), and
anchoring MTs in synaptic terminals during giant-synapse formation (Godenschwege et al.,
2006). However, given the complexity of these roles, and its orthology to four human genes,
it is not easy to see which neuroglian functions are most relevant for HSP.
PLP1 (SPG2)
Mutations in proteolipid protein 1 (PLP) cause Pelizaeus-Merzbacher disease (PMD) and
X-linked complicated spastic paraplegia (SPG2). SPG2 has milder symptoms than PMD,
involving demyelination rather than failure to form a myelin sheath (Gencic et al., 1989;
Woodward, 2008). PLP1 is an integral membrane protein and a major component of myelin.
M6 (CG7540) is the single Drosophila orthologue of PLP1 and of two other human
glycoproteins M6a and M6b. Depletion of M6 can lead to mild eye roughness and lowered
phototaxis (Zappia et al., 2012). However, Drosophila M6 may be of limited use to
understand the role of PLP1 in HSP, since Drosophila axons lack a myelin sheath.
CONCLUSION
Studying the function and mutant phenotypes of SPG products is a route to better
understanding both of HSP, as well as the mechanisms that maintain axonal function at
enormous distances from the cell body. Work in Drosophila has made major contributions to
understanding the roles of SPG proteins in processes including ER organization, axon
Maintaining Long Supply Lines 107
transport, and BMP signalling, and thus to the model that the wide spectrum of HSP genes
and phenotypes is due to a more limited number of common disease mechanisms.
Flies now offer great potential for the future. First, new SPGs continue to emerge, and
flies will continue to be an excellent model for understanding the basic function of these
genes. Furthermore, there is now a need to meet the next challenge, of understanding disease
mechanisms. Drosophila, as a simple genetic model system with well-developed tools to
study axonal transport, function and integrity, will increasingly provide insights into
pathomechanisms as well as into basic biological function of SPG proteins.
ACKNOWLEDGMENTS
We thank Niamh O'Sullivan for helpful discussions. B.Y. is supported by a Yousef
Jameel Scholarship.
ABOUT THE AUTHORS
Belgin Yalçın is a PhD student in the Department of Genetics, University of Cambridge.
Her first degree is from Istanbul Technical University, and her Ph.D. project aims to
understand the roles of Drosophila SPG proteins in organization of axonal ER.
Cahir J O'Kane is a Faculty member in the Department of Genetics, University of
Cambridge. He has helped to develop some of the tools used most widely in Drosophila
neurobiology, including enhancer trapping and targeted silencing of neurons by tetanus toxin.
He has a long-standing interest in neuronal membrane traffic, and his work on this aspect of
SPG function includes defining the role of the Drosophila NIPA1/SPG6 homolog in BMP
receptor traffic and signalling, and the role of Drosophila reticulon in organization of axonal
ER.
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 5
DROSOPHILA AS A MODEL FOR CMT PERIPHERAL
NEUROPATHY: MUTATIONS IN TRNA SYNTHETASES
AS AN EXAMPLE
Georg Steffes1 and Erik Storkebaum
1,*
1Molecular Neurogenetics Laboratory, Max Planck Institute
for Molecular Biomedicine, Muenster, Germany
ABSTRACT
Charcot-Marie-Tooth (CMT) disease is characterized by the degeneration of
peripheral motor and sensory neurons, leading to progressive muscle weakness and
wasting, and sensory loss. Electrophysiological and pathological criteria allow the
distinction between demyelinating, axonal and intermediate forms of CMT. The disease
is genetically heterogeneous, with currently more than 30 genes causally linked to CMT.
The molecular underpinnings of the peripheral motor and sensory neuropathy are poorly
understood, and there is no effective drug treatment available. In this chapter, we discuss
the use of Drosophila melanogaster as a genetic model organism for CMT. Major
advantages include the possibility to study the effect of CMT-associated mutant proteins
on motor and sensory neurons in their physiological context, and its suitability to perform
genetic screens. To illustrate the usefulness of Drosophila as a model for CMT, we
highlight forms of CMT that are associated with mutations in tRNA synthetases. These
enzymes ligate amino acids to their cognate tRNA, and therefore catalyze an essential
step in protein synthesis. Mutations in the genes encoding tyrosyl-tRNA synthetase
(YARS), glycyl-tRNA synthetase (GARS), alanyl-tRNA synthetase (AARS), and possibly
lysyl-tRNA synthetase (KARS) and histidyl-tRNA synthetase (HARS) give rise to axonal
and intermediate forms of CMT. Loss of aminoacylation activity per se is not the cause
of the disease, although the possibility that altered subcellular localization of
aminoacylation-active mutants could lead to defects in local protein synthesis cannot be
excluded at the present moment. Current evidence suggests that the disease may be
caused by a gain-of-toxic function mechanism, the molecular nature of which remains
elusive. The future use of Drosophila CMT models in genetic screens for disease-
modifying genes may be of great value to unravel the molecular mechanisms of disease,
and to identify possible therapeutic targets.
Georg Steffes and Erik Storkebaum 122
Keywords: Charcot-Marie-Tooth disease, peripheral motor and sensory neuropathy, axonal
degeneration, tRNA synthetase, aminoacylation activity, genetic screen
INTRODUCTION
Charcot Marie Tooth disease (CMT) is the most common inherited neuromuscular
disorder and has an estimated prevalence of 1 in 2500 individuals (Martyn and Hughes,
1997). CMT is characterized by distal muscle weakness and atrophy, sensory loss, decreased
reflexes, and foot deformities. These classical symptoms are due to degeneration of peripheral
motor and sensory nerves, whereby the longest nerves are preferentially affected. Hence,
CMT is also referred to as hereditary motor and sensory neuropathy. The disease usually
occurs in the first two decades of life and is subsequently slowly progressive over decades
(Dyck, 1993).
CMT is both clinically and genetically heterogeneous. Traditionally, two main forms are
distinguished, namely demyelinating and axonal forms of CMT. Clinically, the distinction is
based on electrophysiological criteria: nerve conduction velocities (NCVs) lower than 38m/s
are classified as demyelinating, whereas NCVs higher than 38m/s are considered axonal
forms (Reilly et al., 2011). Demyelinating forms of CMT are pathologically characterized by
segmental demyelination and remyelination with so called onion bulb formations - concentric
arrangements of supernumerary Schwann cells around an incompletely remyelinated axon.
Demyelinating forms are classified as CMT1 if the inheritance pattern is autosomal dominant,
and CMT4 in case of autosomal recessive inheritance (Patzko and Shy, 2011). Many CMT1-
and CMT4-associated genes are expressed in myelinating Schwann cells but not in neurons.
However, the primary demyelination in these forms of CMT ultimately leads to (secondary)
axonal degeneration, and the classical CMT symptoms can be mainly attributed to this axonal
degeneration, rather than the demyelination itself (Pareyson and Marchesi, 2009). Whereas
demyelinating CMT accounts for the majority of CMT cases, axonal forms (CMT2) give rise
to about 20% of CMT cases (Ajroud-Driss et al., 2011). Electrophysiologically, axonal CMT
is characterized by normal or mildly slowed NCVs, but with reduced compound action
potential amplitudes. Pathologically, evidence of chronic axonal degeneration and
regeneration is found. The majority of CMT2 is autosomal dominant, but autosomal recessive
forms have been described (Pareyson and Marchesi, 2009). More recently, it has become
evident that a clear distinction between demyelinating and axonal forms of CMT is not
always possible, and intermediate forms between CMT1 and CMT2 are recognized. These
forms of intermediate CMT are characterized by intermediate NCVs (25-45 m/s) and
pathological features of both demyelination and axonal degeneration (Nicholson and Myers,
2006). Apart from autosomal dominant and autosomal recessive forms of intermediate CMT,
X-linked CMT often shows intermediate NCVs and pathological evidence of axonal loss and
some demyelination, with few onion bulbs (Kleopa and Scherer, 2006).
Further subdivision of CMT types is based on causative genes or assigned loci. To date,
more than 30 genes have been associated with CMT (Table 1). These genes encode proteins
with often very different molecular functions suggesting that derailment of multiple
Drosophila as a Model for CMT Peripheral Neuropathy 123
molecular pathways can give rise to peripheral motor and sensory neuropathy. Molecular and
cellular pathways that may be involved in CMT molecular pathogenesis include myelination
and myelin maintenance, axonal transport, mitochondrial dynamics, endosomal trafficking,
axon-Schwann cell interaction, transcriptional regulation and protein chaperone activity
(Ajroud-Driss et al., 2011; Patzko and Shy, 2011). To increase complexity even further,
CMT-associated proteins have very different expression patterns and subcellular localizations
(Table 1). Indeed, some CMT gene products are selectively expressed in Schwann cells or
neurons, whereas others are ubiquitously expressed. This raises the question why mutations in
such ubiquitously expressed genes give rise to the specific peripheral neuropathy phenotype.
Moreover, CMT proteins can be localized to the cytoplasm, the nucleus, mitochondria, ER,
endosomes, the plasma membrane, myelin or neuronal cytoskeleton (Pareyson and Marchesi,
2009).
Thus, the exact molecular mechanisms by which CMT-associated genes lead to
peripheral neuropathy are still enigmatic, and there is no effective drug treatment available for
CMT. Clearly, there is an urgent need for better insights into the molecular pathogenesis of
CMT, to identify therapeutic targets, and to test potential therapeutic agents. Animal models
for CMT, including Drosophila models, can be instrumental to achieve these goals.
CELLULAR AND ANIMAL MODELS FOR CMT
Studies on CMT patients and patient-derived samples allow genetic analysis to identify
causative genes, analysis of genotype/phenotype correlation, clinical trials to test the
efficiency of candidate (drug) treatments and neuropathological analysis of post-mortem
tissue samples. However, despite the relevance of directly conducting studies on CMT
patients, such studies have several limitations. These include (i) the fact that invasive studies
are not possible, so that e.g. neuropathology in early disease stages cannot be assessed, (ii) the
often very limited number of patients with certain genetic mutations, and (iii) the fact that
clinical trials are expensive and need to be based on good preclinical evidence in cellular or
animal models. For these reasons, cellular and animal models are of key importance to study
the molecular pathogenesis of CMT.
Cellular Models for CMT
Advantages of cellular models for CMT are the relative ease of studying cellular
processes (e.g. axonal transport), and their suitability for genetic (e.g. using small interfering
RNAs) or pharmacological manipulation and screening. An obvious drawback is that the
physiological, in vivo context is missing when culturing cells in a dish. The use of co-
cultures, e.g. of neurons and Schwann cells, addresses, but does not solve this problem.
Cellular CMT models can be divided into patient-derived and non-human, typically mouse
(model) derived cells.
Georg Steffes and Erik Storkebaum 124
The use of patient-derived cells is obviously very relevant, because one works in the
disease genetic background. Traditionally, blood sample-derived lymphocyte cultures or
fibroblasts obtained from skin biopsy are used. Limitations are that these cells can only be
used if the CMT-associated gene is expressed in lymphocytes or fibroblasts and obviously
these cells are not the affected cell types in CMT. It recently became possible to overcome
these limitations by the use of induced pluripotent stem cells (iPSCs). These cells can be
derived from fibroblasts, allow unlimited proliferation, and can be differentiated into all cell
types of the body, including motor and sensory neurons and Schwann cells (Dolmetsch and
Geschwind, 2011; Marchetto et al., 2011). Although this technology holds great promise for
studying molecular mechanisms of disease and identifying possible therapeutic compounds,
the procedure to generate iPSCs and subsequent differentiation is elaborate, time-consuming,
and technically challenging. Furthermore, at the present moment it is not yet possible to
obtain "pure" cultures of the CMT-relevant cell types, and the obtained cells often correspond
to "embryonic" stages, which is a disadvantage when studying adolescent or adult-onset
diseases (Dolmetsch and Geschwind, 2011; Marchetto et al., 2011). In contrast to patient-
derived cells, derivation of cells from animal models allows the generation of primary
neuronal or Schwann cell cultures (or co-cultures), as well as the derivation of these cell types
from embryonic stem cells (ESCs) or iPS cells. CMT rodent models are typical sources for
these cells.
Animal Models for CMT
CMT animal models have the major advantage that the disease-relevant cell types can be
studied in their physiological context, and that the effect of disease-associated mutations on
animal behaviour and physiology can be evaluated. However, the price to pay for this are the
anatomical and genetic differences between animal models and humans, which become more
prominent with increasing evolutionary distance. Indeed, when constructing an animal model
for CMT, one hopes not only to recapitulate the hallmark disease phenotypes, but also the
cellular and molecular mechanisms that lead to disease in patients. If this is not the case,
studying the animal model in question will never allow to decipher the molecular
pathogenesis underlying the human disease. Therefore, we think it is important that the
animal model has an orthologue of the studied CMT-associated gene, in order to maximize
the chances that the molecular pathways that lead to CMT in humans are conserved in the
animal model. Obviously, even if this is the case, one should keep in mind that the CMT-
associated gene may have acquired additional functions during evolution. If alteration of this
additional function would be the molecular cause of the disease, the animal model will not be
suitable for cracking the "CMT code".
The most popular animal models for neurodegenerative diseases are - with increasing
evolutionary distance - rodent models (mouse and rat), zebrafish, Drosophila melanogaster
and C. elegans. When modelling CMT in such animal models, the genetic approach will be
determined by the mode of inheritance of the CMT form studied.
Table 1. Different forms of CMT and associated genes
Type Subtype OMIM Gene Inheritance Location Molecular function Atypical clinical phenotypes
CMT1
CMT1A 118220 PMP22 autosomal dominant compact myelin myelination, cell growth, differentiation
CMT1B 118200 MPZ autosomal dominant compact myelin cell adhesion
CMT1C 601098 LITAF/SIMPLE autosomal dominant Schwann cells transcription factor
CMT1D 607678 EGR2 autosomal dominant Schwann cells transcription factor
CMT1E 118300 PMP22 autosomal dominant compact myelin myelination, cell growth, differentiation hearing loss
CMT1F 607734 NEFL autosomal dominant neuronal cytoskeleton regulation of axonal diameter, axonal
transport
CMT2
CMT2A1 118210 KIF1B autosomal dominant ubiquitous axonal transport
CMT2A2 609260 MFN2 autosomal
dominant/recessive
mitochondrial membrane
and ER
fusion of mitochondria; mitochondria-ER
interactions
CMT2B 600882 RAB7 autosomal dominant late endosomes regulates vesicular transport
CMT2B1 605588 LMNA autosomal recessive nuclear lamina nuclear stability, chromatin structure and
gene expression
CMT2B2 605589 MED25 autosomal recessive nucleoplasm transcriptional regulation
CMT2C 606071 TRPV4 autosomal dominant cell membrane ion channel, mediates calcium influx diaphragmatic and vocal cord paresis
CMT2D 601472 GARS autosomal dominant cytoplasm, mitochondrial
matrix
protein translation
CMT2E 607684 NEFL autosomal dominant neuronal cytoskeleton regulation of axonal diameter, axonal
transport
CMT2F 606595 HSPB1 autosomal dominant cytoplasm stress resistance, actin and intermediate
filament organization, chaperone activity,
anti-apoptotic activity, proteasome activation
CMT2G 608591 unknown autosomal dominant
CMT2H 607731 GDAP1 autosomal recessive outer mitochondrial
membrane
regulation of mitochondrial dynamics pyramidal features
CMT2J 607736 MPZ autosomal dominant compact myelin cell adhesion hearing loss and pupillary
abnormalities
CMT2K 607831 GDAP1 autosomal dominant/
recessive
outer mitochondrial
membrane
regulation of mitochondrial dynamics
CMT2L 608673 HSPB8 autosomal dominant cytoplasm chaperone activity
Table 1. (Continued)
Type Subtype OMIM Gene Inheritance Location Molecular function Atypical clinical phenotypes
CMT2M 606482 DNM2 autosomal dominant cytoplasm endocytosis
CMT2N 613287 AARS autosomal dominant cytoplasm protein translation
CMT2O 614228 DYNC1H1 autosomal dominant cytoplasm axonal transport
CMT2P 614436 LRSAM1 autosomal
dominant/recessive
cytoplasm E3 ubiquitin-protein ligase
CMT3 CMT3 145900 MPZ, EGR2,
PMP22,PRX
autosomal
dominant/recessive
Schwann cells multiple Dejerine-Sottas neuropathy
CMT4
CMT4A 214400 GDAP1 autosomal recessive outer mitochondrial
membrane
regulation of mitochondrial dynamics
CMT4B1 601382 MTMR2 autosomal recessive cytoplasm phosphatase activity, dephosphorylates
PI3P and PI3,5P2
CMT4B2 604563 SBF2/MTMR13 autosomal recessive cytoplasm pseudophosphatase; dimerizes with
MTMR2, thereby increasing its
enzymatic activity
early-onset glaucoma can occur
CMT4C 601596 SH3TC2 autosomal recessive Schwann cell plasma
membrane and perinuclear
endocytic recycling
compartment
endocytic recycling pathway
CMT4D 601455 NDRG1 autosomal recessive cytoplasm growth arrest and cell differentiation hearing loss
CMT4E 605253 EGR2 autosomal recessive Schwann cells transcription factor congenital hypomyelinating neuropathy
CMT4F 145900 PRX autosomal recessive Schwann cells myelin maintenance
CMT4G 605285 unknown autosomal recessive
CMT4H 609311 FGD4 autosomal recessive cytoplasm guanine nucleotide exchange factor for
the Rho GTPase CDC42; F-actin
binding and crosslinking activity
CMT4J 611228 FIG4 autosomal recessive endosome membrane PI3,5P2 phosphatase
CMT5 CMT5 600361 multiple autosomal dominant pyramidal features
CMT6 CMT6 601152 MFN2 autosomal dominant mitochondrial membrane and
ER
fusion of mitochondria; mitochondria-
ER interactions
optic atrophy
DI-CMT DI-CMTA 606483 unknown autosomal dominant
DI-CMTB 606482 DNM2 autosomal dominant cytoplasm endocytosis
Type Subtype OMIM Gene Inheritance Location Molecular function Atypical clinical phenotypes
Type Subtype OMIM Gene Inheritance Location Molecular function Atypical clinical phenotypes
DI-CMTC 608323 YARS autosomal dominant cytoplasm protein translation
DI-CMTD 607791 MPZ autosomal dominant compact myelin cell adhesion
DI-CMTE 614455 INF2 autosomal dominant cytoplasm actin dynamics focal segmental glomerulonephritis
RI-CMT
RI-CMTA 608340 GDAP1 autosomal recessive outer mitochondrial
membrane
regulation of mitochondrial dynamics
RI-CMTB 613641 KARS autosomal recessive cytoplasm, mitochondria protein translation developmental delay, self-abusive
behaviour, dysmorphic features and
vestibular Schwannoma
CMTX
CMTX1 302800 GJB1 (Cx32) X-linked dominant Schwann cells and
oligodendrocytes
gap junction protein
CMTX2 302801 unknown X-linked recessive
CMTX3 302802 unknown X-linked recessive
CMTX4 310490 unknown X-linked recessive deafness and mental retardation
CMTX5 311070 PRPS1 X-linked recessive purine and pyrimidine biosynthesis optic atrophy, deafness, polyneuropathy
CMT types and subtypes, inheritance pattern, associated genes with their subcellular localization pattern and molecular function, and atypical clinical
phenotypes are listed.
Georg Steffes and Erik Storkebaum 128
In case of a recessive inheritance pattern, the disease is most likely caused by partial or
full loss of gene function. Therefore, inducing loss-of-function mutations in the orthologous
gene may provide an animal model for CMT in these cases. In case of dominant inheritance,
overexpression of the mutant human gene, or of the orthologous gene that carries the
corresponding disease-causing mutation, may result in a suitable CMT animal model. For
both modes of inheritance, "knock-in" models will best recapitulate the genetic situation in
human patients. However, one caveat is that knock-in models carry the inherent risk that the
introduced (often subtle) mutations may not result in CMT-associated phenotypes during the
relatively short life span of the animal model.
All CMT-associated genes known to date have orthologues in mice, and mouse models
are now available for 17 genetic forms of CMT (Table 2). These mouse models often
recapitulate several disease phenotypes, and are invaluable to study cellular and molecular
mechanisms of disease and to evaluate potential pharmacological treatments (Fledrich et al.,
2012). However, because of the high cage costs, space requirements and life cycle duration,
genome-wide unbiased genetic screens to identify disease-modifying genes seem practically
impossible. Given the fact that the molecular pathogenesis of CMT is poorly understood, and
that the disease may be caused by toxic gain-of-function mechanisms, in particular for
dominantly inherited forms of CMT, such genetic screens may be necessary to elucidate the
molecular mechanisms of disease. Indeed, if CMT-associated mutations cause the protein to
interfere with molecular pathways that are distinct from the normal functions of the protein
(e.g. when the mutant protein acquires novel protein-protein interactions), it is very unlikely
that hypothesis-driven research will be able to uncover the disease-causing molecular
mechanisms. Therefore, a key advantage of small non-vertebrate model organisms such as
Drosophila melanogaster and C. elegans is that they allow conducting genetic screens for
disease-modifying genes. Identification of these genes may not only provide insights into the
molecular pathogenesis of CMT, but may also identify putative therapeutic targets.
DROSOPHILA AS A MODEL FOR CMT
Apart from its suitability for genetic screens, experimental advantages of working with
Drosophila are its short life cycle (10 days at 25°C from fertilized egg to reproducing adult),
the ease and cheapness of its culture, the large number of offspring, the ease of genetic
manipulation and the multitude of genetic tools available (Venken and Bellen, 2005).
Furthermore, the organisational principles of the nervous system are remarkably conserved
between flies and vertebrates (including humans).
The Drosophila central nervous system (CNS) consists of brain and ventral nerve cord
(VNC), the latter being the homologous structure to the spinal cord in mammals (Figure 1).
Lower motor neurons in Drosophila have their cell bodies in the VNC, and project axons to
peripheral muscles, where they form neuromuscular junctions (NMJs). Sensory neurons have
their cell bodies in the periphery, and project into the central nervous system. Basic
neurophysiological principles (e.g. conduction of action potentials, transmission of signals by
release of neurotransmitters packaged in synaptic vesicles, the synaptic vesicle cycle, etc.) are
conserved.
Drosophila as a Model for CMT Peripheral Neuropathy 129
Table 2. Mouse models for CMT
Human Gene
Symbol Subtype Human Gene
Mouse
Gene
Mouse model
for CMT
available
"Kind" of model
PMP22
CMT1A,
CMT1E,
CMT3
peripheral myelin protein 22 Pmp22 YES
PMP22 transgenic
mice, spontaneous
Pmp22 point
mutations
MPZ
CMT1B,
CMT2J,
CMT3, DI-
CMTD
myelin protein zero Mpz YES Null and knock-in
LITAF
(SIMPLE) CMT1C
lipopolysaccharide-induced
tumour necrosis factor Litaf NO
Litaf null mice do
not display
peripheral
neuropathy
EGR2
CMT1D,
CMT3,
CMT4E
early growth response 2 Egr2 YES
knock-out and
conditional
knock-out
NEFL CMT1F,
CMT2E
neurofilament, light
polypeptide Nefl YES
NEFL(P22S)
transgenic mice
KIF1B CMT2A1 kinesin family member 1B Kif1b YES
Kif1b
heterozygous
mice
MFN2 CMT2A2,
CMT6 mitofusin 2 Mfn2 YES
Mfn2 T105M
transgenic mice
RAB7 CMT2B RAB7, member RAS
oncogene family Rab7 NO
LMNA CMT2B1 lamin A/C Lmna YES Null
MED25 CMT2B2 mediator complex subunit 25 Med25 NO
TRPV4 CMT2C
transient receptor potential
cation channel, subfamily V,
member 4
Trpv4 NO
GARS CMT2D glycyl-tRNA synthetase Gars YES
ENU-induced
Gars point
mutations
HSPB1 CMT2F heat shock 27kDa protein 1 Hspb1 YES mutant HSPB1
transgenic mice
GDAP1
CMT2H,
CMT2K,
CMT4A,
RI-CMTA
ganglioside-induced
differentiation-associated-
protein 1
Gdap1 NO
HSPB8 CMT2L heat shock 22kDa protein 8 Hspb8 NO
DNM2 CMT2M,
DI-CMTB dynamin 2 Dnm2 NO
knock-in model
for centronuclear
myopathy
available, but no
peripheral
neuropathy model
Georg Steffes and Erik Storkebaum 130
Table 2. (Continued)
Human Gene
Symbol Subtype Human Gene
Mouse
Gene
Mouse
model for
CMT
available
"Kind" of
model
AARS CMT2N alanyl-tRNA synthetase Aars NO
Sticky mouse
displays
cerebellar ataxia
but no
peripheral
neuropathy
DYNC1H1 CMT2O dynein cytoplasmic 1 heavy
chain 1 Dync1h1 YES
missense
mutations in
Loa and Cra1
mice, 9
nucleotide
deletion in Swl
mice
LRSAM1 CMT2P leucine rich repeat and sterile
alpha motif containing 1 Lrsam1 NO
PRX CMT3,
CMT4F periaxin Prx YES Null
MTMR2 CMT4B1 myotubularin related protein 2 Mtmr2 YES Null and E276X
knock-in
SBF2
(MTMR13) CMT4B2 SET binding factor 2 Sbf2 YES Null
SH3TC2 CMT4C SH3 domain and
tetratricopeptide repeats 2 Sh3tc2 YES Null
NDRG1 CMT4D N-myc downstream regulated
gene 1 Ndrg1 YES Null
FGD4 CMT4H FYVE, RhoGEF and PH
domain containing 4 Fgd4 NO
FIG4 CMT4J FIG4 homologue, SAC1 lipid
phosphatase domain containing Fig4 YES
'pale tremor'
mice contain a
homozygous
transposon
insertion in
intron 18 of the
Fig4 gene
YARS DI-CMTC tyrosyl-tRNA synthetase Yars NO
INF2 DI-CMTE inverted formin, FH2 and WH2
domain containing Inf2 NO
KARS RI-CMTB lysyl-tRNA synthetase Kars NO
GJB1 CMTX 1 gap junction protein, beta 1,
connexin32 Gjb1 YES
Null and Cx32
R142W
transgenic mice
PRPS1 CMTX5 phosphoribosyl pyrophosphate
synthetase 1 Prps1 NO
Mouse homologs of CMT-associated genes and available mouse models for the different CMT
subtypes are listed.
Because of its relevance for modelling CMT, the development and anatomy of the
Drosophila neuromuscular system is described in more detail in the next paragraphs.
Drosophila as a Model for CMT Peripheral Neuropathy 131
Figure 1. A, B, The neuromuscular system in Drosophila larvae. (A) Confocal image of an abdominal
segment of the larval body wall showing the innervation pattern of the muscle fibers by motor neurons.
Motor neurons are labelled in magenta and muscle fibers are labelled in green. (B) Schematic diagram
of the innervation pattern of the intersegmental nerve (ISN) and the segmental nerve (SN) pathways;
the transverse nerve (TN) is not displayed. C, D, The adult neuromuscular system. (C) Schematic
representation of the adult nervous system with the brain (B), ventral nerve cord (VNC) and peripheral
nerves. (D) Schematic representation of the adult muscle system. In the head, the rostral retractor is
labelled in orange and the pharyngeal dilators are labelled in purple. In the thorax, the dorsal
longitudinal muscles (DLMs) are labelled in blue, the dorsoventral muscles are labelled in yellow, and
the tergotrochanteral muscle (TTM) is labelled in green. E, Schematic drawing of a cross-section
through a peripheral nerve (Rodrigues et al., 2011).
Georg Steffes and Erik Storkebaum 132
The Drosophila Neuromuscular System
During Drosophila development, two distinct phases of neurogenesis occur: the first
during embryonic development, and the second during larval life and early metamorphosis
(Lin and Lee, 2012). Embryonic neurogenesis begins at stage 9 of 17 embryonic stages, with
the delamination of neuronal stem cells, called neuroblasts, from the ectodermal germ layer.
Neuroblasts subsequently produce a series of ganglion mother cells. At stage 13, the progeny
of ganglion mother cells begins to differentiate into neurons and glial cells (Lin and Lee,
2012). The Drosophila embryo can be subdivided into 3 thoracic (T1-T3) and 8 abdominal
(A1-A8) segments. Each abdominal hemisegment from A1-A7 contains 30 muscle fibers,
which can be subdivided into ventral and lateral musculature (Fernandes and Keshishian,
1999). During embryonic development, the axons of 36 motor neurons per hemineuromer
project through 3 principal nerves into the muscle field: two main nerves, the intersegmental
nerve (ISN) and segmental nerve (SN), and a minor one, the transverse nerve (TN), which
contains two motor axons and projects along the segment border (Landgraf and Thor, 2006).
Embryonic motor neurons mediate embryonic contractions and larval hatching.
In the larval stage, neuromuscular junctions progressively enlarge as the animal grows, so
that by the end of the third instar the muscles are 10 times their embryonic lengths, and motor
neurons show a significant expansion in synaptic branching and bouton number. The larval
neuromuscular system consists of a segmentally repeated array of dorsal, ventral and lateral
muscle fibers, with variations in specific thoracic and abdominal segments (Figure 1A and B).
The motor neurons are segmentally repeated, and mediate larval behaviours such as crawling,
feeding and moulting. Drosophila motor neurons are glutamatergic, whereas the presynaptic
interneurons are predominantly cholinergic (Fernandes and Keshishian, 1999).
During metamorphosis, almost all of the larval musculature is histolyzed and replaced by
proliferating muscle progenitor cells that were set aside by the end of embryogenesis. In
contrast, the vast majority of adult motor neurons derive from functional larval motor
neurons, which are structurally re-specified to innervate adult muscle targets (Fernandes and
Keshishian, 1999). In contrast to motor neurons, almost all larval sensory neurons degenerate
during metamorphosis and are replaced by adult neurons, which develop from imaginal discs.
Likewise, most adult interneurons are formed during metamorphosis, although other
interneurons are remodelled larval interneurons, which may carry out new tasks during
adulthood (Tissot and Stocker, 2000).
In the adult, there are distinct sets of muscles in the head, thorax, and abdomen that
mediate adult-specific motor functions such as flight, walking, feeding and copulation (Figure
1). The largest muscles are located in the thorax, and are involved in walking and flight. Each
hemithorax has about 80 muscle fibers that are divided in dorsal and ventral sets. Much of the
dorsal thorax is occupied by the indirect flight muscles (IFMs), consisting of 13 muscle fibers
per hemithorax. Six of these comprise the dorsal longitudinal muscles (DLMs), which are
innervated by five identified mesothoracic motor neurons. The remaining seven fibers make
up the dorsoventral muscles (DVMs) (Figure 1), which are mononeuronally innervated by
seven motor neurons. DLMs are the wing depressors, whereas DVMs are the wing elevators.
The alternative contraction and relaxation of these muscles generates the wing beat during
flight. The other group of thoracic muscles are the direct flight muscles (DFMs), 17 in
number. DFMs are smaller than IFMs, and are located at the base of the wing. They are
responsible for steering functions of the wing during flight. The largest tubular muscle in the
Drosophila as a Model for CMT Peripheral Neuropathy 133
thorax is the jump muscle, also known as tergotrochanteral muscle (TTM). The TTM is a
ventral mesothoracic muscle, which executes the jump motion and is innervated by the
tergotrochanteral motor neuron (TTMn) (Figure 1). Among the prominent muscles in the
head are the pharyngeal dilators involved in feeding, the rostral retractors, which control
movements of the proboscis, and the ptilinum retractors, which are used during adult
emergence (Figure 1). In contrast to thoracic and head musculature, adult abdominal muscles
have a simple organization and bear a closer similarity to larval musculature (Figure 1). There
are sets of dorsal, ventral, and lateral muscle fibers in segments A1-A6. A few segment-
specific specializations of muscle fibers are also evident in the adult abdomen, including the
male-specific muscle (MSM) in the fifth abdominal segment. The terminal abdominal
segments also show sex-specific muscle fiber patterning variations, and there are specific
muscles associated with the ovary and the testes.
Which Types of CMT Can Be Modeled in Drosophila?
Particularly relevant for CMT is the anatomy of Drosophila peripheral nerves. As can be
seen in Figure 1E, axons are ensheathed by wrapping glia, considered to be analogous to
vertebrate Schwann cells. Peripheral nerve bundles are surrounded by subperineurial and
perineurial glia, which form the blood-nerve barrier (Stork et al., 2008). However, despite the
similarities between Drosophila and vertebrate peripheral nerves, Drosophila peripheral
nerves lack myelin, and do not possess saltatory nerve conduction. In accordance with that,
Drosophila lacks close homologs of all CMT1-associated genes (PMP22, MPZ, LITAF,
ERG2 and NEFL). For these reasons, we think that Drosophila is not a suitable model to
study the demyelinating forms of CMT.
However, we believe that Drosophila is well suited to model axonal and intermediate
forms of CMT, as axonal morphology and function is conserved, and the vast majority of
axonal and intermediate CMT-associated genes have close homologs in Drosophila (Table 3).
Mutants of the Drosophila homologs of CMT-associated genes can provide insight into the
endogenous function of these genes, and - in case of (occasional) recessive inheritance - may
provide a Drosophila CMT model. Remarkably, most of the Drosophila homologs of CMT-
associated genes have not or only to a very limited extent been studied. Only for Drosophila
homologs of LMNA, DNM2 and DYNC1H1 more than 10 original research papers are
available (Table 3).
In case of a dominant inheritance pattern, the disease may be caused by haplo-
insufficiency, a dominant negative mechanism, gain-of-wild-type-function or gain-of-toxic-
function. Expression of a human CMT-mutant protein in Drosophila, or overexpression of the
homologous Drosophila protein with the CMT-associated mutations introduced in the
homologous amino acid residues, may provide a Drosophila CMT model, except in case of a
haplo-insufficient mechanism. Indeed, in the latter case, 50% reduction of gene dosage
(inactivation of one allele by the CMT mutation) is sufficient to cause the disease.
Overexpression of such a protein with a loss-of-function mutation in an otherwise wild-type
Drosophila background (the Drosophila homolog of the disease gene is intact) may not
induce phenotypes.
Georg Steffes and Erik Storkebaum 134
Table 3. Drosophila homologues of CMT-associated genes
Human Gene
Symbol Subtype Human gene name
Drosophila
homologue Gene name CG number
Papers on
gene function
in Drosophila
PMP22
CMT1A,
CMT1E,
CMT3
peripheral myelin
protein 22 NO n.a. n.a. n.a.
MPZ
CMT1B,
CMT2J,
CMT3,DI-
CMTD
myelin protein
zero NO n.a. n.a. n.a.
LITAF
(SIMPLE) CMT1C
lipopolysaccharide
-induced tumour
necrosis factor
NO n.a. n.a. n.a.
EGR2
CMT1D,
CMT3,
CMT4E
early growth
response 2 NO n.a. n.a. n.a.
NEFL CMT1F,
CMT2E
neurofilament,
light polypeptide NO n.a. n.a. n.a.
KIF1B CMT2A1 kinesin family
member 1B YES unc-104 CG8566 4
MFN2 CMT2A2,
CMT6 mitofusin 2 YES
Marf CG3869 5
fzo CG4568 2
RAB7 CMT2B
RAB7, member
RAS oncogene
family
YES Rab7 CG5915 6
LMNA CMT2B1 lamin A/C YES Lam CG6944 41
LamC CG10119 13
MED25 CMT2B2 mediator complex
subunit 25 YES MED25 CG12254 0
TRPV4 CMT2C
transient receptor
potential cation
channel, subfamily
V, member 4
YES
nan CG5842 7
iav CG4536 12
GARS CMT2D glycyl-tRNA
synthetase YES Aats-gly CG6778 1
HSPB1 CMT2F heat shock 27kDa
protein 1 multiple multiple multiple n.a.
GDAP1
CMT2H,
CMT2K,
CMT4A,
RI-CMTA
ganglioside-
induced
differentiation-
associated-protein
1
YES CG4623 CG4623 0
HSPB8 CMT2L heat shock 22kDa
protein 8 multiple multiple multiple n.a.
DNM2 CMT2M,
DI-CMTB dynamin 2 YES shi CG18102 44
AARS CMT2N alanyl-tRNA
synthetase YES Aats-ala CG13391 1
DYNC1H1 CMT2O
dynein
cytoplasmic 1
heavy chain 1
YES Dhc64c CG7507 35
Drosophila as a Model for CMT Peripheral Neuropathy 135
Human Gene
Symbol Subtype Human gene name
Drosophila
homologue Gene name CG number
Papers on
gene function
in Drosophila
LRSAM1 CMT2P
leucine rich repeat
and sterile alpha
motif containing 1
NO n.a. n.a. n.a.
PRX CMT3,
CMT4F periaxin NO n.a. n.a. n.a.
MTMR2 CMT4B1 myotubularin
related protein 2 YES mtm CG9115 1
SBF2
(MTMR13) CMT4B2
SET binding factor
2 YES Sbf CG6939 2
SH3TC2 CMT4C
SH3 domain and
tetratricopeptide
repeats 2
NO n.a. n.a. n.a.
NDRG1 CMT4D
N-myc
downstream
regulated gene 1
YES MESK2 CG15669 0
FGD4 CMT4H
FYVE, RhoGEF
and PH domain
containing 4
YES RhoGEF4 CG8606 2
FIG4 CMT4J
FIG4 homolog,
SAC1 lipid
phosphatase
domain containing
YES CG17840 CG17840 0
YARS DI-CMTC tyrosyl-tRNA
synthetase YES Aats-tyr CG4561 0
INF2 DI-CMTE
inverted formin,
FH2 and WH2
domain containing
YES form3 CG33556 1
KARS RI-CMTB lysyl-tRNA
synthetase YES aats-lys CG12141 0
GJB1 CMTX 1
gap junction
protein, beta 1,
connexin32
NO n.a. n.a. n.a.
PRPS1 CMTX5
phosphoribosyl
pyrophosphate
synthetase 1
YES CG6767 CG6767 0
The number of papers on the function of the Drosophila gene listed in FlyBase are indicated. This
number gives an indication on how well the respective Drosophila genes have been studied.
As already indicated for mouse models, introducing the CMT-associated mutations in the
endogenous locus of the Drosophila orthologue by homologous recombination (knock-in
approach) is an alternative strategy to generate models for both dominantly and recessively
inherited mutations.
Apart from tyrosyl-tRNA synthetase (discussed in the next paragraph), only one other
CMT-associated protein has been expressed in Drosophila, namely mitofusin 2 (Mfn2).
Eschenbacher et al. studied the functional consequences of rare non-synonymous sequence
variants within the heptad repeat 1 (HR1) domain of Mfn2, two of which are predicted to be
potentially damaging based on bioinformatical analysis (M393I and R400Q) (Eschenbacher et
al., 2012). Expression of these mutant Mfn2 proteins in the Drosophila eye results in reduced
eye area, whereas expression of wild-type Mfn2 does not. Furthermore, RNAi knock-down of
the Drosophila Mfn2 homolog Marf also results in a reduced eye area, which could be
rescued by co-expression of wild-type Mfn2, but not by the mutant Mfn2 proteins. Although
Georg Steffes and Erik Storkebaum 136
these findings are interesting, the studied Mfn2 mutations have not been found in CMT
patients, and most of the 31 Mfn2 mutations that were previously linked to CMT are located
in the GTPase domain. It would therefore be interesting to evaluate the effect of CMT-
associated Mfn2 mutations, and not only on eye morphology but also on motor behaviour,
axonal morphology, and electrophysiology.
A DROSOPHILA MODEL FOR CMT ASSOCIATED WITH MUTATIONS IN
TYROSYL-TRNA SYNTHETASE
The only published bona fide Drosophila CMT model to date models dominant
intermediate CMT type C (DI-CMTC), which is caused by mutations in the YARS gene,
encoding tyrosyl-tRNA synthetase (TyrRS or YARS) (Storkebaum et al., 2009). This enzyme
aminoacylates tyrosyl-tRNA (tRNATyr
) with tyrosine in a two-step reaction (Figure 2A). Like
all tRNA synthetases, YARS is expressed ubiquitously and every cell in the body depends on
its aminoacylation activity for protein translation. YARS forms homodimers, and only the
dimeric form is enzymatically active. Interestingly, apart from YARS, dominant mutations in
GARS, AARS and possibly HARS, and recessive mutations in KARS also result in axonal and
recessive intermediate forms of CMT (Abe and Hayasaka, 2009; Antonellis et al., 2003; Del
Bo et al., 2006; Dubourg et al., 2006; James et al., 2006; Latour et al., 2010; Lin et al., 2011;
McLaughlin et al., 2012; McLaughlin et al., 2010; Rohkamm et al., 2007; Vester et al., 2012)
(Table 1). These 5 tRNA synthetases all contain an aminoacylation domain and an anticodon
recognition domain, which are essential for their canonical aminoacylation function (Figure
2B). Apart from these common domains, many tRNA synthetases have acquired additional
functional domains during evolution, conferring non-canonical functions to these proteins
(Brown et al., 2010; Guo et al., 2010). These non-canonical functions are, however, different
for distinct tRNA synthetases. From a genetic point of view, the fact that 5 of the 20
cytoplasmic tRNA synthetase genes are associated with CMT suggests that alteration of a
common function of these enzymes - probably tRNA aminoacylation - may be the cause of
the disease.
Expression of Mutant Tyrosyl-tRNA Synthetase Recapitulates Features of
Human CMT in Drosophila
To generate a model for DI-CMTC, the UAS/GAL4 system was used to express human
YARS (hYARS) in Drosophila. This binary expression system relies on UAS transgenic
lines, which, when crossed to a specific GAL4 transgenic line, will result in transgene
expression in the cell population in which the yeast transcription factor GAL4 is expressed
(Brand and Perrimon, 1993). As a large number of GAL4 lines are available, this expression
system allows transgene expression in virtually any cell type or tissue of interest. Transgenic
lines were generated that allow expression of wild-type or three CMT-associated hYARS
mutants: two missense mutations (hYARS_G41R and hYARS_E196K) and one in frame
deletion that results in the deletion of 4 amino acids in the YARS protein (hYARS_153-
156delVKQV) (Jordanova et al., 2006). As random transgene insertion was used to generate
Drosophila as a Model for CMT Peripheral Neuropathy 137
the transgenic lines, transgene expression levels of several wild-type and mutant hYARS
expressing lines were determined, in order to select lines with similar expression levels for
further studies (Storkebaum et al., 2009). More recently, it became possible to use site-
specific transgenesis, whereby attachment sites (attP/attB) are used to target UAS-transgenes
to specific landing sites in the genome (Fish et al., 2007).
Figure 2. A, Two-step aminoacylation reaction catalysed by tyrosyl-tRNA synthetase. In the first step,
tyrosine is activated by ATP to form a TyrRS (Tyr-AMP) intermediate with simultaneous release of
pyrophosphate PPi. In the second step, the activated tyrosyl moiety is transferred from AMP to tRNATyr
to give rise to Tyr-tRNATyr
and AMP. B, Schematic representation of the 5 tRNA synthetases that have
been implicated in CMT: tyrosyl-tRNA synthetase (YARS), glycyl-tRNA synthetase (GARS), alanyl-
tRNA synthetase (AARS), lysyl-tRNA synthetase (KARS) and histidyl-tRNA synthetase (HARS).
CMT-associated mutations and their positions relative to the protein functional domains are shown.
Mutations represented in green segregate with disease in a pedigree, mutations in black were found in a
single patient, mutations in orange give rise to dominant peripheral neuropathy phenotypes in mice, and
mutations in blue were detected in compound heterozygous state in a single patient with intermediate
CMT. *The AARS E778A mutation was found in a family with rippling muscles and cramps, and also
in one patient with axonal CMT (McLaughlin et al., 2012).**The AARS D893N mutation was found in
a family with dominant distal hereditary motor neuropathy (dHMN) (Zhao et al., 2012).
Georg Steffes and Erik Storkebaum 138
This strategy eliminates confounding influences of the surrounding genomic DNA
environment on transgene expression levels and patterns, making it ideally suited to evaluate
the phenotypic effect of subtle mutations in the transgene (structure/function analysis).
Strong ubiquitous expression of mutant - but not wild-type - hYARS resulted in
developmental lethality, with no or reduced numbers of adult flies eclosing from their pupal
cases. This mutant-selective toxicity was transgene dosage-dependent, so that flies with lower
expression levels could be tested for motor performance. Mutant hYARS expressing flies
displayed motor deficits in a negative geotaxis climbing assay, as well as in a jump and flight
assay. These motor performance deficits were progressive over time. Also neuron-selective
expression of mutant - but not wild-type - hYARS induced motor performance defects,
showing that mutant hYARS is intrinsically toxic to neurons (Storkebaum et al., 2009). As
DI-CMTC is characterized by both demyelination and axonal degeneration (Jordanova et al.,
2003), this finding indicates that the axonal degeneration is not just secondary to
demyelination, as is the case in many demyelinating forms of CMT.
Figure 3. Schematic representation of the Drosophila giant fiber system. For reasons of simplicity, the
representation is unilateral. The giant fiber neuron (GF) has its cell body in the brain, and projects
through the cervical connective to the VNC, where it synapses with the tergotrochanteral motor neuron
(TTMn) and the peripherally synapsing interneuron (PSI). The TTMn innervates the tergotrochanteral
jump muscle (TTM), whereas the PSI synapses in the periphery with the dorsal longitudinal motor
neurons (DLMns), which innervate the dorsal longitudinal muscles (DLMs). The position of
stimulation and recording electrodes for electrophysiological evaluations are indicated. Brain
stimulation activates the GF, which then activates motor neurons. Thoracic stimulation excites the
TTMn and DLMn directly (Godenschwege et al., 2002).
Drosophila as a Model for CMT Peripheral Neuropathy 139
Finally, expression of hYARS transgenes in the giant fiber (GF) system was used to
assess the effect of hYARS expression on axonal morphology and to evaluate the occurrence
of electrophysiological defects. The GF system mediates an escape response consisting of a
jump and subsequent flight (Allen et al., 2006). It consists of the GF neurons, which are 2
symmetrical neurons in the fly CNS that have their cell bodies in the brain and project their
giant axon to the VNC (Figure 3). In the VNC, the GF axons synapse with the peripherally
synapsing interneuron (PSI) and the tergotrochanteral motor neuron (TTMn). The PSI in turn
synapses with the dorsal longitudinal motor neurons that innervate the dorsal longitudinal
flight muscles. The TTMn synapses with the tergotrochanteral "jump" muscle in the legs of
the fly. This organisation of the network ensures that, when a shadow (e.g. of an approaching
predator) falls over the eye of the fly, the visual input will activate the GF neurons, what will
result in a jump followed by flight.
The GF system is ideally suited to study the effects of CMT-associated (mutant) proteins,
as the giant fiber neurons have particularly long axons, the system is well characterized, and
GAL4 driver lines are available that allow expression in all GF system neurons (A307-
GAL4), as well as selective expression in the GF neurons (C17-GAL4) or the TTMn (ShakB-
GAL4). Furthermore, it allows evaluation of GF morphology, either by expressing marker
transgenes, or - preferentially - by dye filling approaches. Finally, it also allows
electrophysiological approaches that evaluate the response latencies between activation of the
GF neurons (by brain stimulation) and the tergotrochanteral or dorsal longitudinal muscles. If
the synapse between the GF neurons and the TTMn or PSI are dysfunctional, the response
latencies will be increased. Similarly, the ability to follow high-frequency (100 Hz) stimuli
one-to-one can be evaluated (Allen et al., 2006). Expression of mutant hYARS proteins in the
GF system was found to induce both terminal axonal degeneration and electrophysiological
defects (Storkebaum et al., 2009). As selective expression in the GF neurons was sufficient to
induce these phenotypes, one can conclude that mutant hYARS has cell-autonomous toxic
effects in neurons. In conclusion, expression of mutant - but not wild-type - hYARS in
Drosophila resulted in progressive motor performance defects, terminal axonal degeneration
and electrophysiological defects. Thus, several hallmarks of human CMT are recapitulated in
the fly model.
The Drosophila genome contains a single gene encoding cytoplasmic tyrosyl-tRNA
synthetase (Aats-tyr, further referred to as dYARS). The dYARS protein is 68% identical and
80% similar at the amino acid level to human YARS (hYARS), and all amino acid residues
that are mutated in DI-CMTC are identical between dYARS and hYARS. Importantly,
overexpression of dYARS transgenes containing the DI-CMTC mutations induced similar
phenotypes as the hYARS transgenes, indicating that the hYARS phenotypes are not simply
the consequence of expressing a human protein in Drosophila (Storkebaum et al., 2009).
Interestingly, the Drosophila DI-CMTC model has been shown to be useful to predict the
pathogenicity of newly identified mutations in the YARS gene. This was illustrated by a novel
K265N substitution in the YARS anti-codon recognition site, which was identified in one
CMT patient and one control individual. Ubiquitous or pan-neuronal expression of a dYARS
transgene carrying the corresponding amino acid change (UAS-dYARS_K264N) did not
induce motor performance defects or developmental lethality, showing that this substitution is
a benign polymorphism (Leitao-Goncalves et al., 2012).
Georg Steffes and Erik Storkebaum 140
Peripheral Neuropathy Phenotypes are Independent of Aminoacylation
Activity of Mutant YARS Proteins
The Drosophila DI-CMTC model was further used to assess the effect of the CMT-
associated mutations on aminoacylation activity. A genetic complementation experiment was
performed, whereby an RNAi transgene targeting dYARS was expressed in sensory organ
precursor (SOP) cells (scabrous-GAL4). These cells give rise to the sensory organs, including
bristles, which are sensory hairs that cover the body of the fly and detect the direction of the
airflow during flight. Expression of dYARS-RNAi in SOPs induced shortening or loss of the
four scutellar bristles on the posterior part of the thorax, which are always long in control
flies. To assess the effect of CMT mutations on aminoacylation activity, hYARS transgenes
were expressed in the dYARS-RNAi background. Expression of wild-type hYARS fully
rescued the bristle phenotype, indicating that dYARS and hYARS are functional homologs.
This again underscores the relevance of expressing hYARS in Drosophila, and is conform
with the observation that dGARS and hGARS are functional homologs (Chihara et al., 2007).
Expression of hYARS_E196K in the Sca-GAL4>dYARS-RNAi background also fully rescued
the bristle phenotype, suggesting that this mutant has retained aminoacylation activity. In
contrast, expression of hYARS_G41R or hYARS_153-156delVKQV did not rescue the
bristle phenotype, which is suggestive for loss of aminoacylation activity. These findings
were confirmed by an in vitro aminoacylation assay and by genetic complementation in S.
cerevisiae (Storkebaum et al., 2009), as well as by a biochemical study (Froelich and First,
2011). From this it was concluded that hYARS_E196K retains aminoacylation activity,
hYARS_153-156delVKQV has severely reduced activity, and hYARS_G41R displays loss of
enzymatic activity. These findings indicate that loss of aminoacylation activity is not
necessary to cause peripheral motor and sensory neuropathy. 50% loss of aminoacylation
activity is also not sufficient to induce peripheral neuropathy phenotypes, as dYARS
hemizygous flies did not develop motor performance defects (Storkebaum et al., 2009).
These findings were rather unexpected, as all identified disease-causing YARS mutations
are located in the aminoacylation domain of the protein, raising the possibility that the disease
could be due to partial loss of aminoacylation activity (Figure 2). Furthermore, the fact that
mutations in 5 different tRNA synthetase genes all give rise to CMT suggests that (partial)
loss of a common function (probably the canonical aminoacylation function) could be the
cause of the disease. However, the findings are consistent with the fact that for both GARS
and AARS, some CMT-associated mutations result in loss of aminoacylation activity,
whereas others do not alter enzymatic activity (Achilli et al., 2009; Antonellis et al., 2006;
Cader et al., 2007; McLaughlin et al., 2012; Nangle et al., 2007; Seburn et al., 2006; Xie et
al., 2007). However, the fact that loss of aminoacylation activity per se is not necessary to
cause the disease does not exclude the possibility that aminoacylation-active mutants may be
mislocalised in peripheral motor and sensory neurons, hence resulting in defects in local
protein translation and terminal axonal degeneration.
Indeed, mislocalisation of mutant YARS and GARS proteins has been reported in mouse
neuroblastoma (N2A), human neuroblastoma (SH-SY5Y) and mouse motor neuron (MN-1)
cell lines (Antonellis et al., 2006; Jordanova et al., 2006; Nangle et al., 2007). However,
another study reports no alterations in subcellular localization of mutant GARS proteins in
ESC-derived motor neurons and spinal cord sections of GarsP234KY/+
mice, as well as in teased
fiber preparations of sciatic nerves of GarsC201R/+
mice (Stum et al., 2011). Also,
Drosophila as a Model for CMT Peripheral Neuropathy 141
AARS_E778A displays a similar subcellular localization as wild-type AARS in MN-1 cells
(McLaughlin et al., 2012). Finally, although HARS_R137Q - but not wild-type HARS -
induces axonal morphology defects and locomotor defects when expressed in C. elegans
motor neurons, HARS_R137Q displays a similar subcellular localization as wild-type HARS.
Thus, further studies are needed to clarify the potential role of subcellular mislocalisation of
mutant tRNA synthetases in CMT pathogenesis. Ultimately, direct assessment of neuronal
protein translation rates in an animal model would be the best way to evaluate the effect of
tRNA synthetase mutations on (local) protein translation in vivo.
Another caveat when interpreting the available data on aminoacylation activity of mutant
tRNA synthetases is that these studies have been performed either in vitro, using purified
proteins, or in Drosophila or S. cerevisiae in vivo, where the endogenous orthologous tRNA
synthetase is either missing or its levels are strongly reduced. As a consequence, these studies
have evaluated the aminoacylation activity of homodimers of mutant tRNA synthetases.
However, since for YARS, GARS and AARS the disease is dominantly inherited,
heterodimers between WT and mutant tRNA synthetase subunits can be formed (Jordanova et
al., 2006). At this moment, no data on aminoacylation activity of such heterodimers has been
reported. A possible involvement of (hetero-)dimerization is also suggested by the fact that all
pathogenic GARS mutations reported so far all localize near the dimer interface (He et al.,
2011; Nangle et al., 2007). The effect of the mutations on dimer formation has been
investigated, and although some mutations do not alter dimer formation, others either
strengthen or weaken dimer formation (He et al., 2011; Marchetto et al.; Nangle et al., 2007).
Overall, the potential role of heterodimers in disease pathogenesis is currently not clear and
deserves further investigation.
Finally, both Drosophila and mouse models for CMT associated with mutations in tRNA
synthetases can be used to study the genetic mechanisms of disease. The fact that dYARS
hemizygous flies or GARS heterozygous mice do not develop peripheral neuropathy
phenotypes argues against haplo-insufficiency as the disease mechanism (Seburn et al., 2006;
Storkebaum et al., 2009). Furthermore, mice heterozygous for ENU-induced P234KY and
C201R mutations in the murine GARS gene display a peripheral neuropathy phenotype,
which is not modified by overexpression of wild-type GARS (Motley et al., 2011). This
argues against both dominant negative and gain-of-wild-type-function mechanisms.
Furthermore, wild-type GARS transgenic mice that are homozygous or transheterozygous for
the GARS mutant alleles have a more severe phenotype than heterozygous mice, indicating
that the phenotypic strength depends on mutant allele dosage (Motley et al., 2011).
Overall, these genetic findings suggest a gain-of-toxic function as the basis of the disease.
The molecular mechanism of such a gain-of-toxic function is currently unknown, but novel,
mutation-induced protein-protein interactions may well be involved. In this respect, the
finding that each of five spatially dispersed GARS mutations induce the same conformational
opening of a consensus area that is mostly buried in the wild-type protein may provide a
structural basis for newly acquired protein-protein interactions (He et al., 2011). Along with
other approaches, genetic screens in Drosophila models for mutant YARS- and GARS-
associated CMT would be ideally suited to unravel the molecular nature of the gain-of-toxic
function mechanism.
Georg Steffes and Erik Storkebaum 142
CONCLUSION
Drosophila may be of great value to model forms of CMT that involve axonal
degeneration. Arguments in favor of Drosophila are that (i) the basic organizational
principles and functional properties of the neuromuscular system are conserved, (ii)
Drosophila homologs for most forms of axonal and intermediate CMT exist, and (iii) the
effect of CMT mutant proteins on neurons can be studied in their physiological context,
whereby behavioral phenotypes, neuronal morphology and electrophysiology can be
evaluated. Finally, the fact that Drosophila CMT models can be used in genetic screens for
disease modifying genes raises the hope that these models may be instrumental in deciphering
the molecular pathogenesis of this incurable disease, and in the identification of possible
therapeutic targets.
As a Drosophila model for CMT associated with mutations in tyrosyl-tRNA synthetase is
the only published Drosophila CMT model to date, there is a window of opportunity to
generate models for many other forms of CMT and to study the function of the Drosophila
homologs of CMT-associated genes in more detail. Thus, the use of Drosophila as a model
for CMT is only in its initial stages, and future expansion and broadening of this field is
expected.
ACKNOWLEDGMENTS
We would like to thank Brigitte Sass for help with the figures, Daniel Banovic and
Hermann Aberle for providing the figure panels illustrating the larval neuromuscular system,
and Tanja Godenschwege for providing the figure illustrating the giant fiber system. We
thank Arzu Celik and Christian Klämbt for critical reading of the manuscript. E.S. is funded
by the state North Rhine Westphalia and supported by a grant from the Frick Foundation for
ALS Research, SFB629 programm project grant from the German Research Council (DFG),
and a Minna-James-Heineman-Stiftung Minerva research grant.
ABOUT THE AUTHORS
Georg Steffes graduated in Biology at the University of Muenster, Germany, and
performed his doctorate research at the same university in the Institute of Neurobiology and
Behavioural Biology. He did postdoctoral research at Sanger Institute in Cambridge, UK, and
at the Max Planck Institute for Molecular Biomedicine, Muenster, Germany. His research
interests are the genetics of sensory systems and neurodegenerative disease modelling.
Erik Storkebaum graduated in Pharmaceutical Sciences at the University of Leuven,
Belgium, and obtained his Ph.D. at the Vesalius Research Center, VIB, Leuven, Belgium. He
has been working as a postdoctoral researcher in the Laboratory for Developmental Genetics,
University of Leuven, Belgium. Since 2010 he is an independent research group leader at the
Max Planck Institute for Molecular Biomedicine, Muenster, Germany. His research aims at
deciphering the molecular pathogenesis of the motor neurodegenerative disorder amyotrophic
lateral sclerosis (ALS) and CMT peripheral neuropathy.
Drosophila as a Model for CMT Peripheral Neuropathy 143
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 6
LESSONS FROM DROSOPHILA IN
NEURODEGENERATION: MECHANISMS OF
TOXICITY AND THERAPEUTIC TARGETS IN SPINAL
AND BULBAR MUSCULAR ATROPHY
Adrienne M. Wang University of Washington, Department of Pathology, Seattle, WA, US
ABSTRACT
Spinal and bulbar muscular atrophy (SBMA) is one of nine polyglutamine (polyQ)
repeat diseases caused by an abnormal expansion of a glutamine tract in the affected
gene. In each of the nine diseases, the expansion occurs in unrelated genes, and affects
distinct neuronal populations. Despite these differences, polyglutamine diseases are all
characterized by the build-up of misfolded proteins into nuclear aggregates, and selective
neuronal loss. Drosophila models of polyglutamine diseases have yielded many insights
into disease pathology. In SBMA, the polyQ expansion occurs in the first exon of the
androgen receptor (AR), leading to a partial loss of endogenous function as well as a
toxic gain-of-function. Studies using fly models of SBMA helped to establish both the
glutamine length-dependent and ligand-dependent toxicity that are characteristic of the
disease. Additionally, flies have given us numerous insights into molecular mechanisms
of toxicity, revealing a myriad of cellular processes that are altered in disease
pathogenesis, such as transcription, RNA processing, axonal trafficking and
mitochondrial function. With such a wide array of cellular mechanisms affected by the
presence of the mutant AR, treating dysfunction in individual pathways is likely to be
ineffective, and a promising therapeutic target is the modulation of the cell’s innate
protein quality control pathways to clear mutant protein upstream of its toxic effects.
While polyglutamine diseases currently lack efficacious treatment, Drosophila is a
promising model organism suited for straightforward genetic and pharmacologic
manipulation in the hunt for therapeutic targets that prevent or halt neurodegeneration.
Keywords: Polyglutamine, androgen receptor, neurodegenerative disease, protein quality
control
Adrienne M. Wang 148
INTRODUCTION
A diverse group of neurodegenerative diseases affecting the aging population are
characterized by accumulations of abnormally processed or mutant proteins that misfold and
aggregate. Among these are nine genetic disorders caused by expansions of a trinucleotide
CAG repeat within the coding regions of disease-causing genes (Table 1). Since CAG codes
for glutamine, this group of diseases is referred to as polyglutamine repeat diseases (polyQ
diseases).
For each of the nine disorders, the polyglutamine expansion occurs within unrelated
genes and selectively affects different neuronal subtypes, yet these polyglutamine diseases
share several clinical and pathological features (Zoghbi and Orr, 2000). As a group,
polyglutamine diseases preferentially affect the basal ganglia, brainstem nuclei, cerebellum,
and spinal motor nuclei, often leading to impaired motor function. Disease onset occurs most
often in middle age despite lifelong expression of mutant protein, and exhibits an inverse
correlation with CAG repeat length. The longer the glutamine expansion, the earlier the
patient becomes symptomatic, and the more acute the disease progression. Unfortunately,
these expanded polyglutamine repeats are also highly unstable, making them prone to genetic
anticipation, in which instability leads to an increase in length with each successive
generation. The expanded repeat is inherited in an autosomal dominant manner in all
polyglutamine diseases except for spinal bulbar muscular atrophy (), which is X-linked,
suggesting that pathology is due in part to a toxic gain-of-function (Zoghbi and Orr, 2000)
The unstable polyglutamine expansion can adopt an abnormal -sheet conformation
leading to the formation of insoluble aggregates, and these polyglutamine diseases are
characterized by the accumulation of nuclear and/or cytoplasmic protein inclusions. These
inclusions are typically composed of the mutant protein, heat shock proteins (HSPs) and
ubiquitin (Li et al., 1998). There is considerable debate in the field as to whether the presence
of aggregates are toxic or protective, or whether they simply reflect the end stage of
accumulation, with the toxic species being the precursors to the aggregates including
oligomers or proteolysed monomers (Adachi et al., 2005; Arrasate et al., 2004; Saudou et al.,
1998). Regardless, the presence of aggregates is associated with the late stages of disease
pathogenesis, implying that the accumulation of misfolded toxic proteins may be a key step in
degeneration.
Drosophila have proven to be an invaluable tool in the study of polyglutamine diseases,
with their single gene mutations lending themselves to direct genetic modelling. Although
outwardly very different from humans, the architecture and function of the fly body and
organs mirrors our own, and numerous cellular processes are conserved between flies and
mammals. Genetically, 50% of fly genes exhibit homology with human genes, and 75% of
human disease-causing genes have a homologue in Drosophila (Bier, 2005). Flies also exhibit
complex behaviour that is reflective of their highly organized neuronal network, allowing
them to be used as a model for cellular and higher-order behavioural studies, both aspects
affected by neurodegeneration. With such genetic similarity in the package of such a
malleable model system, flies offer an attractive system in which to manipulate endogenous
and exogenous gene expression.
Lessons from Drosophila in Neurodegeneration 149
Table 1. Polyglutamine Diseases
Disease Protein
Normal
repeat
length
Pathogenic
repeat
length
Brain region/neurons
affected
SBMA Spinal Bulbar
Muscular
Atrophy
androgen
receptor
6-36 38-62 Lower motor neurons and
dorsal root ganglion
HD Huntington’s
disease
huntingtin 11-34 40-121 Striatum and cortex
DRPLA Dentatorubral-
pallidoluysian
atrophy
atrophin-1 7-34 49-88 Globus pallidus, dentate
and subthalamic nucleus
SCA1 Spinocerebellar
ataxia type 1
ataxin-1 6-39 40-82 Purkinje cells, dentate
nucleus and brainstem
SCA2 Spinocerebellar
ataxia type 2
ataxin-2 15-24 32-200 Cerebellum, pontine nuclei,
substantia nigra
SCA3
(MJD)
Spinocerebellar
ataxia type
3/Machado-
Joseph disease
ataxin-3
13-36 61-84 Substantia nigra, globus
pallidus, pontine nucleus,
cerebellar cortex
SCA6 Spinocerebellar
ataxia type 6
P/Q-type
calcium channel
subunit 1A
4-20 20-29 Cerebellum, brainstem
SCA7 Spinocerebellar
ataxia type 7
ataxin-7 4-35 37-306 Photoreceptor and bipolar
cells, cerebellum, brainstem
SCA17 Spinocerebellar
ataxia type 17
TATA-box-
binding protein
25-42 47-63 Gliosis and Purkinje cell
loss
The UAS/Gal4 system developed by Brand and Perrimon (Brand and Perrimon, 1993) has
proven to be an indispensible tool for fly geneticists. It allows for expression of genes in a
tissue and time-specific manner, where the gene of interest is placed just down-stream of an
up-stream activating sequence (UAS), and binding of GAL4 to the UAS is required for gene
transcription. By driving expression of GAL4 in a tissue or developmental time-specific
manner, researchers can spatially or temporally control expression of the UAS-linked gene of
interest. In particular, the glass-multimer reporter (GMR-Gal4) driver line has been used
extensively in studies of neurodegeneration. This driver is expressed in the fly compound eye,
allowing for expression of UAS linked proteins in photoreceptor neurons and accessory
pigment cells of the developing eye discs. This is an attractive target for expression of toxic
proteins as the eye is non-essential in the fly, and allows researchers to circumvent
developmental or pathological effects of their target protein.
The fly eye is a highly ordered organ, with each eye containing around 800 units called
ommatidia. These ommatidia are organized in a crystalline pattern with interommatidial
bristles studding each junction at a discrete angle (Figure 1, EtOH). Each ommatidium
contains eight photoreceptor neurons, organized in an invariant fashion. Changes or
degeneration of this highly organized structure are readily seen under the light microscope
and are often referred to as a ‘rough-eye phenotype’ due to the roughness introduced when
the lattice of the eye is disrupted, making it particularly useful for genetic screens (Figure 1,
DHT). In fact, numerous genetic screens for modifiers of the polyglutamine induced rough-
Adrienne M. Wang 150
eye phenotype have informed our current hypotheses about mechanisms of toxicity in
polyglutamine diseases, including transcriptional dysregulation, altered RNA processing,
mitochondrial dysfunction, and defects in axonal transport (Bilen and Bonini, 2007;
Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000; Nedelsky et al., 2010).
Flies expressing expanded polyglutamine repeats were the first neurodegenerative models
successfully created in Drosophila using human transgenes (Jackson et al., 1998; Warrick et
al., 1998). Dr. Nancy Bonini’s group at the University of Pennsylvania and Dr. Larry
Zipursky’s group at the University of California, Los Angeles both expressed truncated
fragments of human disease causing genes in the fly, publishing their models in the same
year. Directed expression of truncated fragments of expanded polyQ human ataxin-3 and
huntingtin, the disease causing proteins in spinocerebellar ataxia type 3 (SCA3) and
Huntington’s disease (HD) respectively, recapitulate several aspects of human polyglutamine
disease. These flies show glutamine length-dependent nuclear inclusions and neuronal
degeneration, and were used to demonstrate the glutamine length-dependence of the disease
pathology. These groups used the rough-eye phenotype to monitor degeneration and showed
that targeting expression of the toxic polyglutamine protein to the fly eye leads to
degeneration of the eye as monitored by age-related changes in eye morphology such as loss
of pigment, disruption of the gross ommatidial array and photoreceptor degeneration. Such
early studies helped to establish Drosophila as a highly tractable model of neurodegenerative
disease in vivo.
Figure 1. DHT-induced rough eye phenotype in a fly model of SBMA. Scanning electron micrographs
of eyes from flies expressing the expanded human AR52Q under control of the GMR-Gal4 promoter
fed control food (EtOH, upper panels) or food supplemented with dihydrotestosterone (DHT, lower
panels). High magnification images are shown in panels on the right. DHT induces hormone and
glutamine length dependent degeneration in the fly eye causing a rough-eye phenotype that is
characterized by ommatidial disarray and fusion, in addition to abnormal and extra interommatidial
bristles.
Lessons from Drosophila in Neurodegeneration 151
Figure 2. Androgen receptor domain structure. The AR protein consists of 3 functional domains: the N-
terminal domain containing the activation function-1 (AF-1) surface and the glutamine tract (PolyQ),
the DNA-binding domain (DBD), and the C-terminal ligand-binding domain (LBD) containing the
activation function-2 (AF-2) region. The nuclear localization signal (NLS) is found within the hinge
region that links the DBD and the LBD.
In SBMA, fly models have been indispensible to our understanding of disease pathogenesis,
answering many fundamental questions about requirements for disease initiation and
progression, the extent to which normal androgen receptor (AR) function is involved in
toxicity, and genetic modifiers of toxicity, paving the way for valuable insights into
polyglutamine pathogenesis and therapeutics.
SBMA PATHOLOGY
SBMA is a progressive neuromuscular disorder that is caused by a toxic expansion of the
glutamine tract in the AR (La Spada et al., 1991). This polyglutamine disease affects only
men, and is characterized by degeneration of proximal limb, mouth, and throat muscles in
patients (Kennedy et al., 1968). Symptom onset is typically between 30-60 years of age, but
muscle weakness is often preceded by muscle cramping and tremor. The clinical features of
SBMA correlate with loss of lower motor neurons in the brainstem and spinal cord, with
marked myopathic and neurogenic changes in skeletal muscle (Sobue et al., 1989). Patients
may also exhibit partial androgen insensitivity such as enlarged breasts, testicular atrophy and
decreased fertility (Dejager et al., 2002). Disease progression is slow, but many patients
eventually require assistance to walk, and risk for aspiration pneumonia increases as bulbar
paralysis develops. Currently, very few treatment options exist for SBMA patients, the most
promising of which are testosterone blockade therapies (Banno et al., 2009; Fernandez-
Rhodes et al., 2011; Katsuno et al., 2006b).
The endogenous function of the AR has been well characterized, making it an ideal
context in which to study the effects of a toxic polyQ tract. In SBMA, the toxic glutamine
tract expansion occurs in the first exon of the AR. The repeat length found in the normal
population is 9-36 glutamines, with an expansion to a length between 38 and 62 glutamines in
SBMA patients. In addition to the glutamine tract in the N-terminal transactivation domain,
the AR contains a nuclear localisation sequence (NLS) and a central DNA binding domain
(DBD) linked to the C-terminal ligand-binding domain (LBD) by a hinge region (Figure 2).
Two interaction surfaces, activation function-1 (AF-1) in the N-terminal domain, and
activation function-2 (AF-2) in the LBD, recruit nuclear co-regulators to modulate
transcription. Without ligand, the nuclear localization signal is masked, and the AR is
localized to the cytoplasm where it is held in a high-affinity ligand binding state by molecular
Adrienne M. Wang 152
chaperones. Binding of testosterone to the ligand-binding domain exposes a nuclear
localization signal within the hinge region of the AR, allowing for the trafficking and import
of the AR into the nucleus (Cutress et al., 2008). Once in the nucleus, ligand-bound AR
dimerizes, interacts with co-regulators via exposed AF-1 and AF-2 domains, and binds to
androgen responsive elements (AREs), triggering transcription or repression of androgen-
dependent genes (He et al., 2002; Wong et al., 1993) (Figure 3).
In the presence of an abnormally expanded glutamine tract, the AR suffers from both a
partial loss of endogenous function as well as a toxic gain-of-function. Expansion of the
polyglutamine tract mildly suppresses transcriptional activities of the AR (Chamberlain et al.,
1994; Kazemi-Esfarjani et al., 1995; Lieberman et al., 2002), likely contributing to the partial
testosterone insensitivity seen in SBMA patients. However, patients with other loss-of-
function mutations in the AR gene, as well as mouse models with similar loss-of-function
mutations in the AR, show androgen insensitivity and testicular feminisation without the
motor impairment observed in SBMA (Sato et al., 2003). In addition, evidence for a gain-of-
function by the pathogenic AR is robust.
Figure 3. Mechanisms of toxicity in SBMA. PolyQ AR affects many cellular processes, both nuclear
and cytoplasmic. In the absence of ligand, the nuclear localization sequence (NLS) is masked, and the
AR is retained in the cytoplasm. Heat shock protein-90 (HSP90) stabilizes the receptor, priming it for
ligand binding. Upon binding of ligand, the NLS is exposed, allowing trafficking of the AR into the
nucleus where it leads to toxicity by altering numerous cellular processes. Pathogenic AR with
expanded polyQ tract is prone to misfolding, interaction with heat shock protein-70 (HSP70), and
subsequent degradation by the proteasome. Proteasomal degradation is shown occurring in the
cytoplasm, but also occurs within the nucleus. Abbrevations: DBD: DNA binding domain, LBD: ligand
binding domain, Ub: ubiquitin, ARE: androgen responsive elements, PGC1: PPAR coactivator-1,
DCTN1: dynactin 1, ROS: reactive oxygen species.
Lessons from Drosophila in Neurodegeneration 153
Several SBMA mouse models have been created in which transgenic expression of the
aberrant AR leads to muscular atrophy and motor dysfunction in the continued presence of
the wild-type protein (Abel et al., 2001; Katsuno et al., 2002; McManamny et al., 2002;
Sopher et al., 2004). Similarly, exogenous expression of the mutant AR in transformed cell
lines and primary neurons leads to altered cellular processes and cell death (Merry et al.,
1998; Mhatre et al., 1993). Data from fly models of polyglutamine disease are consistent with
a toxic gain-of-function, as expression of expanded polyglutamine tracts in the absence of
protein context and ectopic expression of the human expanded AR lead to toxicity (Kazemi-
Esfarjani and Benzer, 2000; Kazemi-Esfarjani and Benzer, 2002; Marsh et al., 2000;
Takeyama et al., 2002). Taken together, these data indicate that the polyglutamine tract
expansion in the AR leads to both a toxic gain-of-function and partial loss of normal function,
and suggest that toxic effects of the mutant protein are the main drivers of disease
pathogenesis.
DROSOPHILA MODELS OF SBMA
Although flies do not have a direct AR orthologue, the nuclear hormone receptor system
is well conserved between humans and flies, with fly hormone receptors containing AF-1 and
AF-2 (two activating domains in the human androgen receptor) domains and interacting
proteins. The first Drosophila model of SBMA was created by Dr. Ken-Ichi Takeyama and
colleagues at the University of Tokyo, by expressing a UAS-responsive full-length human
AR with either a normal (AR12Q) or expanded (AR52Q) polyQ tract under control of the
GMR promoter (Takeyama et al., 2002). The authors show that in this system, the human AR
translocates to the nucleus, and can activate GFP when driven by an androgen responsive
element (ARE). Importantly, this is done in a ligand-dependent manner, requiring dietary
ingestion of dihydroxytestosterone (DHT). A later paper from the same group further verified
that modulation of Drosophila homologues of mammalian AR co-regulators alters human AR
transactivation in flies, establishing this model as a functionally relevant and tractable means
to probe normal and abnormal AR function (Takeyama et al., 2004).
When flies ectopically express the human AR in the eye with either a non-toxic
glutamine tract or with an expanded glutamine tract in the absence of DHT, no abnormalities
or degeneration can be seen. This is in contrast with other polyQ disease models where the
expansion occurs in non-hormone responsive genes, exhibiting toxicity in a glutamine length-
dependent manner only (Kazemi-Esfarjani and Benzer, 2000; Tsai et al., 2004; Warrick et al.,
2005), and indicating a hormone requirement for disease progression in SBMA, an idea that
was debated at the time. Two possible mechanisms had been proposed, the first being that
females were protected from disease pathogenesis by random X-inactivation in which one of
the two copies of the X chromosome in females is inactivated, theoretically reducing motor
neuron toxicity by 50%. The second hypothesis posited that the toxicity was ligand-
dependent, and that higher levels of androgens in males increased their susceptibility to
disease pathogenesis (Lloyd and Taylor, 2010). This debate was resolved with help from the
humble fruit fly.
Dr. Takeyama’s model helped to establish the ligand dependence of SBMA. Expression
of non-toxic glutamine repeat length (AR12Q) in the fly eye leads to DHT-dependent nuclear
Adrienne M. Wang 154
translocation and expression of an ARE responsive GFP, without abnormalities or
degeneration. Flies expressing a toxically expanded glutamine tract (AR52Q) in the eye show
the same DHT dependent transactivation, but also exhibit ommatidial disarray, pitting, and
fusion, in addition to abnormal and supernumerary interommatidial bristles when reared on
DHT containing food (Figure 1). The authors establish that these abnormalities are due to
degeneration rather than developmental effects by showing that the rough eye phenotype can
be delayed and induced in naïve adult flies exposed to DHT. These data suggest that
testosterone-dependent activation of the AR is a critical step in disease pathogenesis.
This model is further supported by the fact that deletion of the LBD and constitutive
transactivation of a truncated AR causes degeneration in the absence of DHT (Takeyama et
al., 2002). Consistent with this, retaining the mutant AR in the cytosol by deletion of the NLS
and/or insertion of a nuclear export sequence (NES) abolishes toxicity (Takeyama et al.,
2002). Ligand dependent degeneration was confirmed by a mouse model of SBMA published
simultaneously, as well as in independently-generated fly models (Chevalier-Larsen et al.,
2004; Pandey et al., 2007). In this way, Drosophila served to definitively establish glutamine
length-dependent and hormone-dependent degeneration in SBMA, today considered two
fundamental features of the disease.
In addition to exhibiting the characteristic glutamine length and hormone dependence of
SBMA when mutant AR is expressed in the fly eye, these aspects of the disease are
recapitulated when the mutant polyQ AR is expressed in a variety of tissues. Larvae
expressing the expanded polyglutamine AR in the developing salivary gland exhibit
significant decrease in size in response to DHT, while larvae expressing the expanded
polyglutamine AR in motor neurons show hormone-dependent motor defects as assessed by
larval crawling (Nedelsky et al., 2010). Further, adult flies expressing the expanded polyQ
AR in motor neurons exhibit an eclosion defect when reared on DHT-supplemented food that
is consistent with motor neuron impairment (Wang et al., 2012).
MECHANISMS OF TOXICITY
Although the exact mechanism by which the mutant androgen receptor exerts a toxic
gain-of-function is unclear, the ligand dependence of SBMA highlights the AR’s nuclear
functions in effecting disease pathogenesis. Ligand induced transactivation of the AR is
further implicated in disease pathogenesis in the SBMA fly, with both nuclear import and
DNA binding being an upstream requirement in disease progression (Nedelsky et al., 2010).
This section will discuss several mechanisms of toxicity implicated in SBMA and other
neurodegenerative diseases summarized in Figure 3.
Transcriptional Dysregulation
Several polyglutamine diseases require nuclear localization of the mutant protein and
exhibit altered transcription prior to phenotypic onset (Lin et al., 2000; Wyttenbach et al.,
2001). A spinocerebellar ataxia 1 (SCA1) mouse model in which the polyQ Ataxin-1 is
cytoplasmically retained shows neither the ataxia nor Purkinje cell loss characteristic of the
Lessons from Drosophila in Neurodegeneration 155
disease. Alternatively, allowing nuclear transport in the absence of aggregation leads to
pathogenesis, establishing nuclear events as a requirement for polyglutamine toxicity
(Klement et al., 1998). Similarly, cytoplasmic retention of the AR rescues the disease in
SBMA fly and mouse models, while nuclear localization of an unliganded AR is insufficient
to cause the toxicity seen in the presence of ligand (Montie et al., 2009; Nedelsky et al., 2010;
Takeyama et al., 2002).
The endogenous function of the AR as a nuclear transcription factor lends added weight
to the idea that transcriptional dysregulation underlies disease pathogenesis in SBMA. The
mutant AR abnormally interacts with transcriptional co-regulators, and has been shown to
alter transcription in both fly and cellular models (Irvine et al., 2000; Kazemi-Esfarjani et al.,
1995; Lieberman et al., 2002; Mhatre et al., 1993; Nedelsky et al., 2010). Several of these co-
regulators are transcription factors that modify the acetylation state of the histones that
package and compact nuclear DNA, thus modifying the accessibility of DNA to the
transcriptional machinery. Furthermore, the acetylation state of the AR itself has been
suggested to regulate the subcellular localization and misfolding of the receptor (Thomas et
al., 2004), as well as interaction with co-regulators (Fu et al., 2002).
One of these co-regulators, CREB-binding protein (CBP), is a histone acetyltransferase
and an important co-activator that is functionally sequestered in the nuclear inclusions seen in
cell culture, animal models, and tissue derived from SBMA patients. Trapping CBP in
nuclear inclusions is thought to result in down-regulation of CBP mediated transcription
(McCampbell et al., 2000). In support of this, overexpression of CBP rescues polyQ mediated
toxicity in cell and Drosophila models of polyglutamine disease, restoring normal
transcription and histone acetylation levels (McCampbell et al., 2000; Taylor et al., 2003).
Genetically or pharmacologically inhibiting histone deacetylases rescues lethality and
ommatidial degeneration in a Drosophila model of HD (Steffan et al., 2001), and has also
been shown to decrease the toxicity of a truncated fragment of the expanded AR
(McCampbell et al., 2001), further implicating transcriptional dysregulation as one
pathogenic mechanism in SBMA.
Post-transcriptional Dysregulation
Post-transcriptional changes in gene expression also affect protein expression and can
produce dysfunction in much the same way as altered transcription. Toxic forms of RNA
have been implicated in other repeat expansion diseases, such as in select spinocerebellar
ataxias and in myotonic dystrophy, where trinucleotide expansions in noncoding mRNA
confer neurotoxicity. The expanded repeats confer toxicity and alter RNA splicing by
affecting expression of splicing factors and/or by sequestering RNA binding proteins (Ranum
and Day, 2004). Aberrant RNA processing can have widespread effects on many transcripts,
and can amplify existing transcriptional dysregulation. RNA missplicing has also been shown
to contribute to SBMA pathogenesis in a knock-in mouse, where changes in RNA binding
protein expression and mRNA splicing have been observed (Yu et al., 2009). Expressing
expanded, non-coding CAG repeat RNA in the fly eye leads to disorganized ommatidia and
interommatidial bristles, a rough-eye phenotype which can be modulated by expression of
RNA binding proteins (Mutsuddi et al., 2004). In fact, numerous RNA binding proteins have
been implicated as modifiers of polyglutamine toxicity and have been identified through
Adrienne M. Wang 156
screens using Drosophila (Fernandez-Funez et al., 2000; Kazemi-Esfarjani and Benzer, 2000;
Li et al., 2008). More recently, expanded CAG RNAs have been shown to alter ribosomal
RNA (rRNA) transcription, resulting in apoptosis (Tsoi et al., 2012).
Mitochondrial Dysfunction
Mitochondrial dysfunction has also been implicated in polyglutamine disease
pathogenesis. Glial expression of the mitochondrial anion transporter uncoupling protein 5
(UCP5) increases metabolism and rescues lifespan and motor deficits in a Drosophila model
of HD without altering neuronal loss (Besson et al., 2010). Perturbations in RNA
transcription discussed above result in cell death due to mitochondrial localization of p53, and
subsequent release of cytochrome C and caspases. In vitro and in vivo models of SBMA show
altered expression of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-
1), a transcription factor that governs mitochondrial biogenesis and function. Mitochondria in
cells expressing pathogenic polyQ AR are lower in number and exhibit decreased
mitochondrial membrane potential, occurring in conjunction with higher levels of reactive
oxygen species (ROS) (Ranganathan et al., 2009). Mitochondrial abnormalities are also
detectable in the leukocytes of SBMA patient samples, highlighting the role of mitochondrial
bioenergetics in polyglutamine disease (Su et al., 2010).
Axonal Trafficking
Neurons, and especially motor neurons, with their long axonal projections, are highly
reliant upon fast axonal transport to shuttle nutrients and molecular signals between the
nucleus and the cell periphery, and defects in axonal transport have been reported in several
neurodegenerative and polyglutamine diseases (Gunawardena et al., 2003; Piccioni et al.,
2002; Szebenyi et al., 2003). Transport deficiencies can be caused by both physical blockage
of the narrow axons and by altered protein interactions leading to sequestration of necessary
equipment away from normal function or altered expression. Specific disruption of this
pathway by dynactin mutations causes an inherited form of motor neuron disease (Puls et al.,
2003), and flies expressing polyglutamine tracts in neurons show glutamine length-dependent
organelle blockages (Gunawardena et al., 2003; Lee et al., 2004).
Controversy exists as to the extent of axonal trafficking defects in SBMA models. Axonal
trafficking defects caused by the polyQ AR occur in isolated squid axons, where impaired fast
transport occurs secondary to activation of JNK signalling (Morfini et al., 2009; Szebenyi et
al., 2003). Defects have also been documented in several, but not all, SBMA mouse models
that have been studied. Dynactin 1, a regulator of retrograde axonal transport, is expressed at
lower levels in SBMA transgenic mice that show perturbed axonal trafficking (Katsuno et al.,
2006a). Early deficits in retrograde labelling also occur in both SBMA knock-in mice and a
myogenic mouse model that overexpresses wild-type AR exclusively in muscle (Kemp et al.,
2011). In contrast, human polyQ-expanded AR yeast artificial chromosome (YAC) transgenic
SBMA mice show no change in retrograde transport or dynactin levels (Malik et al., 2011),
suggesting that these defects may not be necessary for the occurrence of a disease phenotype.
Lessons from Drosophila in Neurodegeneration 157
PROTEIN DEGRADATION AS A THERAPEUTIC TARGET IN SBMA
With such a diverse array of cellular processes affected by the presence of the mutant
AR, therapeutic treatments targeting individual pathways are likely to be unsuccessful (Figure
3). The common upstream factor in all the toxic mechanisms is the activation of the abnormal
AR. Therefore, diminishing levels of the misfolded protein could abrogate multiple
downstream effects. Perhaps the most promising route to achieving this is to enhance the
cell’s natural protein quality control pathways. This machinery contains several distinct and
interacting components, including molecular chaperones, the ubiquitin-proteasome system
and autophagy.
The two main protein degradation pathways in eukaryotic cells are the ubiquitin-
proteasome system (UPS) and autophagy. Both of these degradation pathways serve to
modulate protein homeostasis and have been identified as potential therapeutic targets in
neurodegenerative disease. The UPS degrades damaged or misfolded proteins in both the
cytoplasm and nucleus, while autophagy is responsible for bulk degradation of long-lived
proteins and organelles in the cytosol. In SBMA, it is unclear whether the toxic form of the
AR is a monomeric receptor that would be degraded by the UPS, or an oligomeric or
aggregated form which would require clearance through autophagy. However, the nuclear
localization of the testosterone-bound AR in SBMA keeps the toxic receptor sequestered from
the cytosolically located autophagic machinery, suggesting that modulating UPS activity may
provide a promising therapeutic target.
The Ubiquitin Proteasome System
The UPS is a selective and tightly regulated process of degrading soluble cytosolic and
nuclear proteins into short peptide chains for recycling. By selectively degrading short-lived
and misfolded or damaged proteins, the UPS is able to govern localized protein
concentrations, allowing for regulation of cell cycle and growth regulators, signal
transduction, metabolic enzymes and general housekeeping functions (Hershko and
Ciechanover, 1998). Degradation of a protein through the UPS requires 2 steps: the covalent
attachment of multiple ubiquitin molecules and the degradation of the ubiquitinated protein
by the proteasome.
Ubiquitination of the target protein is achieved in an ATP-dependent stepwise process.
Through a series of transient associations of the ubiquitin molecule with the ubiquitin E1-
activating, E2-conjugating, and E3-ligating enzymes, the ubiquitin molecule is conjugated to
a lysine on the targeted substrate (Hershko et al., 1983). This process repeats itself, allowing
for sequential addition of ubiquitins. Once a protein incurs a chain longer than four ubiquitins,
it is targeted to the 26S proteasome for degradation (Chau et al., 1989).
The 26S proteasome is a multi-subunit, multi-catalytic protease found in both the nucleus
and cytoplasm. It is comprised of a 20S core catalytic complex flanked by two 19S regulatory
components forming a barrel-shaped structure (20S) with a lid (19S). In a highly energy
dependent manner, the 19S subunit functions to identify and bind polyubiquitinated proteins,
feeding unfolded substrates through the 20S proteasome for degradation into oligopeptides
ranging from 4-25 amino acids which can then be reused by the cell.
Adrienne M. Wang 158
Components of the UPS, as well as the molecular chaperones HSP70 and HSP40, are
found in the nuclear aggregates of SBMA models and patients, indicating cellular recognition
of the misfolded protein and cellular attempts to degrade the aggregate-prone protein prior to
inclusion formation (Abel et al., 2001; Adachi et al., 2001; Li et al., 1998). This accumulation
of UPS components in aggregates has also been implicated in proteasome inhibition, where
sequestration of the components in inclusions would lead to downregulation of proteasomal
degradation (Bence et al., 2001). However, the activity of the UPS in an SBMA mouse model
is preserved and even up-regulated in late stages of disease (Tokui et al., 2008).
The importance of the proteasome as the primary degradation pathway of the AR is
shown in a Drosophila model of SBMA, where proteasome inhibition further increases the
toxicity of a truncated fragment of the expanded polyQ AR in the fly eye. This suggests that
steroid hormone receptor degradation occurs through the proteasome (Chan et al., 2002), and
implies that methods to stimulate this innate degradation pathway could be a promising
therapeutic strategy for the clearance of toxic polyQ AR. As discussed below, manipulating
the expression or function of certain molecular chaperones and chaperone-dependent
ubiquitin ligases ameliorates the disease phenotype in SBMA mice and flies, consistent with
the notion that clearance of the polyQ AR through the UPS is critically important to the
disease.
Autophagy
The cell’s alternative degradation pathway allows for bulk cytoplasmic degradation of
larger organelles and long-lived proteins. Autophagy, or “self eating”, is a process in which a
cell is able to engulf contents of cytoplasm and deliver them to the lysosome for degradation
and recycling (Klionsky and Emr, 2000). Activation of autophagy involves tightly regulated
stepwise induction of a double membraneous autophagic vesicle that grows to engulf
cytoplasmic components that the proteasome cannot degrade, such as mitochondria and
protein aggregates, as well as long-lived proteins. This mechanism consists of several discrete
steps executed by autophagy related (Atg) proteins that are analogous to the cascade required
to attach ubiquitin to target proteins. Once the autophagosome is completed, it fuses with
lysosomes where the contents of the autophagosome are enzymatically broken down for reuse
by the cell (Ravikumar et al., 2010).
Upstream of the formation of the autophagic vesicle, autophagy is regulated by two main
proteins, the mammalian target of rapamycin (mTOR), which negatively regulates autophagy
(Ravikumar et al., 2004), and Beclin-1, which is required for both autophagosome formation
and autophagic flux (Liang et al., 2006). mTOR is responsive to cellular signals conveying
the energy and nutrient status of the cell to cellular processes. In times of excess, mTOR
inhibits autophagy; inhibition of mTOR through nutrient deprivation or rapamycin treatment
leads to a release of this inhibition, allowing for autophagy induction and an increase in
nutrients via this recycling pathway.
Due to its ability to degrade organelles and cytosolic aggregates, the role of autophagy in
protein aggregation diseases offers a potentially promising therapeutic target. In fly and
mouse models of HD, mTOR inhibition has been shown to be protective against
neurodegeneration through increased autophagy and decreased protein translation (Ravikumar
et al., 2004). A fly model of another polyglutamine disease, SCA3, displays increased
Lessons from Drosophila in Neurodegeneration 159
autophagy and degeneration that is exacerbated when autophagy is inhibited (Bilen and
Bonini, 2007) and inhibiting autophagy in an SBMA fly model increased the severity of the
rough eye phenotype (Pandey et al., 2007). Paradoxically, inhibiting autophagy in a mouse
model of SBMA increases lifespan and decreases muscle wasting (Yu et al., 2011). This
discrepancy may be due to context and model specific differences between ectopic expression
of the mutant AR in the invertebrate eye and endogenous expression of the mutant AR in
mammalian neurons and muscle cells, or due to variations in the extent to which autophagy is
disrupted.
Crosstalk between protein quality control pathways has been suggested, and several lines
of evidence imply cross-regulation and compensation when some aspect of protein
homeostasis is perturbed. Temperature-sensitive dominant-negative proteasome mutant flies
exhibit increased autophagy, a compensatory response that requires the microtubule
associated deacetylase, HDAC-6 (Pandey et al., 2007). Compensatory regulation between
autophagy and another protein quality control pathway, the unfolded protein response (UPR),
has also been implicated. The UPR is an integrated signal transduction pathway that transmits
information about protein folding within the endoplasmic reticulum (ER) lumen to the
nucleus and cytosol to regulate protein synthesis and folding, influencing cell survival (Ron,
2002; Ron and Walter, 2007). The UPR is activated in skeletal muscle in SBMA patients and
mice expressing AR113Q, and deficiencies in this signalling pathway lead to increased
autophagy (Yu et al., 2011), although this link has yet to be shown in Drosophila models.
Molecular Chaperones
In the absence of ligand, the AR resides in the cytoplasm where it forms a multi-protein
heterocomplex, bound to heat shock proteins, co-chaperones, and tetratricopeptide repeat
(TPR)-containing proteins (Pratt and Toft, 1997). The HSPs are molecular chaperones that
bind to the receptor, and either stabilize the AR in a high-affinity state primed for ligand
binding (Fang et al., 1996), or target the AR for degradation by the proteasome. The
upregulation of specific proteins in response to heat shock was first described in Drosophila
salivary cells (Tissieres et al., 1974), and soon after was recognized as a response across
species from bacteria to human (Schlesinger, 1990).
HSP90
HSP90 is an abundant molecular chaperone that controls the maturation, function, and
turnover of its client proteins. The regulation of AR proteostasis by HSP90 makes it an
HSP90 “client” protein, and puts the AR in a class with many other steroid hormone receptors
and signalling molecules. Binding of HSP90 to its client protein is preceded by the binding of
HSP70 to hydrophobic residues found on partially unfolded proteins. Once HSP70 has
primed the steroid binding cleft, HSP90 binds and stabilizes the binding cleft in an open
conformation with high affinity for ligand (Pratt and Toft, 2003). Both HSP70 and HSP90
have intrinsic ATPase activity that is required in the stepwise assembly of the chaperone
machinery. The binding affinity of these molecular chaperones to their protein substrates is
mediated by their nucleotide binding state, with the ATP-bound form being the low-affinity
state and the ADP-bound form being the high-affinity state (Brehmer et al., 2001). Once the
ligand binds to the accessible ligand-binding cleft of the primed AR, interaction with HSP90
Adrienne M. Wang 160
is required to help traffic the client protein to the site of its action, the nucleus (Pratt et al.,
2004; Thomas et al., 2004; Thomas et al., 2006).
HSP90 inhibitors, which target the ATPase activity of HSP90, have been shown to have
beneficial effects in several models of SBMA, but are yet to be tested in Drosophila. 17-AAG
and geldanamycin, both HSP90 inhibitors, ameliorate disease pathology in cell culture and
mouse models of SBMA (Tokui et al., 2008; Waza et al., 2005). These drugs promote
degradation of the mutant AR protein through the ubiquitin-proteasome system by inhibiting
HSP90’s ATP-dependent progression towards the stabilized heterocomplex. They decrease
accumulation of aggregated mutant receptor by enhancing its degradation, and ameliorate the
motor phenotype of SBMA mice (Tokui et al., 2008; Waza et al., 2006). These drugs also
serve to induce a stress response, which upregulates the levels of several heat shock proteins,
including the inducible form of HSP70 (Sittler et al., 2001). However, the beneficial effects of
HSP90 inhibition is independent of the heat shock response, as mouse embryonic fibroblasts
(MEFs) deficient in HSF-1 (the HSP90-regulated transcription factor required to induce a
heat shock response) still clear AR113Q after treatment with HSP90 inhibitors, even in the
absence of a stress response (Thomas et al., 2006).
HSP70
While HSP90 binds to and stabilizes client proteins in their native conformation, HSP70
binds to hydrophobic residues exposed by partially unfolded or misfolded proteins in non-
native conformations, targeting them for degradation by the ubiquitin proteasome system. In
fact, HSP70 is required for both ubiquitination and the subsequent degradation of
ubiquitinated proteins (Bercovich et al., 1997). The best-studied chaperone-dependent
ubiquitin E3 ligase is the CHIP (carboxy terminus of HSP70-interacting protein). CHIP
interacts with HSP70 through its amino-terminal TPR domain and with E2 ubiquitin
conjugating enzymes through a carboxy-terminal U-box domain (Ballinger et al., 1999). In
this manner, it is thought that CHIP is able to initiate the ubiquitination and promote the
degradation of proteins that have been identified as unfolded or misfolded by HSP70. CHIP is
also associated with nuclear inclusions characteristic of polyglutamine disease, including
SBMA (Morishima et al., 2008), and overexpression of CHIP has been shown to rescue both
a Drosophila model of SCA1 (Al-Ramahi et al., 2006) and a mouse model of SBMA (Adachi
et al., 2007). Notably, although CHIP plays an important role in client protein degradation,
other chaperone-dependent ligases, such as Parkin, can function redundantly with CHIP to
promote client protein degradation (Morishima et al., 2008).
Like HSP90 inhibition, HSP70 over-expression has been shown to have beneficial effects
in a variety of neurodegenerative diseases, including the polyglutamine diseases. HSP70, in
conjunction with its co-chaperone HSP40, inhibits aggregation of the truncated huntingtin
protein in both in vitro and cellular and yeast models of polyglutamine aggregation
(Muchowski et al., 2000). HSP70 alters solubility of the abnormal protein, and suppresses
polyglutamine-induced neurodegeneration when overexpressed in SCA3 and SBMA flies
(Chan et al., 2002; Chan et al., 2000; Warrick et al., 1999). Further, when HSP70 is
overexpressed in a mouse model of SBMA, there is decreased aggregated and soluble AR,
indicating that increased levels of HSP70 promote degradation of the AR. Importantly, these
mice also showed improved survival and motor phenotype when compared to SBMA mice
that were not overexpressing HSP70 (Adachi et al., 2003).
Lessons from Drosophila in Neurodegeneration 161
HSP70 interacts with several co-factors or co-chaperones in the course of recognizing,
binding and targeting misfolded proteins to the proteasome. These co-factors serve to
modulate HSP70 activity and substrate binding affinity. In particular, HSP40, a well-
characterized family of HSP70 co-chaperones, affect substrate binding by enhancing HSP70’s
ATPase activity (Freeman et al., 1995). In the opposite direction, Hip (Hsc70-interacting
protein) is a co-chaperone that interacts with the ATPase domain of HSP70/Hsc70 and
stabilizes it in its ADP bound state (Hohfeld et al., 1995), and thus increasing the affinity of
the chaperone for client proteins. Experimental modulation of HSP70 activity has proven
difficult, and small molecules specifically targeting HSP70 have been identified only recently
(Jinwal et al., 2009; Leu et al., 2009; Wang et al., 2010). Due to this, much of the evidence
probing HSP70 as a modulator of neurodegeneration has come from overexpression studies.
More recently, modulation of HSP70 has been shown to be a promising therapeutic target,
where inhibiting HSP70’s ATPase activity genetically, through overexpression of Hip, or
pharmacologically, through small molecule inhibition, enhances client protein ubiquitination,
stimulates clearance of the polyQ AR, and rescues toxicity in a Drosophila model of SBMA
(Wang et al., 2012).
CONCLUSION
Drosophila as a model organism has been invaluable in increasing our understanding of
polyglutamine disease pathogenesis and especially SBMA. These small organisms have given
us a huge insight into the molecular mechanisms of disease pathogenesis and modifiers of
these mechanisms. Their simplified genetic similarity, ease of screening, and powerful
numbers have allowed a much greater understanding of disease mechanisms than would be
possible or ethically viable in mammalian models or humans. However, despite huge
advances in our knowledge of disease aetiology, efficacious therapies remain elusive.
Evidence from numerous models indicates that harnessing cells' protein quality control
pathways to degrade toxic proteins is an attractive therapeutic target. Disease progression
affects numerous cellular pathways, and increasing degradation of the disease-causing protein
may serve to halt disease progression upstream of several pathological features of protein
aggregation diseases. Fly models have proven to be useful tools to probe the effects of
potentially therapeutic compounds, and are ideal for larger scale screens of compounds, easily
providing information about toxicity and beneficial effects. Their usefulness in identifying
potentially therapeutic compounds is illustrated by work done looking at HDAC inhibitors
and small molecule modulators of HSP70 (Steffan et al., 2001; Wang et al., 2012). In both of
these papers, authors were able to identify therapeutic targets using Drosophila models to
verify these targets both genetically and pharmacologically. The potential of the fly to inform
new therapies in neurodegeneration has not been fully explored, but I am convinced they will
prove even more fruitful in the future.
Adrienne M. Wang 162
ACKNOWLEDGMENTS
This chapter has benefited from the comments and edits from several people, including
Dr. Catherine Collins, Dr. Susan Klinedinst and especially Ian Whiteford who gave assistance
in both editing and figure design. I am grateful for your time and help. I am also very grateful
to Joshua Tuininga for his excellent design skills, and to Alissa Tuininga for facilitating this
work. Finally, I would like to thank Dr. Andrew Lieberman for his years of mentorship and
for allowing my investigation to lead me to fly models.
ABOUT THE AUTHOR
Adrienne M. Wang is currently a postdoctoral fellow in Dr. Matt Kaeberlein’s lab at the
University of Washington in Seattle. She received her PhD in Neuroscience from the
University of Michigan, where she worked with Dr. Andrew Lieberman studying protein
quality control in cellular, mouse, and fly models of SBMA. Her current research utilizes
Drosophila to understand the role of mitochondrial quality control in aging and age-related
diseases.
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 7
SPINAL MUSCULAR ATROPHY:
INSIGHTS FROM THE FRUIT FLY
Stuart J. Grice,1 Kavita Praveen,
2,3
A. Gregory Matera2,3,4
and Ji-Long Liu1,
1MRC Functional Genomics Unit, Department of Physiology,
Anatomy and Genetics, University of Oxford, Oxford, UK 2Program in Molecular Biology and Biotechnology,
University of North Carolina, Chapel Hill, NC, US 3Department of Biology, University of North Carolina,
Chapel Hill, NC, US 4Department of Genetics, University of North
Carolina, Chapel Hill, NC, US
ABSTRACT
Spinal muscular atrophy (SMA) is the most common genetic cause of childhood
mortality. SMA is caused by deletion or mutations in the survival of motor neuron 1
(SMN1) gene, resulting in inadequate levels of the SMN protein. Conserved from yeast to
human, the SMN protein is best known for its critical role in small nuclear
ribonucleoprotein biogenesis and RNA splicing. However, one of the puzzles in the SMA
field is how the reduction of SMN, a housekeeping gene, causes SMA, a motor neuron
specific disease. The fruit fly Drosophila melanogaster has proven to be a powerful
model for human biology and disease. Here we discuss recent progress in SMA disease
modelling in the fruit fly, which has provided unprecedented insights into the
pathological mechanism of SMA.
Keywords: Drosophila; Survival motor neuron (SMN); Uridine-rich small nuclear
ribonucleoprotein (U-snRNP)
Corresponding authors: A. Gregory Matera (E-mail: [email protected]. (AGM)) and Ji-Long Liu (E-mail: jilong.liu
@dpag.ox.ac.uk. (JLL)).
Stuart J. Grice, Kavita Praveen, A. Gregory Matera et al. 172
INTRODUCTION
Spinal muscular atrophy (SMA) is the most prevalent genetic cause of infant mortality.
SMA is characterized by the degeneration of motor neurons in the anterior horn of the lower
spinal cord, leading to symmetrical paralysis, atrophy of the proximal muscles, and loss of
motor function. In most cases, SMA is caused by an insufficient amount of the survival of
motor neuron (SMN), a protein best known for its function in the assembly of Uridine-rich
small ribonucleoproteins (U-snRNPs).
SMA AND SMN IN HUMANS
With a carrier frequency of 1 in 50 and an incidence rate of 1 in 6000-10,000, SMA is the
most common autosomal recessive disorder in the population after Cystic Fibrosis (Ogino and
Wilson, 2002). SMA can be classified into three types based on the severity of the phenotype
(Ogino and Wilson, 2002). Type I SMA, also known as Werdnig-Hoffman disease (Nicole et
al., 2002; Ogino S, 2004), is the most common (60%) and the most severe form where
symptoms can be apparent as early as in utero. Affected infants experience progressive
muscle weakness and hypotonia (reduced muscle tone), and die from complications such as
respiratory failure by 2 years of age. Type II SMA patients become symptomatic between 6
and 18 months of age, show developmental motor delays, and although able to sit
unsupported, they are unable to stand or walk. Their life expectancy is between 2-30 years.
Type III SMA is the mildest form with an age of onset after 2 years. Most type III patients are
able to stand and walk, but can become wheelchair bound in adulthood, and have a normal
life expectancy. In 1995, Lefebvre et al. identified a novel gene on the long arm of
chromosome 5, survival of motor neuron 1 (SMN1), as the causative gene in SMA (Bussaglia
et al., 1995; Rodrigues et al., 1995; van der Steege et al., 1995; Chang et al., 1997).
Over 95% of SMA patients have homozygous deletions involving exon 7 of SMN1
(Lefebvre et al., 1995; Campbell et al., 1997). Further characterization of the 5q region
revealed the existence of at least two copies of the SMN gene in most people. The telomere-
proximal copy (SMN1) and the centromere-proximal copy (SMN2) arose as a result of a 500-
kb inverted duplication.
SMN1 generates fully functional full-length SMN protein, whereas SMN2 predominantly
produces a truncated and unstable protein. This is due to a translationally silent C-to-T
transition, which causes regular skipping of exon 7. Mutations in, or loss of, SMN1 lead to
SMA, while loss of SMN2 alone has no adverse effect. SMN2 resides in a labile region of the
chromosome and is often found in duplicate. Since SMN2 produces low levels of SMN,
SMN2 copy number and disease severity are inversely correlated. SMA is a disease caused by
low SMN levels and not loss, which leads to embryonic lethality.
THE DROSOPHILA SMN COMPLEX AND RNP BIOGENESIS
Studies over the last decade have shown that the majority of SMN in mammalian cells
exists as part of a large multimeric complex consisting of eight additional proteins: Gemins 2-
Spinal Muscular Atrophy: Insights from the Fruit Fly 173
8 and Unrip (a cytoplasm-specific complex member) (Baccon et al., 2002; Carissimi et al.,
2006; Charroux et al., 1999; Charroux et al., 2000; Grimmler et al., 2005; Gubitz et al., 2002;
Pellizzoni et al., 2002a). Orthologues for Gemin2 and Gemin3 have been identified in the
Drosophila genome, and shown to function in U-snRNP biogenesis, similar to their
mammalian counterparts (Cauchi, 2011; Cauchi, 2012; Cauchi et al., 2008; Cauchi et al.,
2010; Cauchi et al., 2011; Kroiss et al., 2008; Shpargel et al., 2009).
Although a putative Drosophila orthologue of Gemin5, Rigor Mortis, can be identified
bioinformatically and loss-of-function mutants phenocopy Gemin3 mutations (Gates et al.,
2004; Shpargel et al., 2009), a physical interaction between Gemin5 and SMN has not been
detected in flies (Kroiss et al., 2008).
The best-characterized function of the SMN complex is in the biogenesis of the core
components of the spliceosome, the Uridine-rich small nuclear ribonucleoproteins (U-
snRNPs). The life cycle of the Sm-class U-snRNAs, U1, U2, U4, U11, U12 and U5, involves
both nuclear and cytoplasmic maturation steps. Sm-class U-snRNAs are transcribed by RNA
polymerase II and acquire a 7-methylguanosine (m7G) cap (Cougot et al., 2004).
Following transcription and 3' end processing, the pre-snRNA is bound by the cap-
binding complex (CBC) (Izaurralde et al., 1995). The pre-snRNA then transits through
nuclear structures called Cajal Bodies (CBs), where it is recognized by the phosphorylated
adaptor for RNA export (PHAX) (Frey et al., 1999; Frey and Matera, 1995; Frey and Matera,
2001; Suzuki et al., 2010). PHAX recruits CRM1/RanGTP to the pre-snRNA, and this
complex is exported to the cytoplasm. The pre-snRNA is released upon phosphorylation of
PHAX in the cytoplasm (Ohno et al., 2000).
The SMN complex then facilitates the binding of seven proteins, SmB/B’, SmD1, SmD2,
SmD3, SmE, SmF and SmG (the Sm proteins), onto a conserved motif called the ‘Sm site’ on
the pre-snRNA (Golembe et al., 2005; Meister et al., 2001; Yong et al., 2004; Yong et al.,
2002) in an adenosine triphosphate (ATP) dependent reaction. The SMN complex provides
specificity and speed to this reaction, which can also occur spontaneously and non-
specifically in vitro (Pellizzoni et al., 2002b).
After assembly with Sm proteins, the pre-snRNA is trimmed at the 3’ end (Kleinschmidt
and Pederson, 1987; Seipelt et al., 1999; Will and Luhrmann, 2001) and the m7G cap is
hypermethylated to a trimethylguanosine (m3G) cap by the enzyme TGS1 (Huang and
Pederson, 1999; Mattaj, 1986; Mouaikel et al., 2002). The import adaptor, Snurportin (SPN),
and Importin (Imp) take the partially assembled pre-snRNA, and the SMN complex, into
the nucleus (Huber et al., 1998; Narayanan et al., 2004; Palacios et al., 1997).
Interaction between SMN and Imp, and observations that U-snRNP import is defective
in the presence of some SMN mutations, indicate that SMN may also function in facilitating
snRNA import (Narayanan et al., 2004; Narayanan et al., 2002).
In the nucleus, the snRNA localizes to CBs, where it is released from the SMN complex,
modified, and bound by other U-snRNP-specific proteins. The mature U-snRNP then
localizes to either regions of active transcription and splicing (perichromatin fibrils) (Fakan,
1994) or to nuclear domains called speckles, where U-snRNPs are thought to be ‘stored’
while not participating in splicing (Sleeman and Lamond, 1999).
Stuart J. Grice, Kavita Praveen, A. Gregory Matera et al. 174
DROSOPHILA SMN MUTANTS AS A MODEL FOR SMA
Integral aspects of cell and developmental biology in humans have been conserved in
Drosophila. Approximately 75% of disease-causing loci in humans have homologues in the
fly (Reiter et al., 2001). This conservation allows us to model and study human disorders in
an organism that is likely to respond with similar pathology to disease-causing mutations.
Furthermore, the availability of a sequenced genome, a multitude of genetic tools and a short
generation time make the fruit fly a particularly attractive model organism. Neuromuscular
development in adult flies resembles that of vertebrates (Fernandes and Keshishian, 1999),
thus making Drosophila suited for study of disorders such as SMA.
The most well-conserved regions between human and fly SMN are the Gemin2 binding
site near the N-terminus, the Tudor domain and the YG box (Miguel-Aliaga et al., 2000). The
Drosophila SMN complex participates in the assembly of Sm proteins onto snRNAs,
indicating that the function of human SMN in U-snRNP biogenesis is conserved in the fly
(Rajendra et al., 2007).
In 2003, Chan et al. identified two point mutations in the YG (self-oligomerization) box
of Drosophila SMN (dSMN) through a small-scale ethyl methane sulphonate (EMS)
mutagenesis screen (SmnA and Smn
B). Smn
A animals survived only until the late larval stages
and showed increasing loss of mobility and coordination. Using genetic mosaic techniques to
create homozygous SMN mutants specifically in the germ line of female flies, the authors
showed that survival of zygotic SMN mutants beyond embryogenesis was due to a large
maternal contribution of dSMN (Chan et al., 2003). The SmnA and Smn
B point mutations are
thought to destabilize the protein as flies carrying these mutations have very low levels of
dSMN (Shpargel et al., 2009).
Several additional Smn mutants are available that have been generated via transposon
mediated mutagenesis (Rajendra et al., 2007; Shpargel et al., 2009). These vary in the severity
of their phenotype based on the location of the insertion (Table 1). Specifically, the SmnD and
SmnC mutations are null mutations, as defined by the inability to detect any zygotic dSMN
protein in these animals (Shpargel et al., 2009). Chang et al. (2008) created a new Smn null
mutation by generating a microdeletion derived from a parental line carrying a transposon
insertion upstream of the Smn gene, SmnE. The Smn
X7 deletion removes the promoter, open
reading frame and part of the 3` UTR of Smn (Chang et al., 2008) and homozygous mutants
are lethal at larval stages. A detailed characterization of SmnX7
/SmnD trans-heterozygous
larvae shows that the lethal phase is broad, with majority of larvae dying between 4-5 days
post egg laying. A small fraction of the remaining larvae are able to survive for 2-3 weeks
without undergoing metamorphosis (Praveen et al., 2012). Smn null larvae also display
locomotion defects that have been characterized using a number of different assays (Chan et
al., 2003; Shpargel et al., 2009; Praveen et al., 2012; Imlach et al., 2012).
An SMN hypomorphic mutant was isolated by the imprecise excision of the SmnE
insertion in a screen for adult flies with neuromuscular phenotypes (Rajendra et al., 2007).
These mutants, referred to as SmnE33
(excision 33), are viable and fertile, but unable to fly or
jump. Severe atrophy of the flight muscles was observed in these animals, indicated by a
complete disorganization of the flight muscles.
Table 1. Drosophila SMN mutants
Stuart J. Grice, Kavita Praveen, A. Gregory Matera et al. 176
This was accompanied by routing and branching defects in the dorsal longitudinal motor
neurons of the flight muscles (Rajendra et al., 2007). Interestingly, SmnE33
flies show a
decrease in the levels of dSMN only in thorax, and can be considered a regio-specific
hypomorph.
Several RNAi fly strains targeting Smn have also been developed. Chang et al. (2008),
describe three transgenic lines carrying UAS-based RNAi constructs targeting the N-terminus
(SmnN4
), the C-terminus, (SmnC24
), and the full-length (SmnFL26B
) dSMN. The most severe of
these, SmnN4
, is lethal at early pupal stages when expressed using the actinGAL4 driver
(Chang et al., 2008). The SmnC24
and SmnFL26B
transgenic flies have milder phenotypes with a
proportion of SmnFL26B
animals reaching adulthood. Two additional RNAi fly lines,
SmnGL00581
and SmnJF02057
, targeting Smn have been generated by the Transgenic RNAi
Project (TRiP). The TRiP lines express a short hairpin (SmnGL00581
) and long hairpin
(SmnJF02057
) RNAi constructs targeting the 3' UTR and open reading frame of Smn,
respectively. Preliminary characterization shows that flies expressing SmnJF02057
construct
using a ubiquitous driver die as pupae (our unpublished observations).
Praveen et al. (2012), recently described a new Drosophila model of SMA that mimics a
point mutation identified in SMA patients. The authors created the equivalent of the human
SMA mutation, T274I, in the fly Smn gene (SmnT205I
), and expressed this transgene from its
native Smn promoter in an Smn null background. The majority of SmnT205I
animals die as
pupae, which is consistent with this mutation being associated with milder forms of SMA in
humans. The variety of Drosophila SMA models that exist and that can be made with relative
ease make this model organism very valuable to the study of SMA at an organismal level.
SMA in humans is the result of a decrease in SMN levels below a tolerable threshold.
However, all SMA patients have enough SMN protein produced from the SMN2 locus to
rescue embryonic lethality. Similarly, the considerable amount of maternally contributed
dSMN in flies rescues the embryonic lethality, extending their lives to larval stages and
allowing us to investigate phenotypes relevant to human SMA. In addition, with the
versatility of Drosophila genetic tools it is possible to transgenically express a low level of
dSMN in SMN mutant animals to mimic the human SMA situation.
USING DROSOPHILA TO UNDERSTAND
THE ROLE OF SMN IN DEVELOPMENT
A number of developmental defects have been described in patients with severe SMA
including congenital heart defects, multiple contractures, bone fractures, respiratory
insufficiency and sensory neuropathy (Garcia-Cabezas et al., 2004; Kelly et al., 1999; Menke
et al., 2008; Rudnik-Schoneborn et al., 2008; Vaidla et al., 2007).
As one of the most genetically tractable organisms, Drosophila has contributed a great
deal to the molecular and cellular understanding of development. Drosophila undergoes four
distinct developmental stages over the course of two weeks. The first, embryogenesis,
involves the organogenesis and neurogenesis required for larval life. SMN is maternally-
contributed and is highly expressed in the early embryo (Miguel-Aliaga et al., 2000). Female
fruit flies have a pair of ovaries. In Drosophila melanogaster, each ovary comprises 16-20
finger-like ovarioles, which are strings of oval-shaped egg chambers.
Spinal Muscular Atrophy: Insights from the Fruit Fly 177
There are three major cell types in each egg chamber: 15 nurse cells, 1 oocyte and about
1000 follicle cells. The maternal SMN contribution is supplied by nurse cells in the egg
chamber, which generate large amounts of proteins and RNAs. SMN can be found in CBs in
the nucleus of nurse cells and oocytes (Liu et al., 2006a; Liu et al., 2006b; Liu et al., 2009).
However, SMN predominantly localizes to the cytoplasm and is highly enriched in the U-
snRNP body (U-body) along with other members of the SMN complex (Liu and Gall, 2007;
Cauchi et al., 2010). U-bodies are subcellular structures that contain components of the
spliceosome such as Uridine-rich snRNAs and associated proteins. U bodies invariably
contact cytoplasmic processing bodies (P-bodies), which contain proteins involved in mRNA
processing, RNA silencing and transport (Liu and Gall, 2007).
Knocking out SMN specifically in the germline alters the organization of the P-body,
while, reciprocally, the mutation of P-body components changes the aggregation of SMN in
U-bodies (Buckingham and Liu, 2011; Lee et al., 2009). Furthermore, the nurse cell
chromosomes fail to disperse in Smn and gemin3 mutants – a phenotype also observed in the
mutants of P-body components (Buckingham and Liu, 2011; Cauchi, 2012; Lee et al., 2009).
Chromosome dispersal occurs prior to the generation of maternal proteins and RNAs that will
be loaded into the oocyte ready for embryogenesis (St Johnston et al., 1991). Embryos
derived from egg chambers with SMN selectively removed die very early (Lee et al., 2009),
showing that, as seen with other model organisms, SMN protein is essential at the very early
stages of development (Monani et al., 2000).
Wild-type embryos hatch into larvae, which go through three progressive moults.
Drosophila larvae feed continuously, driving the growth of imaginal tissues as well as a
second wave of neurogenesis required for adult life. In holometabolous insects, imaginal discs
are present in the larvae and are the tissue precursors that form the majority of the adult body
structures. There are 10 sets of imaginal discs, each set producing a body structure (e.g. eyes,
legs and wings). The voracious feeding behaviour of larvae coincides with, and is supported
by, a 10-fold increase of the larval neuromuscular system. The majority of Smn mutants are
larval lethal, display poor growth and have reduced motor function. Morphological defects at
the larval neuromuscular junctions (NMJs) have been reported (Chan et al., 2003; Chang et
al., 2008), however, this phenotype is controversial. Chan et al. (2003) observed enlarged
boutons (the synapse between neuron and muscle) with no change in bouton number in Smn
mutants, while Chang et al. (2008) reported a decrease in the number of boutons with no
change in size. Most recently, Imlach et al. (2012) reported that they did not find any
morphological defects in the NMJs of Smn mutant larvae. They did show an increase in the
amplitude of evoked excitatory postsynaptic potential (eEPSP) at the Smn mutant NMJs
compared to wild-type NMJs. However, the relevance of this phenotype to motor function or
viability of these mutants is unclear.
Although ubiquitously expressed in the larval central nervous system (CNS), SMN
protein is enriched in the postembryonic neuroblasts (pNbs). pNbs generate the neurons and
glia required for the adult during 2nd
and 3rd
larval stages as well as early pupation. With stem
cells having the highest levels of SMN, the protein then forms a concentration gradient in the
CNS that is inversely proportional to the state of differentiation. In Smn mutants, the CNS
displays growth and maturation defects (Grice and Liu, 2011).
SMN deficiency reduces stem cell division and can lead to stem cell loss (Grice and Liu,
2011). The daughter cells of these dividing stem cells have reduced levels of the U-snRNPs
U2 and U5 that are required for splicing.
Stuart J. Grice, Kavita Praveen, A. Gregory Matera et al. 178
In addition, the tight localization of Miranda protein during metaphase at the basal
membrane of the neuroblast is lost (Grice and Liu, 2011). Miranda forms complexes with
RNA-binding proteins, including Staufen, which binds to mRNAs that will become
partitioned into the daughter cells (Broadus et al., 1998; Li et al., 1997). The Miranda-Staufen
complex has been implicated in facilitating the transport of RNP complexes in the neuroblast,
axon and germline (Roegiers and Jan, 2000).
In addition to the nervous system, defects in germline stem cell development have been
observed in Smn mutants. Like in the CNS, germline stem cells are enriched with SMN, and
these levels again form a differentiation/concentration gradient. SMN loss leads to the
retardation of germline stem cell division and promotes premature stem cell loss.
Overexpression of SMN alters the timing of germ cell development in the male germline
(Grice and Liu, 2011).
Furthermore, ectopic expression of SMN in the testis, in the region across the wild-type
SMN gradient, changes the timing of cell differentiation and leads to a build-up of immature
cyst cells (Grice and Liu, 2011). Together, these results suggest that SMN levels need to be
fine-tuned during development, and that alterations in SMN levels can lead to abnormal
development and modify the capability of stem cells to generate new cells during
development and throughout adult life.
Once a critical size is reached, ecdysone expression stimulates pupa formation and the
start of metamorphosis. During pupariation, the adult body plan is generated and the mature,
adult nervous system is formed from immature neurons generated during the larval wave of
neurogenesis.
Both SMN reduction and overexpression have an effect on pupation and metamorphosis.
Ectopic overexpression of human SMN, which is dominant negative in Drosophila, leads to
pupal lethality with malformation of the head, legs and wings (Miguel-Aliaga et al., 2000).
Overexpression of Drosophila wild-type SMN shortens the time to pupation, but at 25oC,
does not have an overall negative effect on survival.
Further growth and pupation defects have been observed in the less severe Smn mutants
(including SmnB), which have an extended larval period but fail to reach the wild-type size,
and form pseudopupae (Shpargel et al., 2009).
THE QUESTION OF CELL-AUTONOMY IN SMA
Whether SMA results from disruption of a cell-autonomous or non-autonomous function
of SMN remains a highly contested area of research.
To this end, researchers have attempted to identify tissues most important in the
pathology of SMA. The approach adopted is to analyse the level of rescue of SMA-like
phenotypes in mice and Drosophila by selective restoration of SMN levels in specific tissues
or cell types.
Studies in the mouse have used the Cre-LoxP system to selectively ablate or restore SMN
through the use of muscle and neuron specific promoters driving Cre recombinase expression
(Cifuentes-Diaz et al., 2001; Vitte et al., 2004; Gavrilina et al., 2008; Park et al., 2010;
Martinez et al., 2012).
Spinal Muscular Atrophy: Insights from the Fruit Fly 179
Figure 1. Overview of Drosophila research into SMN function and SMA.
Initial reports of neuronal (vs. muscular) restoration of SMN having greater effect in
rescuing SMA-like phenotypes in mice are difficult to interpret due to the leaky expression of
Cre in muscle cells (Gavrilina et al., 2008).
In Drosophila, tissue-specific expression of SMN was achieved using the Gal4-UAS
system, where expression of Gal4 in neuronal or muscular tissues can restrict expression of
SMN to those tissues. There are hundreds of Drosophila Gal4 driver strains available making
it easier to find those that express in a highly tissue-specific manner. Using this system, two
groups showed that complete rescue of the lethality resulting from loss of SMN, required
expression of SMN in both neurons and the mesoderm. In addition, expression in the
mesoderm alone resulted in a higher level of rescue than neurons alone (Chan et al., 2003;
Chang et al., 2008). The question of a cell-autonomous role for SMN has recently been
addressed at greater resolution using the Drosophila system. Taking advantage of the large
number of well-characterized Gal4 drivers available, Imlach et al. (2012) restored SMN
expression in Smn null flies in various tissues, including several different neuronal subtypes.
They analysed the impact of SMN restoration on electrophysiological phenotypes at the
neuromuscular junction as well as on muscle size and larval locomotion.
Stuart J. Grice, Kavita Praveen, A. Gregory Matera et al. 180
Surprisingly, they found that restoration of SMN in motor neurons alone did not rescue
any of these phenotypes. However, expressing SMN in cholinergic neurons (including
sensory and interneurons, but not motor neurons) did rescue larval phenotypes. This finding
suggests that SMN can function in a non-autonomous manner to influence other cell types. It
is important to note that rescue in neuronal or muscles cells alone did not rescue the viability
of the null mutants, consistent with previous data from studies in Drosophila. In contrast to
mice, Drosophila have proved especially useful for such analyses due to their relatively short
generation time and affordability, allowing an extensive analysis of several different patterns
of SMN expression.
CONCLUSION AND FUTURE
Drosophila models offer many advantages for understanding the biology of SMA. Many
parallels between the Drosophila SMA models and vertebrate systems have been identified
including defects in neurotransmission and synaptic architecture, as well as the conservation
of SMN binding partners and basic function.
In addition, Drosophila has provided novel insights into SMA including SMN’s function
in muscle structure, stem cell biology and neuronal circuit formation. The Drosophila genetic
tool kit is becoming ever more advanced, giving scope for the continued analysis of the
processes that drive neuronal developmental and homeostasis. In turn, this versatility allows
for an increasingly refined interrogation of the fundamental cellular defects that cause motor
neuron dysfunction, and can help elucidate the many functions of SMN in the cell.
ABOUT THE AUTHORS
Stuart J. Grice is a postdoctoral researcher at the MRC Functional Genomics Unit,
University of Oxford. He works with Dr. Ji-Long Liu on Drosophila models of SMA,
Charcot-Marie-Tooth-neuropathy and autism. He is particularly interested in how defects in
neurogenesis can lead to neurodevelopmental disease.
Kavita Praveen is a postdoctoral fellow in the Program for Molecular Biology and
Biotechnology at the University of North Carolina, Chapel Hill, US. She works with Dr.
Gregory Matera on developing and characterizing novel models of SMA in Drosophila. Her
particular interest is in understanding the aetiology of SMA at the molecular level.
Gregory Matera is a Professor in the departments of Biology and Genetics at the
University of North Carolina, Chapel Hill, US. He is interested in studying the biogenesis and
functions of non-coding RNAs, including small nuclear RNAs, using the Drosophila
melanogaster model system.
Ji-Long Liu is a programme leader at the MRC Functional Genomics Unit, University of
Oxford. He is interested in intracellular compartmentation in Drosophila. Currently his group
studies three aspects related to RNA: cytoophidia, long noncoding RNAs, and SMA
modelling.
These authors contributed equally to this work: Stuart J. Grice, Kavita Praveen.
Spinal Muscular Atrophy: Insights from the Fruit Fly 181
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In: Drosophila Melanogaster Models of Motor Neuron Disease ISBN: 978-1-62618-747-4
Editor: Ruben J. Cauchi © 2013 Nova Science Publishers, Inc.
Chapter 8
GENETIC SCREENS IN DROSOPHILA AND THEIR
APPLICATION IN MOTOR NEURON DISEASE MODELS
Liya E. Jose,1,2
Patrik Verstreken1,*2
and Sven Vilain1,2,†
1VIB Center for the Biology of Disease, Leuven, Belgium 2KU Leuven, Center for Human Genetics and Leuven Research Institute for
Neurodegenerative Diseases (LIND), Leuven, Belgium
ABSTRACT
The dearth of powerful therapeutic treatments for patients diagnosed with motor
neuron diseases (MNDs) emphasizes the significance of research that focuses on
understanding the causes of these diseases. Identification of the genes responsible for
these diseases enables the study of their function in model organisms. Drosophila
melanogaster is renowned for its powerful genetic tools for studying neurological
diseases.
Most human disease genes have at least one functional orthologue in flies and
transgenic expression of human disease genes can recapitulate features of the disease.
The power of Drosophila for unbiased genetic screens to identify novel genes that cause
neurodegeneration or, to identify modifiers of disease genes, holds promise to uncover
the molecular mechanisms and the (parallel) pathways involved in disease progression
and neurodegeneration.
Throughout Drosophila’s rich genetic screen history, different screen approaches
have been used and different phenotypes have been screened for. The aim of this chapter
is to introduce the various screening approaches and phenotypes that have been used in
the past for high-throughput screening of neuronal defects in MND and we will present
additional available genetic tools that have the potential for uncovering further molecular
mechanisms of MNDs.
Keywords: Enhancer, suppressor, RNAi, mutagenesis, phenotypes
* Patrik Verstreken: [email protected], Herestraat 49 - bus 602, 3000 Leuven, Belgium, Tel:
+32 (0) 16 330018, Fax: +32 (0) 16 330589. † Sven Vilain: [email protected], Herestraat 49 - bus 602, 3000 Leuven, Belgium, Tel: +32 (0) 16
330018, Fax: +32 (0) 16 330589.
Liya E. Jose, Patrik Verstreken and Sven Vilain 186
INTRODUCTION
Drosophila is recognized as a powerful model for analysing the function of human
disease genes (Bonini, 2001; Fernandez-Funez et al., 2000; Ghosh and Feany, 2004; Kazemi-
Esfarjani and Benzer, 2000; Marsh and Thompson, 2004; Outeiro et al., 2007). About 75% of
all known human disease genes have a very well conserved fly orthologue (Bier, 2005; Reiter
et al., 2001).
Furthermore, even when the human gene orthologues are not present in the fly genome,
expression of the human gene or its mutant form can recapitulate characteristics of human
disease, suggesting that the underlying pathways are conserved (Feany and Bender, 2000).
These observations emphasize the ability to understand diverse and complex biological
problems associated with neurological disorders through the use of fruit flies including
pathways involved in motor neuron disease (MND) (Auluck et al., 2002; Canizares et al.,
2000; Chang et al., 2008; Chen et al., 2011; Chihara et al., 2007; Djagaeva et al., 2012; Elia et
al., 1999; Estes et al., 2011; Feany and Bender, 2000; Furutani et al., 2005; Hanson et al.,
2010; Joyce et al., 2011; Leitao-Goncalves et al., 2012; Li et al., 2011; Mockett et al., 2003;
Nedelsky et al., 2010; Praveen et al., 2012; Puccio, 2009; Rajendra et al., 2007; Rubin et al.,
2000; Sang and Jackson, 2005; Sasayama et al., 2012; Storkebaum et al., 2009; Trotta et al.,
2004; Watson et al., 2008).
MNDs are neurological disorders characterized by progressive loss of motor neurons that
result in extensive disability and early death (Lambrechts et al., 2007). Despite numerous
pathological studies in humans, mechanistic insights into the pathology and molecular
mechanisms associated with disease onset are still largely lacking (Carrasquillo et al., 2009;
Lesage and Brice, 2009; Mougeot et al., 2009). Loss-of-function studies in combination with
expression of (human) disease proteins in Drosophila and well-designed genetic screens, can
yield further insight into MND at unprecedented resolution (Chang et al., 2008; Kazemi-
Esfarjani and Benzer, 2000; Suzuki et al., 2009). Fruit flies are thus routinely used to screen
for genes that modify MND-associated phenotypes, helping to understand the mechanisms
underlying these diseases.
Genetic screening holds promise for unravelling the molecular mechanisms of disease
pathology, and for identifying novel therapeutic targets. The short generation time (<2 weeks)
and the ability to maintain large collections of mutant Drosophila in the lab has enabled
researchers to screen for mutants that affect development, neuronal function and behaviour
(Benzer, 1967; Pak, 1995; St Johnston, 2002) and more recently also for modification of
phenotypes induced by disease related genes, including MND. Drosophila can be used to find
enhancers and suppressors of Drosophila models of human MND and to uncover the
pathways and molecular mechanisms involved in the pathology of these disease models
(Chang et al., 2008; Sen et al., 2011; Suzuki et al., 2009). An increasing number of
sophisticated genetic screening strategies are being developed that allow to conduct more
focused genetic screens, including analyses in mosaic animals (Xu and Rubin, 1993), or in
animals that use temporal and spatially controlled gene expression (Brand and Perrimon,
1993). Hence, both classical screen approaches but also more advanced methodologies can be
employed to conduct nonbiased genetic screens to identify in vivo modifiers of MND
pathological mechanisms.
Genetic Screens in Drosophila and Their Application … 187
In this chapter, we give an overview of the genetic tools that have been created to
facilitate genetic screens in Drosophila and discuss some of the phenotypes that are amenable
to such screens. Indeed, the choice of ‘screen-phenotype’ is of paramount importance for two
reasons.
First, the phenotype that is screened for defines the pathway(s) in which mutations will
be isolated; choosing a focused (more ‘molecular’) phenotype therefore usually results in the
isolation of genes in a limited number of processes compared to more broad phenotypes (e.g.
behaviour).
Second, if individual flies are being tested in a screen, the phenotype assessed should be
highly penetrant; this is less of an issue when a population of flies that carries a particular
mutation can be assessed. Taking these ‘limitations’ into account, numerous MND-related
phenotypes that have been described are well-suited for screening such as defects in
locomotion, problems with neurotransmission and neurodegeneration, akin to defects seen in
humans (Chang et al., 2008; Li et al., 2010; Watson et al., 2008). In fact, several of these
phenotypes have been used successfully in the past to screen for genetic modifiers (Benzer,
1967; Harris and Stark, 1977; Pak, 1995).
In the next sections we will describe different screening strategies. With respect to MND,
such genetic screens can be used either to isolate mutations with phenotypes similar to the
MND models that exist, or they can be used to isolate modifiers of the phenotypes exhibited
by MND fly models. In the latter case, the screening approach is performed in the background
of the MND model, screening for enhancement or – ideally – suppression of the MND-
model-induced phenotype.
CLASSIC GENETIC SCREENS
Thanks to the discovery of potent mutagens, high-throughput screens in Drosophila
became feasible. Ethyl Methane Sulphonate or EMS1 is a popular mutagen used in
Drosophila (but also other species) due to its effectiveness and relatively low toxicity to flies
(Ashburner, 2005).
However, the compound is relatively non-specific and can result in the disruption of
almost all genes in the genome. EMS creates point mutations or small deletions in the
genome and mapping the molecular lesions created by EMS can be labour intensive.
Nonetheless, the advent of novel mapping technology (Chen et al., 2008; Parks et al., 2004;
Ryder et al., 2007; Ryder et al., 2004; Thibault et al., 2004; Zhai et al., 2003) in combination
with next generation whole genome sequencing (Wang et al., 2010) greatly facilitate mapping
efforts and bring new life to EMS-based mutagenesis screens. Furthermore, it is possible to
use EMS (or other mutagens) to generate and maintain a large collection of flies carrying
mutations as they can be ‘stably maintained’ with balancer chromosomes without the need to
genotype individual animals every generation. Hence, balancer chromosomes facilitate the
upkeep of vast amounts of mutant flies as ‘lab-stocks’.
1 EMS is an alkylating agent and it affects only one strand of the double helix. Since only one DNA strand is
mutated, the mutated DNA strand might segregate during the first zygotic division from the wild-type strand
before mismatch repair changes the wild-type strand to a mutant strand. Therefore the F1 progeny can be
mosaic for the mutation. This only matters in F1 screens where the mutation could be present in the somatic
tissues but absent in the germline, hence, not passed on to the next generation.
Liya E. Jose, Patrik Verstreken and Sven Vilain 188
Another strategy used to disrupt genes involves transposable elements (TEs), which are
DNA sequences that are mobile within the genome of single cells, including germ cells, thus
allowing transmission of novel TE insertions through the germline. While disrupting genes
with TEs is not very efficient in comparison to EMS, the insertion of a TE in a gene provides
a molecular tag such that the disrupted gene can be readily identified.
The most commonly used TEs are P-elements. P-elements insert preferentially at specific
loci, called hot spots, while they fail to insert in some other loci (Bellen et al., 2004;
Spradling et al., 1995). Nonetheless, numerous genes can be disrupted by P-element
mutagenesis and in an attempt to target a larger number of genes, other types of TEs have
been used in fruit flies including piggyBacs (Hacker et al., 2003; Handler and Harrell, 1999;
Horn et al., 2003; Thibault et al., 2004) and Minos elements (Bellen et al., 2011; Metaxakis et
al., 2005; Venken et al., 2011). Given that creating novel TE insertions is relatively
inefficient, it is more straightforward to screen existing collections of TE-bearing flies
generated for example by the Berkeley Drosophila Genome Project (Bellen et al., 2004). It
should be noted that TE insertions are often weak alleles that result in reduced protein
expression while phenotype-causing EMS-induced mutations usually cause more severe gene
disruption.
Nonetheless, the collection of fruit flies that harbour a TE with molecular information of
the TE insertion site is rapidly expanding and can be used to screen for mutants affecting the
biological process of interest without much need to map lesions (however, see ‘second site
hits’ below).
To isolate recessive mutations, the most straightforward, yet also most labour intensive
screening methodology is a classical “F3 screen”. This strategy allows one to generate
multiple (several thousand) populations of flies where all flies within one population carry the
same mutant chromosome. Intercrossing animals in the F2 generation within a population
results in the creation of homozygous animals that can be screened in the F3 generation
(Figure 1.a). This strategy holds the advantage that homozygous lethal or sterile animals can
be screened in the F3 generation while maintaining the mutations heterozygous over a
balancer as a stock.
If a phenotype is observed, the stock is maintained. This crossing strategy has been
extensively and successfully used by numerous fly researchers around the globe (Nusslein-
Volhard and Wieschaus, 1980; Salzberg et al., 1994) and is well-suited for unbiased screening
approaches.
One disadvantage is that only early roles of a given gene can usually be assessed and
potential later functions are not easily revealed, for example because of lethality or severe
developmental defects induced by the mutation. These drawbacks can be circumvented by
using a clonal screen approach where only part of the fly is rendered homozygous mutant
while the rest of the tissues remain heterozygous, thus precluding lethality induced by the
mutation (see below).
While classical F3 screens are labour intensive, the isolation of viable and fertile
recessive mutations on the X chromosome is quite straightforward. Here, virgin female flies
that carry a pair of attached X chromosomes and a Y chromosome (Benzer, 1967) is crossed
to mutagenized males (that carry an X and Y chromosome). Given that the Y chromosome in
the female fly and the X chromosome in the mutagenized male fly segregate independently,
the male offspring will be hemizygous for the mutagenized X chromosome and carry the Y
chromosome they received from their mother (Figure 1.b).
Genetic Screens in Drosophila and Their Application … 189
Figure 1. Examples of crossing schemes used in EMS mutagenesis screens. a) Typical F3 crossing
scheme: Males are mutagenized by EMS and then crossed to female virgin flies carrying a balancer
chromosome. Single balanced flies putatively carrying a mutation (red asterisk) are crossed back to the
balanced flies to create a mutant stock. In the next generation, mutant males and females can be crossed
and homozygous flies can be retrieved in the F3 generation. b) F1 screen for viable hemizygous male
mutant flies: Males are mutagenized and crossed with female flies that carry a compound X
chromosome and that carry a Y chromosome. Since XXX and YY flies are lethal, in the next generation
all male flies will have the mutant X chromosome (red asterisk) from the father and the Y chromosome
from the mother. c) EMS screen for dominant modifiers: males carrying a mutation that yields a
phenotype that can be easily screened for (black asterisk) is mutated and crossed back to females
carrying the same mutation. In the next generation dominant modifiers of the phenotype can be
investigated.
Hence, these males can be screened, mutants isolated and crossed. This strategy allows
the identification of viable and fertile mutants on the X chromosome in a single generation
(Figure 1.b).
CLONAL GENETIC SCREENS
F3 screening strategies are labour intensive and the early roles of some genes precludes
screening for a potential later function. These issues can be circumvented by performing
clonal screens (Figure 2.a-e). Here, homozygous tissue is generated in an otherwise
heterozygous animal. The homozygous mutant ‘patch’ does not interfere with viability or
fertility, but allows to assess the phenotype of a given mutation, and depending on when in
the lifetime of the organism the tissue is rendered homozygous, ‘later’ functions of a given
gene can also be assessed.
To generate such animals, mitotic recombination between homologous chromosomes is
induced using the Flp/FRT system (Xu and Rubin, 1993). Consequently, at least one cell
division is needed to generate homozygous mutant cells using this methodology, precluding
the generation of homozygous mutant tissue in the post-mitotic phase of a cell. Both
chromosomes, the mutagenized chromosome from the father fly and a specially ‘engineered’
chromosome from the mother fly, harbour FRT sites (Flp Recognition Target) (Duffy et al.,
1998; Golic and Lindquist, 1989).
Liya E. Jose, Patrik Verstreken and Sven Vilain 190
Figure 2. Clonal screens in Drosophila melanogaster using Flp. When the two FRT sites are on the
same identical chromosomal position, close to the centromere, inducing expression of Flp will cause
efficient recombination between the FRT sites and exchange of the distal part of the chromosome arms.
a) Mutagenized male flies that carry chromosomes with FRT sites are crossed to female flies that
express Flp and also harbour an FRT site with a dominant marker and a recessive cell lethal mutation.
b) When Flp is not present, mitotic recombination does not occur. c) When the Flp is present, mitotic
recombination between the FRT sites results in cells homozygous for the mutant chromosome, as well
as ‘twin spot’ cells for the recessive cell lethal mutation. The latter cells die, thus resulting in large
patches of homozygous mutant tissue. d) When eyFlp is used, almost the entire eye is rendered mutant
using this system (Newsome et al., 2000; Stowers and Schwarz, 1999). e) Similar experiments can be
done in the thorax of the flies, where yellow mutant clones can be generated (removing the bristles in
this drawing).
During normal mitosis one chromatid from each homologous chromosome would
segregate into each daughter cell upon cell division (Figure 2.b); however, the tissue specific
expression of Flipase (Flp) following DNA replication induces recombination between the
FRT sites such that now, identical chromatids segregate into one daughter cell (Figure 2.c).
The ‘erroneous’ segregation of chromosomes results in cells homozygous for the
mutagenized chromosome or cells homozygous for the ‘engineered’ chromosome. During
subsequent cell divisions, a homozygous patch of cells is created. The latter can carry a
visible marker (e.g. GFP or w+) and a recessive cell lethal mutation such that homozygous
cells die. The patch of remaining cells that carry the homozygous mutant chromosome can be
Genetic Screens in Drosophila and Their Application … 191
screened for phenotypes. While more complicated in setup, this screening strategy holds the
advantage that homozygous mutant tissue can be screened in the F1 generation (see previous
footnote on EMS, specifically the possibility that EMS creates mosaic animals) and recessive
homozygous lethal as well as viable mutants can be isolated. Furthermore, numerous
transgenic Flp expressing flies have been generated including an eye specific Flp (ey3.5-Flp)
(Iris Salecker and colleagues), an eye/optic lobe expressing Flp (ey-Flp) (Newsome et al.,
2000), a thorax Flp (Ubx-Flp) (Jafar-Nejad et al., 2005), and heat-shock inducible Flp (hs-
FLP) (Duffy et al., 1998), allowing for tissue specific screens (Figure 2.d and 2.e).
The Flp/FRT system has been used very extensively in the Drosophila eye using ey-Flp
(Newsome et al., 2000), where the Flp cDNA was placed under transcriptional control of an
eye-specific promoter from the eyeless (ey) gene. Despite the fact that the ey promoter
fragment used is predominantly expressed in the developing eye, expression was also
detected in the optic lobes (Newsome et al., 2000). If photoreceptor (PR)-specific expression
is desired, the more restricted ey3.5-Flp may be a better choice (Hiesinger et al., 2005).
Screening using the ey-Flp system has allowed to isolate novel genes involved in axon
guidance (Newsome et al., 2000), neurotransmitter release (Babcock et al., 2003; Stowers and
Schwarz, 1999; Verstreken et al., 2003), mitochondrial function (Guo et al., 2005; Stowers et
al., 2002; Verstreken et al., 2005) and neurodegeneration (Bayat et al., 2012; Simpson et al.,
2009; Zhai et al., 2006). While such clonal screens have extensively been performed to isolate
novel players in the aforementioned processes, limited work has been done to exploit the
methodology to isolate novel players in relation to MNDs, yet the technology holds potential
to identify novel key players in MND-relevant pathways. Unfortunately, it may be difficult to
generate homozygous mutant motor neuron clones in fruit flies; however, given the
conservation of many pathways across neuronal subtypes, it may be possible to screen other
types of neurons (e.g. PRs) to discover novel concepts and pathways governing specific
aspects of MND in fruit flies.
DOMINANT GENETIC SCREENS
The methodologies described so far aim at generating homozygous mutants or tissue that
can be used to screen for modification of MND-relevant phenotypes. However, a very
popular and successful strategy involves screens for dominant modification of a phenotype
(Raftery et al., 1995; Simon et al., 1991) including MND-relevant phenotypes. Provided the
heterozygous mutants alone do not modulate the phenotype screened, the isolated mutants are
dominant enhancers or suppressors of the process under investigation (Figure 1.c) (reviewed
in St Johnston, 2002). Enhancers of lethal mutants can also be found through screening for
synthetic lethality. Synthetic lethality is a phenomenon by which hetero-allelic combinations
of two interacting genes leads to organismal lethality while each heterozygous mutant is
viable. Since only one generation is needed to screen for synthetic lethality it is a quick way
to screen for interactors of lethal mutations and has been used in the past to find modifiers in
an MND model (Chang et al., 2008). Enhancer/suppressor screens have proven their merit in
the past in the isolation of pathway members involved in developmental signalling and they
are now also making their way into neurodegenerative disease research (Chang et al., 2008;
Rimkus et al., 2008; Sen et al., 2011; Vos et al., 2012). In this context, dominant suppressors
Liya E. Jose, Patrik Verstreken and Sven Vilain 192
are particularly interesting as drug targets: only one mutant allele is sufficient for suppression
(or enhancement).
While dominant screens may be performed with collections of TEs or EMS-induced
mutations (Chang et al., 2008; Rimkus et al., 2008; Sen et al., 2011; Vos et al., 2012), it is
also possible to use a collection of overlapping deficiencies for dominant suppression or
enhancement of a phenotype (Weiss et al., 2012). This methodology holds two advantages:
First, one deficiency uncovers tens to hundreds of genes at the same time and in a single
cross, the involvement of all genes is tested at once. Second, modifiers are usually contained
within the interacting deficiency thus saving time on mapping compared to EMS-induced
mutations. To identify the causative modifying gene within the deficiency, the individual
genes within the deficiency can then be knocked-down or mutants in these genes can be
tested for phenotype modification. Despite this and several other successful deficiency
screens, a number of important drawbacks need to be taken into account as well: first, the
genes contained within a deficiency sometimes interact, confounding the observed effect;
and, second, similar to EMS and TE bearing chromosomes, the chromosomes that harbour the
deficiency may also contain additional ‘second site’ mutations, and these may also modulate
the phenotype under investigation, thus complicating the identification of the causative gene.
This particular issue may be circumvented by performing linkage analysis to test if the
modifier (suppression or enhancement of the phenotype) is genetically linked to the
deficiency. Hence, while deficiency screening for dominant modifiers allows for the rapid
identification of modifying loci, pinpointing the individual gene that modifies the phenotype
is not always trivial and it is essential to complement the isolation of modifying genes with
rescue experiments where a wild-type copy of the identified gene, ideally expressed under
endogenous promoter control, is placed back into the mutant background.
UAS/GAL4-BASED SCREENS
It is often desirable to generate mutant tissue in an otherwise wild-type background. As
described above, mitotic recombination is a powerful tool, but tissue specific Flp expression
is needed and at least one (and ideally more than one) cell division is required. Hence the
observed phenotype could be the sum of early phenotypes, occurring early after cell division
and later independent phenotypes occurring at a later stage; earlier phenotypes can even
preclude the detection of later phenotypes. Expression of transgenes via the UAS/Gal4 system
alleviates many of these drawbacks. Gal4, a yeast transcription factor that does not have a
deleterious effect in Drosophila (Fischer et al., 1988), binds UAS (Upstream Activator
Sequence) and activates transcription of downstream responder constructs (genes, RNAi)
(Brand and Perrimon, 1993). When flies bearing a transgene that allows for tissue specific or
temporally controlled Gal4 expression are crossed to flies bearing a transgene with a UAS
responder construct, this construct will be transcribed in a tissue- and time-controlled manner.
The UAS/Gal4 system allows control of expression levels, since the system is inherently
temperature-sensitive with minimal activity at low temperature (16°C) and maximum activity
at high temperature (29°C) (Duffy, 2002). However, it is important to note that function and
morphology of the fly NMJ are temperature dependent and carefully designed control
experiments are needed. Further, numerous collections of Gal4 and UAS-responder lines exist
Genetic Screens in Drosophila and Their Application … 193
and countless variations to control the expression of Gal4 have been developed (reviewed in
Duffy, 2002). Hence, the UAS/Gal4 system enables researchers to manipulate gene expression
(overexpression or RNAi-mediated knockdown; see below) in a spatially- and temporally-
controlled manner.
Different libraries of transgenic flies have been generated for overexpression of a large
collection of genes in the genome. Several TEs that have been used in gene disruption
projects harbour UAS sites. If such a TE inserts in the vicinity of a gene, the insertion itself
may disrupt gene expression, but adding Gal4 may induce (overactivate) gene expression
(Figure 3.a). Relevant TE collections are the EP P-elements, the “Exelixis collection” (XP
and WH lines) and the “GS” collection (available at the Bloomington Drosophila Stock
Centre [BDSC] and Harvard Medical School) (Beinert et al., 2004; Bellen et al., 2004; Rorth,
1996; Staudt et al., 2005; Thibault et al., 2004; Toba et al., 1999). Using these TE collections
it becomes possible to perform MND-modifier screens based on overexpression. Such a
screen was recently successfully used to screen for modification of a hypomorphic smn allele.
smn is the Drosophila orthologue of Survival Motor Neuron1 (SMN1), mutations in which
cause spinal muscular atrophy (SMA). Screening the “Exelixis collection” identified several
modifying loci, including members of the BMP and FGF signalling pathways, and further
studies indeed linked defective BMP and FGF signalling to the defects observed in smn
mutant NMJs (Chang et al., 2008; Sen et al., 2011). Several other overexpression screens
have also been performed in relation to MND (Guo et al., 2011; Li et al., 2010) and similarly,
over- or mis-expression screens for genes that modulate axon guidance and synaptogenesis at
the larval neuromusculature have been performed (Kraut et al., 2001). While conceptually
straightforward, it is important to take into account that gene expression may be forced in
ectopic tissues and mis-expression artifacts should be controlled for. Furthermore, TEs that
bear UAS sites are often located in between genes, and it is not always clear which gene or
group of genes are being overexpressed. Nonetheless, mis- or over-expression screens are
fairly simple to set up as well as execute and numerous tools are already available.
The advent of genome-wide RNAi collections to target almost all genes in the
Drosophila genome not only addresses the limitations of limited mutability of particular
genomic regions that are refractory to EMS or TE insertion, but also allows researchers to
perform temporally and spatially controlled knockdown of gene expression (Fortier and
Belote, 2000; Kennerdell and Carthew, 1998; Kennerdell and Carthew, 2000; Lam and
Thummel, 2000; Martinek and Young, 2000; Misquitta and Paterson, 1999). Hairpin RNAi
constructs against different regions of cDNAs in the Drosophila genome exists and are
expressed under control of the UAS/Gal4 system.
Different collections, optimized for RNAi targeting efficiency, have been constructed and
are available from various stock centres (http://www.shigen.nig.ac.jp/fly/nigfly/;
http://stockcenter.vdrc.at/control/main; http://www.flyrnai.org/TRiP-HOME.html) (Dietzl et
al., 2007; Ni et al., 2009). These RNAi constructs can be used to knockdown gene expression
in the entire animal, but they can also be expressed in specific neuronal networks or neuronal
subtypes using specific Gal4 driver lines (Figure 3.a and 3.b, b). Furthermore, RNAi
expression can be employed to knock-down gene expression in multinuclear cells, including
muscles, relevant to MND. While conceptually simple there are important drawbacks to be
considered. First, numerous RNAi constructs are known to harbour off-target effects that are
not straightforward to assess in vivo, thus complicating interpretation of the results (Dietzl et
al., 2007; Ni et al., 2009; Ni et al., 2008; Perrimon et al., 2010).
Liya E. Jose, Patrik Verstreken and Sven Vilain 194
Figure 3. Using the UAS/Gal4 system to perform tissue specific overexpression experiments or target
the gene function with RNAi. a) Crossing the UAS-RNAi collection or a UAS-GeneX collection with
flies that express Gal4 in a tissue specific manner (green in the drawings) allows one to generate flies
that harbour tissue specific knock-down or overexpression of a gene in a tissue specific manner.
b) Neuronal specific Gal4 lines allow for gain- or loss-of-function experiments in subsets of neurons.
One way to control for off-target effects is to conduct rescue experiments using strategies
that allow for the translation of normal wild-type protein from mRNA that is not recognized
by the RNAi machinery (Kondo et al., 2009; Langer et al., 2010; Schulz et al., 2009). Second,
in most cases, RNAi expression causes only partial gene knockdown that may be insufficient
to produce or affect the phenotype.
One solution can be to co-express the RNAi with Dicer2, improving the effectiveness of
some RNAi lines, but Dicer2 expression also increases the incidence of off-target effects
(Dietzl et al., 2007). Although genome-wide RNAi based screens are labour intensive, the
methodology allows to screen sub-collections, for example RNAi lines targeting kinases,
phosphatases etc. Given the ease to apply RNAi in Drosophila, it has in recent years become
a popular tool that can be additionally used to conduct screens.
SCREENABLE PHENOTYPES
When performing large-scale screens it is important to be able to rely on phenotypes that
are specific enough for the process under investigation, yet robust and sensitive such that they
can be easily and quickly quantified, thus limiting the number of individual animals that need
to be analysed.
Genetic Screens in Drosophila and Their Application … 195
A very commonly used neuronal system to conduct genetic interaction screens is the
Drosophila compound eye since it is not essential for viability or fertility. The fly eye is
composed of an array of 800 ommatidia, each containing eight PR neurons seven of which are
visible in tangential sections (Katz and Minke, 2009; Montell, 2012).
Many genes involved in cell-fate determination and differentiation will yield a rough-eye
phenotype when overexpressed or mutated in the fly eye (Brumby et al., 2002; Chanut et al.,
2000; Firth et al., 2000; Mao and Freeman, 2009; Thao et al., 2012). Similarly, many disease
genes when mis-expressed in the eye induce a rough-eye phenotype. However, it is not
always clear how this phenotype arises, whether the defects are caused by abnormal mis-
expression and what the relationship of the eye phenotype to the disease gene is. Nonetheless,
this phenotype has often been used in modifier screens, also in relation to MND-relevant
genes (Suzuki et al., 2009).
Spinobulbar muscular atrophy (SBMA) is a toxic gain-of-function polyglutamine disease
caused by expanded trinucleotide repeats in the androgen receptor (AR) gene. Expression of
full-length AR with expanded poly-glutamine repeats in the Drosophila eye causes a rough-
eye phenotype that is dependent on the presence of the ligand and worsens when an AR
protein with longer repeats is expressed (Chan et al., 2002; Pandey et al., 2007; Takeyama et
al., 2002).
The toxicity of the mutant protein seen in SBMA patients can thus be recapitulated by
eye-specific expression in flies and this tool makes for a good model to screen for genes that
are involved in mutant AR-induced toxicity (Chan et al., 2002). Screening such expanded AR
expressing flies using a set of 2000 genes that were co-overexpressed in the eye (GS strains)
revealed that the Drosophila homolog of the Retinoblastoma protein, Rbf, is a modifier of
AR-dependent SBMA (Suzuki et al., 2009). Thus, by screening 2000 flies for modulation of
AR-induced neurodegeneration in flies, a link between polyQ-AR toxicity and
Retinoblastoma protein function was proposed.
Rough-eye phenotypes can also be the result of degeneration of cells other than PRs,
including the cornea and pigment cells. PR-specific degeneration can be easily assessed using
a rapid optical neutralization technique called the deep pseudo-pupil method that does not
require dissection, antibodies or dyes (Kirschfeld and Franceschini, 1968) and was introduced
in studies of neurodegenerative disease in Drosophila (Jackson et al., 1998). PRs elaborate a
membranous organelle, the rhabdomere which is a photosensitive structure and within one
ommatidium, the rhabdomeres are organized in a very stereotypic manner. Degeneration of
the PRs can thus be assessed by analysing the rhabdomeric organization and structure. In the
deep pseudo-pupil assay, the virtual images of a number of rhabdomeres are superimposed
and the properly organized rabdomeric structure is seen as a magnification of a single
ommatidium (Figure 4.e). Mis-organization or degeneration of the ordered array of
rhabdomeres results in disruption of the pseudo-pupil image, signifying morphological
(degenerative) defects.
However, subtle defects in one or a few ommatidia may not be obvious to detect using
this methodology. Nonetheless, this assay enables fast screening of the gross morphology of
ommatidia, signifying retinal degeneration in a somewhat more subtle manner than screening
for external rough-eye phenotypes.
A commonly used readout to assess the functionality of the PRs is the electroretinogram
(ERG) (Figure 4.f) (Heisenberg, 1971; Pak, 1995), which allows for very fast and efficient
measurement of the electrical response of the fly eye to light stimulus. ERGs measure
Liya E. Jose, Patrik Verstreken and Sven Vilain 196
differences in extracellular potential between PRs and the fly body during a short light
stimulus (e.g. 1s).
In controls, ERG recordings show a de- and re-polarization of the PRs, reflecting an
intact phototransduction mechanism of the PRs, and ‘on’ and ‘off’ transients at the onset and
conclusion of the light stimulus. The presence of the ‘on’ and ‘off’ transients indicate that the
PRs can activate postsynaptic neurons while the depolarization of the PRs signifies the
presence of healthy PR cells that efficiently depolarize in response to light. Recording ERGs
is relatively fast and this assay has been used to screen vast amounts of flies to isolate mutants
involved in neuronal communication (‘on’ and ‘off’ defects) (Hiesinger et al., 2005; Zhai et
al., 2003) and in neurodegeneration (depolarization defects) (Bayat et al., 2012). In relation to
MND, screens based on ERG recordings led to the identification of the fly orthologue of elp3,
a gene whose expression levels are associated with ALS in humans (Simpson et al., 2009).
Hence, ERG recordings are a valuable high-throughput tool in the search for genes that affect
neuronal communication and survival.
Figure 4. Drosophila melanogaster behaviour, retinal morphology and neuronal function can be used to
study mutations affecting it, in a relative fast and easy way. a) During Drosophila melanogaster
development various behavioural traits can be studied. b) During the larval stage, crawling of the larvae
can be assessed whereas c) during the adult state, climbing or flight can be investigated. d) Schematic
of larval fillet preparation with larval motor neurons in grey reaching the muscles and neuromuscular
junction (inset), which allows for functional studies of various synaptic processes. e) Schematic of a
Drosophila melanogaster compound eye with the typical trapezoid shape of the rhabdomeres as can be
assayed with the pseudo pupil method. f) Schematic of the setup used to record electroretinograms
(ERGs) of the fly eye and example of a wild-type ERG.
Genetic Screens in Drosophila and Their Application … 197
Other assays that are often used to describe neurodegenerative phenotypes in flies are
lifespan assays (Van Voorhies et al., 2004) and assays to study sensitivity to oxidative stress
(Shukla et al., 2011) that is often associated with neuronal degeneration. Sensitivity to
oxidative stress can be assayed by exposing the flies to rotenone or paraquat, both of which
are inhibitors of mitochondrial complex I (Li et al., 2003; Sriram et al., 1997); hydrogen
peroxide, which is a more general oxidative stress inducer (Nickla et al., 1983; Wang et al.,
2008); or menadione sodium bisulfite, a mild oxidative stress inducer that persist longer when
mixed in the food and is effective at inducing chronic oxidative stress (Jordan et al., 2012).
Assaying survival in the presence or absence of these compounds is relatively easy, but time-
consuming. Furthermore, the genetic interactors identified will likely improve organismal
health but may yield quite broadly defined and potentially less specific components of the
pathways under study. With respect to MND, secondary screens are thus likely needed to
further group interactors into relevant and defined pathways.
Assays that are more directly linked to the motor system encompass locomotion and
activity-related tests as well as morphological and functional analyses of neuromuscular
endplates. Several external stimuli can be used to trigger locomotion related responses in
adult fruit flies or in larvae, including light (Borst, 2009), gravity (Kamikouchi et al., 2009),
temperature (Sayeed and Benzer, 1996), humidity (Liu et al., 2007), odours and taste
(Vosshall and Stocker, 2007) as well as sound (Kamikouchi et al., 2009) (Figure 4.a-c). More
directly assessing motor behaviour, screens based on flight (Benzer, 1973; Drummond et al.,
1991; Pesah et al., 2004; Vos et al., 2012), locomotion geotaxis, phototaxis (Armstrong et al.,
2006; Desroches et al., 2010; Gargano et al., 2005; Hirsch, 1959; Inagaki et al., 2010;
Kamikouchi et al., 2009; Le Bourg and Buecher, 2002; Pak et al., 1969; Seugnet et al., 2009;
Strauss and Heisenberg, 1993; Toma et al., 2002; Vang et al., 2012), as well as activity
monitoring over a prolonged period using an activity monitoring system (TriKinetics)
(Pfeiffenberger et al., 2010) have been successfully used and several of these assays are well-
described in video-based publications (Ali et al., 2011; Chiu et al., 2010; Nichols et al.,
2012).
The third instar larval NMJ has emerged as a model for neuronal cell biology
(Featherstone and Broadie, 2000), and also in the context of MND, this synapse has yielded
invaluable insight into the mechanisms of several diseases (Chang et al., 2008; Pennetta et al.,
2002; Ratnaparkhi et al., 2008; Sen et al., 2011; Sherwood et al., 2004; Xia et al., 2012).
Several features of this synapse are very stereotyped and could be used in a screen setting as
well (Collins and DiAntonio, 2007). Indeed, several models of MND show defects at this
synapse, for example mutations in fly VAP33, the fly orthologue of ALS8 (Nishimura et al.,
2004) show morphological and functional changes at the NMJ (Pennetta et al., 2002) and the
protein has been linked to Ephrin signalling (Tsuda et al., 2008) that is implicated in ALS
(Van Hoecke et al., 2012). While in most cases, larvae need to be dissected to gain access to
the neuromusculature, it is also possible to visualize NMJs through the cuticle by virtue of
transgenic expression of GFP fusion proteins (Rasse et al., 2005; Zito et al., 1999), thus
making screening of vast numbers of mutants possible using this system. However, given
practice, assays based on dissected preparations in combination with live imaging to monitor
synaptic function (Verstreken et al., 2008) or morphological assays based on expression of
GFP fusion proteins (Pilling et al., 2006) or immunohistochemistry are feasible as well in
medium-throughput screens. Such assays have indeed been used in the past to isolate mutants
that affect the NMJ morphology (Aberle et al., 2002) as well as NMJ synaptic function
Liya E. Jose, Patrik Verstreken and Sven Vilain 198
(Featherstone et al., 2000; Rohrbough et al., 2004) and are attainable as well to use in the
context of MND (Figure 4.d).
CONCLUSION
Several phenotypes have been observed in models of MND in fruit flies that are
particularly amenable to genetic screening. Phenotypes range from larval lethal, pupal lethal,
retinal degeneration to defects in locomotion behaviour and specific defects at the NMJ.
Given the considerations discussed above, different types of screens either focused on NMJ
dysfunction, or using alternative tissues such as the PRs are feasible and may yield invaluable
insights into ’the pathways that lead to MND.
ACKNOWLEDGMENTS
We apologize for the omission of any relevant publication due to the specific focus of
this chapter. We are grateful to Tillman Achsel for critical reading of the manuscript and
Helena Renders for help. Work in the Verstreken lab is supported by an ERC starting grant,
FWO grants, IWT, FCT, the research fund KU Leuven, the Hercules foundation and VIB. SV
is an FWO fellow and LJ is supported by VIB.
ABOUT THE AUTHORS
Liya Elsa Jose obtained her masters degree in 2006 from Amrita University in India. In
2006 she joined a Biotech firm in India and worked as Junior Research Fellow since 2009. In
2009 she got an International VIB PhD Scholarship and joined the lab of Prof. Patrik
Verstreken (Laboratory of Neuronal Communication) to pursue her PhD. After joining the lab
she studied the role of elp3 in the synapse and she is presently focusing on the role of elp3 in
motor neuron disease.
Patrik Verstreken performed a post-doc at Howard Hughes Medical Institute, BCM,
Houston, USA. He performed several Ethyl Methane Sulphonate (EMS)-based screens to
identify novel genes involved in neuronal communication. He is a group leader at VIB since
2007 and his lab focuses on the study of several genes that affects synaptic communication in
health and disease. Researchers in his lab use novel EMS screens to isolate novel components
of this process. Interestingly, several genes that his group has identified have been linked to
neurological disease; including Parkinson’s disease and amyotrophic lateral sclerosis (ALS),
further underscoring the central involvement of neuronal communication in neuronal disease.
Sven Vilain obtained his Ph.D. in 2009 at the Katholieke Universiteit Leuven, Leuven,
Belgium. At the Center for Human Genetics, in the laboratory of neurogenetics led by
Bassem Hassan, he studied the function of the gene atonal by means of a genetic screen and
he participated in the creation of a novel toolkit for the study of atonal. After joining the
Laboratory of Neuronal Communication led by Patrik Verstreken, he studied the role of
mitochondrial Complex I in the Parkinson’s disease related gene pink1 and he participated in
Genetic Screens in Drosophila and Their Application … 199
a genetic screen for dominant modifiers of pink1 mutants. Currently he is focusing on the
synaptic function of the Parkinson’s disease related gene LRRK.
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INDEX
A
AAA, 26, 51, 87, 89, 91, 94, 105, 112, 113, 115, 116
access, xi, 197
accessibility, 155
accounting, 4
acetylation, 71, 99, 155, 164, 168
acid, 10, 12, 14, 99, 108, 110, 139
acidic, 39
actin dynamics, 102, 127
action potential, x, 97, 122, 128
activity level, 205
adaptation, 43
adenosine triphosphate, 173
adhesion(s), 9, 12, 16, 44, 90, 93, 106, 108, 109, 111,
125, 127, 204
adipose, 98
ADP, 159, 161
adulthood, 132, 172, 176
adults, 2
aetiology, 80, 161, 180
age, ix, 14, 18, 25, 57, 60, 99, 148, 150, 151, 162,
172, 201
age-related diseases, 162
aggregation, 12, 16, 41, 59, 60, 61, 75, 76, 77, 81,
82, 83, 109, 155, 158, 160, 161, 163, 165, 166,
168, 169, 177, 202, 203, 210
aging population, 148
agonist, 42
akinesia, 3
Aldrich syndrome, 112
allele, 23, 69, 99, 133, 141, 192, 193, 201
ALS2, 5, 7, 17, 25, 88, 92, 103, 113, 115
alsin, 5, 7, 25, 33, 55, 58, 88, 92, 103, 109, 110, 115
alters, 31, 74, 153, 160, 177, 178
amino acid(s), x, 6, 8, 16, 28, 38, 53, 66, 94, 95, 101,
103, 121, 133, 136, 139, 157
amplitude, 54, 177
amyotrophic lateral sclerosis (ALS), v, viii, ix, xii, 1,
2, 3, 4, 5, 6, 7, 8, 9, 13, 19, 20, 21, 22, 23, 24, 25,
26, 28, 29, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,
54, 55, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 103, 11, 142, 196, 197, 198, 201, 202,
203, 204, 206, 209, 210
amyotrophy, 2, 13, 16, 27, 86
anatomy, x, 130, 133
anchoring, 51, 106
androgen, xi, 18, 19, 147, 149, 151, 152, 153, 154,
162, 163, 164, 165, 166, 167, 168, 169, 195, 201,
204, 208
angioplasty, 48
anterograde transportation, 100
anticodon, 136, 144
antigen, viii, 36, 42
AP-5 complex, 104
apoptosis, ix, 45, 58, 61, 70, 72, 82, 109, 156, 164,
168, 203, 210
Arabidopsis thaliana, 114
arrest, 168
aspiration, 151
aspiration pneumonia, 151
assessment, 141, 146
assets, 70
ataxia, 8, 11, 28, 70, 81, 117, 130, 149, 150, 154,
167, 199, 200, 205
atlastin, 12, 87, 92, 94, 95, 96, 97, 102, 110, 115,
116, 119
ATP, 70, 105, 137, 157, 159, 160, 173, 183
atrophy, vii, xi, 1, 2, 3, 4, 17, 18, 20, 22, 25, 26, 27,
28, 30, 37, 52, 122, 126, 127, 143, 147, 148, 149,
151, 153, 162, 163, 164, 165, 166, 167, 168, 171,
172, 174, 181, 182, 183, 184, 193, 195, 200, 204,
205, 207
attachment, 137, 157
attractant, 44
autism, 180
Index 212
autopsy, 58
autosomal dominant, 2, 6, 10, 11, 13, 15, 17, 26, 28,
34, 46, 94, 95, 96, 97, 98, 99, 100, 101, 102, 105,
110, 111, 112, 119, 122, 125, 126, 127, 148
autosomal recessive, xi, 2, 6, 10, 11, 13, 17, 18, 29,
98, 99, 101, 102, 103, 104, 105, 113, 114, 122,
125, 126, 127, 172
avian, 17
axon, viii, ix, x, 12, 14, 15, 16, 29, 31, 32, 44, 48, 51,
54, 75, 85, 86, 96, 97, 99, 101, 103, 106, 110,
111, 113, 114, 119, 122, 123, 139, 178, 191, 193,
203, 204
axon regeneration, 96, 111
axonal Charcot-Marie-Tooth disease, vii, 1, 24, 27,
144
axonal degeneration, xi, 7, 12, 14, 18, 25, 86, 122,
138, 139, 140, 142, 146
axonal microtubule-based transport, x, 85
axonal pathology, 15
axons, ix, x, 1, 3, 4, 11, 14, 15, 16, 20, 28, 30, 67, 75,
85, 86, 95, 96, 97, 100, 101, 105, 106, 112, 115,
128, 132, 133, 139, 143, 156, 205
B
Baars, 113
bacteria, 114, 159
basal ganglia, 148
base, 36, 132
Belgium, xii, 142, 185, 198
beneficial effect, 160, 161
benign, 139
Berardinelli-Seip congenital lipodystrophy, 98, 111
bias, 65
biochemistry, 47, 50, 53
bioinformatics, 67
biopsy, 124
biosynthesis, 127
biosynthetic pathways, 107
biotechnology, 203
blood, 18, 41, 124, 133, 146
blood-brain barrier, 18, 146
BMA, 17
body fat, 74, 98
bone, x, 11, 70, 85, 176, 182, 184
bone morphogenetic protein (BMP), x, 85
boutons, 40, 68, 69, 95, 96, 177
brain, 8, 14, 36, 52, 62, 78, 98, 99, 103, 113, 114,
116, 128, 131, 138, 139, 199, 201, 207, 208
brainstem, 3, 148, 149, 151
branching, 11, 31, 78, 109, 132, 176
breakdown, 164
budding, 104
by-products, 58
C
Ca2+, 42, 48, 51, 70
calcium, 11, 15, 39, 49, 54, 96, 97, 125, 149
CAM, 109, 110, 111
cancer, 70, 76, 77
candidates, 47
carboxyl, 39, 113, 208
cargoes, x, 7, 51, 85, 100, 101
cascades, 9, 15, 42, 43
caspases, 156
catabolism, 8, 14
catalysis, 6
catalytic activity, 109
cation, 13, 101, 129, 134
CBP, 155
cDNA, 191
cell biology, 53, 180, 181, 182, 184, 197, 205, 207
cell body, ix, 4, 85, 86, 100, 106, 138
cell culture, 124, 155, 160, 168
cell cycle, 88, 108, 157, 206
cell death, 18, 21, 31, 45, 81, 98, 99, 153, 156, 162,
170
cell differentiation, 126, 145, 178
cell division, 94, 177, 178, 182, 189, 190, 192
cell fate, 74, 143, 163, 181, 200
cell line(s), 61, 77, 140, 153
cell membranes, 98
cell signaling, 143
cell size, 205
cell surface, 12, 15, 43
central nervous system,
central nervous system (CNS), 14, 27, 28, 44, 51, 77,
90, 95, 128, 139, 145, 177, 178
centromere, 172, 190
centrosome, 94
ceramide, 39, 41, 53
cerebellum, 15, 148, 149
cerebral palsy, 29
cerebrospinal fluid, 77
certification, 47
challenges, vii, ix, 36, 85, 86
chaperones, 11, 16, 45, 76, 152, 157, 158, 159, 161,
167
cheese, 98, 108, 113, 115
childhood, 144, 171
children, 2
cholesterol, 14, 99, 118
chromatid, 190
chromosome, 7, 49, 50, 53, 75, 78, 80, 113, 144,
156, 169, 172, 188, 189, 190, 199, 202
Index 213
circadian rhythms, 200
classes, 4
classification, 3, 117
clathrin, 15, 31, 103, 104
cleavage, 61, 66, 83, 105
clinical presentation, 58
clinical symptoms, 2
clinical trials, ix, 123
clustering, 40, 42, 118
coding, 8, 65, 94, 148, 155, 180
codon, 94, 139
cognitive function, vii
cognitive impairment, 103
collaboration, 59
communication, 40, 86, 196, 198
compaction, 16
compensation, 14, 159
competition, 42
complement, 192
complexity, vii, 44, 59, 106, 123
complications, 172
composition, 14, 32, 76
compound eye, 149, 195, 196
compounds, 71, 72, 98, 124, 161, 197
conduction, x, xii, 14, 122, 128, 133
conjugation, 163, 200
consensus, 141
conservation, 65, 71, 95, 110, 174, 180, 191
contracture, viii, 1, 2, 3, 4, 17
controversial, 177
controversies, 74
COOH, 164
coordination, 174
copper, 6, 17, 18, 79
copulation, 132
cornea, 195
corpus callosum, 32, 103, 117
correlation, 27, 123, 148
correlations, 52
cortex, 3, 149
cortical neurons, 11, 31
courtship, 79
cross-sectional study, 79
crystal structure, 47
crystalline, 149
CST, 2, 3, 11, 12
cues, viii, 35
culture, 128
cure, 58
cuticle, 197
CYP7B1, 9, 14, 32, 89, 93, 99, 117, 118
cyst, 178
cystic fibrosis, xi
cytochrome, 99, 114, 117, 156
cytochrome P450, 99, 117
cytokinesis, 31, 102, 109, 116
cytoplasm, 8, 19, 62, 63, 67, 69, 70, 100, 123, 125,
126, 127, 151, 152, 157, 158, 159, 173, 177
cytoplasmic phase, xi
cytoplasmic tail, 106
cytoskeleton, 2, 9, 10, 12, 16, 17, 42, 44, 47, 71, 95,
123, 125
D
database, 52
defects, viii, xi, 8, 11, 12, 15, 20, 25, 33, 35, 37, 40,
43, 44, 45, 46, 58, 66, 68, 69, 72, 79, 94, 97, 99,
100, 115, 116, 119, 121, 138, 139, 140, 141, 150,
154, 156, 168, 174, 176, 177, 178, 180, 183, 185,
187, 188, 193, 195, 196, 197, 198, 205
deficiency(ies), 14, 16, 18, 19, 29, 30, 33, 107, 156,
159, 177, 192, 206, 209
degenerate, 132
degradation, ix, 5, 6, 9, 12, 13, 16, 17, 20, 37, 61, 67,
70, 72, 90, 102, 105, 113, 152, 157, 158, 159,
160, 161, 163, 165, 167, 169
dementia, 2, 6, 7, 11, 24, 26, 31, 70, 78, 79, 86, 117
demyelination, 14, 29, 106, 122, 138
dendrites, 97, 100, 101, 112
dendritic spines, 62, 65, 76
dephosphorylation, 47
depolarization, 196
deprivation, 158
depth, 100, 105
deregulation, 9
detectable, 156
detection, 192, 202, 207, 208
detoxification, 204
developmental process, 43
dimerization, 100, 141, 169
disability, 1, 2, 29, 104, 186
discs, 132, 149, 177
disease gene, vii, ix, 13, 46, 65, 74, 124, 133, 185,
186, 195, 200
disease model, 79, 142, 153, 171, 186, 210
disease progression, xi, 148, 153, 154, 161, 185
diseases, vii, ix, x, xi, 4, 36, 66, 77, 85, 109, 124,
147, 148, 150, 154, 155, 156, 158, 160, 161, 169,
185, 186, 197
disorder, xi, 18, 29, 36, 37, 46, 57, 58, 111, 114, 122,
142, 144, 151, 169, 172
distal hereditary motor neuropathy (dHMN), viii, 4,
20, 137
distress, 19, 25
distribution, 16, 26, 43, 102, 104, 115, 144, 145, 167
Index 214
diversity, 86, 145
DNA, viii, 4, 5, 8, 19, 23, 31, 37, 55, 57, 58, 61, 65,
78, 79, 103, 117, 138, 151, 152, 154, 155, 168,
169, 182, 187, 188, 190, 207
DNA damage, 65, 168, 207
DNA repair, 103, 117
domain structure, 70, 151, 182
dopaminergic, 209
dosage, 53, 133, 138, 141, 205
double helix, 187
down-regulation, 155
drawing, 131, 190
drug discovery, 66, 79
drug targets, 36, 192
drug treatment, x, 121, 123
drugs, 160
D-serine, 8
dyes, 195
dynamin, 12, 15, 34, 87, 95, 112, 115, 129, 134
E
egg, 128, 174, 176, 177, 184
electrodes, 138
electron, 15, 43, 45, 150, 209
elongation, 44
embryogenesis, 40, 132, 174, 176, 177
embryonic stem cells (ESCs), 124
EMG, 54
EMS, xii, 174, 187, 188, 189, 191, 192, 193, 198
encoding, x, 7, 8, 12, 14, 18, 19, 23, 25, 32, 33, 55,
58, 76, 104, 111, 112, 115, 117, 119, 121, 136,
139
endocrine, 118, 164
endoplasmic reticulum (ER), viii, 35, 41, 85, 86, 159
endosomal traffic, x, 5, 7, 11, 13, 85, 86, 88, 89, 92,
93, 103, 123
endosomes, 7, 11, 15, 88, 89, 94, 102, 110, 123, 125
energy, 43, 105, 157, 158, 163
energy supply, 105
engineering, 209
England, 48, 49, 50, 54
environment, 73, 138
environmental conditions, 67
environmental factors, 57, 58, 59, 70
enzymatic activity, 126, 140
enzyme(s), x, 6, 14, 16, 17, 18, 19, 58, 99, 105, 121,
136, 143, 157, 160, 173
epigenetics, 59
epilepsy, 50, 86
equipment, 156
ER stress, viii, 12, 35, 37, 41, 45, 46, 47, 52, 53, 81,
96, 101
ERLIN2, 10, 21, 90, 93, 108
ESCRT-III, 11, 90, 94, 102, 116, 119
eukaryotic, 48, 157, 164
eukaryotic cell, 157
evidence, viii, xi, 1, 4, 11, 12, 14, 16, 20, 24, 42, 44,
45, 46, 60, 65, 68, 70, 71, 77, 121, 122, 123, 145,
152, 159, 161, 209
evolution, 124, 136
excision, 174
excitotoxicity, 8, 12, 37, 42
exclusion, 8
exocytosis, 7, 202, 206
F
families, vii, 12, 108, 116, 119
family members, 39, 49, 116
fasciculation, 4, 106
fat, 62, 74, 96, 98
fat body, 96, 98
fatty acid 2-hydroxylase (FA2H), 99
fatty acids, 99, 114
fertility, 151, 189, 195
fertilization, 40, 55
fiber(s), 55, 131, 132, 133, 138, 139, 140, 142
fibrinolysis, 48
fibroblasts, 124, 160
filament, 16, 24, 125
financial, 72
financial support, 72
fish, 59
fission, 15, 41, 43
flight, xii, 132, 138, 139, 140, 174, 176, 196, 197
fluorescence, 50
follicle, 177
food, 150, 154, 197
formation, 6, 7, 8, 9, 12, 14, 16, 24, 25, 49, 51, 53,
61, 65, 76, 94, 96, 102, 106, 109, 110, 111, 114,
115, 118, 141, 144, 148, 158, 163, 168, 178, 180,
181, 182, 202, 204, 205, 206, 208
fractures, 176, 182, 184
Fragile X mental retardation protein (FMRP), 61
fragments, 41, 62, 66, 150
frameshift mutation, 102
functional analysis, 44
functional changes, 197
funding, 20, 72
fused in sarcoma (FUS), viii, 8, 37, 80, 207
fusion, 7, 15, 43, 44, 52, 88, 96, 114, 115, 125, 126,
150, 154, 197
Index 215
G
gait, 11, 12, 106
ganglion, 132, 149
GEF, 88, 91, 103
gene expression, xi, xii, 8, 19, 54, 61, 67, 74, 77, 80,
125, 143, 148, 155, 163, 169, 186, 193, 200
gene silencing, 201, 203
gene targeting, 199
gene therapy, 47
genes, vii, viii, x, xii, 1, 5, 6, 7, 8, 9, 11, 14, 15, 16,
17, 18, 19, 20, 37, 38, 39, 45, 55, 58, 62, 65, 71,
81, 85, 86, 87, 92, 97, 106, 107, 109, 121, 122,
123, 125, 128, 130, 133, 134, 136, 140, 142, 147,
148, 149, 150, 152, 153, 164, 166, 181, 182, 185,
186, 187, 188, 189, 191, 192, 193, 195, 196, 198,
199, 201, 203, 208, 209
genetic background, 68, 76
genetic disease, 117
genetic disorders, 86, 148
genetic diversity, 20
genetic factors, 36, 58
genetic mutations, 123
genetic screening, 186, 198
genetics, 14, 21, 48, 49, 52, 55, 74, 75, 76, 77, 78,
79, 81, 82, 112, 142, 143, 144, 163, 182, 183,
184, 200, 201, 202, 203, 204, 205, 207, 208
genome, 44, 62, 66, 70, 77, 81, 117, 128, 137, 139,
173, 174, 186, 187, 188, 193, 194, 201, 202, 203,
204, 205, 207
genomic regions, 193, 201
genomics, 202, 206
genotype, 52, 123, 187, 208
geotaxis, 138, 197, 201, 202, 208
germ cells, 188
germ layer, 132
germ line, 174
Germany, x, 39, 121, 142
gerontology, 201
gestation, 18
glaucoma, 126
glia, ix, 45, 61, 71, 98, 133, 163, 177
glial cells, 1, 21, 60, 61, 62, 63, 109, 132
globus, 149
glomerulonephritis, 127
glucose, 39
glutamate, 12, 37, 40, 41, 42, 60, 77
glutamic acid, 201
glutamine, xi, 147, 148, 150, 151, 152, 153, 154,
156, 167, 168, 169, 195
glycine, 8, 61, 62, 64, 80
glycoproteins, 106
glycosylation, 12
Golgi, 5, 7, 11, 14, 39, 53, 95, 102, 104, 105, 115,
116
grants, xiii, 198
granules, 61, 63, 65, 67, 73
gravity, 197, 202, 203
growth, viii, xii, 11, 12, 35, 40, 44, 49, 68, 69, 76,
77, 82, 88, 100, 102, 106, 107, 110, 113, 125,
126, 129, 134, 157, 177, 178, 199, 210
growth arrest, 126
growth cones, 100
growth factor, 77, 82
growth rate, 100
GTPase, 12, 15, 34, 87, 95, 96, 111, 115, 119, 126,
136, 202
GTPases, 12, 91, 112, 116
guanine, 7, 25, 33, 54, 55, 91, 103, 113, 115, 118,
126, 207
guanine exchange factor (GEF), 91, 103
guidance, viii, 35, 44, 51, 54, 106, 113, 191, 193,
203, 204
gynecomastia, 3, 18
H
hairless, 200
hairpins, 96
HDAC, 159, 161
health, 197, 198
hearing loss, 99, 125, 126
heart disease, 184
heat shock protein, 11, 16, 20, 27, 105, 148, 152,
159, 160, 163, 165, 167
heat shock protein 60 (HSP60), 11, 105
hereditary spastic paraplegia (HSP), vii, 4
heterogeneity, x, 30, 85, 86, 145
histone, 71, 155, 168
histone deacetylase, 71, 155
histones, 155
history, vii, 145, 185
HIV-1, 61
homeostasis, 9, 19, 39, 41, 49, 90, 115, 118, 157,
159, 180
homologous chromosomes, 189
hormone, 150, 153, 154, 158, 159, 164, 169
host, 58
hot spots, 188
hotspots, 108, 109
HSP60, 11, 89, 93, 105
hub, 112
human, vii, ix, x, xi, 21, 27, 33, 35, 39, 40, 41, 45,
47, 48, 52, 53, 54, 57, 59, 62, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 76, 79, 80, 82, 83, 85, 86, 95,
99, 100, 105, 106, 110, 111, 112, 114, 117, 123,
Index 216
124, 128, 133, 136, 139, 140, 143, 144, 146, 148,
150, 153, 156, 159, 162, 163, 164, 165, 168, 171,
174, 176, 178, 182, 183, 184, 185, 186, 200, 201,
202, 204, 206, 208, 209
human brain, 143
human genome, vii
human immunodeficiency virus, 79
humidity, 197
hydrocephalus, 26, 106, 113
hydrogen, 58, 197
hydrogen peroxide, 58, 197
hydrolysis, 118
hydrops, 3
hyporeflexia, 14
hypothesis, 37, 44, 57, 59, 60, 61, 70, 71, 128, 153
I
ideal, 151, 161
identification, ix, 9, 16, 37, 59, 61, 62, 65, 109, 142,
183, 189, 192, 196, 208, 209
identity, 66, 92, 93
image, 131, 195
images, 150, 195
immune system, 21
immunoglobulin, 25, 40, 108
immunoglobulin superfamily, 108
immunohistochemistry, 197
immunophilins, 167
immunoreactivity, 81
impairments, 86
in utero, 16, 172
in vitro, 41, 58, 60, 96, 140, 141, 160, 163, 164, 173,
183, 207
in vivo, vii, 33, 41, 58, 61, 66, 70, 78, 81, 96, 123,
141, 146, 150, 156, 164, 186, 193, 206, 207
incidence, 58, 172, 194
India, 198
individuals, 37, 57, 77, 122
inducer, 81, 197
induction, 47, 54, 76, 99, 158, 169, 201
infant mortality, 18, 172
infantile-onset ascending hereditary spastic
paraplegia (IAHSP), 103
infants, 2, 172
ingestion, 153
inheritance, 6, 11, 18, 122, 124, 127, 128, 133
inherited disorder, 9, 18
inhibition, 4, 43, 61, 99, 102, 158, 160, 161, 169
inhibitor, 47, 70, 96, 111, 165, 169, 203
initiation, 151
injury, viii, ix, 20, 36, 43
inositol, 11, 19, 39, 70, 77
insect(s), 105, 146, 177, 202
insertion, 130, 136, 154, 174, 188, 193, 209
integration, 75, 168
integrity, 32, 39, 86, 97, 107, 210
Intellectual Disability, 21
intellectual impairment, 86
interface, 115, 141, 145
interference, 203, 204
interneuron, 138, 139
interneurons, 3, 132, 180
interphase, 182
intervention, 46
intron, 130
introns, 62, 65
inventions, 144
ions, 6
iron, 99
iron accumulation, 99
Islam, 106, 111, 112
isolation, 187, 188, 191, 192
issues, 189
Italy, 78
J
Japan, 47
Jordan, 165, 197, 202
K
KIF1A, 88, 92, 100, 101, 110, 113, 114
KIF5A, 10, 12, 15, 30, 33, 88, 92, 100, 110, 114, 116
KIF5B, 100
KIF5C, 100, 113
kinase activity, 209
kinesin, 10, 13, 30, 112, 116, 201, 205
kinesin-I, 100
kinetics, 210
L
L1CAM, 9, 12, 90, 93, 106, 111
labeling, 209
laminar, 3
larvae, xii, 95, 100, 131, 154, 174, 177, 196, 197
larval development, 182
larval stages, 174, 176, 177
lateral sclerosis, vii, 1, 2, 3, 4, 17, 24, 36, 48, 50, 57,
58, 73, 74, 76, 77, 78, 103
LDL, 49
Index 217
lead, viii, ix, xi, 9, 19, 37, 40, 46, 58, 61, 62, 85, 86,
97, 99, 100, 101, 102, 106, 121, 123, 124, 153,
158, 159, 162, 172, 177, 178, 180, 198
learning, 47, 199, 203
legs, 16, 139, 177, 178
lending, 148
lesions, 54, 187, 188
leucine, 104, 130, 135
leukemia, 165
leukocytes, 156
leukodystrophy, 99, 105, 114
life cycle, 128, 173
life expectancy, 57, 172
lifetime, 189
ligand, viii, xi, 35, 42, 43, 44, 54, 109, 119, 147, 151,
152, 153, 154, 155, 159, 195, 201, 209
light, viii, xii, 13, 16, 100, 129, 134, 149, 195, 196,
197
limb weakness, 3
lipid metabolism, viii, 2, 9, 10, 11, 17, 20, 46
lipids, 14, 39, 86, 90, 118
lipodystrophy, 98, 107, 111, 117
liposomes, 96
liver, 14, 21, 99
liver failure, 99
localization, xi, 40, 51, 62, 63, 67, 69, 76, 77, 102,
104, 109, 115, 116, 119, 121, 127, 140, 151, 152,
154, 155, 156, 157, 165, 178, 181, 182
loci, x, 6, 11, 57, 58, 59, 85, 104, 122, 174, 188, 192,
193, 201
locomotor, 11, 12, 15, 17, 59, 66, 68, 69, 71, 82, 141,
199, 200, 201, 208
locus, 21, 31, 48, 58, 118, 135, 176, 200
longevity, 66, 209
Lou Gehrig's disease, 4
lumen, 98, 159
lunapark/Lnp-1, 101
lymphocytes, 124
lysine, 157, 163
lysosome, 7, 21, 158
M
machinery, ix, 15, 62, 63, 75, 85, 86, 96, 143, 155,
157, 159, 167, 194
majority, vii, 8, 11, 61, 62, 64, 66, 122, 132, 133,
172, 174, 176, 177
mammalian cells, 50, 66, 102, 172
mammals, 42, 95, 96, 128, 148
man, 24, 114
management, 145
manipulation, xi, 45, 123, 128, 146, 147, 165
mapping, 23, 187, 192, 199, 200
MASA (Mental retardation, Aphasia, Shuffling gait
and Adducted thumbs) syndrome, 106
maspardin, 11, 89, 93, 105
mass, 41, 205
mass spectrometry, 41
matrix, 11, 105, 112, 125
measurement, 195
median, 14
medical, 52, 182, 183, 184
medicine, vii, 36, 48, 50, 54, 201, 209
membranes, 7, 96, 115, 118
memory, 79, 199
menadione, 197
mental retardation, 11, 61, 65, 77, 82, 95, 98, 127
mentorship, 162
mesoderm, 179
messenger RNA, 74
metabolism, ix, 6, 8, 10, 14, 39, 41, 50, 53, 57, 58,
59, 62, 74, 89, 90, 93, 98, 156, 163
metabolites, 43
metamorphosis, 132, 174, 178
metaphase, 178
meter, 86
methodology, 188, 189, 191, 192, 194, 195
methylene blue, 169
Mg2+, 101, 111
mice, 2, 6, 7, 11, 12, 14, 15, 16, 19, 21, 23, 24, 25,
27, 31, 33, 37, 42, 45, 47, 53, 54, 59, 62, 75, 81,
82, 86, 100, 109, 116, 128, 129, 130, 137, 140,
141, 156, 158, 159, 160, 162, 165, 168, 170, 178,
179, 180, 183, 207
microcephaly, 29
microinjection, 40
microRNA, 8, 77
microscope, 149
microtubule (MT), 86
migration, 12, 42, 106
miniature, 40
mitochondria, viii, 11, 15, 24, 35, 41, 43, 44, 47, 52,
55, 58, 87, 100, 101, 102, 103, 105, 113, 115,
116, 118, 123, 125, 126, 127, 156, 158, 202, 205,
208, 209
mitochondrial DNA, 43
mitosis, 116, 190
mixing, 43
model system, x, 2, 57, 58, 59, 65, 107, 148, 180
modelling, vii, 20, 65, 71, 72, 99, 124, 130, 148, 180
models, vii, viii, ix, xi, xii, 20, 27, 32, 43, 45, 54, 59,
60, 65, 67, 68, 70, 71, 72, 76, 77, 79, 80, 81, 97,
119, 121, 123, 124, 128, 129, 135, 136, 141, 142,
144, 147, 150, 151, 152, 153, 154, 155, 156, 158,
159, 160, 161, 162, 165, 166, 167, 176, 180, 186,
187, 197, 198, 199, 201, 203, 205, 207, 209
Index 218
molecular biology, 20, 47, 55, 202
molecular medicine, 81, 204
molecular oxygen, 58
molecular weight, 8
molecules, 65, 74, 109, 157, 159, 161
monomers, 148
morphogenesis, 12, 15, 52, 78, 115, 116
morphology, xii, 11, 12, 15, 16, 37, 43, 44, 62, 68,
69, 76, 94, 95, 118, 133, 136, 139, 141, 142, 144,
150, 192, 195, 196, 197
mortality, 59, 82, 171
mosaic, 174, 186, 187, 191
mother cell, 132
motif, 8, 13, 27, 39, 50, 51, 62, 64, 91, 130, 135,
164, 173
motor activity, 100
motor behavior, 24, 109
motor control, 4
motor neuron degeneration, vii, viii, ix, xii, 7, 14, 15,
19, 20, 21, 25, 30, 43, 48, 59, 68, 75, 106, 165,
168, 169, 207
motor neuron disease, 4, 6, 27, 30, 33, 48, 50, 55, 57,
58, 59, 61, 62, 71, 72, 79, 116, 156, 167, 185,
186, 198
motor neurons, vii, viii, ix, xi, 1, 2, 4, 6, 12, 14, 19,
20, 22, 35, 36, 42, 43, 44, 45, 49, 53, 58, 60, 61,
66, 67, 68, 69, 72, 81, 86, 98, 113, 128, 131, 132,
138, 139, 140, 149, 151, 154, 156, 172, 176, 180,
183, 186, 196
motor system, xii, 85, 197
mRNA, ix, 8, 19, 29, 30, 46, 61, 62, 63, 65, 71, 73,
76, 77, 80, 144, 155, 177, 181, 184, 194
mRNAs, 19, 61, 65, 67, 78, 178
mtDNA, 51
muscle atrophy, xii, 3, 18, 36, 58, 59
muscles, 14, 18, 43, 49, 96, 128, 131, 132, 137, 138,
139, 151, 172, 174, 176, 180, 193, 196
muscular dystrophy, 32
muscular tissue, 179
mutagen, 187
mutagenesis, xii, 174, 185, 187, 188, 189, 202
mutant, viii, ix, x, xi, 2, 6, 12, 15, 16, 18, 19, 22, 23,
25, 26, 27, 32, 33, 35, 37, 40, 41, 43, 44, 45, 46,
48, 51, 52, 54, 59, 63, 65, 66, 67, 68, 69, 75, 76,
77, 78, 81, 82, 85, 86, 95, 98, 100, 106, 113, 114,
121, 128, 129, 133, 135, 137, 138, 139, 140, 141,
142, 143, 144, 145, 147, 148, 153, 154, 155, 157,
159, 160, 162, 163, 174, 176, 177, 182, 186, 187,
188, 189, 190, 191, 192, 193, 195, 201, 202, 204,
209
mutant proteins, x, 37, 121, 142, 148
mutation, viii, x, 7, 19, 21, 23, 28, 31, 32, 35, 37, 39,
40, 41, 43, 45, 48, 50, 51, 52, 53, 55, 59, 61, 64,
73, 76, 81, 97, 99, 104, 110, 111, 112, 114, 115,
116, 117, 128, 133, 137, 141, 143, 145, 146, 174,
176, 177, 187, 188, 189, 190, 200, 202, 204, 206
myelin, x, 14, 15, 16, 23, 27, 28, 86, 90, 99, 106,
110, 111, 114, 115, 119, 123, 125, 126, 127, 129,
133, 134
myelin metabolism, 111
myelin sheath, x, 86, 99, 106, 119
myelination, viii, x, 2, 14, 15, 28, 123
myocardial infarction, 48
myopathy, 70, 129
myosin, 40, 52
N
NAD, 210
National Academy of Sciences, 53, 54, 73, 74, 76,
77, 79, 118, 182, 183, 199, 202, 203, 204, 206,
207, 208, 210
necrosis, 129, 134
negative effects, 95
nematode, 39, 47, 55
nerve, 4, 14, 98, 122, 128, 131, 132, 133, 183
nervous system, viii, x, 23, 30, 43, 44, 71, 98, 109,
117, 128, 131, 177, 178
networking, 52
neural development, 119
neurobiology, 47, 75, 107, 200
neuroblastoma, 140
neuroblasts, 132, 177
neurodegeneration, xi, 12, 15, 19, 20, 47, 50, 59, 70,
73, 74, 75, 78, 81, 82, 98, 99, 108, 114, 147, 148,
149, 158, 160, 161, 163, 164, 166, 167, 168, 169,
170, 185, 187, 191, 195, 196, 199, 200, 201, 205,
206, 208, 209, 210
neurodegenerative diseases, 7, 46, 47, 60, 124, 148,
154, 160, 201, 204
neurodegenerative disorders, vii, xi, 60, 70, 71, 169
neurofilaments, 16
neurogenesis, 132, 176, 177, 178, 180
Neuroglian, 109, 111
neurological disease, 1, 2, 14, 36, 145, 185, 198
neurologist, viii, 36
neuromuscular function, vii, 116
neuromuscular junction (NMJ), 95
neuronal cells, 96
neuronal circuits, xii, 50
neuronal stem cells, 132
neurons, vii, ix, x, xii, 1, 3, 4, 6, 11, 12, 15, 16, 19,
36, 42, 44, 48, 59, 60, 61, 62, 63, 65, 66, 67, 68,
69, 71, 82, 85, 86, 98, 100, 107, 111, 114, 116,
118, 121, 122, 123, 124, 128, 131, 132, 138, 139,
140, 141, 142, 143, 145, 146, 149, 153, 154, 156,
Index 219
159,165, 177, 178, 179, 180, 184, 191, 194, 195,
196, 202, 209
neuropathy, viii, x, xi, 1, 2, 3, 4, 14, 17, 18, 20, 23,
24, 26, 27, 28, 31, 33, 34, 98, 108, 115, 116, 118,
119, 121, 122, 123, 126, 137, 140, 141, 143, 144,
145, 146, 176, 180, 208
neuropathy target esterase (NTE), 98
neuropeptides, 108
neurophysiology, 54
neuroscience, xii, 36, 46, 48, 52, 54, 73, 78, 79, 80,
81, 82, 200, 203, 206, 208, 209
neurosecretory, 117
neurotoxicity, 70, 75, 76, 77, 78, 155, 163, 169, 201,
202, 208
neurotransmission, 180, 187
neurotransmitter, 191
neurotransmitters, x, 128
neurotrophic factors, 8
next generation, 187, 189
NH2, 164, 169
NIPA1, 9, 11, 30, 88, 92, 94, 101, 102, 107, 108,
111, 118
NMDA receptors, 8, 48, 49
NTE, 90, 93, 98, 113
nuclear membrane, 48
nuclear stability, 125
nuclei, 3, 148, 149
nucleocytoplasmic shuttling, ix, 63, 70
nucleolus, 183
nucleoplasm, 125
nucleoprotein, 8
nucleus, xi, 8, 19, 61, 63, 67, 70, 109, 123, 149, 152,
153, 156, 157, 159, 160, 168, 173, 177
null, 7, 43, 66, 69, 95, 129, 174, 176, 179, 180
nutrient, 158, 181
nutrients, 156, 158
O
obesity, 74, 168
oligodendrocyte, 14
oligodendrocytes, 14, 99, 127
oligomerization, 113, 119, 174
oligomers, 96, 117, 148
omission, 198
ommatidium, 149, 195
oocyte, 40, 42, 48, 49, 52, 55, 177
oogenesis, 119, 181
organ, 109, 140, 149, 202
organelle(s), 6, 11, 15, 20, 43, 86, 102, 108, 156,
157, 158, 195
organism, vii, x, 15, 46, 66, 69, 121, 147, 161, 174,
176, 189
organs, 140, 148
orthologue, viii, ix, 66, 95, 96, 97, 98, 99, 100, 101,
102, 103, 104, 105, 106, 115, 124, 135, 153, 173,
185, 186, 193, 196, 197, 204
ovaries, 176
overlap, 15, 16, 18, 42, 58
ovulation, 52
ox, 171
oxidative damage, 6, 60
oxidative stress, 1, 7, 20, 58, 75, 197, 202, 207
oxygen, 204, 205
P
p53, 156
Pak3, 95
palliative, 58
paralysis, vii, 3, 4, 12, 16, 58, 110, 151, 172
paraplegin, 11, 24, 89, 93, 105
paresis, 6, 125
parkinsonism, 7
pathogenesis, 11, 17, 20, 21, 27, 35, 36, 39, 42, 43,
44, 45, 46, 55, 74, 80, 123, 124, 128, 141, 142,
147, 148, 151, 153, 154, 155, 156, 161, 167, 204
pathology, viii, ix, xi, 4, 6, 7, 8, 15, 22, 35, 36, 37,
40, 41, 43, 45, 46, 54, 57, 58, 59, 60, 67, 68, 69,
71, 72, 94, 95, 101, 104, 144, 147, 148, 150, 160,
163, 170, 174, 178, 186
pathophysiological, 66
pathophysiology, viii, ix, xii, 67, 68, 72, 167
pathways, vii, viii, ix, x, xi, 1, 3, 6, 11, 12, 20, 22,
35, 36, 37, 39, 40, 41, 42, 44, 46, 59, 65, 70, 71,
72, 85, 103, 111, 123, 124, 128, 131, 147, 157,
159, 161, 163, 185, 186, 191, 193, 197, 198, 200,
201, 208
Pelizaeus-Merzbacher disease (PMD), 106
peptide chain, 157
peptides, 166
peripheral nervous system, 111, 114
peripheral neuropathy, 14, 16, 21, 123, 129, 130,
137, 140, 141, 142, 143, 145
permeability, 49
pharmacological treatment, 128
pharmacology, 201
PHB, 91
phenocopy, 173
phenotype(s), ix, xii, 1, 11. 14. 8, 21, 23, 29, 30, 36,
40, 42, 43, 44, 51, 52, 65, 66, 67, 68, 69, 70, 71,
74, 86, 95, 96, 99, 100, 103, 104, 106, 111, 113,
115, 123, 124, 125, 126, 127, 128, 133, 137, 139,
140, 141, 142, 143, 149, 150, 154, 155, 156, 158,
159, 160, 162, 163, 170, 172, 174, 176, 177, 178,
Index 220
179, 180, 185, 186, 187, 188, 191, 192, 194, 195,
197, 198, 199, 200, 201, 206
phosphate, 17, 39
phosphatidylcholine, 98, 115, 119
phospholipids, 14
phosphorylation, 48, 58, 61, 82, 173, 183
phototaxis, 106, 197
physical interaction, 44, 173
physiological psychology, 202
physiology, vii, viii, xi, xii, 124, 167, 202
placebo, 164
plasma membrane, 12, 15, 50, 91, 102, 123, 126
plasmid, 144
plasticity, 201
platform, 144, 203
playing, 9
PLP, 91, 106
PLP1, 9, 14, 90, 93, 106
PLS, vii, 2, 3, 4, 6, 17, 103
point mutation, 35, 94, 129, 174, 176, 187
polarization, 196
polymerase, 74, 173
polymorphism(s), 44, 76, 139
polypeptide, 9, 129, 134
population, xi, 51, 78, 94, 136, 151, 172, 187, 188
pregnancy, 18
premature death, 2, 14, 18
preparation, 196
priming, 152
principles, vii, x, 128, 142
private sector, 47
probe, 153, 161
progenitor cells, 132
progressive bulbar palsy (PBP), vii, 4
progressive muscular atrophy (PMA), vii, 4
progressive neurodegenerative disorder, 35
project, 3, 107, 128, 132, 139, 142, 199, 207
proliferation, 8, 43, 124, 182
proline, viii, 37, 41
promoter, 21, 65, 114, 150, 153, 162, 174, 176, 191,
192
propagation, 20
proteasome, ix, 5, 6, 9, 61, 70, 72, 125, 152, 157,
158, 159, 160, 161, 169
protective role, 60
protein chaperone activity, x, 20, 123
protein folding, ix, 70, 72, 89, 105, 159
protein kinases, 91
protein misfolding, 16, 60
protein synthesis, x, 62, 121, 159
protein-protein interactions, 9, 128, 141
proteolipid protein, 9, 14, 27, 31, 91, 106, 111
proteolipid protein 1 (PLP), 106
proteolysis, 89
proteome, 75
proteostasis, viii, 2, 9, 16, 20, 60, 159
protrudin, 51, 89, 93
pruning, 30
pupa, 178
purification, 62, 63, 164
pyramidal cells, vii, 3
pyrimidine, 127
pyrophosphate, 130, 135, 137
Q
quality control, xi, 11, 147, 157, 159, 161, 162
R
Rab, 15
radicals, 58
reactions, 89
reactive oxygen, 6, 152, 156, 203
reactivity, 33
reading, 142, 174, 176, 198
receptors, viii, 11, 28, 35, 37, 40, 41, 42, 43, 44, 46,
49, 51, 54, 102, 114, 116, 153, 159, 169, 209
recognition, 8, 27, 61, 64, 136, 139, 158
recombination, 135, 189, 190, 192, 202
recommendations, iv
recruiting, 112
recycling, 126, 157, 158
redistribution, 70
redundancy, 97, 167
REEP1, 10, 12, 17, 18, 22, 34, 94, 97, 115, 120
reflexes, 4, 36, 122
regeneration, 96, 111, 122
relaxation, 132
relevance, 45, 97, 123, 130, 140, 165, 177
remodelling, 16, 44
remyelination, 122
repair, 8, 187
repellent, 44
replication, 190
repression, 61, 65, 152, 164
repressor, 61, 78
requirements, 128, 151
researchers, xii, 149, 178, 186, 188, 193
residues, 39, 94, 101, 103, 104, 133, 139, 159, 160
resistance, 125
resolution, 47, 50, 179, 186, 200, 205
respiration, 43
respiratory failure, 172
Index 221
response, 19, 44, 45, 47, 53, 54, 61, 66, 67, 98, 99,
129, 134, 139, 154, 159, 160, 168, 195, 196
restoration, 178, 179, 180
retardation, 106, 178
reticulon-2, 94
reticulum, viii, x, 33, 35, 36, 41, 47, 48, 49, 52, 53,
54, 55, 85, 86, 108, 114, 115, 117, 118, 159, 167,
168
retina, 70
ribosomal RNA, 156
ribosome, 115
risk, 24, 57, 60, 70, 75, 128, 151, 163, 183
risk assessment, 183
RNA, viii, ix, 2, 5, 6, 8, 17, 18, 19, 20, 23, 27, 30,
32, 37, 57, 58, 59, 60, 61, 62, 63, 64, 66, 67, 70,
71, 73, 74, 75, 76, 78, 80, 81, 122, 147, 150, 155,
156, 165, 167, 170, 171, 173, 177, 178, 180, 181,
182, 203, 204
RNA processing, viii, ix, 2, 5, 6, 8, 17, 18, 63, 64,
81, 147, 150, 155
RNA splicing, 8, 61, 75, 155, 170, 171
RNAi, xii, 31, 62, 65, 68, 69, 77, 81, 95, 117, 135,
140, 176, 185, 192, 193, 194, 201, 203, 204, 205,
207
RNAs, 62, 65, 123, 156, 177, 180, 184
rodents, 57, 59
root, 149
roughness, 106, 149
routes, 201
S
safety, 164
salivary gland, 98, 154, 169
salivary glands, 169
science, 47, 51, 205
secretion, viii, 20, 41, 45, 46, 109
secretory vesicles, 100
segregation, 182, 190
seipin, 12, 33, 89, 93, 98, 110, 112, 114, 117, 118,
119
selectivity, 163
self-assembly, 167
sensing, 52, 202, 203
sensitivity, 197, 202, 205
sensory systems, 142
sequencing, 50, 61, 62, 71, 76, 110, 187, 209
serine, viii, 8, 37, 41
serum, 41
services, 64
sex, 133, 165
shape, 94, 120, 196
shock, 10, 13, 17, 24, 26, 129, 134, 152, 159, 160,
162, 168, 191
short-term memory, 207
showing, 46, 95, 131, 138, 139, 154, 177
siblings, 183
signal transduction, 157, 159
signaling pathway, 48, 205
signalling, viii, x, 7, 9, 11, 15, 18, 19, 36, 40, 41, 42,
43, 44, 45, 47, 52, 65, 85, 86, 88, 94, 96, 97, 101,
102, 103, 107, 118, 156, 159, 167, 191, 193, 197,
204
signals, 44, 52, 64, 97, 111, 128, 156, 158
signs, 4
skeletal muscle, 3, 4, 11, 43, 49, 51, 55, 151, 159,
170
skin, 124
SLC33A1, 10, 28, 89, 93, 99, 112, 114, 116
SNP, 200
sodium, 197
solubility, 160, 163
solution, 194
spartin, 11, 31, 90, 93, 94, 102, 108, 109, 110, 116,
118
spastic, vii, x, 1, 2, 3, 4, 6, 9, 10, 11, 12, 21, 22, 23,
24, 26, 29, 30, 31, 32, 34, 85, 86, 102, 103, 104,
106, 107, 108, 109, 110, 111, 112, 113, 114, 115,
116, 117, 118, 119, 209
spastic paraplegia gene (SPG), 85
spasticity, x, 3, 4, 12, 86
spastin, 12, 32, 87, 89, 92, 94, 95, 96, 97, 102, 108,
109, 110, 114, 115, 116, 117, 118, 119, 207, 209
spastizin, 11, 89, 92, 103, 104, 115
spatacsin, 7, 11, 32, 88, 92, 103, 104, 115, 117
species, viii, 6, 20, 38, 39, 148, 152, 156, 159, 187,
203
spectroscopy, 50
sperm, viii, 35, 36, 39, 40, 42, 43, 47, 50, 52, 55
SPG1, 9, 26, 90, 93, 106, 113
SPG10, 10, 30, 88, 92, 100, 116
SPG11, 5, 7, 10, 32, 88, 92, 103, 117
SPG12, 10, 87, 92, 96
SPG13, 10, 25, 89, 93, 105, 112
SPG15, 10, 25, 89, 92, 103, 104, 111
SPG17, 10, 89, 93, 98
SPG18, 10, 21, 90, 93, 105
SPG2, 9, 90, 93, 106
SPG20, 10, 30, 90, 93, 102, 108, 110, 113, 116
SPG21, 10, 31, 89, 93, 105
SPG30, 88, 92, 100, 101, 113
SPG31, 10, 87, 92, 97
SPG33, 89, 93
SPG35, 10, 24, 90, 93, 99, 110
SPG39, 10, 90, 93, 98
Index 222
SPG3A, 9, 87, 92, 95, 115, 119
SPG4, 9, 87, 92, 94, 95, 108, 111, 114, 117
SPG42, 10, 28, 89, 93, 99, 114
SPG47, 10, 22, 104
SPG48, 10, 31, 103, 104, 117
SPG5A, 9, 89, 93, 99
SPG6, 9, 30, 88, 92, 101, 102, 107, 111
SPG7, 10, 89, 93, 105
SPG8, 10, 32, 88, 92, 102, 118
sphingolipids, 99, 119
spinal cord, vii, 3, 4, 6, 9, 12, 19, 36, 45, 47, 53, 55,
62, 94, 128, 140, 151, 172
spinal muscular atrophy (SMA), vii, xi, 4, 20, 183,
193
spindle, 16, 110
spine, 62, 76
spinobulbar muscular atrophy (SBMA), vii
spliceosomal Uridine-rich small nuclear
ribonucleoprotein biogenesis, xi
stability, 24, 61, 71, 113, 118, 165, 209
stabilization, 63
state, 137, 142, 151, 155, 159, 161, 177, 196
stem cells, 124, 145, 177, 178
sterile, 13, 130, 135, 188
steroids, 89, 99
sterols, 14
stimulation, xi, 138, 139
stimulus, 195, 196
stock, 188, 193
stoichiometry, 19
storage, 61, 98
strategy use, 188
stress, viii, ix, 5, 8, 12, 16, 33, 35, 37, 41, 45, 46, 47,
49, 52, 53, 60, 61, 63, 65, 67, 74, 75, 76, 78, 79,
80, 81, 96, 101, 125, 160, 167, 169, 181, 197, 205
stress granules, 8, 61, 63, 65, 67, 74, 75, 76, 78, 80
stress response, ix, 37, 45, 160, 167
structural protein, 14
structure, vii, 16, 39, 44, 47, 51, 69, 106, 115, 118,
125, 128, 138, 143, 149, 157, 177, 180, 183, 184,
195, 209
strumpellin, 11, 88, 92
substitution, 139
substitutions, 61, 95
substrate(s), 70, 99, 157, 159, 161, 163, 164
suppression, xii, 163, 165, 187, 192, 199, 201, 203,
207
surface area, 94
surveillance, 184
survival, xi, 19, 20, 22, 27, 43, 47, 50, 60, 66, 68, 69,
72, 75, 81, 82, 100, 159, 160, 171, 172, 174, 178,
181, 183, 196, 197, 205, 209
susceptibility, 1, 20, 42, 59, 153, 200, 203
swiss cheese, 98, 108, 113
symptoms, 4, 11, 85, 86, 99, 106, 122, 172
synapse, 3, 45, 49, 62, 68, 69, 70, 78, 86, 101, 106,
111, 113, 115, 139, 144, 177, 197, 198, 201, 206,
210
synaptic plasticity, 61, 79
synaptic strength, 95
synaptic transmission, 39, 111, 199
synaptic vesicles, 100, 101, 128
synaptogenesis, 193, 203
syndrome, viii, 1, 2, 3, 4, 9, 17, 26, 29, 30, 31, 33,
36, 65, 74, 79, 98, 99, 106, 108, 111, 113, 117,
119, 145
synthesis, xi, 39, 114, 121, 169, 210
T
Taiwan, 145
target, 4, 10, 14, 19, 65, 71, 78, 98, 105, 108, 113,
114, 115, 116, 118, 119, 137, 147, 149, 157, 158,
159, 160, 161, 188, 193, 194, 201
tau, 5, 7, 26, 165
TCC, 103, 104
techniques, 36, 174
technologies, 146
technology, 124, 187, 191
telangiectasia, 206
telomere, 172
temperature, 192, 197
temporal lobe, 50
temporal lobe epilepsy, 50
terminals, 16, 19, 40, 68, 71, 99, 106
testing, 2, 72, 183
testis, 73, 78, 96, 178
testosterone, 19, 151, 152, 154, 157
tetanus, 107
therapeutic agents, 123
therapeutic approaches, viii, xi
therapeutic interventions, 2
therapeutic targets, xi, 20, 95, 121, 123, 128, 142,
147, 157, 161, 186
therapeutics, xii, 32, 59, 151, 165, 201
therapy, ix, 26, 36, 47, 75, 169
thorax, 131, 132, 140, 176, 190, 191
thyroid, 169
time frame, 81
tissue, xii, 6, 33, 65, 71, 78, 123, 136, 149, 155, 177,
179, 189, 190, 191, 192, 194, 206, 207, 208
topology, 99, 114
toxic effect, xi, 139, 147, 153
toxicity, xi, 6, 8, 22, 32, 37, 48, 54, 60, 66, 67, 68,
69, 70, 76, 77, 79, 81, 83, 94, 138, 147, 150, 151,
Index 223
152, 153, 154, 155, 158, 161, 163, 165, 166, 167,
187, 195, 199, 200, 201, 202, 203, 205
toxin, 107
trafficking, viii, ix, x, xi, 2, 5, 6, 7, 9, 10, 11, 13, 15,
16, 17, 18, 24, 25, 39, 40, 49, 52, 85, 86, 87, 88,
89, 92, 93, 101, 102, 108, 109, 111, 113, 115,
119, 123, 147, 152, 156, 167, 168, 169, 204, 206
traits, 196
trajectory, 44
transcription, 8, 18, 48, 62, 63, 65, 125, 126, 136,
147, 149, 151, 154, 155, 156, 160, 168, 169, 173,
192, 201
transcription factors, 155
transcriptional regulation, ix, x, 8, 65, 88, 123, 125,
166
transcripts, 63, 67, 71, 155, 181
transduction, 204
transformation, 202
transgene, 54, 136, 138, 139, 140, 144, 176, 192
translation, ix, 15, 16, 18, 19, 45, 61, 62, 63, 65, 75,
78, 109, 125, 126, 127, 136, 140, 141, 143, 158,
182, 194, 200
translocation, 154
transmission, x, 39, 128, 188
transport, viii, ix, x, 2, 6, 7, 10, 11, 12, 13, 15, 16,
17, 18, 19, 20, 24, 25, 32, 33, 39, 43, 50, 51, 52,
53, 58, 60, 62, 63, 65, 71, 76, 85, 86, 88, 89, 90,
92, 94, 97, 100, 101, 102, 105, 107, 108, 110,
111, 112, 113, 114, 115, 117, 118, 123, 125, 126,
150, 155, 156, 164, 165, 166, 168, 177, 178, 184,
202, 205, 208
transport processes, 71
transportation, 100
treatment, 47, 58, 115, 147, 151, 158, 160
tremor, 130, 151
trial, 119, 163, 164
triggers, 7, 14, 51
Troyer Syndrome, 102
turnover, 110, 159
tyrosine, xi, 42, 48, 49, 51, 54, 136, 137, 201, 207
U
ubiquitin, 9, 10, 16, 19, 37, 60, 102, 110, 112, 126,
148, 152, 157, 158, 160, 163, 164, 169, 208
ubiquitin-proteasome system, 10, 37, 157, 160, 163
underlying mechanisms, 71
unfolded protein response (UPR), 98, 159
United Kingdom (UK), x, xi, 85, 142, 171
United States, viii, xi, 21, 53, 54, 73, 74, 76, 77, 78,
79, 82, 83, 109, 116, 118, 165, 166, 167, 169,
182, 183, 198, 199, 202, 203, 204, 206, 207, 208,
210
University of Cambridge, x, 85, 107
V
vacuolation, 98
validation, 72
variations, 132, 133, 159, 193, 202
vascular endothelial growth factor (VEGF), 8
vector, 202
VEGF expression, 8
versatility, 176, 180
vertebrates, 43, 105, 128, 145, 174
vesicle, x, 7, 11, 15, 16, 20, 51, 52, 53, 54, 101, 108,
111, 112, 113, 115, 128, 158, 183, 202, 204, 206,
209
vision, 200, 205
vulnerability, xii, 30, 80
W
walking, 50, 69, 132
water, 204
weakness, vii, x, 3, 4, 6, 9, 11, 14, 16, 18, 19, 36, 86,
121, 122, 151, 172
wealth, 20, 46
worms, 38, 41, 42, 57, 59
X
X chromosome, 153, 188, 189, 199
X-inactivation, 153
X-linked hydrocephalus, 106, 113
Y
Y chromosome, 188, 189
YAC, 156, 168
yeast, 38, 39, 44, 50, 59, 72, 74, 79, 96, 110, 119,
136, 156, 160, 171, 183, 192, 202
yield, 186, 195, 197, 198
Z
ZFYVE27, 89, 93
zinc, 6, 8, 62, 79, 91, 104
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