microRNA: potenziali biomarkers

nelle malattie del motoneurone

Pia BernasconiU.O. Neurologia IV –

Neuroimmunologia e Malattie Neuromuscolari

Amyotrophic Lateral Sclerosis (ALS)

Fatal neurodegenerative disease characterized by the progressive loss of both upper andlower motor neurons.

Brown&Al-Chalabi N Engl J Med 2017 Al-Chalabi et al. Lancet Neurol 2016

Identification of multiple genes associated with ALS

About 10% of ALS cases are familial and the remaining cases are sporadic.

Brown&Al-Chalabi N Engl J Med 2017

Seven out of 25 ALS-associated genes are linked to RNA metabolism

microRNA biogenesis & ALS-associated genes

Hawley et al. Front Mol Neurosci 2017

Spinal muscular atrophy (SMA)

Autosomal recessive disease characterized by progressive loss of motor neurons and muscleatrophy. It is caused by homozygous deletion or mutation of survival motor neuron 1 (SMN1)gene, but all SMA patients carry one or more SMN2 copies, modulating disease severity.

Bowerman et al. Dis Model Mech 2017Wirth et al. Curr Opin Genet Develop 2013

SMN1 and SMN2 function and regulation of expression

Singh et al. Biochim Biophys Acta 2017

SMN1 is involved in RNA metabolism, trafficking of RNA–binding proteins, small nuclear RNP biogenesis, alternative splicing

microRNAs in motor neuron function

miR-9: promotes differentiation of neuralprogenitor cells into spinal motor neuronsmiR-218: establishes motor neuron identity

Hawley et al. Front Mol Neurosci 2017

Motor neuron differentiation

Cytoskeletal integritymiR-146a, miR-524, miR582, miR-b1336,and miR-b204: favor cytoskeletonmaintenance

NMJ Function and plasticity

miR-8: modulates NMJ connectivity,synaptic plasticitymiR-310, miR-313, miR-153: modulatesynaptic vesicle releasemiR-206: favors NMJ regeneration

Regeneration

miR-9, miR-124: contribute to development, regeneration, cell survival miR-375: contributes to development and cell survivalmiR-183: contributes to neurite growth

microRNAs involved in ALS pathogenesis

NO NGF

Motor neuron

Oligodendrocytes

Altered axon transport

Misfolded proteins

Mitochondrial dysfunction

Mitochondrial dysfunction

O2-O2-

Proteasome inhibition

Muscle atrophy and regeneration

Astrocyte

NO ROS

miR-124amiR-218miR-9

ER stress

Altered neuromuscular junctions

miR-206miR-1miR-133

miR-9miR-133miR-219

miR-155miR-365

miR-125a

Astrocyte

miR-146amiR-524-5pmiR-582-3p

miR-29a

miR-31miR-23miR-206miR-b1336miR-b2403

miR-125a

Microglia

AchR

Synaptic vesicles

IL1IL6

TNFα

AchR

Misfolded proteins

miR-125a

Boillée et al. Neuron 2006; Rinchetti et al. Mol Neurobiol 2018

Motor neuron

Oligodendrocytes

Altered axon transport

-

Muscle atrophy and regeneration

Astrocyte

miR-9

miR-124

miR-132

miR-431

miR-375

Altered neuromuscular junctions

miR ???

Astrocyte

miR-206

miR-2

microRNAs involved in SMA pathogenesis

miR-206

miR ???

miR-183

miR-431

miR ???

miR ???

Microglia

Magri et al. J Cell Mol Med 2018

G93A-SOD1 mice

• The G93A-SOD1 transgenic mouse model over-expresses the G93A mutated human SOD1 gene

• The onset of disease appears at 12th week of life with hind limb tremor; at about 18th week of life the animals present muscle paralysis and atrophy of hind limbs

G93A-SOD1 transgenic mouse model of ALS

Marcuzzo et al. Exp Neurol 2011, 2017

Marcuzzo et al. Exp Neurol 2014

Involvement of neural and cell cycle-related microRNAs in

neurogenesis in ALS?

miR-124a miR-9 miR-19a miR-19b

Nestin

Lu

mb

ar

spin

al

cord

The increase of nestin-positive cells in thelumbar spinal cord of G93A-SOD1 at week 18did not correlate with an increase ofneurogenesis confirmed by miRNA analysis.

G93A-SOD1WT-SOD1B6.SJL

Neurospheres

Differentation

Neurospheres epSPC

G93A-SOD1 mice

Modified from Xu et al. Stem Cell 2016

Oct4+ epSPC

Self-Renewal

Neurons

Oligodendrocytes

Motoneurons

Astrocytes

+Shh

+Ra

Neurospheres

GFAP

b-TUBIII

O4

Hb9/b-TUBIII

Neural stem cells

Neurons

Neurospheres

G93A-SOD1

Mimicking microRNA could induce neuronal differentiation and support motor neuron regeneration in vivo

miR-124a miR-9 miR-19a miR-19b

Marcuzzo et al. Exp Neurol 2014

Marcuzzo et al. Mol Brain 2015

Marcuzzo et al. Mol Brain 2015

Re-establishing microRNA expression to normal levels could be a new therapeutic approach to ALS

Altered expression of neural and cell cycle-related microRNAs in specific brain regionsmight contribute to ALS pathogenesis in G93A-SOD1 mice

iPSc

Marcuzzo et al. J Transl Sci 2018

miR-432miR-629miR-1289miR-520miR-330miR-376miR-657miR-451miR-155miR-490

Marcuzzo et al. J Transl Sci 2018

MicroRNAs as novel molecules for future development of patient-specific miRNA-based therapeutic strategies

MicroRNA profiling in iPSCs from an IAHSP patient reveals miR-451, miR-432, miR-376 as modulators of pluripotency and neuronal

differentiation

Altered expression of miR-451, miR-432 and miR-376 mightcontribute to motor neuron pathogenesis in the IAHSP patient

Marcuzzo et al. J Transl Sci 2018

Marcuzzo et al., manuscript in preparation

*

MKNK2 FOXJ 3JAG 1 SMURF 1

*

SOX 9 DLX2

miR-124 miR-219

miRNA/mRNA targets Spinal cord Kariya et al., PlosS One 2008

Altered expression of miR-124/SOX9 in both ALS and SMA animal models suggests a crucial

role of this microRNA/mRNA target pair in motor neuron death

Altered expression of miR-219/FOXJ3 in SMA animal model suggests a specific involvement of this microRNA/mRNA target pair

in SMA

miRNAs and skeletal muscle (myo-miRs)

Coenen-Stass et al. Trends Mol Med 2017

Pegoraro et al. J Neurol Sci 2017

myo-miRs are differentially expressed in ALS muscles

Pegoraro et al. J Neurol Sci 2017

Kovanda et al. Sci Reports 2018

• 13 ALS patients: 5 bulbar and 8 spinal symptoms at onset

• Mean age at biopsy: 56.7 years• Mean disease duration: 12.4 months

• 11 ALS patients (2 fALS): 3 bulbar and 8 spinal symptoms at onset

• Mean age at biopsy: 62.2 years• miRNAs are predicted to target proteins

involved in normal processes and various muscle disorders and indicate muscle tissue is undergoing active reinnervation/compensatory attempts.

miR

-13

3A

miR

-13

3B

miR

-1m

iR-2

06

miR

-20

6m

iR-1

33

Am

iR-1

33

Bm

iR-1

day 7 day 21

day 7 day 21

day 7 day 21

day 7 day 21

Onset

Pre-sympt

Sympt Pre-sympt

Sympt Pre-sympt

myo-miRs are differentially expressed in muscles of MND animal models

Biomarker: definition

A biological, genetic or biochemical factor that is objectively measuredand evaluated as an indicator of normal biologic processes,pathological processes or pharmacologic response to a therapeuticintervention.

A good biomarker must have a high prognostic and predictive value,i.e. be able to predict a disease, its progression or treatment efficacy.

The characteristics required for a good biological marker are:a)a specific correlation with the disease;b)adequate predictability on the type of treatment and the response;c)the possibility of carrying out the determination accurately, in a shorttime;d)be relatively insensitive to sampling errors.

Are miRNAs good biomarkers?

- They are ideal biomarkers because they are abundant, stable in storedbiofluid samples (blood, CSF), easy to discover and measure with feasibleprotocols (RNA-seq, qPCR);

- Changes of their expression levels might reflect a distinct cell physiologystate or damage to a specific organ/tissue (e.g. neurodegeneration,inflammation, muscle regeneration, fibrosis) (diagnostic biomarkers);

- Their expression levels can be assessed to monitor disease progression(e.g. poor versus better survival) (prognostic biomarkers) and to predictthe efficacy of a therapy (‘personalized medicine’) (predictivebiomarkers);

- Some miRNAs can be in common to different diseases (e.g. miR-124 ormiR-206 for ALS and SMA) or specific (e.g. miR-219 for SMA animalmodel).

Serum miRNAs discriminate between fALS or asymptomatic carriers and healthy controls

Freischmidt et al. Brain 2014

Matamala et al. Neurobiol Aging 2018

Matamala et al. Neurobiol Aging 2018

Validation of circulating miRNAs in mutant SOD1 Tg mice and …

Matamala et al. Neurobiol Aging 2018

G86R-SOD1

G93A-SOD1

… in ALS patients

Matamala et al. Neurobiol Aging 2018

miRNAs as therapeutic candidates

Therapies based on miRNAs are being developed based on:

- Inhibition of specific miRNA molecules if their expression levels arepathologically elevated (miRNA antagonists or antimiRs, single stranded RNA);

- Supplementation of those miRNAs whose cellular expression is insufficient orlacking (miRNA mimics, double-stranded small RNA).

Rupaimoole et al. Nat Rev Drug Discov 2017

Centro de Investigacion

Principe Felipe (CIPF),

Laboratory of Neuronal

Regeneration, Valencia, Spain

Victoria Moreno-Manzano

Fondazione IRCCS Istituto Neurologico C. Besta

Università degli Studi di Milano

Dip. di Scienze Farmacologiche

e Biomolecolari

Prof.ssa Mariarita Galbiati

Prof. Angelo Poletti

Universidad de Valencia,

Unidad de Neurobiología

comparada,Valencia, Spain

José Manuel García-VerdugoDepartment of

Neurophysiopathology

Giulia Bechi

Massimo Mantegazza

Neurology IV

Stefania Marcuzzo

Silvia Bonanno

Claudia Malacarne

Claudia Barzago

Sara D’Alessandro

Paola Cavalcante

Renato Mantegazza

Scientific Department

Matteo Figini

Alessandro Scotti

Ileana Zucca

Ludovico Minati

Dimos Kapetis

Barbara Galbardi

Neuroradiology

Maria Grazia Bruzzone

Unit of Child Neurology

Giovanna Zorzi

Neurology VII

Denise Locatelli

Cinzia Cagnoli

Fondazione IRCCS Cà Granda, Ospedale

Maggiore Policlinico, Department of

Physiopathology and Transplants, University of

Milan

Monica Nizzardo

Michela Taiana

Stefania Corti

Acknowledgement