Superoxide Dismutase 1 and Amyotrophic Lateral Sclerosis

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS New Series No. 981 — ISSN 0346-6612 — ISBN 91-7305-936-6

Superoxide Dismutase 1 and Amyotrophic Lateral Sclerosis

by

P. Andreas Jonsson

Department of Medical Biosciences, Clinical Chemistry and Pathology Department of Pharmacology and Clinical Neuroscience, Neurology

Umeå University Umeå 2005

Cover illustration: Lumbar spinal cord section from an ALS patient with sclerosis of the corticospinal tract. Klüver–Barerra staining. Kindly provided by Dr. Thomas Brännström Copyright © 2005 P. Andreas Jonsson ISSN 0346-6612 ISBN 91-7305-936-6 Printed by Print & Media Umeå, 2005

Things should be made as simple as possible – but not any simpler

Albert Einstein

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ABSTRACT Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons in the spinal cord, brain stem and motor cortex, leading to paralysis, respiratory failure and death. In about 5% of ALS cases, the disease is associated with mutations in the CuZn-superoxide dismutase (hSOD1) gene. As a rule, ALS caused by hSOD1 mutations is inherited dominantly and the mutant hSOD1s cause ALS by the gain of a noxious property.

The present study focused on two hSOD1 mutations with widely differing characters. In Scandinavia, ALS caused by the D90A mutation is inherited in a recessive pattern. Elsewhere, families with dominant inheritance have been found. The properties of D90A mutant hSOD1 are very similar to those of the wild-type protein. The G127insTGGG (G127X) mutation causes a 21 amino acid C-terminal truncation which probably results in an unstable protein.

The aim of this thesis was to generate transgenic mice expressing D90A and G127X mutant hSOD1s and to compare these mice with each other and with mice expressing other mutant hSOD1s, in search of a common noxious property. The findings were also compared with the results from studies of human CNS tissue.

The cause of the different inheritance patterns associated with D90A mutant hSOD1 was investigated by analyzing erythrocytes from heterozygous individuals from dominant and recessive pedigrees. There was no evidence that a putative protective factor in recessive pedigrees acts by down-regulating the synthesis of D90A mutant hSOD1.

In cerebrospinal fluid, there was no difference in hSOD1 content between homozygous D90A patients, ALS patients without hSOD1 mutations and controls. hSOD1 cleaved at the N-terminal end was found in both controls and D90A patients, but the proportion was significantly larger in the latter group. This indicates a difference in degradation routes between mutant and wild-type hSOD1.

Both D90A and G127X transgenic mice develop an ALS-like phenotype. Similar to humans, the levels of D90A protein were high. The levels of G127X hSOD1 were very low in the tissues but enriched in the CNS. Similarly, in an ALS patient heterozygous for G127X hSOD1, the levels of the mutant protein were overall very low, but highest in affected CNS areas. Despite the very different levels of mutant hSOD1, both D90A and G127X transgenic mice developed similar levels of detergent-resistant aggregates in the spinal cord when terminally ill. Surprisingly, mice overexpressing wild-type hSOD1 also developed detergent-resistant aggregates, although less and later. Most of the hSOD1 in the CNS of transgenic mice was inactive due to deficient copper charging or because of reduced affinity for the metal. The stabilizing intrasubunit disulfide bond of hSOD1 was partially or completely absent in the different hSOD1s. Both these alterations could increase the propensity of mutant hSOD1s to misfold and form aggregates.

The results presented here suggest that the motor neuron degeneration caused by mutant hSOD1s may be attributable to long-term exposure to misfolded, aggregation-prone, disulfide-reduced hSOD1s and that the capacity to degrade such hSOD1s is lower in susceptible CNS areas compared with other tissues. The data also suggest that wild-type hSOD1 has the potential to participate in the pathogenesis of sporadic ALS. Keywords: aggregates, ALS, amyotrophic lateral sclerosis, cerebrospinal fluid, disulfide-reduced, inclusions, misfolded, protective factor, SOD1, transgenic

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CONTENTS

ABBREVIATIONS 1

PREFACE 3

REVIEW OF THE LITERATURE 4

Amyotrophic Lateral Sclerosis – The Disease 4 Clinical signs and symptoms of ALS 4

El Escorial criteria 4 Motor neuron degeneration in ALS 5 Involvement of CNS non-motor areas in ALS 5

Epidemiology 7 Incidence 7 Age at onset 7 Survival 7

Neuropathology 7 General features 7 Inclusion pathology in ALS 8

Etiology and pathogenesis 8 Glutamate excitotoxicity 9 Intermediate filaments 9 Environmental and lifestyle-related risk factors 10 Familial ALS 10 Genetic risk factors 14

Superoxide Dismutase 1 – The Protein 19 Free radicals and superoxide dismutases 19 Structure 21 Properties 22

Expression of SOD1 22 Chemical properties 22 Enzymatic function 23

Copper chaperone for superoxide dismutase 23 Superoxide Dismutase 1 and ALS 25

Features of ALS caused by mutant hSOD1s 25 hSOD1 mutations 25 Pattern of inheritance 25 Epidemiology 25 Phenotype of mutant hSOD1-FALS 25

Properties of mutant hSOD1s 26 Mechanism of disease 27

SOD1 transgenic mice 28 Apoptosis 30

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Mitochondrial pathology 31 Oxidative stress 32 Mutant hSOD1-containing aggregates and inclusions 33

D90A mutant hSOD1 36 D90A mutant hSOD1 in recessive ALS pedigrees 36 ALS in individuals heterozygous for D90A mutant hSOD1 37 Properties of the D90A protein 37

G127X mutant hSOD1 37

AIMS 39

METHODOLOGY 40

Human samples 40 Sample collection 40 SOD1 genotyping 40 Ethical considerations 40

SOD1 transgenic mice 41 The mouse as a model system 41 Generation of transgenic mice 42 Other mice studied 43 Handling of mice 44

Keeping mice 44 Genotyping 44 Breeding 45

Sample collection 45 Ethical considerations 45

Homogenization of samples 45 Generation of antibodies 46 Analysis of SOD isoenzymes 47

SOD activity 47 Direct spectrophotometric method 47 Pyrogallol method 48 Nitroblue tetrazolium gel assay 48

SOD ELISAs 48 Blotting procedures 49

Immunoblotting 49 Northern blotting 49 Southern blotting 50 2D-gel electrophoresis 50

Immunohistochemistry 50 Human histopathology 50

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Mouse histopathology 50 Staining procedure 50

Other experiments 51 Effect of postmortem time 51 Fibroblast culture experiments 51 Analysis of SOD1 aggregates 51

Analysis of detergent-resistant aggregates 51 Analysis of detergent-soluble aggregates 52

Stabilities of human and murine SOD1s 52 Supplementation of homogenates with Cu 53 Cytochrome oxidase assay 53 Inductively coupled plasma atomic emission spectrometry 53 Recombinant hSOD1s 53 Total protein analysis 53 Hemoglobin analysis 54

Statistical calculations 54

RESULTS AND DISCUSSION 55

Paper I 55 No alteration in D90A mutant hSOD1 levels in recessive pedigrees 55

Paper II 56 CSF hSOD1 levels are unaltered in ALS 56 Increased levels of cleaved hSOD1 in D90A ALS patients 58

Papers III-V 59 Phenotypes of mutant hSOD1 transgenic mice 59 Similar mRNA levels – widely differing protein levels 60 Inactive SOD1 in the CNS of transgenic mice – CCS deficiency? 61 Disulfide-reduced SOD1 in high-level hSOD1 transgenic mice 62 Similar amounts of detergent-resistant aggregates in terminal mice 63 Histopathology 64 SOD1 aggregates and motor neuron toxicity 65 Could wt-hSOD1 cause ALS? 67

CONCLUSIONS 68

ACKNOWLEDGMENTS 69

REFERENCES 71

APPENDIX – PAPERS I-V 111


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ABBREVIATIONS aa amino acid ALS amyotrophic lateral sclerosis ALS-PDC ALS Parkinson dementia complex bp base pairs CCS copper chaperone for superoxide dismutase cDNA complementary DNA cM centimorgan CNS central nervous system COX cytochrome C oxidase CSF cerebrospinal fluid CT computed tomography DNA deoxyribonucleic acid DTPA diethylenetriamine pentaacetic acid EAAT2 excitatory amino acid transporter 2 EDTA ethylenediamine tetraacetic acid EMG electromyography FALS familial ALS fMRI functional magnetic resonance imaging GDP guanosine diphosphate GFAP glial fibrillary acidic protein GTP guanosine triphosphate HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HMW high molecular weight hSOD1 human SOD1 HSP heat shock protein JALS juvenile ALS kb kilo base pairs kDa kilo Dalton LBHI Lewy body-like hyaline inclusion LMN lower motor neuron LOD log of odds Mb mega base pairs MND motor neuron disease MRI magnetic resonance imaging mRNA messenger RNA mSOD1 murine SOD1 mutSOD1 mutant SOD1 NCBI National Center for Biotechnology Information NF-H heavy neurofilament NF-L light neurofilament NF-M medium neurofilament PAGE polyacrylamide gel electrophoresis PBP progressive bulbar palsy PBS phosphate buffered saline

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PCR polymerase chain reaction PET positron emission tomography PLS primary lateral sclerosis PMA progressive muscular atrophy RFLP restriction fragment length polymorphism RNA ribonucleic acid RT-PCR reverse transcriptase PCR SALS sporadic ALS SDS sodium dodecyl sulfate SMA spinal muscular atrophy SOD1 CuZn-superoxide dismutase SPECT single photon emission computed tomography SSCP single strand conformational polymorphism UMN upper motor neuron wt-hSOD1 wild-type hSOD1 ww wet weight


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Preface The topic of this thesis is superoxide dismutase 1 and amyotrophic lateral sclerosis. Within this framework account, I have attempted to provide an overview of past and current research in the field, ranging from the more clinical aspects of the disease to the molecular studies of wild-type and mutant superoxide dismutase 1. I have focused on some areas of great interest, such as hereditary factors, the biophysical properties of wild-type and mutant superoxide dismutase 1, aggregation of mutant superoxide dismutase 1, mitochondrial pathology, apoptosis and glutamate excitotoxicity. Other important research areas have been covered in less detail. They include the areas of autoimmunity, axonal transport, inflammation, neurotrophic factors, viral infections and recent therapeutic developments. In the results and discussion section, the results from the papers included in the thesis are summarized and briefly discussed. The papers included in the thesis are enumerated with the following Roman numerals throughout the thesis. I. P. Andreas Jonsson, Åsa Bäckstrand, Peter M. Andersen, Johan Jacobsson,

Matthew Parton, Chris Shaw, Robert Swingler, Pamela J. Shaw, Wim Robberecht, Albert C Ludolph, Teepu Siddique, Veronica I Skvortsova, Stefan L. Marklund. CuZn-superoxide dismutase in D90A heterozygotes from recessive and dominant ALS pedigrees. Neurobiology of Disease (2002), 10, 327-333.

II. Johan Jacobsson, P. Andreas Jonsson, Peter M. Andersen, Lars Forsgren,

Stefan L. Marklund. Superoxide dismutase in CSF from amyotrophic lateral sclerosis patients with and without CuZn-superoxide dismutase mutations. Brain (2001), 124, 1461-1466.

III. P. Andreas Jonsson, Karin S. Graffmo, Thomas Brännström, Peter Nilsson,

Peter M. Andersen, Stefan L. Marklund. Motor neuron disease in mice expressing the wild type-like D90A mutant superoxide dismutase-1. In manuscript.

IV. P. Andreas Jonsson, Karin Ernhill, Peter M. Andersen, Daniel Bergemalm,

Thomas Brännström, Ole Gredal, Peter Nilsson, Stefan L. Marklund. Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis. Brain (2004), 127, 73-88

V. P. Andreas Jonsson, Karin S. Graffmo, Peter M. Andersen, Thomas

Brännström, Mikael Lindberg, Mikael Oliveberg, Stefan L. Marklund. Disulfide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models. Submitted.

Papers I, II and IV were reprinted with the permission of Elsevier Ltd (I) and Oxford University Press (II, IV).

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REVIEW OF THE LITERATURE

Amyotrophic Lateral Sclerosis – The Disease

Clinical signs and symptoms of ALS Amyotrophic lateral sclerosis or ALS is a progressive neurodegenerative disease mainly affecting lower motor neurons in the spinal cord and brain stem and the upper motor neurons of the precentral cortex. The disease was first described in the mid-19th century by Aran, Charcot and others1-3. The symptoms of ALS are usually restricted to the motor system and should include symptoms from both upper and lower motor neurons. The degeneration of motor neurons causes weakness and muscular wasting ultimately leading to death, often due to respiratory failure. Symptoms and signs from the lower motor neurons include weakness, atrophy, cramps, fasciculations and the suppression of reflexes, while upper motor neuron symptoms are characterized by weakness, spasticity, stiffness and brisk reflexes. El Escorial criteria: Diagnostic criteria for ALS were not established until 1990, when the World Federation of Neurology established the El Escorial criteria4, to be used for the diagnosis of ALS in a research setting. These criteria were updated in 1998 at Airlie House, Virginia, USA,5 and mainly rest on clinical, neurophysiological and neuropathological examinations. For the diagnosis of ALS, the El Escorial/Airlie House criteria require the presence of:

A1 evidence of lower motor neuron (LMN) degeneration by clinical,

electrophysiological or neuropathological examination, A2 evidence of upper motor neuron (UMN) degeneration by clinical

examination, and A3 progressive spread of symptoms or signs within a region or to other

regions, as determined by history or examination,

together with the absence of: B1 electrophysiological or pathological evidence of other disease processes

that might explain the signs of LMN and/or UMN degeneration, and B2 neuroimaging evidence or other disease processes that might explain the

observed clinical and electrophysiological signs.

According to these criteria, ALS is classified into various categories depending on the certainty of the diagnosis:

1. Clinically definite ALS 2. Clinically probable ALS 3. Clinically probable ALS – laboratory supported 4. Clinically possible ALS


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In clinical practice, however, these strict criteria are less useful because a patient must have a fairly advanced disease to fulfill the criteria for clinically definite or probable ALS at diagnosis6 and about 10% of patients never fulfill these criteria7.

Motor neuron degeneration in ALS: Since the diagnosis of ALS requires both UMN and LMN symptoms, it excludes related disorders, such as progressive muscular atrophy/bulbar palsy (PMA/PBP) and primary lateral sclerosis (PLS), where only lower and upper motor neurons respectively are affected. Since PMA/PBP and PLS probably share many etiological mechanisms with ALS or form a disease continuum with ALS7-11, the term motor neuron disease (MND) is often used to include these disease entities12,13.

ALS patients have often been ascribed preserved ocular muscular functions, as well as normal function of the voluntary sphincters of the bladder and bowel, suggesting that the CNS motor areas innervating these muscles, the oculomotor nuclei (cranial nerves III, IV, VI) and the nucleus of Onufrowicz, are spared in ALS14-16. There are, however, some reports of complete or incomplete ophthalmoplegia in ALS patients17-19, especially in cases where the disease duration has been prolonged due to mechanical ventilation18,20-22. Upon testing, more subtle eye movement abnormalities can also be detected in ALS patients19,23,24. In ophthalmoplegic cases, histopathological evidence of neuronal loss and gliosis in the oculomotor nuclei has been seen17,20, but at present it is unclear whether the dysfunction is nuclear, supranuclear, or both18,25.

There are also some reports of ALS patients suffering from incontinence or other sphincter disturbances, but this appears to be a fairly infrequent problem26,27. Current evidence, however, suggests that the motor neurons innervating the sphincters could be sub-clinically affected in ALS. Histopathological studies have shown that inclusions characteristic of ALS (Bunina bodies, Lewy body-like hyaline inclusions and ubiquitin-positive inclusions – see below) frequently occur in the Onufrowicz nucleus of ALS patients but without neuronal loss28,29. Further evidence supporting the involvement of these motor neurons in ALS comes from electrophysiological studies. EMGs of the external anal sphincter in a small group of ALS patients without sphincter disturbances showed mild abnormalities in 9 of 16 patients, suggesting that these motor neurons were indeed affected30. However, in overall terms, it appears as if these motor neurons, for unknown reasons, are more resistant than other motor neurons. Involvement of CNS non-motor areas in ALS: There is now also substantial evidence that the degenerative process in ALS is not limited to motor neurons. Although dementia has been described as being relatively rare in ALS26,27,31, it has now become evident that more subtle cognitive changes often occur in ALS patients. Results from neuropsychological testing have demonstrated that cognitive impairment in ALS could be as frequent as 30-60% of ALS patients32-34. These cognitive deficits are possibly more common in ALS patients with bulbar symptoms32,34. Most typically, the deficit is of the frontotemporal type with defects in verbal and non-verbal fluency, abstract reasoning, mental flexibility and judgment32-

34. Parkinsonism in ALS is rare, but there are some reports of ALS patients suffering from Parkinsonian symptoms such as bradykinesia, rigidity and gait abnormalities.

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Tremor is only seen in a few of these patients and the efficacy of levodopa treatment is often poor31,35-37.

Neuroimaging has provided additional support for the involvement of brain extra-motor areas in ALS. Although studies using conventional MRI and CT scans of the CNS have shown conflicting results and therefore are difficult to interpret38, other neuroimaging techniques have provided evidence that suggests the involvement of regions beyond the motor areas of the brain. PET, SPECT and fMRI studies have shown reduced blood flow in extra-motor areas, particularly the frontal lobe, in the brains of ALS patients, with a greater reduction in cognitively impaired ALS patients39-42. Similarly, Lloyd and co-workers used PET and saw a decrease in the amount of GABAA receptors in the prefrontal cortex and other areas of the brain in ALS patients without any signs of cognitive impairment43. Also using PET, Turner demonstrated a global reduction of 5-HT1A receptors in the brain44 and activation of microglia in the prefrontal cortex, pons, motor cortex and thalamus of non-demented ALS patients45. Finally, Takahasi and co-workers, also using PET, saw a negative correlation between disease duration and the uptake of 6-fluorodopa in ALS patients without clinical signs of extrapyramidal symptoms, suggesting a reduction in the number of neurons in the substantia nigra46.

Degenerative processes are also seen in tissue sections of non-motor brain areas of ALS patients. These alterations appear to be more common or more pronounced in patients that are cognitively impaired. For example, there are several reports of ubiquitin-positive inclusions that are negative for tau and α-synuclein in neurons in the frontal lobe neocortex and the dentate gyrus of the hippocampus47-52. Other reports include cortical spongiosis and gliosis in non-motor brain areas of ALS patients47,48,50,51 and cases with degeneration of the substantia nigra without evidence of Lewy bodies have been described51,53.

In the difficult task of cell counting in the brain, there are few reports from ALS patients. Maekawa and co-workers used an antibody for non-phosphorylated neurofilament heavy (NF-H) as a marker for pyramidal neurons and found neuronal loss not only restricted to the motor areas of the CNS but also in the frontal and cingulate cortex of ALS patients54. Gredal et al., on the other hand, were not able to detect any neuronal loss in the motor cortex of ALS patients using a stereological 3-dimensional technique55. Additional studies in this context appear to be needed. In ALS patients with prolonged disease duration due to the use of mechanical ventilation, widespread neuronal degeneration is often seen20,21. To summarize; the preponderance of evidence suggests that ALS should be regarded as a multisystem disease, affecting many different parts of the CNS but with a predilection for motor neurons in the spinal cord, brain stem and motor cortex. This implies that ALS represents one end of a spectrum of neurodegenerative disease entities that only differ by their particular susceptibility to cell toxic events. It therefore seems possible or even likely that disease mechanisms in other neurodegenerative diseases could also apply to ALS and that knowledge gained about the pathogenic mechanisms in ALS also could be applied to other neurodegenerative diseases. It also seems possible that therapies developed for ALS or other neurodegenerative disorders could be used for the treatment of a range of different neurodegenerative diseases.


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Epidemiology A large number of epidemiological studies of ALS have been published. The diagnostic criteria, study designs, methodologies and the populations studied vary considerably, making comparisons difficult. However, some conclusions can be drawn from these studies56-58. Incidence: In most studies, the incidence of ALS has been reported to be between 1.5-2.5 per 100,000 people a year and the occurrence of ALS appears to be geographically uniform, at least in Europe and North America, where most studies have been conducted58-63. Some clustering of a special form of ALS has previously been seen in the western Pacific island of Guam and in areas of the Kii peninsula of Japan (ALS Parkinson dementia complex – ALS-PDC), but now the incidence in these two regions appears to have declined64-66. There is a tendency towards an increase in incidence in later years, especially among women. One explanation for this increase is that it is a consequence of an increase in life expectancy and/or a more complete assessment of cases, but it is also possible that this represent a true increase in incidence56-58,66-68.

ALS is somewhat more common in men than in women and studies indicate a ratio of around 1.4:17,27,31,58,59,61,63,69-73. The gender ratio decreases at higher age, however, and approaches 1:1 after the age of 6531,57,67,70,71. The reason for this is not known, but it has been speculated that the difference in lower age groups may be due to hormonal differences.

Age at onset: The age at onset shows great variation, ranging from juvenile forms of ALS (JALS) to patients contracting the disease at a very advanced age, but the incidence appears to peak in the 6th to 8th decades of life58,61,63,74 and the mean age of onset in most studies is between 56 and 63 years of age27,31,60,61,71-73,75. In analogy to a gender ratio of 1:1 in older age groups, it appears as if the mean age of onset is higher for women59,60,70,71,76,77.

Survival: Survival has been described in the form of both mean and median values in the literature. However, there appears to be little difference between these two measures and the mean/median survival from onset of symptoms to death is frequently reported to be between 2.5 and 3 years7,27,31,60,61,71-73,76,78. In about 25% of cases, the presenting symptom is bulbar7,27,31,58,69-72,76,78 and these patients show a somewhat more rapid disease progression, as do older patients26,27,31,56,57,69,70,72,73,77,78. With regard to survival, it is likely that these two factors, at least in part, should be regarded as confounding variables, since bulbar onset appears to be more common in the elderly69,72.

Neuropathology General features: The key neuropathological feature of ALS is a loss of motor neurons which reflects the clinical symptoms of the patient. Macroscopically, this loss can be manifested as atrophy of the spinal cord, ventral roots, and, in a few patients, the precentral gyrus79. Microscopically, loss of motor neurons is often most discernable in the lumbar and cervical ventral horns of the spinal cord but in patients with mainly bulbar symptoms motor neuron loss could be more or less confined to

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the motor nuclei of the brainstem. Upper motor neurons loss is often more difficult to appreciate80, most likely due to the normal low “density” of motor neurons in the motor cortex. Upper motor neuron degeneration is, however, reflected by myelin pallor in the cortico-spinal tract80 (hence amyotrophic lateral sclerosis, also see cover illustration). Remaining ventral horn motor neurons are often shrunken and show signs typical for apoptosis79. Additional neuropathological features of ALS include reactive gliosis and the occurrence of different kinds of inclusions with varying degree of specificity for ALS. Inclusion pathology in ALS: The following inclusions are known to occur in the spinal cords of ALS patients:

• Bunina bodies: These are small (1-4 µm) eosinophilic granular inclusions within the cytoplasm of ventral horn motor neurons. They consist of an amorphous material with tubules and vesicular structures and stain for cystatin C. There are indications that this inclusion is derived from lysosomes. Bunina bodies are perhaps the most ALS-specific inclusion, but not all ALS patients have this type of inclusion52,79-81.

• Skein-like inclusions: The appearance of this inclusion is similar to

skeins, hence the name. Skein-like inclusions are only detected when stained for ubiquitin (see below). The inclusions contain bundles of filaments with granules, 15-25 nm in diameter. Although characteristic for ALS they are not specific for the disease79,81,82.

• Spheroids: These are eosinophilic and argyrophilic inclusions, primarily

found in the proximal axons and perikaryon, that contain phosphorylated neurofilaments (see below). This type of inclusion can be as large as 20 µm, but smaller spheroids also exist and are sometimes referred to as globules. The specificity of these inclusions is low and spheroids/globules can be seen in normal subjects but the number appears to be increased in ALS79,80.

• Lewy body like hyaline inclusions (LBHI): LBHIs exists in both neurons

and astrocytes and are hyaline inclusions with a peripheral halo similar to the Lewy bodies seen in Parkinson disease. Initially it was believed that this type of inclusion was specific for certain types of familial ALS but it is now clear that LBHIs also can occur in also in sporadic ALS83-85. The inclusions are 3-15 µm in diameter and are composed of 15-25 nm granule-coated fibrils and, in the periphery, of 10 nm neurofilaments 79,81,83. Besides neurofilaments, LBHIs also stain for other proteins (see below).

Etiology and pathogenesis The cause of ALS is still largely unknown. When it comes to the pathogenesis of ALS, a number of disease mechanisms have been proposed and some of the most studied are discussed here.


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Glutamate excitotoxicity: Glutamate is one of the most important excitatory amino acids in the CNS. At high concentrations, this amino acid is known to be toxic to neurons, probably by overexciting the cells. The toxic mechanism is likely to involve cellular calcium influx, the production of reactive nitrogen and oxygen species and the initiation of the apoptosis machinery, as has been recently reviewed86.

Because of its inherent toxic properties and experimental findings indicative of an abnormal glutamate metabolism in ALS patients, glutamate has attracted a great deal of interest from ALS researchers in the last couple of decades. There are several reports suggesting increased levels of glutamate in extracellular compartments, such as blood87,88 and cerebrospinal fluid (CSF)89-91, and reduced levels of glutamate in CNS tissues, representing the intracellular compartment, of ALS patients92-94. The results in this area of research have, however, been conflicting88,89,95,96, possibly as a result of the difficulties in measuring glutamate.

Albeit conflicting results, it has been suggested that ALS is associated with a shift in glutamate from the intracellular to the extracellular space, possibly resulting in the exposure of glutamate receptors on neurons to abnormally high concentrations of extracellular glutamate. In support of these findings, a deficiency in the capacity to transport glutamate from the extracellular to the intracellular space was demonstrated in ALS patients97 and, subsequently, a selective loss of the astrocytic glutamate transporter, EAAT2, in affected CNS areas of ALS patients was reported98-100. It was therefore suggested that the loss of EAAT2 could be responsible for the altered glutamate metabolism in ALS patients. The reasons for the loss of EAAT2 protein were (are) not known, since the levels of EAAT2 mRNA in the motor cortex of ALS patients was not significantly altered101 and screening for mutations in the EAAT2 gene gave no explanation102-104. In 1998, one report suggested that the low levels of EAAT2 protein in ALS patients were due to aberrantly spliced forms of EAAT2 mRNA105. Later studies have, however, reported a similar degree of alternatively spliced EAAT2 mRNA also in controls106-109.

Glutamate binds to three different types of receptors: NMDA, AMPA and kainate receptors. One of these receptors, the AMPA receptor, has been particularly implicated in ALS. For example, it was recently shown that the mRNA editing of the GluR2 subunit of the AMPA receptor was defective in the motor neurons of ALS patients110. Perhaps the most convincing evidence that glutamate participates in the process of motor neuron degeneration in ALS comes from the anti-glutamatergic drug riluzole. Clinical trials with this drug have shown a positive effect on survival and riluzole is so far the only drug approved for the treatment of ALS111. The precise mechanism of this drug is uncertain, but one of its actions is probably to inhibit presynaptic glutamate release112,113. Intermediate filaments: In differentiated neurons, five different intermediate filament proteins are expressed – three neurofilament proteins, peripherin and α-internexin. The three different subunits of neurofilaments are termed heavy, medium and light (NF-H, NF-M and NF-L) in reference to their molecular weight114. The functions of the intermediate filaments are both to participate in axonal transport and to uphold axonal caliber. Intermediate filaments have been implicated in ALS pathogenesis by the occurrence of conglomerates (spheroids, see above) of 10 nm phosphorylated neurofilaments in the proximal axons, perikaryon and, somewhat more diffuse, in the somas of motor neurons. These conglomerates can also contain

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peripherin. It has been suggested that the occurrence of these conglomerates is a consequence of poor axonal transport80.

A number of transgenic mouse strains null for or overexpressing the different neurofilament proteins have been generated114,115. Generally, these mouse strains do not develop an overt MND phenotype but in some strains an axonopathy and motor neuron dysfunction is seen, e.g. in mice that are null for NF-L116 or both NF-M and NF-H117 and in mice overexpressing peripherin118 or NF-H119. In mice overexpressing L394P mutant murine NF-L a more overt MND phenotype is seen with motor neuron loss and neurogenic muscular atrophy120.

Surprisingly, deletion of NF-L121 or overexpression of NF-H122 in the G85R and G37R, respectively, hSOD1 mouse models of ALS (see below) resulted in accumulations of neurofilaments in the perikarya and, importantly, significantly prolonged survival. The reasons for these surprising results are not known but the results indicate that the stoichiometry between the different intermediate filaments is important for motor neuron survival and that perikaryal accumulations of neurofilaments in some situations actually could be beneficial (e.g. by acting as a “phosphorylation sink” 114,123. Environmental and lifestyle-related risk factors: Many epidemiological studies have been performed to try to identify environmental and lifestyle-related risk factors for ALS. Generally, these studies are hampered by the heterogeneity of ALS and the relative rarity of the disease. A number of such risk factors have, however, been suggested; they include exposure to heavy metals and trace elements (such as lead, mercury and selenium), exposure to chemicals, working with leather, welding, heavy labor, sports activities, physical trauma (including surgery), electrical trauma, farming, smoking, alcohol, excess body mass and participation in the Persian Gulf war. Despite a plethora of studies in this area, the results have been far from conclusive and today the only established risk factors are age, gender (see above) and hereditary factors (see below), which have recently been reviewed57,66-68,124,125. Familial ALS: In at least 5-10% of ALS cases, there is a family history of ALS in at least two blood relatives126 and in these instances the disease is regarded as familial (FALS)7,31,58,60,61,70,73,75,127. In most cases, FALS is indistinguishable from sporadic ALS (SALS), but it is generally recognized that FALS has a few years earlier onset, more equal gender distribution and perhaps a higher frequency of concurrent dementia, Parkinsonism and sensory disturbances. Histopathologically, the involvement of the posterior columns, dorsal spinocerebellar tracts and Clarke’s nucleus is probably also more frequent in FALS127-130. In certain types of FALS, however, a more divergent phenotype is seen (see below).

Today, 10 different gene loci (Table 1) have been linked to familial ALS (FALS). In four of these loci, the gene responsible for the disease has been identified. Two of these genes are involved in JALS, a condition that is relatively rare. In JALS, onset should take place before the age of 25 and the progression of the disease is generally much slower131,132. The other two disease-causing genes have been found in families with classical ALS (ALS1) or a heterogeneous ALS/SMA disease (ALS8). The 10 different genetic loci are discussed below.


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Table 1. Familial ALS loci

Disease Type Locus Gene Inheritance References

ALS1 Classical ALS 21q22.11 SOD1 Dominant/Recessive 133

ALS2 JALS, PLS, HSP 2q33.1 ALS2 Recessive 134,135

ALS3 Classical ALS 18q21 – Dominant 136

ALS4 JALS, slow progression 9q34.13 SETX Dominant 137

ALS5 JALS 15q15.1-q21.1 – Recessive 138

ALS6 Classical (and FTD?) 16q12.1-q12.2 – Dominant 139-141

ALS7 Classical 20ptel-p13 – Dominant 140

ALS8 Heterogeneous ALS/SMA 20q13.33 VAPB Dominant 142

ALS-X Late onset Xp11-q12 – X-linked dominant 143

ALS/FTD Classical ALS and/or FTD 9q21-q22 – Dominant 144

FTD = frontotemporal dementia, HSP = hereditary spastic paraplegia

ALS1: In 1991, the first genetic locus linked to ALS was reported by Siddique and co-workers. They found linkage to a 10 cM region of chromosome 21q22.1-q22.2 in families with autosomal dominant ALS145. A few years later, in a pivotal paper by Rosen et al., it was shown that mutations in the gene encoding for superoxide dismutase 1 (hSOD1) was responsible for the disease in these families133. This discovery gave rise to a surge in research into the pathogenesis of ALS and mutations in this protein are also the topic of this thesis. A detailed description of mutant SOD1 (mutSOD1) and ALS is given below. ALS2: The second locus linked to ALS was reported in 1994, when linkage to an 8 cM large region of chromosome 2q33-q35 was shown in a consanguineous Tunisian family with juvenile-onset, recessive ALS. The ALS in this family was predominantly a slowly progressing UMN disease, but there were also signs of LMN involvement131,146. In 2001, it was shown that a one base-pair deletion in the ALS2 gene encoding for alsin was responsible for the disease in this family. An additional two families with mutations in ALS2 were also reported, both of whom had the diagnosis juvenile-onset PLS (i.e. without signs of LMN involvement)134,135. Since then, another six mutations in the ALS2 gene have been discovered, all in families with infantile recessively inherited ascending spastic paraplegia147-149. So far, all the detected mutations result in the truncation of the protein and it appears as if the mutant protein is highly unstable and rapidly degraded150. These findings and the recessive inheritance pattern make it likely that mutant alsin causes motor neuron disease by a loss of function. The function of the alsin protein is not completely known, but alsin contains three guanine exchange factor (GEF) domains that are generally involved in the cycling of GTP/GDP for GTPases (presumably Rab5)151. The protein also contains several membrane occupation and recognition nexus (MORN) motifs, which suggests binding to membranes134,135, and recent studies have suggested that alsin is involved in endosome dynamics150,151. The reason for the particular vulnerability of motor neurons is not known and there is no clue in the

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expression pattern of alsin, since the highest expression appears to be in the cerebellum, a part of the CNS that does not appear to be affected in ALS2135,150,151.

Alsin knockout mice have recently been reported. These mice show signs of cerebellar dysfunction, such as impaired motor coordination and motor learning deficits and an elevated anxiety response. There were, however, no neuropathological signs of motor neuron disease152.

The importance of ALS2 mutations in ALS remains to be evaluated, but so far it seems as if mutant alsin is not a common cause of ALS, even in atypical cases with young onset, slow progression and predominantly UMN symptoms153,154. ALS3: In 2002, one family fulfilling the El Escorial criteria for adult onset ALS was described and linkage to an approximately 8 Mb large region of chromosome 18q21 with a LOD score of 4.5 was shown136. The clinical picture was classical ALS but with a somewhat early onset (mean 45 years of age), but, as stated above, this is commonly seen in FALS. The mean disease duration for this family was 5 years and the inheritance pattern was autosomal dominant. The gene responsible for the disease has so far not been identified. ALS4: This is an apparently rare autosomal dominant form of ALS with childhood or adolescent onset. There are signs of both upper and lower motor neuron lesions and there are also some indications of the involvement of sensory neurons. Bulbar and respiratory symptoms are not seen. The disease progression is very slow, with an apparently normal life expectancy, and there is a large variation in the severity of symptoms155,156. ALS4 should thus be regarded as atypical ALS. There are three known ALS4 families with linkage to chromosome 9q34 and it was recently shown that missense mutations in the SETX (senataxin) gene co-segregated with the disease137. Interestingly, families afflicted by another neurodegenerative disease, autosomal recessive ataxia-oculomotor apraxia type 2 (AOA2), also carry mutations in this gene and at present it is unclear whether there is any relationship between these two disease entities157,158. The function of the senataxin protein is not known, but it has been suggested that senataxin is involved in DNA/RNA maintenance137,157. The mechanism by which mutant senataxin causes disease is not known and both loss and gain of function are conceivable137. ALS5: Patients with ALS5 suffer from JALS and, in 1998, linkage to a 6 cM large region of chromosome 15q15-q22 was reported in families from Germany and Tunisia138. The inheritance pattern was recessive and patients suffered from both spinal and bulbar symptoms. Both UMN and LMN signs were present. The Tunisian patients had a slow disease progression, while the German family had a much more rapid progression. Signs of mental retardation were also seen in the two German siblings131,138. No further reports of JALS linked to chromosome 15 have been published. ALS6: In the August 2003 issue of the American Journal of Human Genetics, three papers were published that showed linkage of autosomal, dominant, adult-onset ALS in four families to chromosome 16q12.1-12.2. The symptoms displayed were mainly classical ALS, but signs of frontotemporal lobe dementia were seen in some


13

patients139. The chromosomal region of interest spans about 4.5 Mb139, but so far the gene responsible for ALS6 has not been reported. ALS7: In their 2003 paper, Sapp and his co-workers also reported linkage to chromosome 20ptel in one family fulfilling the El Escorial criteria for adult-onset ALS140. The maximum LOD score was 3.46 and the haplotype under scrutiny covers around 1 Mb140. It should be noted that the linkage analysis was only based on two affected individuals in the same generation of a family. No further data on this family have been published. ALS8: In 2004, Nishimura and co-workers described a Caucasian Brazilian family with adult-onset ALS and dominant inheritance and linkage to chromosome 20q13 was demonstrated159. Later that year, they were able to reveal that a missense mutation in the VAPB (vesicle-associated membrane protein-associated protein B) gene segregated with disease in the family. In this report, an additional six families were also reported, all showing the same mutation in the VAPB gene142, and a founder effect in the mid-15th century is suspected160. The clinical phenotype was heterogeneous. Three families had a slowly progressing atypical ALS, three families had symptoms corresponding to adult-onset spinal muscular atrophy (SMA) and, in one family, patients suffered from both atypical and classical ALS. It is believed that the function of the VAPB protein is to act during ER-Golgi transport and secretion142. It is not known why a mutation in this ubiquitously expressed protein is toxic to motor neurons and so far, no more data have been published on ALS and mutation(s) in VAPB. ALS-X: One family consisting of 11 affected individuals with linkage to the X chromosome has been described in one abstract. There was no male-to-male transfer. The maximum LOD score was 3.8, which was close to the maximum LOD score for this family143. No additional data concerning this family have been described. ALS-FTD: There are many reports on the nosological overlap between ALS and frontotemporal dementia (see above). The co-existence of ALS and frontotemporal dementia has also been reported in FALS. In 2000, Hosler et al. showed linkage between dominantly inherited ALS/FTD and a 17 cM large region of chromosome 9q21-q22. Five families in which individuals had ALS and/or frontotemporal lobe dementia were reported. The ALS disease seen in these families was reported to be classical ALS144. No further reports of ALS-FTD linked to chromosome 9q21-q22 have been published. There are two other genes involved in familial neurodegenerative diseases that are of potential importance for ALS. Dynactin is a multiprotein complex involved in axonal transport. Heterozygous mutations in the gene for the p150 subunit of the dynactin complex (DCTN1) have been linked with a LMN disease with prominent bulbar symptoms similar to spino-bulbar muscular atrophy (SBMA or Kennedy’s disease – a hereditary disease caused by trinucleotide repeats in the androgen receptor gene)161. Mutations in the DCTN1 gene have occasionally also been detected in ALS patients162. Interestingly, perturbation of proteins involved in the dynactin complex in mice (e.g. Loa or Cra1 mice and transgenic mice overexpressing the p50 subunit of

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the dynactin complex – dynamitin) results in a motor neuronopathy with features similar to ALS163,164. It is unclear whether alterations in this gene should be regarded as direct causative genes with reduced penetrance or merely as a genetic risk factor.

The other gene is tau, which encodes a microtubule-associated protein. Mutations in this gene cause dominantly inherited FTDP-17 (frontotemporal dementia with Parkinsonism linked to chromosome 17), which is, as the name implies, a disease characterized by frontotemporal dementia and Parkinsonism165. Rarely, signs of motor neuron degeneration are also seen in these patients166,167. Tau is also found in the neurofibrillary tangles of Alzheimer’s disease patients. In analogy to what is seen for dynactin, transgenic mice overexpressing mutant tau suffer from motor neuron loss and have a phenotype similar to ALS168. It is, however, important to mention that tau inclusions and neurofibrillary tangles are not usually seen in classical ALS, although patients with the western pacific ALS-PDC display tau-positive inclusions82,169. There is, however, little evidence that the tau gene is involved in ALS-PDC170,171, although one study has indicated that the tau loci could be a susceptibility factor for ALS-PDC172. The genetic heterogeneity in all forms of FALS has thus proved to be much larger than previously anticipated and only a minority of FALS cases can so far be associated with any of these loci. It is therefore evident that there are several additional loci to be discovered140,173. Even though some of the mutant genes involved in FALS are very rare causes of ALS, much could probably be learnt from these discoveries with regard to motor neuron biology and pathology. It is also possible or even likely that some of these proteins are involved in SALS, as is discussed below. Genetic risk factors: In addition to the genes involved in familial forms of ALS, a number of polymorphic genes have been proposed as causative genes or susceptibility factors in ALS. For many of these genetic risk factors, studies have not been conclusive, due mainly to small sample sizes and ethnical heterogenity in the population. Further investigations using larger sample sizes are therefore needed. The main findings of some of the most studied of these genes are summarized below. APEX: This is an enzyme involved in DNA repair as an endonuclease. Olkowski detected missense mutations in the coding region of this gene in six of eleven ALS patients. No mutations were seen in five controls174. A much larger study consisting of 153 ALS patients and 58 controls was performed by Hayward and colleagues. In addition to rare polymorphisms in the gene in both controls and ALS patients, they detected a significantly higher frequency of a homozygous D148E polymorphism in SALS patients than in controls, suggesting that this polymorphism may be a risk factor for ALS175. Tomkins and co-workers, on the other hand, were unable to find any significant differences when investigating 84 ALS patients and 154 controls. Some rare polymorphisms of unknown importance were seen in both ALS patients and controls, also in this study176. In the brains of ALS patients, the amount and activity of APEX have been reported to be reduced177. APOE: The ε4 allele of APOE (APOE4 – apolipoprotein E) is significantly over-represented in Alzheimer’s disease and has been associated with an early onset of the


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disease, which could also be the primary effect of this genotype, as has recently been reviewed178,179. It has therefore been speculated whether this allele could also be a risk factor for ALS and several studies have addressed this aspect.

The first published study was that of Mui and colleagues, who screened 72 SALS and 98 FALS patients and 60 unaffected relatives for the APOE4 allele. In this study, they were unable to show any increased risk associated with the APOE4 allele180. Similar negative findings have subsequently been confirmed by others181-188. However, some studies have suggested that the APOE genotype could be important for the age at onset. Li and co-workers demonstrated a later onset of ALS with an increasing number of APOE2 alleles181 and Moulard et al. showed that, in bulbar onset ALS, the APOE4 allele was associated with an earlier onset187. Other studies have been unable to verify a correlation between APOE genotype and age at onset of symptoms, although all stratifications were not made180,182-186. Some studies have suggested that the APOE genotype is important for disease progression, with the APOE4 allele associated with a more rapid disease progression183 or the APOE2 allele with a slower course187, but survival was not controlled for other factors in these studies. Other studies, some of which have performed a multivariate analysis, have been unable to detect significant differences180,182,188. Lacomblez et al., however, seeing no association between genotype and survival, showed that a low value of plasma apoE correlated positively with survival188. It has also been suggested that the APOE4 allele is over-represented in ALS patients with bulbar instead of spinal onset182,187, but again this has not been seen in other studies184-186.

All these studies are relatively small, a frequent problem when studying ALS, and of different methodological qualities. This and the contradictory results make it difficult to draw safe conclusions. However, it seems reasonable to assume that, if the APOE genotype has any importance in ALS, the overall effect of the genotype is small. CNTF: Ciliary neurotrophic factor (CNTF) is a protein whose main function appears to be to promote neuronal survival189. In the spinal cords of ALS patients, reduced levels of this neurotrophic factor have been reported190, although levels in the sciatic nerves of patients were unchanged191. A one base pair insertion in the intronic sequence of this gene, resulting in a null mutant, has previously been described. The null mutant exists as a polymorphism in the population, with a few percent being homozygotic for null CNTF192. Because of the role played by CNTF in neuronal survival and the motor neuron loss seen in CNTF knockout mice193 it has been speculated if the null mutant could be a susceptibility factor for ALS. In a small study consisting of 49 FALS patients, Orrell and co-workers were not able to show any such association194 and similar results were demonstrated by Giess and colleagues in an unspecified group of 98 ALS patients195. However, in the latter study, all the ALS patients who were homozygotic null for CNTF had younger onset (42, 14 and 23 years of age) suggesting that the null mutant is a risk factor for early-onset disease195. In the largest study so far, comprising 400 ALS patients (351 SALS and 49 FALS) and 217 controls, there were no differences, either in frequency or disease onset, between ALS patients and controls196. It thus appears unlikely that the CNTF null mutation is of major importance in ALS pathogenesis.

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HFE: Autosomal recessive hemochromatosis is caused by mutations in the HFE gene. Two mutations are particularly prevalent, H63D and C282Y with C282Y being associated with a higher risk for iron accumulation. Interestingly, mutations in this gene have also been associated with an earlier onset of Alzheimer’s disease197. Wang and colleagues investigated the prevalence of HFE mutations in 121 ALS patients and 133 controls and found a significant association between the H63D mutation and ALS. In the larger study of Goodall et al., comprising 379 ALS patients and 400 controls, a similar significant association with H63D (odds ratio 1.85 for one mutant allele), but surprisingly not with C282Y, was seen198. In a previous study by Yen and co-workers, who screened 51 ALS patients and 47 controls for these two polymorphisms, no such association was seen199. The reasons for these discrepancies are not known but the latter study is of smaller size and is possibly also hampered by ethnical heterogeneity. The mechanism by which H63D HFE could increase the risk to develop ALS is not known, but it has been speculated that the mutation could lead to an altered iron transport cross the blood-brain barrier since the H63D mutation is known to affect binding to the transferrin receptor198. Interestingly, the HFE locus is located on chromosome 6p21.3 close to the major histocompatibility complex (MHC) region. One possibility is therefore that the H63D mutation is in linkage disequilibrium with a HLA haplotype that increases the risk to develop ALS200. This would of course implicate the immune system in ALS pathogenesis. NFH: The central domain of the five different intermediate filaments expressed in neurons is conserved, but in the N and C-terminal ends of the proteins (also known as head and tail respectively) there are less conserved domains. At the C-terminal end of NF-H and NF-M, there are multiple repeats of amino acids Lys-Ser-Pro or KSP repeats. In humans, the number of KSP repeats in NF-H is polymorphic with either 44 or 45 (previously stated 43 or 44) repeats. The phosphorylation of these repeats is important for neurofilament spacing and axon caliber and probably also for axonal transport, since hyperphosphorylation decreases transport velocity114,201,202.

Three studies have suggested that alterations in this domain of the NFH gene are associated with SALS. Figlewicz and co-workers reported heterozygous deletions in 5 of 356 ALS patients (1.4%), while none of the 306 controls carried a deleted copy. However, healthy relatives of ALS patients with mutant NFH also carried the mutant gene203. Tomkins and colleagues screened 164 ALS patients and found one heterozygous insertion in one SALS patient causing an extra four KSP repeats. No alterations were seen in 209 age-matched controls204. In the largest study so far, 530 ALS patients and 366 controls were screened for mutations using SSCP and RFLP. Four different tail deletions in three SALS patients and one FALS patients were seen. It could not be determined whether the mutation in the FALS patient segregated with disease. No linkage to the NFH locus at chromosome 22q12.2 has, however, been reported in FALS. Deletions in three unaffected relatives of these patients and in two controls were also detected. There was no association between the 44 or 45 repeats polymorphism and ALS. In two smaller studies, no association between mutations in the NFH gene and ALS were seen205,206.

In the study by Vechio and colleagues, NFL and NFM were also investigated without significant associations. Garcia and co-workers recently sequenced the NFL, NFM and NFH genes, but excluded the KSP region of NFH in 100 SALS, 100 FALS patients and 100 controls. They found a few different mutations in all three genes in


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both controls and ALS patients. The data were somewhat difficult to interpret due to the small sample size, but it was suggested that mutations in neurofilaments could be possible risk factors207. A similar picture has been reported for another intermediate filament, peripherin208,209.

In all, the collective studies suggest that mutations in the KSP domain of NFH may be a rare susceptibility factor for sporadic ALS. Two negative studies have been published, but they appear to have been underpowered, considering that KSP mutations in NFH only appear in about 1% of ALS cases (and in 0.2% of controls). Findings from studies in transgenic mice and histopathology from ALS CNS tissue (see above) add further support for the role of neurofilaments in motor neuron degeneration. An interesting observation is that heterozygous mutations in the NFL gene are linked to Charcot-Marie-Tooth type 2E114,201,202. SMN1/SMN2/NAIP: The survival motor neuron protein (SMN) is a protein that is most probably involved in the assembly of small nuclear ribonucleoproteins in a protein complex called Gems210. The SMN gene exists in two almost identical copies on chromosome 5q13. The telomeric copy of the gene has been named SMN1 and the centromeric SMN2. Homozygous deletions or conversions of SMN1 are responsible for more than 95% of spinal muscular atrophy (SMA) cases, but curiously it also appears as if the phenotype of SMA is correlated to the copy number of SMN2211. Because of the degeneration of lower motor neurons seen in SMA, it has been speculated if deletions or other major alterations in the SMN1 or SMN2 genes could be associated with ALS. There are today clear evidence that homozygous deletions of the SMN1 gene are not associated with ALS212-218. However, a study by Corcia and colleagues reported an association between ALS and one or three copies of the SMN1 gene213. No such association was, however, seen in the study by Veldink et al.214. On the other hand, this latter group reported that homozygous SMN2 deletions were four times more common in ALS than in controls and that these patients also had a significantly shorter survival214. Moulard and co-workers also reported an association with SMN2 but only in a subgroup of patients with a pure LMN disease217, which also was seen by Echaniz-Laguna et al.219. Other studies have not been able to confirm these findings212,213,216.

The gene for the neuronal apoptosis inhibitory protein (NAIP) is in the close vicinity of the SMN genes and has also been implicated in SMA pathogenesis, since this gene is deleted in a large proportion of SMA cases. However, there is no evidence of the involvement of this gene in ALS212,214-218.

The data so far have thus been contradictory and difficult to evaluate and larger studies are needed to draw safer conclusions about the role of these genes in ALS pathogenesis. SOD2: Manganese superoxide dismutase or SOD2 is one of three human superoxide dismutases the main function of which is to catalyze the degradation of the superoxide anion (see below). SOD2 is a mitochondrial protein, but the gene for SOD2 is located on chromosome 6q25.3. After translation, the SOD2 protein is transported to the mitochondrial matrix, guided by a signal peptide where the protein is metallated with manganese220. The finding that mutations in the hSOD1 gene cause ALS and that SOD2 knockout mice develop a dramatic phenotype with, among other things, limb weakness221 made it plausible that mutant SOD2 could be associated

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with ALS. Parboosingh and colleagues screened the 3’ end of the SOD2 gene using single strand conformation analysis (SSCA) and found no association with ALS222. Tomblyn et al., on the other hand, sequencing the coding region, found homozygosity for an Ala-9Val substitution in the mitochondrial targeting sequence in 11 of 20 SALS patients and 3 of 10 controls223. Although this study was very small, this suggested an association between the Ala-9Val polymorphism and ALS. This finding was further investigated by Van Landeghem et al., who, in a somewhat larger study involving 72 ALS patients and 136 controls, came to the opposite conclusion. Here, the Ala allele was associated with a greater risk of contracting ALS224. Tomkins et al., on the other hand, detected no significant association between ALS and this polymorphism or any other mutations in the gene, but there was a trend towards a lower frequency of Ala in ALS cases (p=0.08)225.

The significance of the Ala-9Val polymorphism is not fully understood. In vitro, the Val allele has been shown to be less efficiently imported into the mitochondrial matrix leading to lower SOD2 activity, but this has not yet been shown in vivo226-228.

The frequency of the polymorphism shows great ethnic variability225 and this probably explains the divergent results in these studies and also stresses the importance of choosing the correct controls. All in all, the current evidence does not favor the involvement of this gene in ALS pathogenesis. VEGF: Vascular endothelial growth factor (VEGF) is a growth factor for the vasculature that promotes neovascularization. The expression of this gene is partly regulated by hypoxia. A deletion of the hypoxia–response element in the promoter region of this gene in mice surprisingly caused a disease-phenotype similar to motor neuron disease229. It was unclear whether the motor neuron death was caused by a decreased vascularization or by a reduction of a more direct neurotrophic effect of VEGF.

This finding suggested that polymorphisms in the promoter region of the human VEGF gene could be associated with ALS. In a large study by Lambrechts et al., it was reported that homozygosity for two specific haplotypes in the VEGF promoter were significantly more frequent in ALS patients than in controls. These haplotypes lead to decreased levels of plasma VEGF and indeed ALS patients also had lower levels of plasma VEGF, reaching only ~50% of controls230. It was estimated that the “at risk” haplotypes increased the risk of contracting ALS by 80%. There were no other polymorphisms in other parts of the VEGF gene that were associated with ALS. Two smaller studies have since investigated the “at risk” VEGF polymorphisms and their relationship to ALS. Terry and co-workers saw a trend similar to that reported by Lambrechts et al., but the association between the haplotypes and ALS was not significant. Brockington and colleagues, on the other hand, reported no association between the VEGF promoter haplotype and ALS231.

Other studies have also investigated VEGF levels in ALS patients. Contrary to the report by Lambrechts et al., two smaller studies have reported unchanged232 or higher233 levels of VEGF in plasma and serum respectively of ALS patients. The considerably smaller sample sizes in these studies make the results difficult to evaluate, however.

Currently, the evidence favors VEGF promoter polymorphism as a risk factor for ALS. Although the increase in risk seen by Lambrechts et al. was not large, it was


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suggested that the “at risk” haplotypes could be responsible for as many as 5-10% of ALS cases230, which is similar to that of mutant SOD1. There are several other polymorphic genes that have been suggested as risk factors or susceptibility genes for ALS; they include CYP2D6234, MAOB235, LIF236 and PSEN1237. Only single studies have investigated these genes and the significance of these genes remains to be determined. To summarize: besides the genes involved in FALS, the only truly established risk factors for ALS are age and gender. A number of environmental and lifestyle-related factors have been proposed to provoke the disease, as have a number of polymorphic genes, but in most cases the data relating to these factors are still inconclusive. However, environmental and genetic risk factors cannot be totally dismissed. Since the vast majority of ALS cases are sporadic, it is more than likely that, in sporadic ALS, a combination of environmental and genetic factors provoke the disease, perhaps in conjunction with the wild-type forms of proteins involved in FALS in analogy to what is seen in the case of Alzheimer’s disease, Parkinson’s and Creutzfeldt-Jakob disease, for example, where proteins clearly involved in sporadic cases are found mutated in familial cases238.

Superoxide Dismutase 1 – The Protein

Free radicals and superoxide dismutases Originally described as a copper-containing protein with unknown function, “hemocuprein” was first isolated from bovine erythrocytes by Mann and Keilin in 1939239. The function of this protein was unknown until 1969, when McCord and Fridovich240 reported an enzyme, also purified from bovine erythrocytes, which catalyzed the dismutation of superoxide radicals (O2•-) to hydrogen peroxide (H2O2) and oxygen (O2) (Reaction 1). This enzyme was shown to be identical to the protein purified back in 1939 and was now renamed superoxide dismutase (EC 1.15.1.1) to better reflect the enzymatic function of the protein.

(Reaction 1) 2O2•- + 2H+ → O2 + H2O2 The superoxide radical is, as the name implies, a free radical, meaning that the molecule has an unpaired electron241. The unpaired electron makes the molecule more prone to react with other molecules (e.g. proteins and lipids) by oxidizing, reducing, or in other ways modify the molecules. There is therefore an obvious need for the removal of free radicals. Superoxide dismutases are the enzymes responsible for the removal of superoxide. The bulk of the superoxide radicals are formed in the electron-transport chain of mitochondria but superoxide can also be formed by enzymes such as nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, xanthine oxidase, nitric oxide synthase and peroxidases220. Despite its name, superoxide is not as reactive as many other radicals although it is known to react with for example ascorbate, glutathione and FeS cluster proteins. Instead, superoxide main toxic potential lays in the formation of more reactive intermediates such as

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metal

catalyst

peroxynitrite (ONOO-, Reaction 2, also see below) or the hydroxyl radical (OH•) by the metal (Me) catalyzed Haber-Weiss reaction220,241 (Reactions 3 and 4 and the combined Reaction 5).

(Reaction 2) NO + O2•- → ONOO-

(Reaction 3) O2•-+ Me2+ → O2 + Me+ (Reaction 4) H2O2 + Me+ → OH• + OH- + Me2+

(Reaction 5) O2•- + H2O2 → O2 + OH• + OH-

Since the discovery by McCord and Fridovich of CuZn-superoxide dismutase (CuZn-SOD or SOD1), two additional human SODs have been reported. As has already been mentioned, manganese-containing superoxide dismutase (Mn-SOD or SOD2) is found in the mitochondrial matrix242,243. The third human superoxide dismutase is extracellular superoxide dismutase (EC-SOD or SOD3)244. Like SOD1, this SOD is, a copper- and zinc-containing metallo-enzyme. SOD3 has a high homology to the C-terminal part of SOD1245, but, unlike SOD1, the protein is mainly found in the extracellular environment of tissues with a particular affinity for heparan sulfate proteoglycans on cell surfaces246. Tissue SOD3 levels are also more variable. In humans, high levels of SOD3 are, for example, seen in the blood vessel walls, thyroid gland, pancreas and lung tissue, while lower levels are seen in the CNS247,248. As previously discussed, the Ala-9Val polymorphism in the targeting sequence of SOD2 has been suggested as a susceptibility factor for ALS. SOD3 has so far not been implicated in ALS.

Although hydrogen peroxide does not have an unpaired electron (and therefore is not a free radical) it still has the ability to oxidize substrates. However, similar to superoxide, the main toxic potential of hydrogen peroxide lies in the formation of the hydroxyl radical from hydrogen peroxide and reduced transition metal ions (Me+; Reaction 4), particularly Fe2+ in a reaction known as the Fenton reaction. The hydroxyl radical is highly reactive with almost everything and there is therefore a need to remove hydrogen peroxide. Another advantage of the removal of hydrogen peroxide is that the inactivation of SOD by hydrogen peroxide is reduced. In humans, several enzymes are involved in the removal of hydrogen peroxide. In the peroxisomes, the heme containing enzyme catalase decomposes hydrogen peroxide to water and oxygen (Reaction 6). Another important family of enzymes is the peroxidases, which remove hydrogen peroxide by oxidizing another substrate (S in Reaction 7). Perhaps the most important member of this family is the selenium-containing enzyme glutathione peroxidase (GPX), which catalyzes the oxidation of the substrate glutathione (GSH, Reaction 8). Other proteins involved in the removal of hydrogen peroxide include the thioredoxins and peroxyredoxins220,241.

(Reaction 6) 2H2O2 → 2H2O + O2

(Reaction 7) SH2 + H2O2 → S + 2H2O

(Reaction 8) 2GSH + H2O2 → GSSG + 2H2O


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As has been discussed previously, SOD2 knockout mice have a severe phenotype with early postnatal lethality, probably due to the very high production of superoxide in the mitochondria221,249. A similar devastating phenotype is not seen for SOD1 null mice250-252, although a range of phenotypic features has been reported. For example, an increased vulnerability to axonal injury250, a mild axonopathy253, a reduced female fertility251,252, a propensity to develop cataracts254, an age-dependent hearing loss255 and a slight decrease in life span, possibly due to the development of hepatocellular carcinoma256.

Structure The crystallographic structure of hSOD1 was first reported in 1992 by Parge and co-workers257. In overall terms, the structure was very similar to that of bovine SOD1, which had been thoroughly studied previously258,259. The structure of holo wt-hSOD1 has subsequently been refined at 1.8 Å resolution260 and there is also an abundance of structural data for other variants of SOD1, including mutSOD1s.

SOD1 is a hydrophilic ellipsoidal dimeric protein about 30x40x70 Å in size. Each subunit consists of 153 amino acids and ligands one copper and one zinc ion. The general structure of the subunit is that of a flattened Greek-key β-barrel. The β-barrel is formed by eight antiparallel β-strands joined by seven external loops (Fig. 1). Loops I, II and V are short β-hairpin connections between adjacent β-strands. Loop IV (residues 49-84) makes up the disulfide bond, the zinc-binding domain and part of the active site cavity. Loop VI (residues 102-114) together with the smaller loop III (residues 37-40) provides the Greek-key connection across the β-barrel, while loop VII (residues 121-144) is an electrostatic loop implicated in substrate attraction.

N

1 2 874563

C

I

II

III

IV

V

VI

VII

Fig. 1 Schematic view of the SOD1 structure as adapted from 257,258. β-sheets are depicted as open arrows and loops as lines. The β-sheets are enumerated in sequential order using Arabic numerals and the loops are enumerated using Roman numerals.

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The copper ion is coordinated by the four histidine residues at positions 46, 48, 63 and 120 and also binds a water molecule. Zinc is also coordinated by the histidine residue at position 63 but also by histidine residues 71 and 80 and the aspartate at position 83. The metal ions are buried inside the protein and only the active site copper ion is solvent exposed. To reach the copper ion, the superoxide radical has to pass through a narrow cavity in the protein which, in the end, at the site of the copper ion, is only about 3 Å in diameter. Several negatively charged residues surrounding the cavity and positive charges in the cavity provide an electrostatic field gradient, driving the negatively charged superoxide radical to the copper ion. This arrangement is likely to be important for the high rate of catalysis seen for SOD1261 (2x109 M-1s-1). The purpose of the narrow cleft is probably to avoid unwanted redox reactions between the copper ion and other larger molecules. Zinc does not participate in the redox reactions, but is important for the stability of the enzyme262.

There are four cysteines in the hSOD1 protein at positions 6, 57, 111 and 146. The cysteines at positions 57 and 146 form an intrasubunit disulfide bond, a rare feature for a protein mainly residing in the reducing intracellular environment. The bond is of structural importance and provides (un)folding constraints for the protein (see below).

Properties Expression of SOD1: The hSOD1 gene is located on chromosome 21q22.11 (NCBI) and spans 11 kb of DNA. It consists of 5 exons separated by four introns (Fig. 2) and produces two transcripts 0.7 and 0.9 kb in size263. Although the gene contains several regulatory elements264,265, the transcription of the gene in vivo does not appear to be “actively” regulated (unlike SOD2 and SOD3)220 and SOD1 have even been regarded as a housekeeping gene. In spite of this, activity levels differ widely in different organs. In humans, the highest levels of hSOD1 activity are seen in the liver and kidney, while the brain only contains intermediate levels248,266. The levels of hSOD1 within the human CNS have been measured with in situ hybridization and immunohistochemistry and hSOD1 expression appears primarily in neurons267-270 but could also be seen in glia269-271. There is, however, no clear correlation between the amount of hSOD1 in different neuronal populations with the susceptibility to mutant hSOD1s267,268.

SOD1 is mainly a cytosolic protein, but there are also indications, mainly from animal studies, that SOD1 can localize to other cellular compartments such as the mitochondrial intermembrane space243,272-275, peroxisomes273,275-278, lysosomes 275,276,279 and in the nucleus268,276,277. Results in this area have, however, been conflicting. Chemical properties: The hSOD1 dimer has a molecular weight of approximately 32 kDa with a pI of 4.7-4.9280. hSOD1 is therefore negatively charged at physiological pH, which could be important for substrate attraction as described above. The activity of the enzyme is, however, almost pH independent in the range of 5.3-9.5241.

SOD1 is unusually resistant to proteolysis, thermal inactivation and inactivation by chemical denaturants such as 4% SDS (sodium dodecyl sulfate) and 10 M urea262,281. Nor does long-term storage at 5°C appear to reduce activity247. Known


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inhibitors of the protein are hydrogen peroxide, cyanide, azide and dietyldithiocarbamate282. Interestingly, the high stability of SOD1 is largely dependent on the metal occupancy262,283-286 and the disulfide status of the protein287. In fact, disulfide reduction and zinc depletion of copper-deficient wild-type hSOD1 reduced the relative melting temperature from 74.6°C to 42.9°C (a reduction of 31.7°C). These modifications are cooperative, since disulfide oxidation or zinc loading alone only increased the relative melting temperature by 6.9°C and 15.5°C respectively. This means that the melting temperature of disulfide-reduced apo hSOD1 is only slightly higher than the physiological temperature in humans (~37°C)288.

The disulfide status and metal occupancy of hSOD1 also appears to be of importance for the dimerization of the protein, since disulfide-reduced apo hSOD1 appears at least in part as a monomer. Metal depletion or disulfide reduction alone are not sufficient to monomerize the protein in vitro285,287-289. Enzymatic function: As described above, the enzymatic function of SOD1 is to dismute the superoxide anion to hydrogen peroxide and water (Reaction 1). This is done in two steps. First, copper is reduced to Cu+ by the superoxide radical (Reaction 9). During this step, copper, when reduced, disassociates from the imidazolate ring of H63 and the water molecule. At the same time, the histidine ligand is protonated. Later, in the second step of the dismutation reaction, the copper ion is oxidized by an additional superoxide radical (Reaction 10) and re-associates with H63 and the water molecule290,291.

(Reaction 9) Cu2+ + O2•- → Cu+ + O2

(Reaction 10) Cu+ + O2•- + 2H+ → Cu2+ + H2O2 The protons in Reaction 10 are probably donated from a water molecule and from the H63 residue. At low O2•- concentrations, the rate-limiting step of the reaction is probably the diffusion rate of the superoxide radical and at higher concentrations the transfer of the protons290.

Copper chaperone for superoxide dismutase Due to the high toxic potential of free copper, this cation is tightly regulated in cells. It has been estimated that the amount of intracellular free copper is less than one copper ion per cell292. Since the levels of free copper ions in the cells are so low, there is an obvious need for copper transporters. In humans, the copper transporter responsible for the copper loading of SOD1 is termed copper chaperone for superoxide dismutase (CCS)293. Much of the research on CCS has been done on yeast CCS, but most of these data also appear to be valid for the human protein. Human CCS consists of 274 amino acids and sequence analysis has suggested that this protein consists of three distinct domains294. This view has subsequently been confirmed by X-ray crystallography of domains I and II of yeast CCS295 and of domain II of human CCS296.

The N-terminal domain (domain I) consists of residues 1 to 85 and shows high homology to the copper chaperone ATOX1, which is a protein involved in the

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transport of copper to copper ATPases in the trans-Golgi network (ATP7A and ATP7B, or the Menkes and Wilson disease proteins). This domain contains an MXCXXC motif that is known to bind copper. The second domain of CCS (residues 86-234) is highly homologous to SOD1. Interestingly, a single mutation in this region of CCS (D201H) is sufficient to give CCS superoxide dismutase activity297. The third and shortest domain (residues 235-274) is located at the C-terminal end of the protein. This domain is unique to CCS and, like domain I, this domain also contains a copper-binding CXC motif.

A model for the copper loading of SOD1 has been proposed whereby domain I of CCS acquires copper, probably from the CTR1 copper transporter in the plasma membrane298. Domain II of CCS then transiently docks with zinc-charged SOD1 monomer and creates a disulfide-linked heterodimer that subsequently results in the transfer of copper from domain III to SOD1299-302. Currently, the relationship and the handling of copper between the two copper-binding domains are unclear. The heterodimeric formation between human SOD1 and human CCS has been confirmed with X-ray crystallography303 and is not surprising, considering the high homology of SOD1 and CCS domain II. Human SOD1 is also relatively promiscuous and can dimerize with SOD1s from quite distantly related organisms304. Interestingly, studies in yeast have suggested that CCS is also important for the formation of the intrasubunit disulfide bond of SOD1. The exact mechanism for this is not known, but it probably involves the exchange of the intermolecular disulfide bond between SOD1 and CCS to an intramolecular disulfide bond in SOD1301.

The expression of human and mouse CCS mirrors that of SOD1 and the protein, like SOD1, appears to be mainly cytosolic. Levels of SOD1 are, however, 12 to 30 times higher than CCS305. Recent findings in yeast suggest that about 1-5% of CCS is located, together with SOD1 (see above), in the mitochondrial intermembrane space. Intriguingly, the uptake of SOD1 into mitochondria appears to depend on CCS, since CCS null yeast has lower levels of SOD1 in the mitochondrial intermembrane space306. The form of SOD1 that preferentially enters the mitochondria is (monomeric) disulfide-reduced apo SOD1 which, within the intermembrane space, is disulfide oxidized by CCS which helps retain SOD1 within the mitochondria. The copper loading of SOD1 by CCS is not necessary for retention, but SOD1 most probably has to be zinc loaded to interact with CCS301,307. The mechanism by which disulfide-reduced SOD1 and CCS enter the mitochondria or the reason why the disulfide oxidized enzyme is retained is not known308. Presumably, the function of the mitochondrial intermembrane space SOD1 is to detoxify superoxide radicals generated within the mitochondria, possibly as a second line of defense, where SOD2 is the first line.

CCS knockout mice are viable and show no overt phenotype. Like SOD1 knockout mice, these mice suffer from reduced female fertility and are extra sensitive to the free radical-generating substance paraquat. As expected, the SOD1 activity levels are dramatically decreased to about 15-30% of those of control mice, which corresponds to the decrease in copper loading of SOD1 that is seen309. It has been suggested that the residual SOD1 activity seen in CCS null mice depends on reduced glutathione for the insertion of copper into SOD1310.


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Superoxide Dismutase 1 and ALS

Features of ALS caused by mutant hSOD1s hSOD1 mutations: At present, 119 different mutations in the hSOD1 gene have been associated with ALS (P.M. Andersen personal communication). Most of the mutations are missense mutations311, but some mutations cause alterations in the reading frame leading to C-terminal truncations and in some cases the insertion of amino acids312. Mutations have been found in all five exons of the hSOD1 gene and are not restricted to a specific part of the folded protein313. Relatively few mutations have, however, been found in exon 3 and the N-terminal part of exon 2 is so far completely devoid of mutations311. There are some examples of intronic mutations that cause aberrant splicing of the hSOD1 transcript314,315. Silent mutations with no amino acid alteration have been reported in some ALS patients, but the importance of these mutations is not known. Pattern of inheritance: ALS caused by hSOD1 mutations is inherited dominantly, with the exception of ALS caused by the D90A mutation that is inherited both as a recessive and a dominant trait (see below). The penetrance of ALS caused by mutSOD1 is high. A large US study reported a penetrance of 92% at the age of 70316. Some mutations, e.g. the I113T mutation317, have, however, been associated with reduced penetrance in FALS. Interestingly, the I113T mutation is also surprisingly frequently seen in “SALS” 318-321, but it appears as if some of these “SALS” cases actually represent FALS with reduced penetrance322. A few I113T families with high penetrance have, however, been reported316. Epidemiology: In FALS, about 20% of the families have a mutation in the hSOD1 gene316,323-328. The incidence of mutSOD1-FALS could thus be calculated at about 2% (20% × 10%) of ALS cases, which is often the number given in the literature. In reality, however, the incidence is probably much higher, since hSOD1 mutations are seen in about 3-7% of apparently sporadic ALS cases319,324,329. In a recent large US study, comprising 2,045 unspecified ALS patients, the frequency of hSOD1 mutations in ALS was 7.2%, which supports the notion that about 5% of SALS cases are associated with hSOD1 mutations311. It is likely that some of these SALS cases represent FALS with reduced penetrance, as discussed above, but there is also a possibility of de novo mutations330. The most common mutation worldwide is the D90A mutation that exists as a polymorphism in the northern part of Scandinavia (see below). This mutation is probably also responsible for more ALS cases than any other mutation, given the reduced penetrance of this mutation (P.M. Andersen personal communication). In the US, the A4V mutation is the most common mutation and is found in about 50% of mutSOD1-FALS cases311,316,318. Elsewhere in the world, there are only a few reports of this mutation311. The high prevalence of this mutation in the North American population is probably the result of a founder effect during the early colonization of North America, since a FALS pedigree described as early as 1880 by William Osler, later turned out to carry this mutation316. I113T appears to be the third most common mutation311,325. Phenotype of mutant hSOD1-FALS: In overall terms, ALS caused by mutSOD1s is indistinguishable from sporadic classical ALS316,331 but similar to adult-onset FALS

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linked to other genes (Table 1); the mean onset is somewhat earlier than for SALS316,325,332 and the gender ratio is closer to 1:1316,325. The clinical phenotype is dominated by lower motor neuron symptoms311,324,332 and in some patients extramotor involvement is evident324. Neuropathologically, mutSOD1-FALS patients are similar to SALS patients, but dorsal column involvement and LBHI are possibly more common and the corticospinal tract is less affected in analogy to the predominant LMN symptoms. In the spinal cord, inclusions containing SOD1 have been described, but similar inclusions have also been described in a few SALS cases9,79,80,333.

In general, there is a poor correlation between genotype and phenotype for most mutant hSOD1s. Clinical characteristics, age at onset and disease duration differ widely between and among the different mutant hSOD1s, even within the same family316,324,325,333. Environmental and/or other genetic factors most probably influence the phenotype. There are, however, some exceptions. Patients carrying the A4V mutation generally present with a rapidly progressing mainly lower motor neuron disease with a mean survival of only about one year316,324,334-336 and recessively inherited D90A ALS is associated with a slowly progressing stereotypic somewhat atypical phenotype (see below). Recent studies on recombinant proteins have also reported an association between the stability of the protein and disease duration (see below). Genotype-phenotype studies are, however, generally hampered by the small number of patients for most mutations.

Properties of mutant hSOD1s The biochemical and biophysical properties of mutSOD1 have been investigated in a number of studies and have been shown to vary widely. In general, most mutSOD1s appear perturbed compared with wt-hSOD1. For example:

• The SOD activities of mutSOD1 in ALS patients range from 0% to close to 100% (see 333 for references).

• Several mutSOD1s showed decreased resistance to proteolytic treatment

with proteinase K, but G37R, G93C and I113T appeared to be as resistant as the wild-type protein337.

• In cells transfected with mutant and wild-type hSOD1, the half-life was

considerably shorter for most mutant hSOD1s, but some mutant hSOD1s, e.g. H46R and H48Q, appeared to be almost as stable as wt-hSOD1337-340.

• There are indications that the zinc-binding affinity341 and metal ion

specificities342 are weakened in mutSOD1 and some mutSOD1s also show a significant reduction in both copper and zinc content compared with wt-hSOD1 (sometimes referred to as metal-binding region mutSOD1s). Other mutSOD1s, however, have a metal-binding capacity similar to wt-hSOD1 (wild-type like mutSOD1s)343.

• “Metal-binding region” mutSOD1s have been shown to be more sensitive

to disulfide reduction, which may lead to decreased resistance to


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proteolytic digestion. Some mutSOD1s are, however, almost as resilient to disulfide reduction as wt-hSOD1 (e.g. D90A and G93A)344.

• The thermal stability of as isolated mutant and wild-type hSOD1s has

been evaluated with differential scanning calorimetry and found to reflect the metal-binding capacity of the different mutSOD1s (see above). Most mutant hSOD1s, however, display a decrease in stability compared with wt-hSOD1345,346. In cases of disulfide-reduced, metal-deficient hSOD1, the mutant protein even unfolds at temperatures well below the physiological temperature in humans (~37°C)288. However, the melting point for as isolated D90A and G93A was not that unlike wt-hSOD1345 and a recent study by Rodriguez and colleagues shows that some disulfide-reduced apo mutSOD1s (e.g. H48Q and D101N) have melting temperatures very similar to wt-hSOD1347.

• The unfolding behavior of disulfide-reduced wild-type and mutant

hSOD1s when exposed to guanidinium chloride or urea was evaluated by Lindberg et al. using circular dichroism. In this case, all mutSOD1s displayed a reduction in stability compared with wt-hSOD1, more when deficient in metal. The difference between the wild-type protein and D90A was, however, not that great284. Similar results have also been reported by others346,348.

• Lindberg et al. also measured the folding rates of monomeric and dimeric

mutant hSOD1s using stopped flow technique, with urea as the denaturant, and compared the folding rates with wt-hSOD1. They were able to divide the mutants into three groups – one group that affected the stability of the monomer (class 1), the second group included one mutant (L144F) that only affected the stability of the dimer interface (class 2) and the third and dominant group included the mutants that affected both the stabilities of the monomer and the dimer interface (class 1+2). Interestingly, an association between the stability of the mutant protein and disease duration was suggested349.

• Compared with wt-hSOD1, mutSOD1s have a greater affinity for phenyl-

sepharose and bind 8-anilino-1-napthalene-sulfonic acid (ANS) better suggesting an increase in hydrophobic surfaces350,351.

Mechanism of disease It is generally agreed that mutSOD1s cause ALS by the gain of a toxic property rather than a loss of enzymatic function. The main evidence for this is that:

1. The disease is inherited dominantly.

2. The D90A mutation retains full SOD activity in human erythrocytes and CNS.

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3. Some mutSOD1 transgenic mice develop a phenotype similar to MND, despite higher than normal SOD1 activity.

4. SOD1 knockout mice do not develop a MND phenotype.

The nature of this toxic property of mutSOD1 is not known, but a number of disease mechanisms have been proposed and some of them are discussed below. SOD1 transgenic mice: To facilitate research in this area, a number of model systems have been used to study the mutant protein and its consequences (as discussed in the methodology section). Transgenic mice and rats expressing different mutSOD1s have been very valuable for our understanding of the disease and the noxious property of mutSOD1s. A number of such strains have been generated and some of them are summarized in Table 2.

Table 2. Mutant SOD1 transgenic rodents

Mutation Promoter Specie MND phenotype Reference

A4V hSOD1 gene hSOD1 Mouse No 352

G37R hSOD1 gene hSOD1 Mouse Yes 353

G37R hSOD1 cDNA PRNP1 Mouse Yes 354

G37R hSOD1 cDNA NFL2 Mouse No 355

H46R hSOD1 gene hSOD1 Mouse Yes 356

H46R hSOD1 gene hSOD1 Rat Yes 357

H46R/H48Q hSOD1 gene hSOD1 Mouse Yes 358

H46R/H48Q/H63G/H120G hSOD1 gene3 hSOD1 Mouse Yes 359

G85R hSOD1 gene hSOD1 Mouse Yes 360

G85R hSOD1 cDNA THY14 Mouse No 361

G86R mSOD1 gene mSOD1 Mouse Yes 362

G86R mSOD1 cDNA GFAP5 Mouse No 363

D90A hSOD1 gene hSOD1 Mouse Yes This thesis

G93A hSOD1 gene6 hSOD1 Mouse Yes 352

G93A hSOD1 gene hSOD1 Rat Yes 357,364

G93A hSOD1 cDNA THY14 Mouse No 361

L126Z hSOD11 gene7 hSOD1 Mouse Yes 365

L126delTT hSOD1 gene8 hSOD1 Mouse Yes 366

G127insTGGG hSOD1 gene9 hSOD1 Mouse Yes This thesis

1 In the CNS, expression is mainly in neurons and glia 2 Expression in neurons only 3 Also known as the “Quad” mutant 4 Expression in neurons only. Green fluorescent protein containing construct 5 Expression in astrocytes only 6 Both high and low expressing lines are frequently used367 7 Introns 3 and 4 were deleted in the construct. Stop at 126 8 Also with a FLAG containing construct. Stop at 131 9 Stop at 133


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The neuropathology of these mouse strains mainly recapitulates the findings in ALS patients, including motor neuron loss, reactive gliosis, LBHIs, spheroids and ubiquitin-positive inclusions (including skein-like inclusions). Some deviating features are, however, seen. Perhaps the most conspicuous of these is the presence of vacuoles seen in the CNS of G93A, G37R and even wt-hSOD1368 transgenic mice and rats. These vacuoles are thought to emanate from swollen mitochondria353. Interestingly, transgenic mice and rats expressing mutSOD1s that are deficient in copper binding (e.g. H46R, H46R/H48W and G85R) do not develop these vacuoles. Another deviating feature is the lack of Bunina bodies in the transgenic mice.

In mutSOD1 transgenic mice, there is an gene-dosage effect on the phenotype, since mice that are homozygous for the transgenic insertion show a much more rapid disease progression354,366,367, while mice with a loss of copy numbers have a protracted disease course367. Interestingly, double G93A/wt-hSOD1 transgenic mice have been reported to have an accelerated disease progression compared to G93A hSOD1 alone, suggesting that wt-hSOD1 itself could be harmful368,369 (see also Results and Discussion). This was not, however, seen in G85R/wt-hSOD1 transgenic mice370. It has been suggested, although this was not convincingly demonstrated, that this difference was due to the stabilization of the G93A, but not the G85R protein, by heterodimerization with wt-hSOD1369. A gene-dosage effect may also be relevant in humans. In the literature, there is one report of an ALS patient homozygous for mutant SOD1 (besides the special case of recessively inherited D90A discussed below). This patient, who was homozygous for the N86S mutation, suffered from an aggressive form of ALS and died at the age of just 13 years with a very short disease duration of only 14 weeks371. Most interestingly, two recent studies have reported that hSOD1 gene silencing with RNA interference (RNAi) in G93A transgenic mice dramatically improves onset, progression rate and survival372,373. A similar strategy could be feasible in humans and could potentially be a new therapeutic alternative for ALS patients.

The cell type(s) in which mutSOD1s are toxic is(are) not known. The specific expression of mutSOD1 in astrocytes363 or neurons355,361 in transgenic mice has failed to provoke disease. Studies of mutSOD1 chimeric mice374 and glial and neuronal co-culture experiments375 have suggested that mutSOD1 have to be present in multiple cell types to cause motor neuron degeneration. Concerns have, however, been raised about the expression levels of mutSOD1 in strains with cell-specific expression of mutSOD1365. An absolute amount of mutant protein was only given in the report from Pramatarova et al. and, compared with our own measurement of hSOD1 in the spinal cord of D90A transgenic mice, the levels of NFL regulated H46R355 correspond to about one quarter of the levels of D90A mutant SOD1 in homozygous D90A mice (Papers III, V). The levels of THY1 regulated G85R have now also been re-calculated to be less than 5% of endogenous mouse SOD1 (mSOD1)376. These levels of the mutant protein could possibly be too small to cause disease. A recent study by Wang and colleagues showed that the expression of mutSOD1 in muscle cells, astrocytes and neurons but not microglia is sufficient to cause disease354. This finding most likely excludes microglia as a primary participant in the motor neuron death mechanisms.

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Apoptosis: Today, there is a considerable amount of evidence supporting a role for apoptosis (or programmed cell death) in ALS pathogenesis377,378. Some of the main evidence is briefly summarized below.

In neuronal cells transfected with mutSOD1s, there are signs of DNA fragmentation characteristic of (although not exclusively seen in) apoptosis379-382, as well as signs of caspase activation375,383,384 in distressed mutSOD1-expressing cells. Furthermore, treatment with caspase inhibitors381-383 or co-expression with the anti-apoptotic protein Bcl2 attenuates cell death in these cells381.

In tissues from mutSOD1 transgenic mice (and humans), it is difficult to detect apoptotic cells, since the process itself is rapid and little trace can be discerned after the process is over. The daily rate of motor neuron loss is probably also quite low. The morphology of cells undergoing apoptosis in tissues is also somewhat unspecific. This may be the reason why some studies of apoptosis in mutSOD1 transgenic mice have been negative385,386. It might therefore be a better choice to rely more on biochemical investigations in order to study apoptosis in vivo. The activation of caspases in neurons (and possibly also glia) in the CNS of mutSOD1 transgenic mice has been reported by several groups383,384,387-391. Interestingly, in one study, the activation of caspase-1 was seen at just one month of age in G85R mice, with the activation of caspase-3 occurring much later384. Vukosavic et al. reported similar results of the temporal activation of caspases in G93A mice390. This sequence of activation is also in agreement with the known activation cascade of caspases389. Dominant negative inhibition of caspase-1 in neurons significantly prolonged survival in G93A mice392, as did intracerebroventricular treatment with the broad caspase inhibitor zVAD-fmk388. The caspase-1 downstream mediator interleukin-1β (IL-1β) has also been reported to be upregulated in the spinal cords of mutSOD1 (G37R) transgenic mice, although on an IL-1β null background there was no difference in survival393. G93A transgenic mice on a caspase 11 knockout background resulted in a decrease in caspase 1- and 3-like activities, but did not lead to enhanced survival387. For this reason, caspase 11 does not appear to be needed to generate the MND phenotype of G93A transgenic mice. The lack of effect could represent redundancy in which other caspases may compensate for the lack of caspase 11.

Cytochrome C is important in mitochondrial-associated caspase activation and it has been shown that this protein translocates from the mitochondria to the cytosol in the spinal cords of G93A mice (see also below). One caveat is naturally that these mice have distended vacuolated mitochondria which could possibly interfere with the analysis. However, wt-hSOD1 transgenic mice, which also have vacuolated mitochondria, did not show any signs of cytochrome C translocation389.

The levels of the anti-apoptotic proteins Bcl2 and Bcl-xL have been shown to be decreased in spinal cord neurons of mutSOD1 transgenic mice, while pro-apoptotic Bak and Bax are increased394-396. A recent study also indicated that both wild-type and mutant hSOD1s were able to bind to Bcl-2. However, in mitochondrial preparations from mutSOD1 transgenic mice, only mutSOD1s co-precipitated with Bcl-2 and it was suggested that aggregated mutSOD1 could entrap Bcl-2 and thereby promote apoptosis396. Several reports have shown that the overexpression of Bcl2 in mutSOD1 transgenic mice attenuates caspase activation and the amount of unbound Bax, resulting in a significant prolongation in survival390,394,397.


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In humans, there are few reports of apoptosis in mutSOD1-FALS. Pasinelli and colleagues investigated an A4V patient and showed that hSOD1 in this patient bound to Bcl-2. In sporadic ALS cases without hSOD1 mutations, the activation of caspases has been seen in the spinal cord388 and more specifically in motor neurons391. The levels of proteins in the Bcl-2 family in ALS patients are more difficult to evaluate, but there are signs of upregulation of Bak and Bax and downregulation of Bcl-2 in the spinal cords of ALS patients398-400. Finally, there is one report suggesting translocation of cytochrome C from the mitochondria to the cytosol in motor neurons of ALS patients389. Mitochondrial pathology: In several of the mutSOD1 transgenic mouse strains, there is evidence of vacuolated mitochondria (see above). The temporal occurrence of these vacuoles appears to precede the occurrence of symptoms in mutSOD1 transgenic strains, which suggests the involvement of these organelles in mutSOD1-FALS pathogenesis401. The vacuolation of mitochondria has been ascribed to the entry of hSOD1 into the intermembrane space of mitochondria402,403. Vacuolization could therefore be dependent on CCS. However, overexpression of mutSOD1 on a CCS null background did not show any “gross differences” in histopathology404.

There are several ways in which injured mitochondria may be toxic to motor neurons. One possibility is that cytochrome C in the intermembrane space leaks into the cytosol and thereby initiates apoptosis (see also above)403,405,406. Another possibility is that the normal mitochondrial function becomes impaired. In mitochondrial fractions from the CNS of G93A mice, a decrease in the maximum respiratory rate and a lowering of mitochondrial complex activities have been reported406-409. A similar decrease in cytochrome C oxidase (COX or complex IV) activity was also seen in the motor neurons of ALS patients by staining tissue sections for COX activity410. In CNS homogenates from ALS patients, the results have been conflicting. Two studies have shown a decrease in COX activity411 in the spinal cord of ALS patients but not when corrected for the amount of mitochondria by measuring citrate synthase412. In the brains of ALS patients, studies of COX activity have been negative, while complex I-III activities have shown variable results409,413. It is, however, possible that the defect in energy metabolism is only a secondary effect of already injured neurons. The fact that the effect on respiratory enzymes and rate was especially prominent in symptomatic mice is compatible with this theory406,407. Other proposed mitochondrial disease mechanisms include the association between mitochondria and mutSOD1-containing aggregates in mutSOD1 transgenic mice (see above and below)376,396, with a subsequent decrease in Bcl-2 (see above)396.

The findings described above suggested that treatment with mitochondrial protective agents would be beneficial. Indeed, supplementation with creatine, which could function as an energy buffer in cells and/or inhibit mitochondrial transition pore openings, prolonged survival in G93A mice and signs of free radical damage were attenuated414. Similarly, treatment with the anti-inflammatory and anti-apoptotic drug minocycline, which has also been shown to inhibit mitochondrial cytochrome C release415, significantly enhances survival in G37R416 and G93A415,417 transgenic mice. A drug cocktail using both these substances has shown an even greater effect on survival418.

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In human clinical trials, creatine has failed to prove effective419,420 and a minocycline phase III study is currently ongoing421. The failure of creatine suggests that the mitochondrial abnormalities seen in some mutSOD1 transgenic strains are not relevant to ALS pathogenesis. There are also other reasons why the vacuolated mitochondrial may prove to be irrelevant.

• Not all mutSOD1 transgenic strains develop mitochondrial vacuoles (see above).

• Vacuoles are almost never seen in spinal cord sections from ALS

patients422.

• Mice transgenic for wt-hSOD1 also develop vacuoles but do not develop overt MND402.

Oxidative stress: It has been suggested that aberrant copper chemistry at the active site of mutSOD1s could be responsible for motor neuron death. Two substrates have been proposed for the copper ion, nitric oxide and hydrogen peroxide.

Nitric oxide can react with the superoxide radical and form the highly reactive peroxynitrite (Reaction 2) Peroxynitrite could subsequently, by widening of the active site cavity of mutSOD1, react with Cu2+ to form a nitronium intermediate (Reaction 11) that could nitrate tyrosine residues of proteins (Reaction 12), which could be harmful to neurons by blocking phosphorylation sites, for example423,424.

(Reaction 11) Cu2+ + ONOO- → CuO···NO2+

(Reaction 12) CuO···NO2

+ + Tyr-H → Cu2+ +HO- + Tyr-NO2 Another route in which nitric oxide reacts with a superoxide intermediate of SOD1 to create peroxynitrite has also been suggested (Reactions 13 and 14)425.

(Reaction 13) Cu+ +O2 → Cu2+···O2•-

(Reaction 14) Cu2+···O2•- + NO → Cu2+ + ONOO- Increased levels of free nitrotyrosine have been observed in the spinal cords of both mutSOD1 transgenic mice424,426 and ALS patients with and without hSOD1 mutations427. In spinal cord sections of both transgenic mice and ALS patients, there is also evidence of increased levels of nitrotyrosine424,427-429. With regard to the levels of protein-bound nitrotyrosine in the spinal cords of mutSOD1 mice and ALS patients, findings have been contradictory426,430.

Importantly, the survival of G93A mice that are null for the inducible431 or neuronal432 nitric oxide synthase is similar to that of ordinary G93A mice, suggesting that the levels of nitric oxide are not important in ALS pathogenesis. The levels of nitric oxide in these mice were, however, not reported. The other proposed substrate is hydrogen peroxide. It is known that SOD1 can function as a peroxidase and thereby generate highly reactive and potentially toxic


33

hydroxyl radicals, similar to the Fenton reaction (Reaction 4). Wideau-Pazos and co-workers measured the hydroxyl radical production of recombinant A4V and G93A mutSOD1s using a DMPO spin trap assay and found an increase in hydroxyl radical formation compared with wt-hSOD1. Similar to the peroxynitrite hypothesis, they speculated that mutation could confer a widening of the active site cavity that would give the substrate greater access to the active site433. Evidence supporting an increase in hydroxyl radical formation from mutSOD1s has also been seen in the striatum of G93A transgenic mice using microdialysis434. Other groups have reported similar results435,436, but these results have also been questioned, since not all groups have noted such differences between the wild-type and the mutant enzyme281,426,437,438. The DMPO spin trap assay has also been criticized, since it may not be specific for free hydroxyl radicals438,439. Today, the active site copper hypothesis is less favored. Some of the evidence speaking against the hypothesis has already been mentioned, but there is also additional evidence. For example:

• Some mutSOD1 have very low copper affinity343,345.

• Knocking out the CCS gene in mutSOD1 transgenic mice does not attenuate disease404.

• Mice transgenic for the artificial “Quad” mutant that is lacking all four

copper-binding histidines still develop MND disease359. Mutant hSOD1-containing aggregates and inclusions: One conspicuous finding in the spinal cords of older mutSOD1 transgenic mice is the occurrence of neuronal366,440 and astrocytic360,370,441 SOD1-containing inclusions. These inclusions can be found in the soma as well as in the neuropil and are sometimes associated with LBHIs366,440,442. Not all inclusions, however, stain for SOD1441. Interestingly, similar inclusions (predominantly LBHIs) are also seen in the spinal cords of ALS patients with442,443 and without SOD1 mutations84,85,422. These inclusion also stain positive for other proteins; they include ubiquitin360,422,440,441,443,444, neurofilaments422,440,443, CCS441,442, heat shock proteins441,444,445, proteasome 20S441, NEDL1446, dorfin447 and the glial fibrillary acidic protein (GFAP)360,441. Some spinal cord inclusions also stain with thioflavin, which is a specific stain for fibrillary aggregates that are rich in β-sheets354,358,359. Such staining, although very weak, was also seen for in vitro aggregated protein filaments of mutSOD1s346,348,350.

In spinal cord homogenates of symptomatic mutSOD1 transgenic mice, SOD1-containing, detergent-resistant aggregates can be detected by filter trap assays354,358,359,444,448 or by different centrifugal techniques354,359,365,444,449,450. In the detergent-resistant aggregates, high molecular weight (HMW) species of SOD1 are seen, suggesting the enrichment of these species in aggregates354,359,451. Similar detergent-resistant aggregates have also been seen in an A4V patient359. It is probable that these aggregates, at least in part, represent the SOD1-containing inclusions discussed above.

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Three major proteolytic systems participate in the turnover of proteins. The ubiquitin/proteasome pathway functions both as a degradation route for short-lived (regulatory) proteins and as a quality control system in which mis/unfolded proteins are degraded by the proteasome proteolytic complex after being covalently tagged with polyubiquitin452. The process of ubiquitination is not known in detail but depends on three types of enzymes. An E1 ubiquitin-activating enzyme activates ubiquitin in an ATP dependent manner. Subsequently, the activated ubiquitin is transferred to the substrate by binding to an E2 ubiquitin-conjugating enzyme. The ubiquitin containing E2 enzyme then interacts with an E3 ubiquitin-protein ligase that is responsible for substrate recognition and ligation of ubiquitin to the substrate. The polyubiquitinated substrate is then recognized by the 19S proteasome of the 26S proteasomal complex, deubiqutinated, unfolded and degraded by the 20S proteasome453-455. An increasing number of proteins are, however, recognized to be degraded by the 26S proteasome without prior ubiquitination and are, in some cases, degraded directly by the 20S proteasome without processing by the 19S proteasome456.

Calpains are a family of calcium-dependent cysteine proteases that participate in the degradation of proteins in the membrane or in the cytosol. The regulatory mechanisms of calpains are poorly understood457.

The third system is the autophagic/lysosomal system which is thought to play a major role in the turnover of long-lived proteins. Autophagy occurs when a membrane surrounds a part of the cytoplasm, creating an autophagosome, and then fuses with lysosomes where proteins are degraded by lysosomal proteases. Although mainly a non-selective process, autophagy can in some instances be more specific, for example by eliminating injured organelles. The signals regulating the process of selective autophagy are, however, unknown. The rate of autophagy could be regulated both positively and negatively458.

Little is known about the way wild-type SOD1 is degraded. In vitro, it has been demonstrated that apo hSOD1 is degraded by the proteasome in an ubiquitin-independent manner. Additional data comes from cell culture studies using proteasome inhibitors where it has been demonstrated that the wild-type protein is at least partly digested by the proteasomal system340,459 but also that the system is responsible for the digestion of a much larger proportion of the mutant enzyme340,451,460. In these cell-culture experiments, proteasome inhibition causes a change in the distribution of the mutant protein into inclusions or aggregates451,460 and decreases cell viability459,461. The overexpression of mutSOD1 in cells has also been associated with a decrease in proteasome activity460,462, but the results in this area are conflicting459 and a similar decrease in proteasome activity was not seen in the spinal cords of G93A mice, even though the levels of some proteasomal subunits were altered449. Interestingly, the overexpression of dorfin, one of two known mutSOD1 E3-ubiquitin ligases (the other one is NEDL1446) and also appearing in LBHIs of ALS patients and mice, in mutSOD1-expressing cells was shown to enhance survival and decrease the amount of inclusions447.

There is also evidence to support a role for the phagocytic/lysosomal system in the degradation of SOD1. For example, immuno-electron microscopy has shown that SOD1 can be localized to lysosomes276, as well as autophagocytic vesicles463, and SOD1 was enriched in lysosomal fractions from density gradient centrifugation275,279,


35

although the amount of SOD1 that was associated with lysosomes in some studies probably is an overestimation. Heat shock proteins (HSP) are a group of highly conserved proteins that are induced in response to environmental stress by infection, ultraviolet radiation, chemicals, oxidative stress and of course heat, among other things. The HSP proteins are grouped in families according to their size (HSP 100, HSP90, HSP 70 etc.) and the smallest are referred to as the small HSPs (e.g. HSP27, HSP10 and αB-crystalline). Ubiquitin has also been included in this group of proteins. The classical role of HSPs is to function as molecular chaperones and to participate in the folding of proteins but also to help in the handling of misfolded proteins. Another function is to mediate in the degradation of proteins464,465.

Studies of cells expressing mutSOD1s have indicated that HSPs could be upregulated in response to the expression of mutSOD1. For example, Bruening and co-workers showed that mutSOD1-expressing NIH 3T3 cells had higher levels of HSP27, HSP70 and αB-crystalline, which corresponded well to the increase in chaperoning activity seen in these cells466. Studies of mutSOD1 transgenic mice have shown a more complex picture. In the same paper, Bruening et al. reported a decrease in chaperoning activity in the lumbar spinal cords of presymptomatic G93A mice and it was therefore suggested that the abundant mutSOD1 protein depleted chaperones in ALS-affected areas466. Similar results were also reported by Tummala et al. investigating spinal cord homogenates from G93A but also G85R mice467. When examining the levels of the different HSPs, a temporal pattern of expression has been reported. In the spinal cords of presymptomatic mutSOD1 transgenic mice, the levels of HSP25/27 and αB-crystalline are equal to or lower than those of control mice, but the levels increase in symptomatic mice, possibly as a result of gliosis354,359,365,444,467-

469. The levels of HSP40, HSP60, HSP70 and HSP90 have been reported to be mainly unchanged359,445,467-469. The pattern of expression of the small HSPs suggested that the high levels of expression of these proteins in glia protected these cells in mutSOD1 transgenic mice365, while motor neurons had lower levels444. In spinal cord sections of ALS patients, the relative HSP27 intensity was similar to that of controls444,468.

It is difficult to understand why the spinal cords of mutSOD1 transgenic mice display a reduction in chaperone activity, but at the same time HSP levels are increased. Tummala et al. investigated this enigma and suggested that the lack of concordance could be due to the inactivation of HSPs by mutSOD1 and reported that, by adding recombinant mutSOD1s to spinal cord lysates, chaperone activity is inhibited dose dependently. In their assay, however, the possibility was not excluded that the mutSOD1s were preferentially chaperoned by HSPs rather than the reporter protein, catalase467.

There is also more direct evidence of an interaction between mutSOD1 and HSPs. Co-immunoprecipitation experiments in cells transfected with different hSOD1s have shown that HSP25, HSP40, HSP70 and αB-crystalline interact with mutSOD1 but not wt-hSOD1307,450. HSPs have also been found in mutSOD1-containing aggregates354 and inclusions441,444, but Maatkamp et al. failed to see a more direct association between HSP25 and mutSOD1444.

Interestingly, in an in vitro assay of mutSOD1 aggregation, it was found that αB-crystalline attenuated the formation of aggregates365. Similarly, Bruening and

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colleagues transfected a primary motor neuron culture with G93A and HSP70 and saw a decrease in inclusions and toxicity compared with cells expressing G93A alone466. Subsequent cell-culture studies have provided similar results382,470. Although overexpression of HSP70 alone in G37R, G85R and G93A mice did not alter disease445, treating G93A mice with arimoclomol, an inducer of HSPs, results in a remarkable improvement and an increase in mean survival from 125 days to 153 days471.

Recently, mutations in the two small HSPs, HSP27 and HSP22 was linked to two dominantly inherited neuromuscular diseases, Charcot-Marie-Tooth 2F 472 and distal hereditary motor neuropathy type II 473 respectively. It is well known that almost all proteins, including proteins completely consisting of α-helical structures, can form fibrils under certain circumstances474. The way this process occurs in the case of mutSOD1 is not known in detail. For the protein to form aggregates unfolding (or misfolding) of the protein is a necessity. As has been discussed above, SOD1 is considerably more prone to unfold in its disulfide-reduced apo form and, when mutated, the protein is even less stable. Different in vitro aggregation assays have also shown that the form of the protein most prone to aggregate is the monomeric mutant apo protein348,350,359,475. Strikingly, Stathopulos et al. were able to demonstrate an inverse correlation between the thermal stability of mutated and wild-type apoSOD1s and the propensity to aggregate346. The importance of disulfide reduction in aggregation has not been reported.

One example of the way SOD1 aggregation may occur was given by Elam et al., who examined the crystal structure of apo S134N and H46R. They showed that these mutSOD1s crystallized into three different crystal forms through interactions between loops and the β-barrel. The filamentous structure was formed by conformational changes in loop IV and VII (Fig. 1) that caused the deprotection of the edges of β-strands 5 and 6, providing new contact surfaces leading to oligomerization476. Electron and atomic force microscopy of in vitro aggregated mutSOD1 have revealed both amorphous aggregates346,350 and more fibrillary structures346,348,350,477. Interestingly, similar fibrils, 15-25 nm in diameter and immunoreactive for SOD1, have also been seen in the hyaline inclusions of ALS patients478 and mutSOD1 transgenic mice478,479. Intriguingly, it has also been reported that metal-depleted wt-hSOD1 also aggregates, but that wt-hSOD1 was comparatively less prone to form fibrillary aggregates than mutSOD1348,350,475,477.

D90A mutant hSOD1 D90A mutant hSOD1 in recessive ALS pedigrees: Of all hSOD1 mutations identified so far, only D90A cause ALS with a recessive pattern of inheritance480. In the northern part of Scandinavia, this mutation exists as a polymorphism with an allele frequency of about 2% and, in Finland alone (population 5,236,611, Dec 31, 2004, Tilastokeskus), there should be approximately 99,000 carriers of this mutation481. Patients homozygous for the D90A allele have also been found in other parts of the world including Germany482, France483, Italy328,484, Russia485, Canada486 and the USA311. The penetrance of the disease in recessive D90A pedigrees has been calculated to 95% at the age of 70 years487. The mean age at onset is 44 years, ranging from 20-94 (!) years. The homozygous D90A patients suffer from a


37

characteristic stereotypic phenotype. Onset is characterized by stiffness and cramps in the legs and a feeling of general fatigue. Lumbar back pain is common. Paresis always starts in the lower limbs and then slowly progresses. Later, symptoms from the upper limbs and the brain stem nuclei are seen. UMN symptoms are abundant. Some patients also experience paresthesias and micturition problems. Mean survival has been calculated to 13 years, but some patients have a disease duration of more than 20 years488. ALS in individuals heterozygous for D90A mutant hSOD1: Most fascinating and probably unique to genetics, this mutation has also been associated with ALS in dominant FALS pedigrees311,489 and the mutation has also been found in SALS in geographical locations where the mutation is otherwise rare, suggesting that the mutation in these cases are disease-causative although with low penetrance319,485,489. The dominant D90A ALS cases display a more variable and aggressive clinical picture, similar to that of SALS.

It has been proposed that the phenotype in recessive D90A pedigrees is modified by a cis-acting protective factor close to the D90A hSOD1 gene490 that could act by downregulating D90A expression in recessive cases491. Despite considerable efforts, this putative protective factor has not yet been identified. Intriguingly, it appears as if both dominant and recessive cases are all descended from a single founder some 895 generations ago (~20,000 years) and that the recessive haplotype arose about 63 generations ago (~1,500 years)491. Properties of the D90A protein: The SOD1 activity in hemolysates from homozygous D90A individuals is very similar (~92%) to the SOD1 activity of controls480 and for the purified enzymes there were no significant differences in copper and zinc binding or specific activity between wild-type and D90A hSOD1. On denaturing gel electrophoresis, the mobility of the D90A protein is somewhat higher than that of the wild-type protein281.

The properties of the protein in vitro have also been shown to be very similar to those of wt-hSOD1. For example:

• The metallation, specific activity and the copper coordination are similar to wt-hSOD1343.

• Metal-charged D90A exhibits a high degree of thermal stability, close to

that of the wild-type protein345.

• The D90A protein is also almost as stable as the wild-type protein when exposed to denaturants such as urea or guanidinium chloride284.

• The disulfide bond of D90A mutant hSOD1 is nearly as stable as the

bond in the wild-type protein when exposed to reductants344.

G127X mutant hSOD1 Only one family has been reported with the Gly127insTGGG mutation (abbreviated G127X). This mutation inserts a repeat of four nucleotides (TGGG), which leads to

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five novel amino acids and a premature stop codon resulting in a 21 amino acid truncation (128-Gly, 129-Gln, 130-Arg, 131-Trp, 132-Lys, 133-STOP). In the first report, the insertion was wrongly stated to be an insertion of ACCC, which is actually the anti-sense of TGGG, and to put a stop codon at position 132 when the correct number is 133492.

This Danish family has subsequently been described in more detail. Both UMN and LMN symptoms, as well as bulbar and spinal symptoms, are known to occur in G127X ALS patients. Reduced sense of vibration has also been reported, although cutaneous sensation was normal. The range of age at onset is 40-60 years of age and the disease duration has been ~3 years. Erythrocyte SOD1 activity was about 40% of that of controls324.


39

AIMS The main aim of this thesis was to investigate the mechanisms by which mutSOD1s cause ALS. The strategy was to investigate mutSOD1s with widely differing properties and compare these mutSOD1s with each other and with wild-type hSOD1 in the search for a common noxious property of mutSOD1s. The more specific aims were: • To investigate whether the putative protective factor in recessively inherited

D90A FALS acts by downregulating synthesis, influencing degradation or in other ways altering D90A mutSOD1 (Paper I).

• To measure the levels of the three different SODs in cerebrospinal fluid from

sporadic, familial and mutSOD1-FALS patients and controls (Paper II). • To generate D90A and G127X mutSOD1 transgenic mice (Papers III, IV). • To compare the in vivo properties of wild-type hSOD1 and wild type-like D90A

mutSOD1 to find a clue to the toxicity of this mutSOD1 (Paper III) • To validate the G127X mice model by comparing the findings in the model with

findings in an ALS patient carrying the same mutation (Paper IV). • To study and compare the biochemical properties of mutant and wild-type

hSOD1s in transgenic mouse strains (Papers III-V).

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METHODOLOGY A summary of the main materials and methods used in this thesis and some considerations relating to the methodology are given below.

Human samples

Sample collection Blood was drawn into EDTA or acid citrate dextrose (ACD) containing vacuum tubes. Samples were then centrifuged and packed erythrocytes, buffy coat and plasma were collected separately and stored at -80°C until further analyzed (Papers I, II, IV).

CSF was collected using methods used in ordinary clinical practice. Samples were immediately frozen and stored at -80°C until further analyzed. Cases with headache or other neurological symptoms without underlying neurological disease served as controls (Paper II).

Skin punch biopsies were taken from a patient carrying the G127X SOD1 mutation to establish a fibroblast culture. The fibroblasts were grown in Ham’s F10 containing 10% fetal calf serum (Paper IV).

An autopsy on the same G127X patient (Paper IV) was performed within 20 hours after death. The whole brain and spinal cord were removed and frozen at -80°C. Further dissection of the tissue for analysis was performed on an aluminum plate at -20°C. For histological studies, pieces were immersion-fixed in 4% paraformaldehyde, 100 mM sodium phosphate, pH 7.4. As controls, five patients with no known neurological disease were used. The ages and postmortem times of these patients were similar.

Most of the individuals and/or their families in Papers I, II, IV have been described previously319,324,482,485,488,489.

SOD1 genotyping All the patients in Papers I, II, IV were screened for hSOD1 mutations. DNA was purified from buffy coats using the Nucleon BAAC2 kit (Amersham). The hSOD1 gene was analyzed with PCR and SSCP according to Andersen et al.480. Primers and PCR conditions were according to Table 3. The D90A allele was also identified by restriction enzyme cleavage of exon 4 PCR products using Fnu4HI, since this mutation introduces a new site for this restriction enzyme.

Ethical considerations All the human samples were collected with informed consent and the studies adhered to the tenets of the World Medical Association Declaration of Helsinki. All the studies were approved by the medical research ethics committee at Umeå University and in Paper IV also by the ethics committee at the University of Copenhagen, Denmark.


41

Table 3. Primers and PCR conditions used for genotyping

Exon Direction Oligonucleotide sequence Product length

1 Forward 5’-TTC CGT TGC AGT CCT CGG AA-3’ 158 bp

Reverse 5’-CGG CCT CGC AAC ACA AGC CT-3’

2 Forward 5’-TTC AGA AAC TCT CTC CAA CTT-3’ 208 bp

Reverse 5’-CGT TTA GGG GCT ACT CTA CTG T-3’

3 Forward 5’-CAT AAT TTA GCT TTT TTT TCT TCT TC-3’ 138 bp

Reverse 5’-CCA TTG ATT ACA AGA GTT AAG C-3’

4 Forward 5’-CAT CAG CCC TAA TCC ATC TGA-3’ 236 bp

Reverse 5’-CGC GAC TAA CAA TCA AAG TGA-3’

5 Forward 5’-GTT ATC TTC TTA AAA TTT TTT ACA G-3’ 133 bp

Reverse 5’-CTC AGA CTA CAT CCA AGG GA-3’

PCR conditions were: Exon 1: 95°C for 15’ then 35 cycles of 94°C for 1’, 65°C for 1’, 72°C for 1’ ending with 72°C for 10’ Exons 2-5: 95°C for 15’ then 35 cycles of 94°C for 1’, 55°C for 1’, 72°C for 1’ ending with 72°C for 10’

SOD1 transgenic mice

The mouse as a model system In Sweden, about 200 people each year succumb to ALS. A fraction of these patients are autopsied, thereby making CNS tissue from ALS patients, especially those carrying SOD1 mutations, scarce. To be able to study the motor neuron pathology of ALS, we therefore have to rely on different model systems. These results then have to be compared with what we see in ALS patients. In our studies, we have used transgenic mice overexpressing different kinds of hSOD1s as a model system. The mouse has several advantages, for example:

• The physiology of the mouse is similar to that of humans. • Mice have a rapid rate of reproduction. • Mice are relatively easy to handle. • The complete genome of the mouse has been sequenced.

An additional advantage of using these model systems is that tissues and cells can be sampled early in the disease process, which for natural reasons is impossible in humans. The model systems also give us the opportunity to manipulate cells and animals both genetically and chemically.

There are also other model systems which have been used to study mutSOD1 and ALS. They include in vitro models, such as cell-culture experiments and tissue-slice experiments, and in vivo models using other mutSOD1 transgenic animals, such as Caenorhabditis elegans, Drosophila melanogaster and Rattus norvegicus.

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All these models have their advantages and disadvantages. Differentiated neurons do not divide and short-lived primary neuronal cultures or more artificial tumor cell lines therefore have to be used for cell culture experiments. These cells typically have a relatively low expression of mutSOD1, but toxicity is still seen in a matter of days, suggesting that the pathogenic process in the cell cultures could be different. Moreover, the system usually only includes one cell type which is different from the situation in the spinal cord. One advantage of this system is that it is easy to manipulate and that the biochemical properties of proteins are easy to study. Tissue slices more closely resemble the environment in the spinal cord since the neurons here are supported by glial cells, but the conditions are still fairly artificial. This system is also comparatively easy to manipulate and analyze. The transgenic expression of mutSOD1 in C. elegans and D. Melanogaster has been difficult to evaluate since no overt neurological phenotype has been seen, possibly due to the short life span of these animals493-495. Transgenic rats expressing mutSOD1 have also been generated, with a phenotype very similar to that of transgenic mice. The only difference between the mouse and the rat models appears to be the size of the animals. The size of rats could in some instances be an advantage. For example, some therapeutic trials may be more easily performed on a larger animal and the sampling of tissue and fluids from rats could also be easier and richer. The larger size of rats could, however, make them more difficult to handle in some situations. One recent development is the use of chick embryos to study the effect of mutSOD1 in the spinal cord496.

Generation of transgenic mice The human SOD1 gene263 (Fig. 2) was chosen for the generation of transgenic mice to mimic the transcriptional regulation seen in humans. Subsequent studies by others have shown that cDNA constructs provoke disease as well354, but that the choice of promoter could be important since constructs that only express mutant SOD1 in certain cell types have failed to cause an ALS-like phenotype355,361,363 (see also above). The level of expression is also important354,366,367.

Amino acids:

Basepairs:

Restriction enzymes: Eco

RI, 1

Bam

HI, 1

1564

634

9325

811

5

7304

4742

705

4647

7375

9432

82

311

11

564

3

56-79

1

1-23 119-153

4

80-118

2

24-55

5

Nco

I, 6

698

Nco

I, 7

215

Nco

I, 9

031

Bcl

I, 6

267

Bcl

I, 9

592

Pst

I, 5

74

Pst

I, 2

978

Pst

I, 8

625

Pvu

II, 4597

Nsi

I, 2

588

Nsi

I, 8

256

Nsi

I, 9

891

HindI

II, 7708

HindI

II, 9152

HindI

II, 9962

Fig. 2 The human SOD1 gene. Coding regions are depicted in white with exon numbers and non-coding regions in grey. All cleavage sites for the restriction enzymes used for cloning and mutagenesis are shown. An 11.6 kb fragment containing the whole human SOD1 gene was cloned into the plasmid vector pGEM3 (Promega). Subcloning was performed using a pSP73-P


43

vector (Promega). Mutagenesis and construct for the D90A transgenic mice was performed according to Fig. 3 and according to Fig. 4 for the G127X mice.

An exon 4 PCR fragment from a

D90A patient was ligated into a

PvuII-PstI subclone of the human

SOD1 gene using HindIII-NsiI.Exon 5 was added by ligating

a PstI-BamHI fragment of the

SOD1 gene to the subclone.

3 42

Pst

I, 8

625

Pvu

II,

4597

Nsi

I, 8

256

HindI

II, 7708

D9

0A

4

Nsi

I, 8

256

HindI

II, 7708

D9

0A

Bam

HI, 1

1564

3 42 5

Pst

I, 8

625

Pvu

II, 4597

Nsi

I, 8

256

HindI

II, 7708

D9

0A

Eco

RI, 1

Bam

HI, 1

1564

31 42 5

Pst

I, 8

625

Pvu

II, 4597

Nsi

I, 8

256

HindI

II, 7708

D9

0A

Finally the PvuII-BamHI fragment was ligated

into the pGEM vector containing the whole

SOD1 gene using PvuII and BamHI.

Fig. 3 Mutagenesis and construct for D90A transgenic mice. Some cleavage sites of the restriction enzymes have been omitted. PCR primers for D90A were: 5'-CAC TAG CAA AAT CAA TCA TCA-3' and 5'-TCT TAG AAT TCG CGA CTA ACA ATC-3' and for G127X: 5'-AAA GTA AGA GTG ACT GCG GAA CTA-3' and 5'-CTG GCA AAA TAC AGG TCA TTG A-3'. The entire 11.6 kb mutated SOD1 genomic fragment was then excised with EcoRI and BamHI, electroeluted and used for microinjections in ova from C57Bl6/CBA mice. Transgenic mice were identified by Southern blots and then mated with C57Bl/6JBom (B6) mice.

Other mice studied In addition to the D90A and G127X transgenic mice, five other transgenic strains were studied. From Jackson Laboratories, a high (G93AGur) and a low (G93AGurdl) expressing line of G93A transgenic mice were acquired352. The low-expressing G93A line is derived from the G93AGur strain but has a reduced copy number. Copy number loss is known infrequently to occur in this strain367. Wild-type hSOD1 transgenic mice352, strain N1029, were also acquired from Jackson Laboratories.

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G85R transgenic mice360 were generously donated by Dr D.W. Cleveland. The construct of these strains was almost identical to that of D90A and G127X mice. SOD1 null mice were generously donated by Dr A.G. Reaume250. Non-transgenic littermates were used as controls.

An exon 5 PCR fragment from a

G127X patient was ligated into a

PstI-BamHI subclone of the human

SOD1 gene using NcoI-BclI.

The mutated PstI-BamHI fragment

was then ligated with a PvuII-PstIsubclone of the human SOD1.

Finally the PvuII-BamHI fragment was ligated

into the pGEM vector containing the whole

SOD1 gene using PvuII and BamHI.

5

Nco

I, 9

031

Bcl

I, 9

592

G127X

Bam

HI, 1

1564

5

Nco

I, 9

031

Bcl

I, 9

592

Pst

I, 8

625

G127X

Bam

HI, 1

1564

3 42 5

Nco

I, 9

031

Bcl

I, 9

592

Pst

I, 8

625

Pvu

II, 4597

G127X

Eco

RI, 1

Bam

HI, 1

1564

31 42 5

Nco

I, 9

031

Bcl

I, 9

592

Pst

I, 8

625

Pvu

II, 4597

G127X

Fig. 4 Mutagenesis and construct for G127X transgenic mice. Some cleavage sites of the restriction enzymes have been omitted.

Handling of mice Keeping mice: Transgenic mice were kept at the animal facilities at Umeå University and the Swedish Defense Research Agency (FOI). Mice were maintained under a 12-hour light/dark cycle at 22°C and fed standard laboratory and water ad libitum. For experiments using a copper-enriched diet, chow enriched with 400 ppm copper (Lactamin) was used, starting at weaning. Controls were fed the same chow without enrichment. Genotyping: G93A, G85R, wild-type hSOD1, SOD1 null and D90A transgenic mice were identified by ELISA or SOD activity measurement. Heterozygous G127X mice were identified with PCR of tail DNA497, since the G127X protein is non-reactive in the ELISA and does not have any SOD activity.


45

The zygosity of D90A mice was determined by the amount of SOD1 in erythrocytes, as determined by the ELISA corrected for the amount of hemoglobin. D90A mice that could not safely be determined to be hetero- or homozygous were not used. The age at bleeding could be important when differentiating between hetero- and homozygous D90A mice, since older mice have lower levels of mutant SOD1 in erythrocytes than younger ones (see Paper III). Care was therefore taken to bleed the mice directly after weaning, at about three weeks of age. Breeding: It is well known that the mouse background could be important for the phenotype of transgenic mice and this also appears be the case for mutSOD1 transgenic mice498,499. In the studies presented here, mice were backcrossed into C57Bl/6JBom (B6) mice for at least 4 generations. G93A, G85R, wild-type hSOD1 and heterozygous G127X and D90A transgenic mice were continuously mated with B6 mice (Bomholtgård). G127X homozygous mice were maintained by homozygous mating and homozygous D90A mice were bred by mating two D90A heterozygous mice. The breeding of SOD1 null mice has previously been described500.

Sample collection Mice intended for biochemical analysis were sacrificed by an overdose of sodium pentobarbital injected intraperitoneally. Following dissection, pieces of tissue were frozen in liquid N2 and kept at -80°C.

For histopathological investigations, mice were anesthetized by an intraperitoneal injection of midazolam, fentanyl and fluanisone and subjected to perfusion fixation through the heart with 4% paraformaldehyde, 75 mM sodium phosphate (pH 7.2), and then stored in paraformaldehyde containing phosphate buffer until further dissected.

Animals were studied at 2, 50, 100, 200, 400, 600 and 700 days (if applicable) of age (± 5%) and when terminal.

Ethical considerations Mice were handled according to Swedish laws and regulations and the studies were approved by the animal ethics committee in Umeå. Animal care and experiments were carried out in accordance with European Communities Council Directive (86/609/EEC). Care was always taken to avoid mice suffering unnecessarily. For humane reasons, mice were regarded as terminal when they were so ill that they were no longer able to reach the food in their cages.

Homogenization of samples Generally, tissues were homogenized in 25 volumes of PBS (10 mM K phosphate, pH 7.0, in 150 mM NaCl with EDTA-free Complete (Roche Diagnostics) antiproteolytic cocktail added, using an Ultraturrax (IKA) for two minutes. This was followed by the sonication of the homogenate using a Sonifier Cell Disruptor (Branson) for 1 minute.

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For the analysis of detergent-resistant aggregates (Papers III, IV, V), the PBS was supplemented with 0.1% NP40 (Roche Diagnostics) and, in studies of the status of the intramolecular disulfide bond (Paper V), 20 mM iodoacetamide was added to the buffer. For the quantification of tissue SOD1 and CCS content and activities, a buffer containing 50 mM K phosphate, pH 7.4, 3 mM DTPA, 0.3 M KBr and Complete with EDTA was used instead. The high concentration of KBr in this buffer increases the extraction of EC-SOD (SOD3) two to threefold248 and this buffer is therefore usually used for activity measurements in the laboratory.

Human packed erythrocytes were lysed by the addition of 20 volumes of 5 mM Na-HEPES buffer, 50 µM EDTA, pH 7.5 and, for mice, EDTA anticoagulated blood was lysed with 10 volumes of buffer. No precipitation of hemoglobin was performed unless otherwise stated.

Generation of antibodies The SOD1 and CCS primary antibodies used in this thesis were polyclonal rabbit antibodies raised against keyhole limpet hemocyanin (KLH) conjugated peptides. All peptides have a cysteine on the N or C-terminal end of the peptide to be able to link the peptide to KLH or to a sulfolink coupling gel which was used for the purification of the antibodies. A list of the antibodies, the peptide sequence and their reactivity can be seen in Table 4. The purification process was performed in two steps. First, the rabbit antisera were purified on a protein A sepharose (Amersham), which was followed by purification on the sulfolink coupling gel (Pierce) with the corresponding peptides coupled. Table 4. Antibodies generated

Antibody Peptide sequence Reactivity

wt-hSOD1 aa 4-20 CAVCVLKGDGPVQGIINF Human/mouse SOD1

aa 24-39 ESNGPVKVWGSIKGLTC Human SOD1

aa 43-57 HGFHVHEFGDNTAGC Human/mouse SOD1

aa 57-72 CTSAGPHFNPLSRKHG Human/mouse SOD1

aa 80-96 CHVGDLGNVTADKDGVAD Human/mouse SOD1

aa 100-115 CEDSVISLSGDHCIIGR Human/mouse SOD1

aa 131-153 CNEESTKTGNAGSRLACGVIGIAQ Human/mouse SOD1

wt-mSOD1 aa 24-36 ASGEPVVLSGQITC Mouse SOD1

G127X hSOD1 aa 111-132 “Agaz” CIIGRTLVVHEKADDLGGQRWK G127X/wt-SOD1

aa 123-132 “Inger” CADDLGGQRWK Human G127X

Human CCS aa 252-270 CWEERGRPIAGKGRKESAQP Human/mouse CCS


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The five novel amino acids seen in the G127X protein made it possible to generate G127X specific antibodies to be used for immunoblots and immunohistochemistry. Since the antigenicity of only five amino acids is very low, some wild-type sequence was also included in the peptides used for immunization (Fig. 5). The wild-type reactivity of the antibodies is then blocked with wt-hSOD1 that has been denatured by exposure to 6 M guanidinium chloride and 3 mM DTPA followed by dialysis. Two G127X specific antibodies were generated, the longer (Agaz antibody) shows some reactivity to wt-hSOD1, while the shorter (Inger antibody) has no such reactivity.

L S G D H C I I G R T L V V H E K A D D L G G Q R W K .111 132123

Agaz ab

Inger ab

tgactc tca gga gac cat tgc atc att ggc cgc aca ctg gtg gtc cat gaa aaa gca gat gac ttg ggt ggg caa agg tgg aaa

Fig. 5 C-terminal end of the G127X protein. Novel amino acids have a light grey background. The four nucleotides insertion is underlined.

Analysis of SOD isoenzymes

SOD activity SOD activity was determined using three different assays. The direct spectrophotometric method using KO2 (Papers I-V), the pyrogallol method (Paper IV) and the nitroblue tetrazolium gel assay (Papers I, IV). Direct spectrophotometric method: Most assays of the superoxide anion are indirect assays because of the very rapid spontaneous disproportionation of the anion radical in aqueous solutions at neutral pH. Under alkaline conditions, however, the radical is much more stable and can be measured using an ordinary spectrophotometer at 250 nm. In this assay, an excess of O2•- is produced by KO2 at pH 9.5 and the decay of the superoxide radical is continuously measured using a spectrophotometer. The addition of SODs catalyzes the decay of O2•- to hydrogen peroxide and oxygen in a first-degree order and, by measuring the speed of the decay and after correction for the very slow second-degree spontaneous disproportionation of O2•-, the SOD activity can be calculated. In the assay, one unit is defined as the enzymatic activity that yields a decay of O2•- at a rate of 0.1 s-1 in a 3 ml buffer501,502. One unit in the assay corresponds to about 4.3 ng wild-type hSOD1503 or 8.6 ng human SOD3504 or 65 ng bovine SOD2501.

For the differentiation between SOD2 and the other two SODs, 3 mM of cyanide is added to the buffer which almost completely inactivates Cu-containing SODs. Currently, discrimination between SOD1 and SOD3 activity in this assay is not possible. The assay is very sensitive for the activity of CuZn-SODs, but, for the

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detection of SOD2, the assay is much less sensitive and only about 10% of that for CuZn-SODs. Pyrogallol method: Since the alkali conditions used in the direct spectrophotometric method could inactivate unstable SOD1 mutants (such as G127X SOD1), a second method was used to measure the SOD activity in G127X mice (Paper IV). This method is based on the auto-oxidation of pyrogallol, at pH 7.8, which is dependent on O2•- and the auto-oxidation is thus inhibited by SODs. The process is recorded using a spectrophotometer at 420 nm and one unit in the assay is defined as the amount of SOD that inhibits the auto-oxidation by 50%. Like the direct spectrophotometric method, SOD2 is measured by adding 1 mM cyanide. One unit in this assay corresponds to about 650 ng hSOD1 or 700 ng bovine SOD2505,506. Nitroblue tetrazolium gel assay: The third method that was used for measuring SOD activity was a gel-based method. One advantage of this method is that the migration pattern or the isoelectric point of the native enzyme is directly seen. The method was first developed by Beauchamp and Fridovich507, but to enhance the sensitivity some minor modifications were made. The assay is a negative assay where SODs inhibit the reduction of nitroblue tetrazolium to formazan by O2•- in the gel, causing an achromatic zone in an otherwise blue gel. The superoxide radical is generated by the auto-oxidation of riboflavin by light. To increase the sensitivity of the assay, the concentration of nitroblue tetrazolium was lowered from 2.45 mM to 0.25 mM and a 200 mM TRIS-cacodylate buffer pH 7.8 was used.

Here we used isoelectric focusing using Immobiline (Amersham) gels to separate the proteins. In Paper I, a pH gradient of 4.5-5.4 was used and equal amounts of hemolysate SOD activity were applied for all cases. In Paper IV, a gradient of pH 3.5-9 was used and brains from 100-day-old mice were homogenized in 10 volumes of pH 7.0 PBS and centrifuged at 20,000 g for 30 minutes. Twenty, 40 and 60 µl of the supernatant were then separated by isoelectric focusing. Focusing conditions were according to the manufacturer’s descriptions.

Gels were then scanned in a Fluor-S Multiimager (Bio-Rad) and quantified using Quantity One software (Bio-Rad). The quantification of SOD activity using this negative method has limited accuracy, but the values in Paper I are in reasonably good agreement with the expected values.

SOD ELISAs A hSOD1 enzyme-linked immunosorbent assay (ELISA) (Papers II-V) was used to quantify hSOD1, mainly for mouse genotyping purposes. This assay has been previously described by Behndig et al.508. The hSOD1 ELISA is highly specific for native wt-hSOD1 and the ELISA therefore shows no reactivity to G127X hSOD1 and very low reactivity to G85R hSOD1. The reactivity to G93A hSOD1 is, however, high. Initially, D90A hSOD1 had a somewhat lower reactivity in the ELISA503. The use of other secondary antibodies, however, circumvented this problem and the ELISA is now just as reactive to wt-hSOD1 as D90A hSOD1. The ELISA shows no cross-reactivity towards mSOD1.


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To detect SOD3, the ELISA described by Karlsson and Marklund was used509 (Papers II, IV). The SOD3 ELISA shows no cross-reactivity to hSOD1. For conversions to activity units, 8.6 ng/U was assumed504.

Blotting procedures

Immunoblotting Samples were solubilized 1:1 in 2 × SDS-PAGE (SDS-polyacrylamide gel electrophoresis) sample buffer (100 mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 20% glycerol, 4% SDS and bromphenol blue), heated for 5 min at 95°C and then separated on SDS-PAGE gels (Bio-Rad). The gels were then electroblotted onto nitrocellulose membranes (Paper I) or polyvinylidine difluoride (PVDF) membranes (Amersham) (Papers II-V). Blots were probed with the SOD1 and CCS antibodies described above. In Paper I 125I-labeled protein A was used to detect primary antibodies and the bands were visualized using a Bio-Rad GS-525 phosphorimager and quantified using Molecular Analyst software (Bio-Rad). In Papers II-V, horseradish peroxidase-conjugated anti-rabbit IgG antibodies or biotinylated anti-rabbit IgG antibody and horseradish peroxidase-conjugated streptavidin (Amersham) were used for detection and chemiluminescence was generated using substrate from Pierce or Amersham. Bands were visualized on film or by using a Fluor-S Multiimager or Chemidoc imager and Quantity One software (Bio-Rad).

For the quantification of SOD1 by immunoblotting (Paper III, IV, V), wild-type hSOD1 with the concentration determined by quantitative amino acid analysis281 was used as the original standard. For the quantification of mSOD1, the 24-36 mSOD1 specific antibody was used and in G127X mice the 131-153 antibody.

In the CNS from the human G127X ALS patient, the mutant-specific Inger antibody was used for the determination of the mutant enzyme (Paper IV). In this case, G127X mutant SOD1 in a transgenic mouse brain homogenate previously quantified against the original standard was used as standard.

In general, quantifications by immunoblots were run at least in duplicate.

Northern blotting Total RNA was prepared from mouse brain using the Trizol reagent (Invitrogen), according to the manufacturer’s description. Electrophoresis was performed using the NorthernMax-Gly kit (Ambion) and RNA was blotted onto Hybond XL nylon membranes (Amersham). Blots were probed using ULTRAhyb hybridization buffer (Ambion) and the MegaPrime random labeling kit (Amersham). Bands were visualized with a Bio-Rad GS-525 Molecular Imager and quantified using Molecular Analyst software (Bio-Rad). Samples were normalized against β-actin (Clontech). SOD1 mRNA was detected using a purified PCR fragment as a probe. Primers were 5'-GCG TGG CCT AGC GAG TTA TG-3' and 5'-ATC CTT TGG CCC ACC GTG TTT TCT G-3' yielding a 248-bp amplicon of exons 1-3. The PCR amplicon was purified using High Pure PCR Product Purification kit (Roche Diagnostics) (Papers III, IV, V).

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Southern blotting Mouse DNA was prepared from tails using standard methods and cleaved with BglI. DNA was then separated on 0.7% agarose gels and blotted onto Hybond XL nylon membranes (Amersham). The EcoRI-BamHI fragment of the SOD1 gene described above was used as the probe. The labeling and visualization of bands were as described for northern blots. The copy number was determined by quantification against known amounts of the EcoRI-BamHI fragment (Papers III, IV).

2D-gel electrophoresis Spinal cords from 100-day-old and terminal G127X mice were homogenized in 8 M urea, 4% CHAPS and 2% Bio-Lyte 3-10, swollen into 7-cm non-linear pH 3-10 immobilized pH-gradient strips (Bio-Rad) and electrofocused according to the manufacturer’s instructions. The strips were then top loaded onto 12.5% SDS-PAGE gels, electrophoresed and finally electroblotted onto polyvinylidine difluoride membranes and immunoblotted as described above with the 24-39 anti-hSOD1 antibody and an anti-ubiquitin antibody (Sigma) (Paper IV).

Immunohistochemistry

Human histopathology Pieces from the frontal, temporal and precentral cortical regions of the brain and the cervical, thoracic and lumbar spinal cord were dissected from the G127X patient. The pieces were then immersion fixed in paraformaldehyde as described above and embedded in paraffin wax and sectioned for histopathological analysis (Paper IV). Mouse histopathology For histopathological analysis, five mice of each age and genotype were studied. After fixation, the CNS was dissected in situ and the cervico-thoracic and thoraco-lumbar borders, as well as the junction between the brain stem and spinal cord, were defined with guidance by the ventral roots. The spinal cord was then divided into three parts corresponding to the cervical, thoracic and lumbosacral levels respectively. Each of these levels was then equally divided into five pieces which were separately embedded in paraffin wax. The uppermost piece of each level and the second lowermost piece of the lumbosacral level were sectioned and used for the histopathological investigations (Papers III-V).

Staining procedure Single- and double-labeling immunohistochemistry was performed using the Ventana AEC and alkaline phosphatase red immunohistochemistry system, using the protocol of the manufacturer, and was preceded by microwave irradiation of the sections in citric acid buffer for 3x5 minutes. Antibodies used for SOD1 staining (Papers III-V) were mainly the 4-20 and 131-153 anti-SOD1 peptide antibodies described above. Tests were performed with the other anti-hSOD1 peptide antibodies on one terminal mouse of each strain, which gave similar results as the 4-20 and 131-153 anti-hSOD1 peptide antibodies. In studies of the G127X protein, the 131-153 hSOD1 antibody


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was used as a negative control, since this antibody does not recognize G127X. For the detection of the G127X protein, the two G127X-specific antibodies were used. These antibodies do not stain non-G127X tissue. Antibodies to the following proteins were also used:

• GFAP Dako (Papers III-V) • Ubiquitin Dako (Paper IV) • UCH-L1 Ultraclone (Paper IV) • αB-crystallin Novocastra (Paper IV)

Sections were also stained with hematoxylin/eosin, Klüver-Barerra, modified Bielschowsky and periodic acid-Schiff (Papers III-V).

Other experiments

Effect of postmortem time To mimic the effect of the storage/postmortem time of a deceased human body on the various assays carried out on the patient (Paper IV), pieces of brain from G127X transgenic mice were put aseptically into small tubes and kept at 37°C for 6 h, followed by room temperature for 6 h, and then at 4°C. Immediately thereafter and after different times, tubes were snap frozen to -80°C for subsequent thawing and analysis.

Fibroblast culture experiments Fibroblasts were grown in RPMI 1640 (Roswell Park Memorial Institute) containing 10% fetal calf serum, 70 µg/ml bensylpenicillin and 2 mM L-glutamine. To inhibit the proteasome, cells were incubated with 10 or 20 µM of two different irreversible proteasome inhibitors, lactacystin or 3-nitro-4-hydroxy-5-iodophenylacetyl-LLL-vinylsulfone (NLVS) (Calbiochem) for 48 hours510. Cultures without proteasome inhibitor served as controls. Cells were then lysed by sonication in 50 mM K phosphate, pH 7.4, 3 mM DTPA, 0.3 M KBr and Complete with EDTA and immunoblotted using the Agaz antibody. Equal amounts of fibroblast lysate SOD activity were applied to the gels. For analysis of aggregates, fibroblasts lysed in PBS pH 7.0 without detergent were centrifuged once at 20,000 g for 30 minutes and then analyzed by immunoblotting using the Agaz and the 131-153 hSOD1 antibodies (Paper IV).

Analysis of SOD1 aggregates Analysis of detergent-resistant aggregates: Brain and spinal cord samples from the mice were homogenized in 25 volumes of PBS (pH 7.0) with 0.1% of the detergent Nonidet P40 (NP40) added (Roche Diagnostics). Homogenized samples were then centrifuged at 20,000 g for 30 minutes at 4ºC. The supernatants were removed and the pellets were resuspended and sonicated in double the original volume of homogenizing solution, followed by centrifugation. This washing step was repeated 5

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times. Following the last wash, the pellets were resuspended and sonicated in 1 × SDS-PAGE sample buffer. The samples were then analyzed by immunoblotting, using the 24–39 hSOD1 specific antibody (Papers III-V). Analysis of detergent-soluble aggregates: Tissues from G127X transgenic mice were homogenized in 25 volumes of PBS (pH 7.0) without detergent added and centrifuged at 20,000 g for 30 minutes at 4ºC. The supernatants were carefully removed and the pellets were resuspended in PBS pH 7.0 with 0.1% NP40. Resuspended pellets were then analyzed with immunoblotting using the 24-39 and 131-153 hSOD1 antibodies. The relative amounts of G127X SOD1 and mSOD1 respectively in the pellets were quantified against dilutions of the original homogenate in immunoblots. The proportion (percentage) of pelleted mSOD1 (processomes) is then subtracted from the proportion (percentage) of pelleted G127X and this difference (Fig. 6) is termed detergent-soluble aggregates (Paper IV).

20%

40%

100%

70%

60%

20%

40%

100%

70%

60%

20%

40%

100%

70%

60%

Pelleted

G127X

Pelleted

mSOD1

Detergent-

soluble

G127X Fig. 6 The figure describes the calculations of detergent-soluble aggregates. The proportion of pelleted mSOD1 (black) is subtracted from the proportion of pelleted G127X (grey), giving the proportion of detergent-soluble aggregates. In this case, the amount of detergent-soluble aggregates corresponds to ~32%.

Stabilities of human and murine SOD1s Pools of packed erythrocytes from three D90A homozygous individuals, three control individuals and three control C57Bl/6J mice were mixed with 1.6 volumes of 37.5/62.5 vol/vol chloroform/ethanol at -20ºC to precipitate and remove the hemoglobin of erythrocytes. After vortexing and centrifugation (2,500 g, 10 min), 200 μl of the SOD1-containing upper phase was added to 400 μl 3.75 M guanidinium chloride in 0.1 M Na-HEPES, pH 7.4, with 3 mM DTPA and incubated at 37ºC. The denaturation was stopped after different times by adding 50 μl aliquots of the mixture to 400 μl 50 mM Na-HEPES pH 7.4 with 0.25% bovine serum albumin, followed by analysis of the SOD activity.


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For analysis of the thermal stabilities, 100 μl of the chloroform/ethanol upper phase was added to 200 μl Na-HEPES, pH 7.4, with 3 mM DTPA and the tube was placed in a 70ºC water bath. After different times, 50 μl aliquots were added to the HEPES buffer at room temperature and analyzed as described above (Paper III).

Supplementation of homogenates with Cu The tissues were homogenized in 25 volumes of the PBS as described above. To the homogenates, CuSO4 (1 mM) or an equal volume of PBS was added. Homogenates were then incubated overnight at 4ºC, followed by the analysis of SOD activity. Three mice of each strain were analyzed. Non-transgenic mice and SOD1 knockout mice were used as controls (Paper V).

Cytochrome oxidase assay The activity of cytochrome oxidase (Complex IV) was measured in spinal cord homogenates of mice from the high-expressing SOD1 transgenic strain and non-transgenic mice of different ages according to Birch-Machin et al.511. In this method, the oxidation of cytochrome C by adding potassium ferricyanide was followed with a spectrophotometer at 550 nm (Paper V).

Inductively coupled plasma atomic emission spectrometry Tissues were dissected with stainless steel tools with powder-free gloves and stored at -80ºC until further analyzed. Test tubes used for analysis were rinsed with nitric acid and MilliQ water (Millipore). Weighed tissue pieces were put in the test tubes and then solubilized using 50% nitric acid, 50% hydrogen peroxide and finally diluted 5 times with MilliQ water. Samples were then analyzed using inductively coupled plasma atomic emission spectrometry (ICP) on a PE Optima 3000XL (PerkinElmer) (Paper V).

Recombinant hSOD1s Recombinant human SOD1 variants were co-expressed with the copper chaperone for superoxide dismutase (CCS) in E. coli and purified as previously described512. CuSO4 (3 mM) and ZnSO4 (30 μM) were added to the culture medium. The metal contents were determined by graphite furnace atomic absorption (Paper V).

Total protein analysis Total protein was measured using a Coomassie Brilliant Blue G-250 assay (Bio-Rad), standardized with human serum albumin513 (Papers I-V).

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Hemoglobin analysis Total hemoglobin was determined with a standard cyanomethemoglobin assay (Bioreagens) using a spectrophotometer at 540 nm (Papers I, III-V).

Statistical calculations For statistical calculations SPSS or Microsoft Excel software were used. As a measure of variance of means, standard deviation (SD) (Papers I-V) was used. When comparing two groups (Papers I, II, V), the non-parametric Mann-Whitney U test was used and, when comparing multiple groups (Paper II), one-way ANOVA (analysis of variance) was used and, if significant, Tukey’s post-hoc test was performed to determine differences between groups. The significance of correlation (Paper II) was calculated with Spearman’s rho test.


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RESULTS AND DISCUSSION

Paper I

No alteration in D90A mutant hSOD1 levels in recessive pedigrees It has been suggested that the recessive inheritance pattern seen for D90A mutSOD1 is due to a tightly linked protective factor that could act by downregulating the mutant protein491. To investigate this and to further explore the effects of a putative protective factor, erythrocytes from heterozygous individuals from recessive and dominant D90A families and sporadic heterozygous D90A ALS patients were analyzed. Families were considered recessive if no D90A heterozygote in the family had developed ALS and dominant if heterozygous individuals, including family members, had contracted the disease. In all, erythrocytes from 21 heterozygous individuals from 17 different Scandinavian, German and US recessive families were examined. The dominant cases were eight individuals from six different families from Belgium, Great Britain and Russia. Six of these individuals had ALS. As controls, 13 healthy individuals were analyzed simultaneously. There was no difference in erythrocyte SOD1 activity between “recessive” and dominant individuals (56.4 ± 5.4 U/mg Hb and 56.6 ± 2.7 U/mg Hb respectively). The heterozygous individuals had a somewhat lower activity than the controls (59.5 ± 3.4 U/mg/Hb p < 0.05). The D90A to wild-type ratio was measured with immunoblots that are able to distinguish between the wild-type and the D90A allele. Here, too, there was no difference (0.78 ± 0.10 vs. 0.81 ± 0.09). These data suggest a specific activity of D90A that is 114% of the wild-type enzyme which is in perfect agreement with previous findings in homozygous D90A patients that also reported a 14% increase in the specific activity for the D90A protein503. The turnover of hSOD1 in homozygous and heterozygous individuals therefore appears to be similar. To analyze whether the protective factor altered the quaternary structure of SOD1, isoelectric focusing and SOD activity staining were performed. In homozygous D90A and wt-hSOD1 patients, multiple bands were seen, in agreement with previous published results280,514,515. The reason for this banding pattern is not known, but there are several possibilities, such as (de)metallation514, carbonylation514, (de)acetylation516. phosphorylation517,518, or oxidation of cysteines to cysteine-sulfinic acid519 or by disulfide-linked glutathione520. However, some of these post-translational modifications appear less likely. For example, copper deficiency inactivates the protein and other modifications do not necessarily confer charge differences.

To aid in the analysis of the banding pattern, we analyzed the available fractions of previously ion-exchange purified enzyme from homozygous D90A patients and controls281. Fractions I and III were of equal molecular weight according to mass spectrometry, while fraction II showed a heterodimeric pattern and was suggested to be partly glutathionylated. On zymograms, fraction I only displayed one band, while the other two fractions contained several bands. Thus it could not be concluded that glutathionylation was responsible for the multiple banding patterns. The D90A

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mutations confer the protein a more positive charge, which is also reflected on the zymograms. As expected, the heterozygous individuals displayed a heterodimeric formation between the wild-type and D90A subunits. After inspecting and quantifying the zymograms, we were not able to discern any difference in the banding pattern between recessive and dominant cases. There was therefore no evidence that the putative factor in recessive D90A cases acts by downregulating synthesis, influencing degradation or by altering the quaternary structure of the D90A protein. One caveat when it comes to this study is of course that only two of the dominant cases were documented familial cases. It may be that the sporadic ALS cases carried the recessive allele and for unknown reasons contracted the disease. However, Scandinavia aside, the recessive D90A allele is very rare. In the United Kingdom, which is in the proximity of Scandinavia, aberrant hSOD1 patterning (such as that seen for the D90A mutation) was found in 7 individuals out of 11,237 tested (corresponding to a frequency of 62 per 100,000 people)521. In Russia outside the Kola Peninsula, 12 of 3,093 tested (corresponding to a frequency of 388 per 100,000 people) had aberrant hSOD1 patterning. This mutation is therefore very rare, although the possibility of it occurring in ALS from other causes could not be excluded. The two Belgian dominant FALS individuals were also very similar to the other D90A individuals in the amount and structure of the D90A protein.

Another concern may be that here we have investigated erythrocytes not CNS tissue. The transcriptional regulation of SOD1 is poorly understood but may be different in the CNS. To conduct a similar study on CNS tissue would, however, be practically impossible due to difficulties obtaining CNS tissue from individuals with hSOD1 mutations. So far, we have analyzed CNS tissue from six homozygous D90A ALS patients and the preliminary results suggest that the hSOD1 levels in the ventral horns of these patients are very similar to those of controls (K.S. Graffmo et al., unpublished results). Additionally, our studies of CSF from homozygous D90A patients showed that SOD1 levels were similar to those of controls (see Paper II below).

Paper II

CSF hSOD1 levels are unaltered in ALS In other neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease, the proteins that are found to be mutated in familial cases are clearly also involved in sporadic cases. For example, in Alzheimer’s disease, accumulations of Aβ are seen in the amyloid plaques characteristic of the disease and, in certain familial cases, the disease is linked to mutations in the amyloid precursor protein (APP)238. Curiously, Alzheimer’s disease patients have been associated with a paradoxical decrease in Aβ1-42 in CSF522. It is conceivable that similar alterations may also pertain to ALS and SOD1. This notion was investigated by analyzing CSF from controls (n=37), SALS (n=54), FALS (n=12) and mutSOD1-FALS (n=14) patients in terms of SOD content and molecular forms of hSOD1.


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SOD content was analyzed by measuring both cyanide-sensitive and -insensitive activity and the levels of hSOD1 and SOD3 were evaluated with ELISAs. FALS and SALS cases had somewhat higher hSOD1 protein levels compared with controls. However, the mean age was also lower in the control group and there was a significant correlation between age and hSOD1 levels (p<0.001 for all individuals without mutSOD1). Assuming a linear correlation between hSOD1 activity and age in non-mutant hSOD1 individuals, the hSOD1 activity can be roughly described by the equation:

(Equation 1) Activity = 0.33 × Age + 13.8 (U/ml) For controls with a mean age of 52.1, this corresponds to a value of 31.0 U/ml, for SALS 33.9 U/ml (mean age 60.8) and for FALS 35.4 U/ml (mean age 65.6). These values are all reasonably close to the measured values and suggest that the difference seen between controls and ALS patients only reflects the age of the individuals. An age dependent increase in SOD activity in CSF has also previously been reported523. The levels of SOD3, cyanide-insensitive SOD (SOD2) and total protein were unremarkable. Homozygous D90A patients had hSOD1 levels that were very close to those of controls. The hSOD1 levels in CSF samples from patients heterozygous for A89V, S105L and G114A mutSOD1 were clearly lower.

The levels of the CNS intracellular protein content are mirrored in the CSF protein content. Factors contributing to CSF content can, for example, be the levels of protein expression, the degree of cell damage and the turnover rate of proteins. There was no evidence of a general efflux of protein to the CSF, since protein levels were similar between groups. This does not of course exclude the possibility that individual proteins are altered. Previous studies have in some instances reported such differences in ALS, although the results have not been clear cut. For example, the levels of VEGF have been reported to be both lower232 and unchanged524 (with widely differing values), whereas tau levels have been reported to be both higher525 and unchanged526. Others have reported an increase in NF-L527 and PEDF528 and a decrease in Aβ1-42529.

In our study, we were not able to find any evidence that the levels of hSOD1, SOD2, or SOD3 were altered in ALS. This is different from what has been reported from more acute conditions, such as stroke, meningitis and encephalitis530,531, where hSOD1 levels can be several times higher in the CSF. This probably reflects the slow progression and chronic nature of ALS.

As has been discussed and investigated in Paper I, it has been suggested that the putative protective factor in recessively inherited D90A ALS acts by downregulating the D90A protein491. Similar to the study of erythrocytes (Paper I), we found no indication that this is the case, since D90A levels in the CSF of homozygous D90A patients is very similar to the SOD1 levels seen in controls.

The levels of SOD in ALS/MND CSF have been investigated in four other studies. Three smaller studies (18, 11 and 10 ALS patients) used different methods to measure total SOD activity, cyanide-sensitive SOD (SOD1 and SOD3) and SOD1 protein and found that ALS correlated with a decrease in SOD activity523,532,533. In the somewhat larger study by Yoshida et al. consisting of 25 MND patients, there was no

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difference in the amount of SOD1 protein compared with controls531, which is in agreement with our study, the largest so far.

Increased levels of cleaved hSOD1 in D90A ALS patients The main finding in this paper was the occurrence of a second, lower molecular weight, hSOD1 reactive band on CSF immunoblots. The molecular weight difference (~2.5 kDa) and the lack of reactivity with an N-terminal antibody suggest that this band represents the remains of a ~20 aa N-terminal cleavage of hSOD1. This band was also seen in CNS homogenates but at much lower levels. It was not a storage artifact, since CSF snap frozen in liquid nitrogen was no different from samples stored over-night at 4°C or at room temperature. The relative amount of the cleaved subunit was significantly higher for homozygous D90A patients than for controls, SALS and FALS cases (e.g. 32% for homozygous D90A patients vs. 24% for controls). Heterozygous carriers of the other hSOD1 mutations (A89V, S105L and G114A) also showed an increase in cleaved hSOD1, but it is difficult to draw any safe conclusions based on single cases. There were no other differences on immunoblots.

As has been discussed above, little is known about the degradation of hSOD1. It is possible that the N-terminal cleaved hSOD1s represent an initial step in the route of degradation of hSOD1 (a proteolytic fragment corresponding of amino acids 1-20 have been observed after proteasomal digestion in vitro534). There are several possibilities why this band is increased in homozygous D90A patients. Previous studies281,503 and Paper III demonstrate that, although D90A is very stable, it is somewhat less stable than wt-hSOD1, suggesting that this mutant is more rapidly degraded than wt-hSOD1, which could contribute to an increase in the amount of proteolytically cleaved hSOD1. An alternative explanation is that the degradation of the mutant hSOD1, for unknown reasons, is slower than that of the wild-type protein or that the proteolytic machinery in these patients is compromised, leading to increased levels of partly digested hSOD1.

The reason why this band is selectively accumulated in CSF is not known. It is possible that the cleaved hSOD1 more easily leaks to the CSF from the CNS tissue or that in cells with a high proportion of the cleaved specie, this fragment is toxic and result in cell death.

C-terminally truncated hSOD1s, like the more common hSOD1 missense mutations, have previously been reported to cause ALS (see above and Paper IV). The shortest ALS associated mutSOD1 reported so far is the K118TGP-Stop mutation that results in a 32 amino acid truncation312,319. This part of the protein cannot therefore be toxic per se. The toxicity of an N-terminally cleaved protein is, however, not known. Interestingly, in transfected cell cultures, hSOD1 fragments consisting of amino acids 1-75, 38-153 and 49-153 were all highly prone to aggregate359 and, in chicken embryo spinal cords, a YFP-tagged hSOD1 peptide consisting of only amino acids 1-36 induced aggregates and TUNEL positive cells496. The situation may thus be similar to Alzheimer’s disease where fragments of the protein are highly amyloidogenic (such as the Aβ1-42)238. One can speculate that the N-terminal proteolytic cleavage of hSOD1 may represent such a fragment that could act as a seed for aggregation.


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Papers III-V In these three papers, the generation of D90A (Paper III) and G127X (Paper IV) mutSOD1 transgenic mice is described and the two strains are compared with other mutSOD1 transgenic strains and a mouse strain expressing wt-hSOD1 (Table 5). Additionally, mutSOD1 in G127X transgenic mice is compared with an ALS patient carrying the same mutation (Paper IV). Table. 5 Umeå data on the survival of hSOD1 transgenic mouse strains

Mutation Line Survival ± SD (days) Expression of hSOD1. Percentage of G93AGur ± SD (n = 3)

D90A 134 mm 407 ± 46 (n = 65) 51% ± 8%

D90A 154 mm 492 ± 84 (n = 6) ND

D90A 134 / 154 495 ± 46 (n = 3) ND

G127X 716 mc 477 ± 88 (n = 39) ND

G127X 716 mm 250 ± 38 (n = 202) 63% ± 8%

G127X 832 mc 213 ± 18 (n = 6) ND

G127X 832 mm 126 ± 4 (n = 10) ND

G85R mc 345 ± 41 (n = 77) 43% ± 6%

G93A Gur mc 124 ± 12 (n = 267) 100%

G93A Gurdl mc 253 ± 31 (n = 113) 50% ± 10%

wt-hSOD1 mc – 60% ± 4%

mm denotes homozygous mice and mc mice hemizygous for the transgene insertion ND = Not determined

Phenotypes of mutant hSOD1 transgenic mice As described above, the D90A and G127X protein is two very different hSOD1 mutations. In actual fact, these two mouse strains also develop somewhat different MND phenotypes. The D90A transgenic mice develop a more slowly progressing MND with progressive paralysis and muscle wasting that usually starts in one hind limb and then slowly progresses over weeks. The disease duration is approximately 50 days. An unusual feature with this strain, which is not observed in other mutSOD1 transgenic mice strains, is the occurrence of a urinary bladder distended by urine in terminal mice. This feature resembles the problems with micturition from which some D90A patients suffer488, which suggests that this feature may be specific to D90A mutSOD1.

The G127X transgenic mice, on the other hand, develop a much more rapidly progressing MND, where the disease duration could be as short as two days. Fore limb onset is common. G93A and G85R mice fall between these two models, as G85R are closer to the G127X mice, while G93A are more similar to the D90A transgenic mice. This possibly reflects the stabilities and the levels of these two proteins, as discussed below. For a given hSOD1 mutation, survival is inversely proportional to the levels of mutSOD1 expression (e.g. homozygotes compared to

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heterozygotes). But between different mutations the fit is not optimal, suggesting that the toxic potential of the mutSOD1 might differ (Table 5 and Fig. 7).

0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50 60 70 80 90 100

G93AGurdl

G127X

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hSOD1 expression (% of G93AGur)

Surv

ival (d

ays)

G93AGur

Fig. 7 The association between the expression levels and the survival of different mutSOD1 transgenic strains, n = 3.

Similar mRNA levels – widely differing protein levels In Paper V, the levels of hSOD1 mRNA in the CNS are compared with the “steady-state” levels of hSOD1 protein. Despite similar levels of expression (Table 5), which should be proportional to the protein synthesis rate, the amounts of hSOD1 protein in the CNS vary widely between strains. The different strains can be divided into two groups, one high hSOD1 level group (wt-hSOD1, D90A, and G93A) with hSOD1 levels of ~500-2,000 µg/g ww and one group (G85R and G127X) with much lower levels, ~50-100 µg/g ww. Interestingly, in the spinal cords of G85R and G127X transgenic mice, a surge in hSOD1 was seen in terminal mice, as previously noted for G85R mice360. Compared with non-CNS tissue, hSOD1 was selectively accumulated in the CNS, which was especially evident in the low-level strains. Even more conspicuously, in the patient carrying G127X mutSOD1, only minute amounts of the mutant protein were detected (Paper IV), but here too mutSOD1 was selectively accumulated is disease susceptible tissues (Fig. 8).


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TL

0.4% 3.7%2.2%0.7%

PG

LV

H

CV

H

TL

PG

LV

H

CV

H

G127X Control Fig. 8 Immunoblot of homogenates from the temporal lobe (TL), precentral gyrus (PG), cervical ventral horn (CVH) and lumbar ventral horn (LVH) of the G127X patient and a patient who died of non-neurological causes. The blot is probed with the G127X-specific Inger antibody. Numbers denote the amount of mutSOD1 as a percentage of the total hSOD1 content of controls. Note the accumulation of mutSOD1 (arrow) and the occurrence of high molecular weight mutSOD1 species (arrowhead) and smearing in the affected ventral horns. These findings suggest that the degradation of misfolded hSOD1 in the CNS is impaired. In fibroblasts from the G127X patient, the G127X protein was not detected (Paper IV), but, if treated with a proteasome inhibitor (NLVS or lactacystin), the mutant protein was readily seen (without signs of ubiquitination). Although proteasome inhibitors are not 100% specific for proteasomal proteolysis, this suggest that at least a portion of the mutant protein is rapidly degraded by the proteasome in agreement with previous findings (see above).

Inactive SOD1 in the CNS of transgenic mice – CCS deficiency? In the CNS of the high-level hSOD1 transgenic mice, SOD1 activity is smaller than might be expected from the amount of protein (Paper V). By incubating the homogenates with copper ions, a large proportion of the activity could be restored. The hSOD1s in these strains are therefore copper-deficient. The reason for this is not completely understood, but it appears as if this is not due to deficient uptake in the intestine, since copper supplementation to mice did not augment activity. Nor did it appear as if the transport of copper into CNS cells was deficient, since the copper-dependent COX enzyme was unaffected. The levels of CCS were upregulated in the CNS of transgenic mice but only modestly (3-5 times the levels of non-transgenic mice). In the liver of transgenic mice, where the hSOD1s is almost fully active, CCS levels were relatively higher (5-10 times the levels of non-transgenic mice) than in

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the CNS, thereby partly providing an explanation for the lack of copper charging in CNS tissue. The reason for this discrepancy is not known and overall, the regulation of CCS is poorly understood.

The two low-level mutSOD1 transgenic strains were also both inactive, but for other reasons. The G127X protein lacks the C-terminal part of the protein and thereby the disulfide bond and part of the active site cleft258. This severe protein defect is probably enough to completely inactivate the protein (Paper IV). G85R mutSOD1 has previously been shown to have very low activity in vitro and in cells337,338,535,536, as well as in G85R transgenic mice360. The low activity has been attributed to the low copper affinity of this mutSOD1337,535,536. Previous findings are in agreement with the 30% activity of G85R seen when supplemented with copper ions. Interestingly, recombinant G85R was found to be fully active relative to its copper content (Paper V and 537). Consequently, in the spinal cord of transgenic mice, G85R is probably not copper charged at all at the active site.

The zinc status of SOD1 in the transgenic mice is very difficult to evaluate and it is not known how SOD1 acquires the zinc ion.

Disulfide-reduced SOD1 in high-level hSOD1 transgenic mice In the strongly reducing intracellular environment, SOD1 has a unique intrasubunit disulfide bond that is of structural importance for the protein (see above). The amount of disulfide-reduced SOD1 was measured using immunoblots on homogenates where the cysteines had been quenched with the alkylating substance iodoacetamide to prevent re-oxidation (Paper V). Two bands, that appeared to differ by ~2 kDa in size, were then seen on non-reducing SDS-PAGE gels. The higher mobility band was referred to as oxidized and the lower mobility band as reduced. Similar differences in mobility have previously been observed for prokaryotic538, yeast301 and human SOD1s288, but the identity of the bands was further ascertained by analyzing recombinant proteins. This was done on recombinant wild-type or G85R hSOD1 with all four cysteines mutated to alanine (and thus disulfide-reduced) or only the cysteines not involved in disulfide formation (Cys6/Cys111) and thus disulfide-oxidized in the oxidizing environment. The amount of disulfide-reduced SOD1 was quantified versus dilutions of homogenate disulfide-reduced with β-mercaptoethanol.

In the spinal cords of high-level hSOD1 transgenic strains, between 8% and 14% of the hSOD1 was disulfide-reduced. The relative amounts of disulfide-reduced hSOD1 in peripheral tissues (liver and kidney) were lower. In low-level lines, “all” hSOD1 was disulfide reduced, but some of the disulfide-reduced hSOD1 had apparently engaged in disulfide coupling with other proteins or with SOD1, as has previously been noted in vitro288. The endogenous mSOD1 showed a reduction pattern similar to that of hSOD1 in the high-level transgenic strains. However, in the low-level strains, all mSOD1 appeared to be disulfide-oxidized. This suggests that disulfide reduction is partly related to the copper content which is probably not limiting in the G127X and G85R mutSOD1 strains. As discussed above, G85R probably has a very low copper affinity, which would explain the propensity of this mutant protein to be disulfide-reduced, although copper deficiency per se does not necessarily confer disulfide reduction. The propensity for disulfide reduction of G85R has also been noted in vitro344.


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Similar amounts of detergent-resistant aggregates in terminal mice Despite the widely differing levels of mutSOD1 protein in the transgenic strains, similar amounts of hSOD1 detergent-resistant aggregates, associated with heavy smearing, accumulated in terminal mice (Papers III-V). In the G127X patient, almost all the mutant enzyme was aggregated, but it was also shown that this mutant protein aggregated rapidly when tissues were stored non-frozen (Paper IV). The occurrence of the detergent-resistant aggregates late in disease makes it unlikely that they are the direct cause of disease, since mutSOD1 transgenic mice show pathological alterations long before. It is, however, possible that aggregates also occur earlier but are solubilized by the detergent used in the assay. This was investigated in G127X mice (Paper IV), as described in the methodology section. Indeed, throughout the life of these mice, there is a burden of detergent-soluble aggregates (or protoaggregates) of about 20% of the total amount of the G127X protein.

Another conspicuous finding is the occurrence of HMW species of mutant but also of wild-type hSOD1 enriched in the spinal cords of transgenic mice (see above and Fig. 9).

Fig. 9 Immunoblot of spinal cord homogenates from 100-day-old hSOD1 transgenic mice using a human SOD1 specific antibody. Equal amounts of homogenate are loaded in each lane. Note that the prominent HMW band has a relative mobility similar to that of the monomeric hSOD1 band. One band, 30-35 kDa in size, is especially prominent. The amounts of this band are similar between strains, despite widely differing levels of hSOD1 protein. The intensity of this band increases slowly with age. Similar species of mutSOD1 are also

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seen in the G127X patient. Under oxidative conditions, this banding pattern on immunoblots is even more striking (see above). It has therefore been proposed that these species represent SOD1 cysteines linked with cysteines on other molecules or other SOD1 subunits288. The exact nature of these species is, however, not known. In Paper IV, investigations were conducted to determine whether ubiquitin was covalently attached to the HMW species of G127X. This was in fact the case, but only in terminal mice and not in the major band of the HMW species. Interestingly, on these 2D immunoblots, the HMW species was readily seen, but, when gels were stained with silver, the spots could not be seen, suggesting that the amount of these species is overestimated (D. Bergemalm personal communication).

Histopathology The spinal cord histopathology was investigated in all transgenic strains at 50, 100, 200, 400, 600 days of age or at the terminal stage (Paper III-V). Similar to the data on hSOD1 protein levels, the strains divided into two groups. The high-level hSOD1 group (wt-hSOD1, D90A, G93AGur and G93AGurdl) demonstrated vacuolization, SOD1 staining in the ventral roots and SOD1-positive inclusions that increase with age with a terminal surge in occurrence. The low-level strains (G85R and G127X) demonstrated fewer large SOD1-positive inclusions, no vacuolization and no SOD1 staining in the ventral roots. There was, however, no obvious difference in motor neuron loss in the two groups at terminal disease and all strains developed similar amounts of small dense granular SOD1-positive inclusions, mainly in the neuropil. The degree of astrocytosis (GFAP-positive cells) was also similar between strains and increased time-dependently. In G127X mice, astrocytes were positive for αB-crystalline (other strains were not investigated).

These results suggest that the vacuolization pathology seen in the high-level strains, but not in the low-level strains, is not required for the ALS-causing toxicity of mutSOD1s. However, a negative influence of the vacuoles on motor neuron survival cannot be excluded. Likewise it appears as if the presence of mutSOD1 in motor neuron axons is not a requirement for toxicity (G85R and G127X mice and also noted in L126Z mice365). It is difficult to discern the type of inclusion corresponding to the detergent-resistant aggregates. The large SOD1-positive inclusions seen in all strains are one candidate, but the amounts of these inclusions differed between the high- and low-level strains whereas the detergent-resistant aggregates demonstrate similar amounts in the spinal cords of terminal mice of all the transgenic strains. Possibly, these inclusions are more amorphous and are easier solubilized in the high-level strains. The occurrence of the small dense granular inclusions was similar between strains and the alteration that differed least between the different strains.

In the G127X patient, motor neuron loss was seen at all levels of the spinal cord. Bunina bodies, LBHIs and skein like inclusions were seen in remaining motor neurons. Both LBHIs and skeins stained with the G127X-specific Inger antibody. There were also small round inclusions in motor neuron somata and the neuropil staining for Inger. No staining with Inger was seen in motor neuron axons. Astrogliosis was especially prominent in the corticospinal tract and astrocytes also often stained with αB-crystalline and some also had LBHIs. Many similarities in the


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histopathological picture were thus noted between the G127X patient and the G127X mice.

SOD1 aggregates and motor neuron toxicity The way mutSOD1 aggregates is far from completely understood. As has been described above, the form of the protein that is most likely to aggregate is the monomeric, disulfide-reduced, metal-deficient protein. In the hSOD1 transgenic mice, disulfide-reduced, copper-deficient hSOD1 exists (Fig. 10), constituting subfractions of the high-level hSOD1s and larger proportions of the low-level hSOD1s. These modifications of hSOD1 transform the protein from an unusually stable enzyme capable of withstanding 10 M urea and 4% SDS, as well as high temperature and long-term storage, to a protein that could (simplistically) unfold when we develop a high fever (melting point 42.9°C)288. Mutations in the hSOD1 protein further destabilize the protein, causing even more protein to un/misfold (see above). A recent report from the Valentine Lab, in which some mutSOD1s, e.g. H46R, H48Q, D101N and D124V, had thermal stabilities similar to the wt-hSOD1, contradicts this last statement347. Like previous results, metal deficiency and disulfide reduction made the protein less stable. In this study (and others), the results relate to studies of the folded protein. Little is known about what happens in vivo when the protein folds and if folding fails.

The in vitro stabilities of the different mutSOD1s have been associated with disease duration349. It is not known why the stability correlates to disease duration and not age at onset, for example. One possibility is that an unknown factor triggers the seeding of an aggregated hSOD1 species and initiates disease. Subsequently, the seed sequesters additional unfolded hSOD1 species. Onset would therefore be more dependent on the putative trigger, while disease progression would be dependent on the recruitment of additional hSOD1 to aggregates. One can only speculate about the nature of this putative trigger, but environmental, life-style related and genetic factors may come into play. The existence of a trigger may also provide an explanation for the differences in penetrance observed for different mutSOD1s. Some mutSOD1 may be less sensitive to the trigger (wt-hSOD1?, see below), which could result in a reduced penetrance. The sensitivity for this trigger may not be related in any way to the stability of the protein.

The reasons for the particular susceptibility of motor neurons are far from clear. It is not a matter of proteins levels, since several organs have higher levels of hSOD1 than CNS, even though it could be argued that motor neurons have higher levels than most other CNS cells (see above). This argument, however, appears not to be valid since ventral horns in humans, in which motor neurons account for ~30% of the volume (somas, axons and dendrites) (T. Brännström personal communication), do not have higher levels of hSOD1 than other parts of the CNS (Paper IV, unpublished observations). One clue is that the CNS accumulates misfolded hSOD1 in transgenic mice and that the spinal cord develops detergent-resistant aggregates (see above). This implicates the proteasome and the recognition of substrates to be degraded as a vulnerability factor (Fig. 10). Another possibility is that the environment in the CNS is such that the aggregation of proteins is favored. CCS levels may be another vulnerability factor, since the regulation of this protein does not appear to be as dynamic in the CNS as in other organs (see above), which could make a larger

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proportion of the SOD1 protein more copper-deficient and disulfide-reduced in the CNS than in other organs. It is possible that the CCS could also have more classical chaperoning properties that could aid in the handling of misfolded SOD1, although this has not yet been shown (this is perhaps also less likely since mice expressing mutSOD1 on a CCS null background do not have a different phenotype404).

In spite of this, it has not yet been convincingly shown that aggregates of hSOD1 are toxic to motor neurons or how they might be toxic. What has been shown is that mutSOD1 readily aggregates in motor neurons. Nevertheless, several downstream toxic mechanisms have been proposed. For example, interactions between aggregates/inclusions of mutSOD1 and the mitochondrial matrix539 and the mitochondrial intermembrane space403 have been shown and it has also been suggested that the mutSOD1 could aggregate on the cytoplasmic surface of mitochondria376. Possibly these accumulations of mutSOD1 could be harmful to the mitochondria. In astrocytes of G85R mice, hSOD1 containing inclusions were seen together with a decrease in astrocytic EAAT2360. In mutSOD1 transgenic mice, accumulations of hSOD1 and neurofilaments (spheroids)540 are seen in perikarya and the proximal axon. Possibly this could lead to cytoskeletal disruption resulting in the impaired axonal transport previously reported in mutSOD1 transgenic mice497,541,542.

Fig. 10 Turnover of SOD1 and hypothetical mechanism of disease. After transcription, multiple forms of SOD1 are formed, disulfide-reduced/oxidized, metallated/unmetallated and monomeric/dimeric. These forms are also in equilibrium with each other. Mutations in the SOD1 protein would shift the equilibrium to the more immature forms that are more likely to misfold. The degradation of the mature protein is most likely to occur via the phagocytic/lysosomal system. The degradation of a misfolded protein would be mediated by the proteasomal system. It is possible that the recognition and degradation of the misfolded SOD1s via the proteasome is slow in the CNS. This could cause the protein to aggregate, leading to cytotoxicity.


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The aggregation of hSOD1 can also lead to proteasome inhibition and a decrease in HSPs (the “quality control machinery”) that could result in an accumulation of misfolded proteins, thereby starting a cascade of aggregation leading to a vicious circle. Possibly, the terminal surge in the hSOD1 aggregates in transgenic mice represents the final breakdown of the “quality control” machinery in the cells.

Little is also known about the form of the aggregate that would confer cytotoxicity. It has been suggested that the large inclusions seen in older hSOD1 transgenic mice are non-toxic aggregates405,543,544 possibly representing aggresomes451. Aggresomes are deposits of aggregates that are gathered by retrograde transport along the microtubules. This could be a way for the cells to handle misfolded and aggregated proteins, possibly by degradation via the autophagic route545. The putative toxic aggregated hSOD1 species could therefore be misfolded oligomers546 of hSOD1 similar to the protoaggregates seen in G127X mice.

Could wt-hSOD1 cause ALS? The similarities between wt-hSOD1 and D90A mutSOD1 led us to investigate the properties of this mutant hSOD1 and to compare it with wt-hSOD1 to search for a clue to the toxicity of this mutant hSOD1 (Paper III). These experiments were performed on homozygous D90A mice and wt-hSOD1 transgenic mice with similar levels of hSOD1 expression and hemizygous D90A mice with lower levels of expression (Table 5). In erythrocytes from transgenic mice, the D90A mutant protein was nearly as stable as the wild-type protein, but both were much less stable than mSOD1 when exposed to denaturants or high temperature. These data relating to the high stability of mSOD1 confirm previous observations304.

Interestingly, wt-hSOD1 transgenic mice also developed detergent-resistant aggregates, although less and later than in terminal mutSOD1 transgenic mice. In this respect, hemizygous wt-hSOD1 transgenic mice fell between homozygous D90A mice, that develop MND, and hemizygous D90A mice, that do not develop MND. The histopathological picture of these three mouse strains supported this view and appeared in the following order with regard to the degree of pathology observed; homozygous D90A mice > wt-hSOD1 transgenic mice > hemizygous D90A mice. This suggests that the aggregation potential of wt-hSOD1 is less than equal to, but more than half of the D90A mutant enzyme, raising the possibility that hSOD1 could be involved in SALS without hSOD1 mutations. This would also be in analogy to what is seen in Alzheimer’s and Parkinson’s diseases (as discussed above).

This interpretation is supported by the paper from Jaarsma and co-workers, who noted that wt-hSOD1 transgenic mice suffered from mild motor deficits and a 20-30% motor neuron loss in two year old mice (when detergent-resistant aggregates start to develop) compared with non-transgenic mice368. Inclusions containing hSOD1 have also been observed in ALS patients without hSOD1 mutations (T. Brännström personal communication and above).

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CONCLUSIONS I. In erythrocytes, there was no evidence that the putative protective factor in

recessive D90A cases acts by downregulating synthesis, influencing degradation or by altering the quaternary structure of D90A hSOD1.

II. In CSF, there was no difference in SOD isoenzyme content between ALS

patients lacking hSOD1 mutations and controls. There was no evidence that the putative protective factor in recessive D90A cases acts by downregulating synthesis the mutant enzyme. CNS contains N-terminally cleaved hSOD1 that is enriched in the CSF. ALS patients who are homozygous for D90A mutSOD1 have higher levels of N-terminally cleaved hSOD1 in the CSF compared with controls. Could the N-terminally cleaved fragment be involved in ALS pathogenesis?

III. D90A hSOD1 transgenic mice develop an ALS-like phenotype similar to that

of ALS patients homozygous for D90A mutSOD1. The in vivo and in vitro properties of D90A hSOD1 are almost indistinguishable from those of wt-hSOD1. Mice overexpressing wt-hSOD1 develop detergent-resistant aggregates similar to those seen in D90A transgenic mice, although less and later. This suggests that wt-hSOD1 could be involved in ALS cases without hSOD1 mutations.

IV. G127X hSOD1 transgenic mice develop an ALS-like phenotype similar to

that of ALS patients heterozygous for G127X mutSOD1. Similar to what is found in a G127X patient, detergent-resistant aggregates and high molecular weight hSOD1 species develop in affected motor areas of the mice. In mice and humans, minute amounts of G127X mutSOD1 are sufficient to cause motor neuron disease.

V. Disulfide-reduced, copper-deficient and aggregation-prone hSOD1 is enriched

in the CNS of hSOD1 transgenic mice. The motor neuron degeneration caused by mutSOD1s may therefore be attributable to long-term exposure to misfolded, aggregation-prone, disulfide-reduced SOD1. Such hSOD1 species should constitute minute subfractions of the stable mutants and larger proportions of the unstable mutants.


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ACKNOWLEDGMENTS I wish to thank: My supervisor, Stefan Marklund, for introducing me to the field and for generously providing support, guidance, and encouragement. Your vast scientific knowledge is truly an inspiration. My assistant supervisors Thomas Brännström and Peter Andersen for sharing their immense knowledge in neuropathology and ALS. Jörgen Andersson, Eva Bern, Ingalis Fransson, Lena Hedman, Karin Hjertkvist, Ann-Charlott Nilsson, Ulla-Stina Spetz and Agneta Öberg for their invaluable work at the lab and for always being so positive and patient. Special thanks to Agneta and Eva for all those terrific western blots. Elisabeth Nilsson and Karin Wallgren for taking so good care of our mice. Karin Graffmo for meticulously keeping track of all of our mice and for nice conversations. Birgitta Nilsson, Iris Svanholm and Monica Sörman for administrative assistance and for being so helpful with all kinds of paper work. Göran Brattsand and Kjell Grankvist for insightful and thought-provoking discussions. Former and present PhD students at the department of Clinical Chemistry; Christine Ahlbeck Glader, Britta Andersson, Parviz Behnam-Motlagh, Anders Behndig, Daniel Bergemalm, Micael Granström, Veronica Janson, David Johansson, Åsa Johansson, Linda Marklund, Mia Sentman, Per Zetterström, Eva Åström, Heather Stewart at the Department of Neurology and Karin Nilsson at the Department of Pathology for contributing to a nice environment and for beings such good sports. Special thanks to former roommates Christine Ahlbeck Glader, Linda Marklund and Åsa Johansson for pleasant discussions about everything under the sun and to Mia Sentman for introducing me in the lab and always being so positive and helpful. Kim Ekblom, Johan Hultdin, Håkan Jakobsson, Kurt Karlsson, Stig Karlsson, Jörn Schneede and Le Thu Trinh at the Clinical Chemistry laboratory for interesting discussions and for contributing to an pleasant atmosphere, especially at lunches. Extra gratitude to Johan Hultdin for patiently answering all thesis questions these last couple of months. Lars Forsgren, Johan Jacobsson, Lena Lundqvist and Margit Lundmark at the Department of Neurology.

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All other co-authors, especially Åsa Bäckstrand, Ole Gredal and Peter Nilsson and for their important contributions. The Mikael Oliveberg group for fruitful collaborations and for providing insights into the SOD1 protein structure and folding dynamics. My friends for continuous support especially Adrian Elmi for always standing up for your friends. My family for love and support. The individuals who donated blood or tissues for analysis, for their contribution to ALS research. The financial support from the Swedish Science Council, the Swedish Brain Fund/Hållsten Foundation, the Swedish Medical Society including Björklunds Fund for ALS Research, the Swedish Associations of Persons with Neurological Disabilities, the Västerbotten County Council, the JC Kempe Memorial Foundation, and the Medical Faculty at the University of Umeå.


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REFERENCES 1. Aran, F.A. (1850) Recherches sur une maladie non encore décrite du système musculaire

(atrophie musculaire progressive). Arch. Gen. Med., 24, 5-35, 172-214.

2. Charcot, J.M. and Joffroy, A. (1869) Deux cas d' atrophie muscularie progressive avec lésions de la substance grise et des faisceaux antéro-latéraux de la moelle épinière. Arch. Physiol. Neurol. Path., 2, 744-760.

3. Desai, J. and Swash, M. (2002) Essentials of diagnosis. In: Motor Neuron Disease, 1-20, Kuncl, R.W. (ed.), Saunders, Edinburgh.

4. Brooks, B.R. (1994) El Escorial World Federation of Neurology criteria for the diagnosis of amyotrophic lateral sclerosis. Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Sclerosis of the World Federation of Neurology Research Group on Neuromuscular Diseases and the El Escorial "Clinical limits of amyotrophic lateral sclerosis" workshop contributors. J. Neurol. Sci., 124 Suppl, 96-107.

5. Brooks, B.R., Miller, R.G., Swash, M., and Munsat, T.L. (2000) El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 1, 293-299.

6. Belsh, J.M. (2000) ALS diagnostic criteria of El Escorial Revisited: do they meet the needs of clinicians as well as researchers? Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 1 Suppl 1, S57-S60.

7. Traynor, B.J., Codd, M.B., Corr, B., Forde, C., Frost, E., and Hardiman, O.M. (2000) Clinical features of amyotrophic lateral sclerosis according to the El Escorial and Airlie House diagnostic criteria: A population-based study. Arch. Neurol., 57, 1171-1176.

8. Meininger, V. (1999) Getting the diagnosis right: beyond El Escorial. J. Neurol., 246 Suppl 3, III10-III12.

9. Ince, P.G., Lowe, J., and Shaw, P.J. (1998) Amyotrophic lateral sclerosis: current issues in classification, pathogenesis and molecular pathology. Neuropathol. Appl. Neurobiol., 24, 104-117.

10. Swash, M., Desai, J., and Misra, V.P. (1999) What is primary lateral sclerosis? J. Neurol. Sci., 170, 5-10.

11. Le Forestier, N., Maisonobe, T., Piquard, A., Rivaud, S., Crevier-Buchman, L., Salachas, F., Pradat, P.F., Lacomblez, L., and Meininger, V. (2001) Does primary lateral sclerosis exist? A study of 20 patients and a review of the literature. Brain, 124, 1989-1999.

12. Swash, M. and Desai, J. (2000) Motor neuron disease: Classification and nomenclature. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 1, 105-112.

13. Brain, W.R. (1962) Diseases of the Nervous System, Oxford university press, Oxford.

14. Walton, J.N. (1977) Brain's Diseases of the Nervous System, Oxford university press, Oxford.

P. Andreas Jonsson

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15. Mannen, T., Iwata, M., Toyokura, Y., and Nagashima, K. (1977) Preservation of a certain motoneurone group of the sacral cord in amyotrophic lateral sclerosis: its clinical significance. J. Neurol. Neurosurg. Psychiatry, 40, 464-469.

16. Mannen, T., Iwata, M., Toyokura, Y., and Nagashima, K. (1982) The Onuf's nucleus and the external anal sphincter muscles in amyotrophic lateral sclerosis and Shy-Drager syndrome. Acta Neuropathol. (Berl), 58, 255-260.

17. Harvey, D.G., Torack, R.M., and Rosenbaum, H.E. (1979) Amyotrophic lateral sclerosis with ophthalmoplegia. A clinicopathologic study. Arch. Neurol., 36, 615-617.

18. Hayashi, H., Kato, S., and Kawada, A. (1991) Amyotrophic lateral sclerosis patients living beyond respiratory failure. J. Neurol. Sci., 105, 73-78.

19. Palmowski, A., Jost, W.H., Prudlo, J., Osterhage, J., Kasmann, B., Schimrigk, K., and Ruprecht, K.W. (1995) Eye movement in amyotrophic lateral sclerosis: a longitudinal study. Ger J. Ophthalmol., 4, 355-362.

20. Hayashi, H. and Kato, S. (1989) Total manifestations of amyotrophic lateral sclerosis. ALS in the totally locked-in state. J. Neurol. Sci., 93, 19-35.

21. Mizutani, T., Aki, M., Shiozawa, R., Unakami, M., Nozawa, T., Yajima, K., Tanabe, H., and Hara, M. (1990) Development of ophthalmoplegia in amyotrophic lateral sclerosis during long-term use of respirators. J. Neurol. Sci., 99, 311-319.

22. Kotchoubey, B., Lang, S., Winter, S., and Birbaumer, N. (2003) Cognitive processing in completely paralyzed patients with amyotrophic lateral sclerosis. Eur. J. Neurol., 10, 551-558.

23. Jacobs, L., Bozian, D., Heffner, R.R., Jr., and Barron, S.A. (1981) An eye movement disorder in amyotrophic lateral sclerosis. Neurology, 31, 1282-1287.

24. Ohki, M., Kanayama, R., Nakamura, T., Okuyama, T., Kimura, Y., and Koike, Y. (1994) Ocular abnormalities in amyotrophic lateral sclerosis. Acta Otolaryngol. Suppl, 511, 138-142.

25. Vaphiades, M.S., Husain, M., Juhasz, K., and Schmidley, J.W. (2002) Motor neuron disease presenting with slow saccades and dementia. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 3, 159-162.

26. Jokelainen, M. (1977) Amyotrophic lateral sclerosis in Finland. II: Clinical characteristics. Acta Neurol. Scand., 56, 194-204.

27. Gubbay, S.S., Kahana, E., Zilber, N., Cooper, G., Pintov, S., and Leibowitz, Y. (1985) Amyotrophic lateral sclerosis. A study of its presentation and prognosis. J. Neurol., 232, 295-300.

28. Bergmann, M., Volpel, M., and Kuchelmeister, K. (1995) Onuf's nucleus is frequently involved in motor neuron disease/amyotrophic lateral sclerosis. J. Neurol. Sci., 129, 141-146.

29. Kihira, T., Yoshida, S., Yoshimasu, F., Wakayama, I., and Yase, Y. (1997) Involvement of Onuf's nucleus in amyotrophic lateral sclerosis. J. Neurol. Sci., 147, 81-88.

30. Carvalho, M., Schwartz, M.S., and Swash, M. (1995) Involvement of the external anal-sphincter in amyotrophic lateral sclerosis. Muscle Nerve, 18, 848-853.


73

31. Haverkamp, L.J., Appel, V., and Appel, S.H. (1995) Natural history of amyotrophic lateral sclerosis in a database population. Validation of a scoring system and a model for survival prediction. Brain, 118 ( Pt 3), 707-719.

32. Lomen-Hoerth, C., Murphy, J., Langmore, S., Kramer, J.H., Olney, R.K., and Miller, B. (2003) Are amyotrophic lateral sclerosis patients cognitively normal? Neurology, 60, 1094-1097.

33. Portet, F., Cadilhac, C., Touchon, J., and Camu, W. (2001) Cognitive impairment in motor neuron disease with bulbar onset. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 2, 23-29.

34. Massman, P.J., Sims, J., Cooke, N., Haverkamp, L.J., Appel, V., and Appel, S.H. (1996) Prevalence and correlates of neuropsychological deficits in amyotrophic lateral sclerosis. J. Neurol. Neurosurg. Psychiatry, 61, 450-455.

35. Zoccolella, S., Palagano, G., Fraddosio, A., Russo, I., Ferrannini, E., Serlenga, L., Maggio, F., Lamberti, S., and Iliceto, G. (2002) ALS-plus: 5 cases of concomitant amyotrophic lateral sclerosis and parkinsonism. Neurol. Sci., 23 Suppl 2, S123-S124.

36. Qureshi, A.I., Wilmot, G., Dihenia, B., Schneider, J.A., and Krendel, D.A. (1996) Motor neuron disease with parkinsonism. Arch. Neurol., 53, 987-991.

37. Desai, J. and Swash, M. (1999) Extrapyramidal involvement in amyotrophic lateral sclerosis: backward falls and retropulsion. J. Neurol. Neurosurg. Psychiatry, 67, 214-216.

38. Kalra, S. and Arnold, D. (2003) Neuroimaging in amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 4, 243-248.

39. Abe, K., Fujimura, H., Toyooka, K., Hazama, T., Hirono, N., Yorifuji, S., and Yanagihara, T. (1993) Single-photon emission computed tomographic investigation of patients with motor neuron disease. Neurology, 43, 1569-1573.

40. Waldemar, G., Vorstrup, S., Jensen, T.S., Johnsen, A., and Boysen, G. (1992) Focal reductions of cerebral blood flow in amyotrophic lateral sclerosis: a [99mTc]-d,l-HMPAO SPECT study. J. Neurol. Sci., 107, 19-28.

41. Abrahams, S., Goldstein, L.H., Kew, J.J., Brooks, D.J., Lloyd, C.M., Frith, C.D., and Leigh, P.N. (1996) Frontal lobe dysfunction in amyotrophic lateral sclerosis. A PET study. Brain, 119 ( Pt 6), 2105-2120.

42. Abrahams, S., Goldstein, L.H., Simmons, A., Brammer, M., Williams, S.C., Giampietro, V., and Leigh, P.N. (2004) Word retrieval in amyotrophic lateral sclerosis: a functional magnetic resonance imaging study. Brain, 127, 1507-1517.

43. Lloyd, C.M., Richardson, M.P., Brooks, D.J., Al Chalabi, A., and Leigh, P.N. (2000) Extramotor involvement in ALS: PET studies with the GABA(A) ligand [(11)C]flumazenil. Brain, 123, 2289-2296.

44. Turner, M.R., Rabiner, E.A., Hammers, A., Al Chalabi, A., Grasby, P.M., Shaw, C.E., Brooks, D.J., and Leigh, P.N. (2005) [11C]-WAY100635 PET demonstrates marked 5-HT1A receptor changes in sporadic ALS. Brain, 128, 896-905.

45. Turner, M.R., Cagnin, A., Turkheimer, F.E., Miller, C.C., Shaw, C.E., Brooks, D.J., Leigh, P.N., and Banati, R.B. (2004) Evidence of widespread cerebral microglial activation in amyotrophic lateral sclerosis: an [11C](R)-PK11195 positron emission tomography study. Neurobiol. Dis., 15, 601-609.

P. Andreas Jonsson

74

46. Takahashi, H., Snow, B.J., Bhatt, M.H., Peppard, R., Eisen, A., and Calne, D.B. (1993) Evidence for a dopaminergic deficit in sporadic amyotrophic lateral sclerosis on positron emission scanning. Lancet, 342, 1016-1018.

47. Mackenzie, I.A. and Feldman, H. (2003) The relationship between extramotor ubiquitin-immunoreactive neuronal inclusions and dementia in motor neuron disease. Acta Neuropathol. (Berl), 105, 98-102.

48. Wilson, C.M., Grace, G.M., Munoz, D.G., He, B.P., and Strong, M.J. (2001) Cognitive impairment in sporadic ALS: a pathologic continuum underlying a multisystem disorder. Neurology, 57, 651-657.

49. Kawashima, T., Doh-ura, K., Kikuchi, H., and Iwaki, T. (2001) Cognitive dysfunction in patients with amyotrophic lateral sclerosis is associated with spherical or crescent-shaped ubiquitinated intraneuronal inclusions in the parahippocampal gyrus and amygdala, but not in the neostriatum. Acta Neuropathol. (Berl), 102, 467-472.

50. Okamoto, K., Hirai, S., Yamazaki, T., Sun, X.Y., and Nakazato, Y. (1991) New ubiquitin-positive intraneuronal inclusions in the extra-motor cortices in patients with amyotrophic lateral sclerosis. Neurosci. Lett., 129, 233-236.

51. Wightman, G., Anderson, V.E., Martin, J., Swash, M., Anderton, B.H., Neary, D., Mann, D., Luthert, P., and Leigh, P.N. (1992) Hippocampal and neocortical ubiquitin-immunoreactive inclusions in amyotrophic lateral sclerosis with dementia. Neurosci. Lett., 139, 269-274.

52. Piao, Y.S., Wakabayashi, K., Kakita, A., Yamada, M., Hayashi, S., Morita, T., Ikuta, F., Oyanagi, K., and Takahashi, H. (2003) Neuropathology with clinical correlations of sporadic amyotrophic lateral sclerosis: 102 autopsy cases examined between 1962 and 2000. Brain Pathol., 13, 10-22.

53. Kato, S., Oda, M., and Tanabe, H. (1993) Diminution of dopaminergic neurons in the substantia nigra of sporadic amyotrophic lateral sclerosis. Neuropathol. Appl. Neurobiol., 19, 300-304.

54. Maekawa, S., al Sarraj, S., Kibble, M., Landau, S., Parnavelas, J., Cotter, D., Everall, I., and Leigh, P.N. (2004) Cortical selective vulnerability in motor neuron disease: a morphometric study. Brain, 127, 1237-1251.

55. Gredal, O., Pakkenberg, H., Karlsborg, M., and Pakkenberg, B. (2000) Unchanged total number of neurons in motor cortex and neocortex in amyotrophic lateral sclerosis: a stereological study. J. Neurosci. Methods, 95, 171-176.

56. Worms, P.M. (2001) The epidemiology of motor neuron diseases: a review of recent studies. J. Neurol. Sci., 191, 3-9.

57. Strong, M. and Rosenfeld, J. (2003) Amyotrophic lateral sclerosis: a review of current concepts. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 4, 136-143.

58. Piemonte and Valle d'Aosta Register for Amyotrophic Lateral Sclerosis (PARALS) (2001) Incidence of ALS in Italy: evidence for a uniform frequency in Western countries. Neurology, 56, 239-244.

59. McGuire, V., Longstreth, W.T., Jr., Koepsell, T.D., and van Belle, G. (1996) Incidence of amyotrophic lateral sclerosis in three counties in western Washington state. Neurology, 47, 571-573.


75

60. Sorenson, E.J., Stalker, A.P., Kurland, L.T., and Windebank, A.J. (2002) Amyotrophic lateral sclerosis in Olmsted County, Minnesota, 1925 to 1998. Neurology, 59, 280-282.

61. Forsgren, L., Almay, B.G., Holmgren, G., and Wall, S. (1983) Epidemiology of motor neuron disease in northern Sweden. Acta Neurol. Scand., 68, 20-29.

62. Traynor, B.J., Codd, M.B., Corr, B., Forde, C., Frost, E., and Hardiman, O. (1999) Incidence and prevalence of ALS in Ireland, 1995-1997: a population-based study. Neurology, 52, 504-509.

63. The Scottish Motor Neuron Disease Research Workgroup (1992) The Scottish Motor Neuron Disease Register: a prospective study of adult onset motor neuron disease in Scotland. Methodology, demography and clinical features of incident cases in 1989. J. Neurol. Neurosurg. Psychiatry, 55, 536-541.

64. Yoshida, S., Uebayashi, Y., Kihira, T., Kohmoto, J., Wakayama, I., Taguchi, S., and Yase, Y. (1998) Epidemiology of motor neuron disease in the Kii Peninsula of Japan, 1989-1993: active or disappearing focus? J. Neurol. Sci., 155, 146-155.

65. Plato, C.C., Garruto, R.M., Galasko, D., Craig, U.K., Plato, M., Gamst, A., Torres, J.M., and Wiederholt, W. (2003) Amyotrophic lateral sclerosis and parkinsonism-dementia complex of Guam: changing incidence rates during the past 60 years. Am. J. Epidemiol., 157, 149-157.

66. Roman, G.C. (1996) Neuroepidemiology of amyotrophic lateral sclerosis: clues to aetiology and pathogenesis. J. Neurol. Neurosurg. Psychiatry, 61, 131-137.

67. Chio, A. (2000) Risk factors in the early diagnosis of ALS: European epidemiological studies. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 1 Suppl 1, S13-S18.

68. Mitchell, J.D. (2000) Amyotrophic lateral sclerosis: toxins and environment. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 1, 235-250.

69. Jablecki, C.K., Berry, C., and Leach, J. (1989) Survival prediction in amyotrophic lateral sclerosis. Muscle Nerve, 12, 833-841.

70. Rosen, A.D. (1978) Amyotrophic lateral sclerosis. Clinical features and prognosis. Arch. Neurol., 35, 638-642.

71. Li, T.M., Alberman, E., and Swash, M. (1990) Clinical features and associations of 560 cases of motor neuron disease. J. Neurol. Neurosurg. Psychiatry, 53, 1043-1045.

72. Eisen, A., Schulzer, M., MacNeil, M., Pant, B., and Mak, E. (1993) Duration of amyotrophic lateral sclerosis is age dependent. Muscle Nerve, 16, 27-32.

73. Norris, F., Shepherd, R., Denys, E., U K, Mukai, E., Elias, L., Holden, D., and Norris, H. (1993) Onset, natural history and outcome in idiopathic adult motor neuron disease. J. Neurol. Sci., 118, 48-55.

74. Majoor-Krakauer, D., Willems, P.J., and Hofman, A. (2003) Genetic epidemiology of amyotrophic lateral sclerosis. Clin. Genet., 63, 83-101.

75. Battistini, S., Giannini, F., Greco, G., Bibbo, G., Ferrera, L., Marini, V., Causarano, R., Casula, M., Lando, G., Patrosso, M.C., Caponnetto, C., Origone, P., Marocchi, A., Del Corona, A., Siciliano, G., Carrera, P., Mascia, V., Giagheddu, M., Carcassi, C., Orru, S., Garre, C., and Penco, S. (2005) SOD1 mutations in amyotrophic lateral sclerosis Results from a multicenter Italian study. J. Neurol., 252, 782-788.

P. Andreas Jonsson

76

76. Jokelainen, M. (1977) Amyotrophic lateral sclerosis in Finland. I: An epidemiologic study. Acta Neurol. Scand., 56, 185-193.

77. Forbes, R.B., Colville, S., Cran, G.W., and Swingler, R.J. (2004) Unexpected decline in survival from amyotrophic lateral sclerosis/motor neurone disease. J. Neurol. Neurosurg. Psychiatry, 75, 1753-1755.

78. del Aguila, M.A., Longstreth, W.T., Jr., McGuire, V., Koepsell, T.D., and van Belle, G. (2003) Prognosis in amyotrophic lateral sclerosis: a population-based study. Neurology, 60, 813-819.

79. Kato, S., Shaw, P., Wood-Allum, C., Leigh, P.N., and Shaw, C. (2003) Amyotrophic lateral sclerosis. In: Neurodegeneration: The molecular pathology of dementia and movement disorders, 350-368, Dickson, D.W. (ed.), ISN Neuropath Press, Basel.

80. Ince, P.G. (2000) Neuropathology. In: Amyotrophic lateral sclerosis, 83-112, Brown, R.H., Jr., Meininger, V., and Swash, M. (eds.), Martin Dunitz, London.

81. Hirano, A. (1996) Neuropathology of ALS: an overview. Neurology, 47, S63-S66.

82. Ikemoto, A., Hirano, A., and Akiguchi, I. (2000) Neuropathology of amyotrophic lateral sclerosis with extra-motor system degeneration: characteristics and differences in the molecular pathology between ALS with dementia and Guamanian ALS. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 1, 97-104.

83. He, C.Z. and Hays, A.P. (2004) Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis. J. Neurol. Sci., 217, 47-54.

84. Matsumoto, S., Kusaka, H., Ito, H., Shibata, N., Asayama, T., and Imai, T. (1996) Sporadic amyotrophic lateral sclerosis with dementia and Cu/Zn superoxide dismutase-positive Lewy body-like inclusions. Clin. Neuropathol., 15, 41-46.

85. Shibata, N., Hirano, A., Kobayashi, M., Sasaki, S., Kato, T., Matsumoto, S., Shiozawa, Z., Komori, T., Ikemoto, A., Umahara, T., and Asayama, K. (1994) Cu/Zn Superoxide Dismutase-Like Immunoreactivity in Lewy Body-Like Inclusions of Sporadic Amyotrophic-Lateral-Sclerosis. Neuroscience Letters, 179, 149-152.

86. Mattson, M.P. (2003) Excitotoxic and excitoprotective mechanisms: abundant targets for the prevention and treatment of neurodegenerative disorders. Neuromolecular. Med., 3, 65-94.

87. Plaitakis, A. and Caroscio, J.T. (1987) Abnormal glutamate metabolism in amyotrophic lateral sclerosis. Ann. Neurol., 22, 575-579.

88. Perry, T.L., Krieger, C., Hansen, S., and Eisen, A. (1990) Amyotrophic lateral sclerosis: amino acid levels in plasma and cerebrospinal fluid. Ann. Neurol., 28, 12-17.

89. Shaw, P.J., Forrest, V., Ince, P.G., Richardson, J.P., and Wastell, H.J. (1995) CSF and plasma amino acid levels in motor neuron disease: elevation of CSF glutamate in a subset of patients. Neurodegeneration., 4, 209-216.

90. Spreux-Varoquaux, O., Bensimon, G., Lacomblez, L., Salachas, F., Pradat, P.F., Le Forestier, N., Marouan, A., Dib, M., and Meininger, V. (2002) Glutamate levels in cerebrospinal fluid in amyotrophic lateral sclerosis: a reappraisal using a new HPLC method with coulometric detection in a large cohort of patients. J. Neurol. Sci., 193, 73-78.


77

91. Rothstein, J.D., Tsai, G., Kuncl, R.W., Clawson, L., Cornblath, D.R., Drachman, D.B., Pestronk, A., Stauch, B.L., and Coyle, J.T. (1990) Abnormal excitatory amino acid metabolism in amyotrophic lateral sclerosis. Ann. Neurol., 28, 18-25.

92. Tsai, G.C., Stauch-Slusher, B., Sim, L., Hedreen, J.C., Rothstein, J.D., Kuncl, R., and Coyle, J.T. (1991) Reductions in acidic amino acids and N-acetylaspartylglutamate in amyotrophic lateral sclerosis CNS. Brain Res., 556, 151-156.

93. Plaitakis, A., Constantakakis, E., and Smith, J. (1988) The neuroexcitotoxic amino acids glutamate and aspartate are altered in the spinal cord and brain in amyotrophic lateral sclerosis. Ann. Neurol., 24, 446-449.

94. Perry, T.L., Hansen, S., and Jones, K. (1987) Brain glutamate deficiency in amyotrophic lateral sclerosis. Neurology, 37, 1845-1848.

95. Camu, W., Billiard, M., and Baldy-Moulinier, M. (1993) Fasting plasma and CSF amino acid levels in amyotrophic lateral sclerosis: a subtype analysis. Acta Neurol. Scand., 88, 51-55.

96. Patten, B.M., Harati, Y., Acosta, L., Jung, S.S., and Felmus, M.T. (1978) Free amino acid levels in amyotrophic lateral sclerosis. Ann. Neurol., 3, 305-309.

97. Rothstein, J.D., Martin, L.J., and Kuncl, R.W. (1992) Decreased glutamate transport by the brain and spinal cord in amyotrophic lateral sclerosis. N. Engl. J. Med., 326, 1464-1468.

98. Sasaki, S., Komori, T., and Iwata, M. (2000) Excitatory amino acid transporter 1 and 2 immunoreactivity in the spinal cord in amyotrophic lateral sclerosis. Acta Neuropathol. (Berl), 100, 138-144.

99. Rothstein, J.D., Van Kammen, M., Levey, A.I., Martin, L.J., and Kuncl, R.W. (1995) Selective loss of glial glutamate transporter GLT-1 in amyotrophic lateral sclerosis. Ann. Neurol., 38, 73-84.

100. Fray, A.E., Ince, P.G., Banner, S.J., Milton, I.D., Usher, P.A., Cookson, M.R., and Shaw, P.J. (1998) The expression of the glial glutamate transporter protein EAAT2 in motor neuron disease: an immunohistochemical study. Eur. J. Neurosci., 10, 2481-2489.

101. Bristol, L.A. and Rothstein, J.D. (1996) Glutamate transporter gene expression in amyotrophic lateral sclerosis motor cortex. Ann. Neurol., 39, 676-679.

102. Meyer, T., Munch, C., Volkel, H., Booms, P., and Ludolph, A.C. (1998) The EAAT2 (GLT-1) gene in motor neuron disease: absence of mutations in amyotrophic lateral sclerosis and a point mutation in patients with hereditary spastic paraplegia. J. Neurol. Neurosurg. Psychiatry, 65, 594-596.

103. Aoki, M., Lin, C.L., Rothstein, J.D., Geller, B.A., Hosler, B.A., Munsat, T.L., Horvitz, H.R., and Brown, R.H.J. (1998) Mutations in the glutamate transporter EAAT2 gene do not cause abnormal EAAT2 transcripts in amyotrophic lateral sclerosis. Ann. Neurol., 43, 645-653.

104. Jackson, M., Steers, G., Leigh, P.N., and Morrison, K.E. (1999) Polymorphisms in the glutamate transporter gene EAAT2 in European ALS patients. J. Neurol., 246, 1140-1144.

105. Lin, C.L., Bristol, L.A., Jin, L., Dykes-Hoberg, M., Crawford, T., Clawson, L., and Rothstein, J.D. (1998) Aberrant RNA processing in a neurodegenerative disease: the cause for absent EAAT2, a glutamate transporter, in amyotrophic lateral sclerosis. Neuron, 20, 589-602.

P. Andreas Jonsson

78

106. Nagai, M., Abe, K., Okamoto, K., and Itoyama, Y. (1998) Identification of alternative splicing forms of GLT-1 mRNA in the spinal cord of amyotrophic lateral sclerosis patients. Neurosci. Lett., 244, 165-168.

107. Meyer, T., Fromm, A., Munch, C., Schwalenstocker, B., Fray, A.E., Ince, P.G., Stamm, S., Gron, G., Ludolph, A.C., and Shaw, P.J. (1999) The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals. J. Neurol. Sci., 170, 45-50.

108. Flowers, J.M., Powell, J.F., Leigh, P.N., Andersen, P., and Shaw, C.E. (2001) Intron 7 retention and exon 9 skipping EAAT2 mRNA variants are not associated with amyotrophic lateral sclerosis. Ann. Neurol., 49, 643-649.

109. Honig, L.S., Chambliss, D.D., Bigio, E.H., Carroll, S.L., and Elliott, J.L. (2000) Glutamate transporter EAAT2 splice variants occur not only in ALS, but also in AD and controls. Neurology, 55, 1082-1088.

110. Kawahara, Y., Ito, K., Sun, H., Aizawa, H., Kanazawa, I., and Kwak, S. (2004) Glutamate receptors: RNA editing and death of motor neurons. Nature, 427, 801

111. Miller, R.G., Mitchell, J.D., Lyon, M., and Moore, D.H. (2002) Riluzole for amyotrophic lateral sclerosis (ALS)/motor neuron disease (MND). Cochrane Database Syst. Rev., CD001447

112. Martin, D., Thompson, M.A., and Nadler, J.V. (1993) The neuroprotective agent riluzole inhibits release of glutamate and aspartate from slices of hippocampal area CA1. Eur. J. Pharmacol., 250, 473-476.

113. Cheramy, A., Barbeito, L., Godeheu, G., and Glowinski, J. (1992) Riluzole inhibits the release of glutamate in the caudate nucleus of the cat in vivo. Neurosci. Lett., 147, 209-212.

114. Lariviere, R.C. and Julien, J.P. (2004) Functions of intermediate filaments in neuronal development and disease. J. Neurobiol., 58, 131-148.

115. Strong, M.J., Kesavapany, S., and Pant, H.C. (2005) The Pathobiology of Amyotrophic Lateral Sclerosis: A Proteinopathy? J. Neuropathol. Exp. Neurol., 64, 649-664.

116. Zhu, Q., Couillard-Despres, S., and Julien, J.P. (1997) Delayed maturation of regenerating myelinated axons in mice lacking neurofilaments. Exp. Neurol., 148, 299-316.

117. Elder, G.A., Friedrich, V.L., Jr., Margita, A., and Lazzarini, R.A. (1999) Age-related atrophy of motor axons in mice deficient in the mid-sized neurofilament subunit. J. Cell Biol., 146, 181-192.

118. Beaulieu, J.M., Nguyen, M.D., and Julien, J.P. (1999) Late onset death of motor neurons in mice overexpressing wild-type peripherin. J. Cell Biol., 147, 531-544.

119. Cote, F., Collard, J.F., and Julien, J.P. (1993) Progressive neuronopathy in transgenic mice expressing the human neurofilament heavy gene: a mouse model of amyotrophic lateral sclerosis. Cell, 73, 35-46.

120. Lee, M.K., Marszalek, J.R., and Cleveland, D.W. (1994) A mutant neurofilament subunit causes massive, selective motor neuron death: implications for the pathogenesis of human motor neuron disease. Neuron, 13, 975-988.


79

121. Williamson, T.L., Bruijn, L.I., Zhu, Q., Anderson, K.L., Anderson, S.D., Julien, J.P., and Cleveland, D.W. (1998) Absence of neurofilaments reduces the selective vulnerability of motor neurons and slows disease caused by a familial amyotrophic lateral sclerosis-linked superoxide dismutase 1 mutant. Proc. Natl. Acad. Sci. U. S. A., 95, 9631-9636.

122. Couillard-Despres, S., Zhu, Q., Wong, P.C., Price, D.L., Cleveland, D.W., and Julien, J.P. (1998) Protective effect of neurofilament heavy gene overexpression in motor neuron disease induced by mutant superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A., 95, 9626-9630.

123. Nguyen, M.D., Lariviere, R.C., and Julien, J.P. (2001) Deregulation of Cdk5 in a mouse model of ALS: toxicity alleviated by perikaryal neurofilament inclusions. Neuron, 30, 135-147.

124. Brooks, B.R. (2000) Risk factors in the early diagnosis of ALS: North American epidemiological studies. ALS CARE Study Group. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 1 Suppl 1, S19-S26.

125. Armon, C. (2001) Environmental risk factors for amyotrophic lateral sclerosis. Neuroepidemiology, 20, 2-6.

126. Stewart, H.G. and Andersen, P.M. (2004) Neurophysiology of hereditary amyotrophic lateral sclerosis. In: Clinical neurophysiology of motor neuron diseases, 543-562, Eisen, A. (ed.), Elsevier, Amsterdam.

127. Li, T.M., Alberman, E., and Swash, M. (1988) Comparison of sporadic and familial disease amongst 580 cases of motor neuron disease. J. Neurol. Neurosurg. Psychiatry, 51, 778-784.

128. Mulder, D.W., Kurland, L.T., Offord, K.P., and Beard, C.M. (1986) Familial adult motor neuron disease: amyotrophic lateral sclerosis. Neurology, 36, 511-517.

129. Camu, W., Khoris, J., Moulard, B., Salachas, F., Briolotti, V., Rouleau, G.A., and Meininger, V. (1999) Genetics of familial ALS and consequences for diagnosis. French ALS Research Group. J. Neurol. Sci., 165 Suppl 1, S21-S26.

130. Strong, M.J., Hudson, A.J., and Alvord, W.G. (1991) Familial amyotrophic lateral sclerosis, 1850-1989: a statistical analysis of the world literature. Can. J. Neurol. Sci., 18, 45-58.

131. Ben Hamida, M., Hentati, F., and Ben Hamida, C. (1990) Hereditary motor system diseases (chronic juvenile amyotrophic lateral sclerosis). Conditions combining a bilateral pyramidal syndrome with limb and bulbar amyotrophy. Brain, 113 ( Pt 2), 347-363.

132. Kunst, C.B. (2004) Complex genetics of amyotrophic lateral sclerosis. Am. J. Hum. Genet., 75, 933-947.

133. Rosen, D.R., Siddique, T., Patterson, D., Figlewicz, D.A., Sapp, P., Hentati, A., Donaldson, D., Goto, J., O'Regan, J.P., Deng, H.X., Rahmani, Z., Krizus, A., McKenna-Yasek, D., Cayabyab, A., Gaston, S.M., Berger, R., Tanzi, R.E., Halperin, J.J., Herzfeldt, B., Van den Bergh, R., Hung, W.Y., Bird, T., Deng, G., Mulder, D.W., Smyth, C., Laing, N.G., Soriano, E., Pericak-Vance, M.A., Haines, J., Rouleau, G.A., Gusella, J.S., Horvitz, R.H., and Brown-RH, J. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis. Nature, 362, 59-62.

134. Yang, Y., Hentati, A., Deng, H.X., Dabbagh, O., Sasaki, T., Hirano, M., Hung, W.Y., Ouahchi, K., Yan, J., Azim, A.C., Cole, N., Gascon, G., Yagmour, A., Ben Hamida, M., Pericak-Vance, M., Hentati, F., and Siddique, T. (2001) The gene encoding alsin, a protein with three guanine-nucleotide exchange factor domains, is mutated in a form of recessive amyotrophic lateral sclerosis. Nat. Genet., 29, 160-165.

P. Andreas Jonsson

80

135. Hadano, S., Hand, C.K., Osuga, H., Yanagisawa, Y., Otomo, A., Devon, R.S., Miyamoto, N., Showguchi-Miyata, J., Okada, Y., Singaraja, R., Figlewicz, D.A., Kwiatkowski, T., Hosler, B.A., Sagie, T., Skaug, J., Nasir, J., Brown, R.H., Jr., Scherer, S.W., Rouleau, G.A., Hayden, M.R., and Ikeda, J.E. (2001) A gene encoding a putative GTPase regulator is mutated in familial amyotrophic lateral sclerosis 2. Nat. Genet., 29, 166-173.

136. Hand, C.K., Khoris, J., Salachas, F., Gros-Louis, F., Lopes, A.A., Mayeux-Portas, V., Brown, J.R., Jr., Meininger, V., Camu, W., and Rouleau, G.A. (2002) A novel locus for familial amyotrophic lateral sclerosis, on chromosome 18q. Am. J. Hum. Genet., 70, 251-256.

137. Chen, Y.Z., Bennett, C.L., Huynh, H.M., Blair, I.P., Puls, I., Irobi, J., Dierick, I., Abel, A., Kennerson, M.L., Rabin, B.A., Nicholson, G.A., Auer-Grumbach, M., Wagner, K., De Jonghe, P., Griffin, J.W., Fischbeck, K.H., Timmerman, V., Cornblath, D.R., and Chance, P.F. (2004) DNA/RNA helicase gene mutations in a form of juvenile amyotrophic lateral sclerosis (ALS4). Am. J. Hum. Genet., 74, 1128-1135.

138. Hentati, A., Ouahchi, K., Pericak-Vance, M.A., Nijhawan, D., Ahmad, A., Yang, Y., Rimmler, J., Hung, W., Schlotter, B., Ahmed, A., Ben Hamida, M., Hentati, F., and Siddique, T. (1998) Linkage of a commoner form of recessive amyotrophic lateral sclerosis to chromosome 15q15-q22 markers. Neurogenetics, 2, 55-60.

139. Ruddy, D.M., Parton, M.J., Al Chalabi, A., Lewis, C.M., Vance, C., Smith, B.N., Leigh, P.N., Powell, J.F., Siddique, T., Meyjes, E.P., Baas, F., de, J., V, and Shaw, C.E. (2003) Two families with familial amyotrophic lateral sclerosis are linked to a novel locus on chromosome 16q. Am. J. Hum. Genet., 73, 390-396.

140. Sapp, P.C., Hosler, B.A., McKenna-Yasek, D., Chin, W., Gann, A., Genise, H., Gorenstein, J., Huang, M., Sailer, W., Scheffler, M., Valesky, M., Haines, J.L., Pericak-Vance, M., Siddique, T., Horvitz, H.R., and Brown, R.H., Jr. (2003) Identification of two novel loci for dominantly inherited familial amyotrophic lateral sclerosis. Am. J. Hum. Genet., 73, 397-403.

141. Abalkhail, H., Mitchell, J., Habgood, J., Orrell, R., and de Belleroche, J. (2003) A new familial amyotrophic lateral sclerosis locus on chromosome 16q12.1-16q12.2. Am. J. Hum. Genet., 73, 383-389.

142. Nishimura, A.L., Mitne-Neto, M., Silva, H.C., Richieri-Costa, A., Middleton, S., Cascio, D., Kok, F., Oliveira, J.R., Gillingwater, T., Webb, J., Skehel, P., and Zatz, M. (2004) A Mutation in the Vesicle-Trafficking Protein VAPB Causes Late-Onset Spinal Muscular Atrophy and Amyotrophic Lateral Sclerosis. Am. J. Hum. Genet., 75, 822-831.

143. Siddique, T., Hong, S.T., Brooks, B.R., Hung, W.Y., Siddique, N.A., Rimmler, J., Kaplan, J.P., Haines, J.L., Brown, R.H.Jr., and Pericak-Vance, M.A. (1998) X-linked dominant locus for late-onset familial amyotrophic lateral sclerosis. Am. J. Hum. Genet., Suppl. A308

144. Hosler, B.A., Siddique, T., Sapp, P.C., Sailor, W., Huang, M.C., Hossain, A., Daube, J.R., Nance, M., Fan, C., Kaplan, J., Hung, W.Y., McKenna-Yasek, D., Haines, J.L., Pericak-Vance, M.A., Horvitz, H.R., and Brown, R.H., Jr. (2000) Linkage of familial amyotrophic lateral sclerosis with frontotemporal dementia to chromosome 9q21-q22. JAMA, 284, 1664-1669.

145. Siddique, T., Figlewicz, D.A., Pericak-Vance, M.A., Haines, J.L., Rouleau, G., Jeffers, A.J., Sapp, P., Hung, W.Y., Bebout, J., McKenna-Yasek, D., Deng, G., Horvitz, H.R., Gusella, J.F., Brown, R.H.Jr., and Roses, A.D. (1991) Linkage of a gene causing familial amyotrophic lateral sclerosis to chromosome 21 and evidence of genetic-locus heterogeneity. N. Engl. J. Med., 324, 1381-1384.

146. Hentati, A., Bejaoui, K., Pericak-Vance, M.A., Hentati, F., Speer, M.C., Hung, W.Y., Figlewicz, D.A., Haines, J., Rimmler, J., Ben Hamida, C., Ben Hamida, M., Brown, R.H.Jr.,


81

and Siddique, T. (1994) Linkage of recessive familial amyotrophic lateral sclerosis to chromosome 2q33-q35. Nat. Genet., 7, 425-428.

147. Eymard-Pierre, E., Lesca, G., Dollet, S., Santorelli, F.M., di Capua, M., Bertini, E., and Boespflug-Tanguy, O. (2002) Infantile-onset ascending hereditary spastic paralysis is associated with mutations in the alsin gene. Am. J. Hum. Genet., 71, 518-527.

148. Devon, R.S., Helm, J.R., Rouleau, G.A., Leitner, Y., Lerman-Sagie, T., Lev, D., and Hayden, M.R. (2003) The first nonsense mutation in alsin results in a homogeneous phenotype of infantile-onset ascending spastic paralysis with bulbar involvement in two siblings. Clin. Genet., 64, 210-215.

149. Gros-Louis, F., Meijer, I.A., Hand, C.K., Dube, M.P., MacGregor, D.L., Seni, M.H., Devon, R.S., Hayden, M.R., Andermann, F., Andermann, E., and Rouleau, G.A. (2003) An ALS2 gene mutation causes hereditary spastic paraplegia in a Pakistani kindred. Ann. Neurol., 53, 144-145.

150. Yamanaka, K., Vande, V.C., Eymard-Pierre, E., Bertini, E., Boespflug-Tanguy, O., and Cleveland, D.W. (2003) Unstable mutants in the peripheral endosomal membrane component ALS2 cause early-onset motor neuron disease. Proc. Natl. Acad. Sci. U. S. A, 100, 16041-16046.

151. Otomo, A., Hadano, S., Okada, T., Mizumura, H., Kunita, R., Nishijima, H., Showguchi-Miyata, J., Yanagisawa, Y., Kohiki, E., Suga, E., Yasuda, M., Osuga, H., Nishimoto, T., Narumiya, S., and Ikeda, J.E. (2003) ALS2, a novel guanine nucleotide exchange factor for the small GTPase Rab5, is implicated in endosomal dynamics. Hum. Mol. Genet., 12, 1671-1687.

152. Cai, H., Lin, X., Xie, C., Laird, F.M., Lai, C., Wen, H., Chiang, H.C., Shim, H., Farah, M.H., Hoke, A., Price, D.L., and Wong, P.C. (2005) Loss of ALS2 function is insufficient to trigger motor neuron degeneration in knock-out mice but predisposes neurons to oxidative stress. J. Neurosci., 25, 7567-7574.

153. Hand, C.K., Devon, R.S., Gros-Louis, F., Rochefort, D., Khoris, J., Meininger, V., Bouchard, J.P., Camu, W., Hayden, M.R., and Rouleau, G.A. (2003) Mutation screening of the ALS2 gene in sporadic and familial amyotrophic lateral sclerosis. Arch. Neurol., 60, 1768-1771.

154. Al Chalabi, A., Hansen, V.K., Simpson, C.L., Xi, J., Hosler, B.A., Powell, J.F., McKenna-Yasek, D., Shaw, C.E., Leigh, P.N., and Brown, R.H., Jr. (2003) Variants in the ALS2 gene are not associated with sporadic amyotrophic lateral sclerosis. Neurogenetics., 4, 221-222.

155. De Jonghe, P., Auer-Grumbach, M., Irobi, J., Wagner, K., Plecko, B., Kennerson, M., Zhu, D., De Vriendt, E., Van, G., V, Nicholson, G., Hartung, H.P., and Timmerman, V. (2002) Autosomal dominant juvenile amyotrophic lateral sclerosis and distal hereditary motor neuronopathy with pyramidal tract signs: synonyms for the same disorder? Brain, 125, 1320-1325.

156. Chance, P.F., Rabin, B.A., Ryan, S.G., Ding, Y., Scavina, M., Crain, B., Griffin, J.W., and Cornblath, D.R. (1998) Linkage of the gene for an autosomal dominant form of juvenile amyotrophic lateral sclerosis to chromosome 9q34. Am. J. Hum. Genet., 62, 633-640.

157. Moreira, M.C., Klur, S., Watanabe, M., Nemeth, A.H., Le, B., I, Moniz, J.C., Tranchant, C., Aubourg, P., Tazir, M., Schols, L., Pandolfo, M., Schulz, J.B., Pouget, J., Calvas, P., Shizuka-Ikeda, M., Shoji, M., Tanaka, M., Izatt, L., Shaw, C.E., M'Zahem, A., Dunne, E., Bomont, P., Benhassine, T., Bouslam, N., Stevanin, G., Brice, A., Guimaraes, J., Mendonca, P., Barbot, C., Coutinho, P., Sequeiros, J., Durr, A., Warter, J.M., and Koenig, M. (2004) Senataxin, the ortholog of a yeast RNA helicase, is mutant in ataxia-ocular apraxia 2. Nat. Genet., 36, 225-227.

P. Andreas Jonsson

82

158. Duquette, A., Roddier, K., McNabb-Baltar, J., Gosselin, I., St Denis, A., Dicaire, M.J., Loisel, L., Labuda, D., Marchand, L., Mathieu, J., Bouchard, J.P., and Brais, B. (2005) Mutations in senataxin responsible for Quebec cluster of ataxia with neuropathy. Ann. Neurol., 57, 408-414.

159. Nishimura, A.L., Mitne-Neto, M., Silva, H.C., Oliveira, J.R., Vainzof, M., and Zatz, M. (2004) A novel locus for late onset amyotrophic lateral sclerosis/motor neurone disease variant at 20q13. J. Med. Genet., 41, 315-320.

160. Nishimura, A.L., Al Chalabi, A., and Zatz, M. (2005) A common founder for amyotrophic lateral sclerosis type 8 (ALS8) in the Brazilian population. Hum. Genet.

161. Puls, I., Jonnakuty, C., LaMonte, B.H., Holzbaur, E.L., Tokito, M., Mann, E., Floeter, M.K., Bidus, K., Drayna, D., Oh, S.J., Brown, R.H., Ludlow, C.L., and Fischbeck, K.H. (2003) Mutant dynactin in motor neuron disease. Nat. Genet.

162. Munch, C., Sedlmeier, R., Meyer, T., Homberg, V., Sperfeld, A.D., Kurt, A., Prudlo, J., Peraus, G., Hanemann, C.O., Stumm, G., and Ludolph, A.C. (2004) Point mutations of the p150 subunit of dynactin (DCTN1) gene in ALS. Neurology, 63, 724-726.

163. Hafezparast, M., Klocke, R., Ruhrberg, C., Marquardt, A., Ahmad-Annuar, A., Bowen, S., Lalli, G., Witherden, A.S., Hummerich, H., Nicholson, S., Morgan, P.J., Oozageer, R., Priestley, J.V., Averill, S., King, V.R., Ball, S., Peters, J., Toda, T., Yamamoto, A., Hiraoka, Y., Augustin, M., Korthaus, D., Wattler, S., Wabnitz, P., Dickneite, C., Lampel, S., Boehme, F., Peraus, G., Popp, A., Rudelius, M., Schlegel, J., Fuchs, H., de Angelis, M.H., Schiavo, G., Shima, D.T., Russ, A.P., Stumm, G., Martin, J.E., and Fisher, E.M. (2003) Mutations in dynein link motor neuron degeneration to defects in retrograde transport. Science, 300, 808-812.

164. LaMonte, B.H., Wallace, K.E., Holloway, B.A., Shelly, S.S., Ascano, J., Tokito, M., Van Winkle, T., Howland, D.S., and Holzbaur, E.L. (2002) Disruption of dynein/dynactin inhibits axonal transport in motor neurons causing late-onset progressive degeneration. Neuron, 34, 715-727.

165. Hutton, M., Lendon, C.L., Rizzu, P., Baker, M., Froelich, S., Houlden, H., Pickering-Brown, S., Chakraverty, S., Isaacs, A., Grover, A., Hackett, J., Adamson, J., Lincoln, S., Dickson, D., Davies, P., Petersen, R.C., Stevens, M., de Graaff, E., Wauters, E., van Baren, J., Hillebrand, M., Joosse, M., Kwon, J.M., Nowotny, P., Che, L.K., Norton, J., Morris, J.C., Reed, L.A., Trojanowski, J., Basun, H., Lannfelt, L., Neystat, M., Fahn, S., Dark, F., Tannenberg, T., Dodd, P.R., Hayward, N., Kwok, J.B., Schofield, P.R., Andreadis, A., Snowden, J., Craufurd, D., Neary, D., Owen, F., Oostra, B.A., Hardy, J., Goate, A., van Swieten, J., Mann, D., Lynch, T., and Heutink, P. (1998) Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature, 393, 702-705.

166. Lynch, T., Sano, M., Marder, K.S., Bell, K.L., Foster, N.L., Defendini, R.F., Sima, A.A., Keohane, C., Nygaard, T.G., Fahn, S., and . (1994) Clinical characteristics of a family with chromosome 17-linked disinhibition-dementia-parkinsonism-amyotrophy complex. Neurology, 44, 1878-1884.

167. Nasreddine, Z.S., Loginov, M., Clark, L.N., Lamarche, J., Miller, B.L., Lamontagne, A., Zhukareva, V., Lee, V.M., Wilhelmsen, K.C., and Geschwind, D.H. (1999) From genotype to phenotype: a clinical pathological, and biochemical investigation of frontotemporal dementia and parkinsonism (FTDP-17) caused by the P301L tau mutation. Ann. Neurol., 45, 704-715.

168. Lewis, J., McGowan, E., Rockwood, J., Melrose, H., Nacharaju, P., Van Slegtenhorst, M., Gwinn-Hardy, K., Paul, M.M., Baker, M., Yu, X., Duff, K., Hardy, J., Corral, A., Lin, W.L., Yen, S.H., Dickson, D.W., Davies, P., and Hutton, M. (2000) Neurofibrillary tangles, amyotrophy and progressive motor disturbance in mice expressing mutant (P301L) tau protein. Nat. Genet., 25, 402-405.


83

169. Perl, D.P. and Hof, P.R. (2005) Western pacific ALS/parkinsonism-dementia complex. In: Neurodegenerative diseases, 827-843, Beal, M.F., Lang, A.E., and Ludolph, A. (eds.), Cambridge University Press, Cambridge.

170. Perez-Tur, J., Buee, L., Morris, H.R., Waring, S.C., Onstead, L., Wavrant-De Vrieze, F., Crook, R., Buee-Scherrer, V., Hof, P.R., Petersen, R.C., McGeer, P.L., Delacourte, A., Hutton, M., Siddique, T., Ahlskog, J.E., Hardy, J., and Steele, J.C. (1999) Neurodegenerative diseases of Guam: analysis of TAU. Neurology, 53, 411-413.

171. Morris, H.R., Steele, J.C., Crook, R., Wavrant-De Vrieze, F., Onstead-Cardinale, L., Gwinn-Hardy, K., Wood, N.W., Farrer, M., Lees, A.J., McGeer, P.L., Siddique, T., Hardy, J., and Perez-Tur, J. (2004) Genome-wide analysis of the parkinsonism-dementia complex of Guam. Arch. Neurol., 61, 1889-1897.

172. Poorkaj, P., Tsuang, D., Wijsman, E., Steinbart, E., Garruto, R.M., Craig, U.K., Chapman, N.H., Anderson, L., Bird, T.D., Plato, C.C., Perl, D.P., Weiderholt, W., Galasko, D., and Schellenberg, G.D. (2001) TAU as a susceptibility gene for amyotropic lateral sclerosis-parkinsonism dementia complex of Guam. Arch. Neurol., 58, 1871-1878.

173. Ostojic, J., Axelman, K., Lannfelt, L., and Froelich-Fabre, S. (2003) No evidence of linkage to chromosome 9q21-22 in a Swedish family with frontotemporal dementia and amyotrophic lateral sclerosis. Neurosci. Lett., 340, 245-247.

174. Olkowski, Z.L. (1998) Mutant AP endonuclease in patients with amyotrophic lateral sclerosis. Neuroreport, 9, 239-242.

175. Hayward, C., Colville, S., Swingler, R.J., and Brock, D.J. (1999) Molecular genetic analysis of the APEX nuclease gene in amyotrophic lateral sclerosis. Neurology, 52, 1899-1901.

176. Tomkins, J., Dempster, S., Banner, S.J., Cookson, M.R., and Shaw, P.J. (2000) Screening of AP endonuclease as a candidate gene for amyotrophic lateral sclerosis (ALS). Neuroreport, 11, 1695-1697.

177. Kisby, G.E., Milne, J., and Sweatt, C. (1997) Evidence of reduced DNA repair in amyotrophic lateral sclerosis brain tissue. Neuroreport, 8, 1337-1340.

178. Laws, S.M., Hone, E., Gandy, S., and Martins, R.N. (2003) Expanding the association between the APOE gene and the risk of Alzheimer's disease: possible roles for APOE promoter polymorphisms and alterations in APOE transcription. J. Neurochem., 84, 1215-1236.

179. Nussbaum, R.L. and Ellis, C.E. (2003) Alzheimer's disease and Parkinson's disease. N. Engl. J. Med., 348, 1356-1364.

180. Mui, S., Rebeck, G.W., McKenna-Yasek, D., Hyman, B.T., and Brown, R.H., Jr. (1995) Apolipoprotein E epsilon 4 allele is not associated with earlier age at onset in amyotrophic lateral sclerosis. Ann. Neurol., 38, 460-463.

181. Li, Y.J., Pericak-Vance, M.A., Haines, J.L., Siddique, N., McKenna-Yasek, D., Hung, W.Y., Sapp, P., Allen, C.I., Chen, W., Hosler, B., Saunders, A.M., Dellefave, L.M., Brown, R.H., and Siddique, T. (2004) Apolipoprotein E is associated with age at onset of amyotrophic lateral sclerosis. Neurogenetics., 5, 209-213.

182. Al Chalabi, A., Enayat, Z.E., Bakker, M.C., Sham, P.C., Ball, D.M., Shaw, C.E., Lloyd, C.M., Powell, J.F., and Leigh, P.N. (1996) Association of apolipoprotein E epsilon 4 allele with bulbar-onset motor neuron disease. Lancet, 347, 159-160.

P. Andreas Jonsson

84

183. Drory, V.E., Birnbaum, M., Korczyn, A.D., and Chapman, J. (2001) Association of APOE varepsilon4 allele with survival in amyotrophic lateral sclerosis. J. Neurol. Sci., 190, 17-20.

184. Siddique, T., Pericak-Vance, M.A., Caliendo, J., Hong, S.T., Hung, W.Y., Kaplan, J., McKenna-Yasek, D., Rimmler, J.B., Sapp, P., Saunders, A.M., Scott, W.K., Siddique, N., Haines, J.L., and Brown, R.H. (1998) Lack of association between apolipoprotein E genotype and sporadic amyotrophic lateral sclerosis. Neurogenetics., 1, 213-216.

185. Bachus, R., Bader, S., Gessner, R., and Ludolph, A.C. (1997) Lack of association of apolipoprotein E epsilon 4 allele with bulbar-onset motor neuron disease. Ann. Neurol., 41, 417

186. Smith, R.G., Haverkamp, L.J., Case, S., Appel, V., and Appel, S.H. (1996) Apolipoprotein E epsilon 4 in bulbar-onset motor neuron disease. Lancet, 348, 334-335.

187. Moulard, B., Sefiani, A., Laamri, A., Malafosse, A., and Camu, W. (1996) Apolipoprotein E genotyping in sporadic amyotrophic lateral sclerosis: evidence for a major influence on the clinical presentation and prognosis. J. Neurol. Sci., 139 Suppl:34-7, 34-37.

188. Lacomblez, L., Doppler, V., Beucler, I., Costes, G., Salachas, F., Raisonnier, A., Le Forestier, N., Pradat, P.F., Bruckert, E., and Meininger, V. (2002) APOE: a potential marker of disease progression in ALS. Neurology, 58, 1112-1114.

189. Sendtner, M. (2005) Neurotrophic factors. In: Neurodegenerative diseases, 94-107, Beal, M.F., Lang, A.E., and Ludolph, A. (eds.), Cambridge University Press, Cambridge.

190. Anand, P., Parrett, A., Martin, J., Zeman, S., Foley, P., Swash, M., Leigh, P.N., Cedarbaum, J.M., Lindsay, R.M., Williams-Chestnut, R.E., and . (1995) Regional changes of ciliary neurotrophic factor and nerve growth factor levels in post mortem spinal cord and cerebral cortex from patients with motor disease. Nat. Med., 1, 168-172.

191. Takahashi, R., Kawamura, K., Hu, J., Hayashi, M., and Deguchi, T. (1996) Ciliary neurotrophic factor (CNTF) genotypes and CNTF contents in human sciatic nerves as measured by a sensitive enzyme-linked immunoassay. J. Neurochem., 67, 525-529.

192. Takahashi, R., Yokoji, H., Misawa, H., Hayashi, M., Hu, J., and Deguchi, T. (1994) A null mutation in the human CNTF gene is not causally related to neurological diseases. Nat. Genet., 7, 79-84.

193. Masu, Y., Wolf, E., Holtmann, B., Sendtner, M., Brem, G., and Thoenen, H. (1993) Disruption of the CNTF gene results in motor neuron degeneration. Nature, 365, 27-32.

194. Orrell, R.W., King, A.W., Lane, R.J., and de Belleroche, J.S. (1995) Investigation of a null mutation of the CNTF gene in familial amyotrophic lateral sclerosis. J. Neurol. Sci., 132, 126-128.

195. Giess, R., Goetz, R., Schrank, B., Ochs, G., Sendtner, M., and Toyka, K. (1998) Potential implications of a ciliary neurotrophic factor gene mutation in a German population of patients with motor neuron disease. Muscle Nerve, 21, 236-238.

196. Al Chalabi, A., Scheffler, M.D., Smith, B.N., Parton, M.J., Cudkowicz, M.E., Andersen, P.M., Hayden, D.L., Hansen, V.K., Turner, M.R., Shaw, C.E., Leigh, P.N., and Brown, R.H., Jr. (2003) Ciliary neurotrophic factor genotype does not influence clinical phenotype in amyotrophic lateral sclerosis. Ann. Neurol., 54, 130-134.


85

197. Candore, G., Balistreri, C.R., Colonna-Romano, G., Lio, D., and Caruso, C. (2004) Major histocompatibility complex and sporadic Alzheimer's disease: a critical reappraisal. Exp. Gerontol., 39, 645-652.

198. Goodall, E.F., Greenway, M.J., van, M., I, Carroll, C.B., Hardiman, O., and Morrison, K.E. (2005) Association of the H63D polymorphism in the hemochromatosis gene with sporadic ALS. Neurology, 65, 934-937.

199. Yen, A.A., Simpson, E.P., Henkel, J.S., Beers, D.R., and Appel, S.H. (2004) HFE mutations are not strongly associated with sporadic ALS. Neurology, 62, 1611-1612.

200. Mullighan, C.G., Bunce, M., Fanning, G.C., Marshall, S.E., and Welsh, K.I. (1998) A rapid method of haplotyping HFE mutations and linkage disequilibrium in a Caucasoid population. Gut, 42, 566-569.

201. Liu, Q., Xie, F., Siedlak, S.L., Nunomura, A., Honda, K., Moreira, P.I., Zhua, X., Smith, M.A., and Perry, G. (2004) Neurofilament proteins in neurodegenerative diseases. Cell Mol. Life Sci., 61, 3057-3075.

202. Al Chalabi, A. and Miller, C.C. (2003) Neurofilaments and neurological disease. Bioessays, 25, 346-355.

203. Figlewicz, D.A., Krizus, A., Martinoli, M.G., Meininger, V., Dib, M., Rouleau, G.A., and Julien, J.P. (1994) Variants of the heavy neurofilament subunit are associated with the development of amyotrophic lateral sclerosis. Hum. Mol. Genet., 3, 1757-1761.

204. Tomkins, J., Usher, P., Slade, J.Y., Ince, P.G., Curtis, A., Bushby, K., and Shaw, P.J. (1998) Novel insertion in the KSP region of the neurofilament heavy gene in amyotrophic lateral sclerosis (ALS). Neuroreport, 9, 3967-3970.

205. Rooke, K., Figlewicz, D.A., Han, F.Y., and Rouleau, G.A. (1996) Analysis of the KSP repeat of the neurofilament heavy subunit in familiar amyotrophic lateral sclerosis. Neurology, 46, 789-790.

206. Vechio, J.D., Bruijn, L.I., Xu, Z., Brown, R.H., Jr., and Cleveland, D.W. (1996) Sequence variants in human neurofilament proteins: absence of linkage to familial amyotrophic lateral sclerosis. Ann. Neurol., 40, 603-610.

207. Garcia, M.L., Singleton, A.B., Hernandez, D., Ward, C.M., Evey, C., Sapp, P.A., Hardy, J., Brown, R.H., Jr., and Cleveland, D.W. (2005) Mutations in neurofilament genes are not a significant primary cause of non-SOD1-mediated amyotrophic lateral sclerosis. Neurobiol. Dis.

208. Gros-Louis, F., Lariviere, R., Gowing, G., Laurent, S., Camu, W., Bouchard, J.P., Meininger, V., Rouleau, G.A., and Julien, J.P. (2004) A frameshift deletion in peripherin gene associated with amyotrophic lateral sclerosis. J. Biol. Chem., 279, 45951-45956.

209. Leung, C.L., He, C.Z., Kaufmann, P., Chin, S.S., Naini, A., Liem, R.K., Mitsumoto, H., and Hays, A.P. (2004) A pathogenic peripherin gene mutation in a patient with amyotrophic lateral sclerosis. Brain Pathol., 14, 290-296.

210. Gubitz, A.K., Feng, W., and Dreyfuss, G. (2004) The SMN complex. Exp. Cell Res., 296, 51-56.

211. Crawford, T.O. (2002) Spinal muscular atrophy. In: Motor Neuron Disease, 97-114, Kuncl, R.W. (ed.), Saunders, Edinburgh.

P. Andreas Jonsson

86

212. Gamez, J., Barcelo, M.J., Munoz, X., Carmona, F., Cusco, I., Baiget, M., Cervera, C., and Tizzano, E.F. (2002) Survival and respiratory decline are not related to homozygous SMN2 deletions in ALS patients. Neurology, 59, 1456-1460.

213. Corcia, P., Mayeux-Portas, V., Khoris, J., de Toffol, B., Autret, A., Muh, J.P., Camu, W., and Andres, C. (2002) Abnormal SMN1 gene copy number is a susceptibility factor for amyotrophic lateral sclerosis. Ann. Neurol., 51, 243-246.

214. Veldink, J.H., Van den Berg, L.H., Cobben, J.M., Stulp, R.P., de Jong, J.M., Vogels, O.J., Baas, F., Wokke, J.H., and Scheffer, H. (2001) Homozygous deletion of the survival motor neuron 2 gene is a prognostic factor in sporadic ALS. Neurology, 56, 749-752.

215. Parboosingh, J.S., Meininger, V., McKenna-Yasek, D., Brown, R.H., Jr., and Rouleau, G.A. (1999) Deletions causing spinal muscular atrophy do not predispose to amyotrophic lateral sclerosis. Arch. Neurol., 56, 710-712.

216. Jackson, M., Morrison, K.E., Al Chalabi, A., Bakker, M., and Leigh, P.N. (1996) Analysis of chromosome 5q13 genes in amyotrophic lateral sclerosis: homozygous NAIP deletion in a sporadic case. Ann. Neurol., 39, 796-800.

217. Moulard, B., Salachas, F., Chassande, B., Briolotti, V., Meininger, V., Malafosse, A., and Camu, W. (1998) Association between centromeric deletions of the SMN gene and sporadic adult-onset lower motor neuron disease. Ann. Neurol., 43, 640-644.

218. Orrell, R.W., Habgood, J.J., de Belleroche, J.S., and Lane, R.J. (1997) The relationship of spinal muscular atrophy to motor neuron disease: investigation of SMN and NAIP gene deletions in sporadic and familial ALS. J. Neurol. Sci., 145, 55-61.

219. Echaniz-Laguna, A., Guiraud-Chaumeil, C., Tranchant, C., Reeber, A., Melki, J., and Warter, J.M. (2002) Homozygous exon 7 deletion of the SMN centromeric gene (SMN2): a potential susceptibility factor for adult-onset lower motor neuron disease. J. Neurol., 249, 290-293.

220. Marklund, S.L. (2005) Endogenous free radicals and antioxidants in the brain. In: Neurodegenerative diseases, 3-17, Beal, M.F., Lang, A.E., and Ludolph, A. (eds.), Cambridge University Press, Cambridge.

221. Lebovitz, R.M., Zhang, H., Vogel, H., Cartwright, J.J., Dionne, L., Lu, N., Huang, S., and Matzuk, M.M. (1996) Neurodegeneration, myocardial injury, and perinatal death in mitochondrial superoxide dismutase-deficient mice. Proc. Natl. Acad. Sci. U. S. A., 93, 9782-9787.

222. Parboosingh, J.S., Rouleau, G.A., Meninger, V., McKenna-Yasek, D., Brown, R.H.J., and Figlewicz, D.A. (1995) Absence of mutations in the Mn superoxide dismutase or catalase genes in familial amyotrophic lateral sclerosis. Neuromuscul. Disord., 5, 7-10.

223. Tomblyn, M., Kasarskis, E.J., Xu, Y., and St Clair, D.K. (1998) Distribution of MnSOD polymorphisms in sporadic ALS patients. J. Mol. Neurosci., 10, 65-66.

224. Van Landeghem, G.F., Tabatabaie, P., Beckman, G., Beckman, L., and Andersen, P.M. (1999) Manganese-containing superoxide dismutase signal sequence polymorphism associated with sporadic motor neuron disease. Eur. J. Neurol., 6, 639-644.

225. Tomkins, J., Banner, S.J., McDermott, C.J., and Shaw, P.J. (2001) Mutation screening of manganese superoxide dismutase in amyotrophic lateral sclerosis. Neuroreport, 12, 2319-2322.


87

226. Hiroi, S., Harada, H., Nishi, H., Satoh, M., Nagai, R., and Kimura, A. (1999) Polymorphisms in the SOD2 and HLA-DRB1 genes are associated with nonfamilial idiopathic dilated cardiomyopathy in Japanese. Biochem. Biophys. Res. Commun., 261, 332-339.

227. Sutton, A., Imbert, A., Igoudjil, A., Descatoire, V., Cazanave, S., Pessayre, D., and Degoul, F. (2005) The manganese superoxide dismutase Ala16Val dimorphism modulates both mitochondrial import and mRNA stability. Pharmacogenet. Genomics, 15, 311-319.

228. Sutton, A., Khoury, H., Prip-Buus, C., Cepanec, C., Pessayre, D., and Degoul, F. (2003) The Ala16Val genetic dimorphism modulates the import of human manganese superoxide dismutase into rat liver mitochondria. Pharmacogenetics, 13, 145-157.

229. Oosthuyse, B., Moons, L., Storkebaum, E., Beck, H., Nuyens, D., Brusselmans, K., Dorpe, J.V., Hellings, P., Gorselink, M., Heymans, S., Theilmeier, G., Dewerchin, M., Laudenbach, V., Vermylen, P., Raat, H., Acker, T., Vleminckx, V., Bosch, L.V., Cashman, N., Fujisawa, H., Drost, M.R., Sciot, R., Bruyninckx, F., Hicklin, D.J., Ince, C., Gressens, P., Lupu, F., Plate, K.H., Robberecht, W., Herbert, J.M., Collen, D., and Carmeliet, P. (2001) Deletion of the hypoxia-response element in the vascular endothelial growth factor promoter causes motor neuron degeneration. Nat. Genet., 28, 131-138.

230. Lambrechts, D., Storkebaum, E., Morimoto, M., Del Favero, J., Desmet, F., Marklund, S.L., Wyns, S., Thijs, V., Andersson, J., van, M., I, Al Chalabi, A., Bornes, S., Musson, R., Hansen, V., Beckman, L., Adolfsson, R., Pall, H.S., Prats, H., Vermeire, S., Rutgeerts, P., Katayama, S., Awata, T., Leigh, N., Lang-Lazdunski, L., Dewerchin, M., Shaw, C., Moons, L., Vlietinck, R., Morrison, K.E., Robberecht, W., Van Broeckhoven, C., Collen, D., Andersen, P.M., and Carmeliet, P. (2003) VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motoneurons against ischemic death. Nat. Genet., 34, 383-394.

231. Brockington, A., Kirby, J., Eggitt, D., Schofield, E., Morris, C., Lewis, C.E., Ince, P.G., and Shaw, P.J. (2005) Screening of the regulatory and coding regions of vascular endothelial growth factor in amyotrophic lateral sclerosis. Neurogenetics., 6, 101-104.

232. Devos, D., Moreau, C., Lassalle, P., Perez, T., De Seze, J., Brunaud-Danel, V., Destee, A., Tonnel, A.B., and Just, N. (2004) Low levels of the vascular endothelial growth factor in CSF from early ALS patients. Neurology, 62, 2127-2129.

233. Nygren, I., Larsson, A., Johansson, A., and Askmark, H. (2002) VEGF is increased in serum but not in spinal cord from patients with amyotrophic lateral sclerosis. Neuroreport, 13, 2199-2201.

234. Siddons, M.A., Pickering-Brown, S.M., Mann, D.M., Owen, F., and Cooper, P.N. (1996) Debrisoquine hydroxylase gene polymorphism frequencies in patients with amyotrophic lateral sclerosis. Neurosci. Lett., 208, 65-68.

235. Orru, S., Mascia, V., Casula, M., Giuressi, E., Loizedda, A., Carcassi, C., Giagheddu, M., and Contu, L. (1999) Association of monoamine oxidase B alleles with age at onset in amyotrophic lateral sclerosis. Neuromuscul. Disord., 9, 593-597.

236. Giess, R., Beck, M., Goetz, R., Nitsch, R.M., Toyka, K.V., and Sendtner, M. (2000) Potential role of LIF as a modifier gene in the pathogenesis of amyotrophic lateral sclerosis. Neurology, 54, 1003-1005.

237. Panas, M., Karadima, G., Kalfakis, N., Psarrou, O., Floroskoufi, P., Kladi, A., Petersen, M.B., and Vassilopoulos, D. (2000) Genotyping of presenilin-1 polymorphism in amyotrophic lateral sclerosis. J. Neurol., 247, 940-942.

P. Andreas Jonsson

88

238. Forman, M.S., Trojanowski, J.Q., and Lee, V.M. (2004) Neurodegenerative diseases: a decade of discoveries paves the way for therapeutic breakthroughs. Nat. Med., 10, 1055-1063.

239. Mann, T. and Keilin, D. (1939) Haemocuprein and hepatocuprein, copper-protein compounds of blood and liver in animals. Proc. Roy. Soc., 126, 303-315.

240. McCord, J.M. and Fridovich, I. (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein). J. Biol. Chem., 244, 6049-6055.

241. Halliwell, B. and Gutteridge, J.M.C. (1999) Free radicals in biology and medicine, Oxford university press, Oxford.

242. Weisiger, R.A. and Fridovich, I. (1973) Superoxide dismutase. Organelle specificity. J. Biol. Chem., 248, 3582-3592.

243. Weisiger, R.A. and Fridovich, I. (1973) Mitochondrial superoxide simutase. Site of synthesis and intramitochondrial localization. J. Biol. Chem., 248, 4793-4796.

244. Marklund, S.L. (1982) Human copper-containing superoxide dismutase of high molecular weight. Proc. Natl. Acad. Sci. U. S. A., 79, 7634-7638.

245. Hjalmarsson, K., Marklund, S.L., Engstrom, A., and Edlund, T. (1987) Isolation and sequence of complementary DNA encoding human extracellular superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A., 84, 6340-6344.

246. Karlsson, K., Sandstrom, J., Edlund, A., and Marklund, S.L. (1994) Turnover of extracellular-superoxide dismutase in tissues. Lab. Invest., 70, 705-710.

247. Stralin, P., Karlsson, K., Johansson, B.O., and Marklund, S.L. (1995) The interstitium of the human arterial wall contains very large amounts of extracellular superoxide dismutase. Arterioscler. Thromb. Vasc. Biol., 15, 2032-2036.

248. Marklund, S.L. (1984) Extracellular superoxide dismutase in human tissues and human cell lines. J. Clin. Invest., 74, 1398-1403.

249. Li, Y., Huang, T.T., Carlson, E.J., Melov, S., Ursell, P.C., Olson, J.L., Noble, L.J., Yoshimura, M.P., Berger, C., Chan, P.H., Wallace, D.C., and Epstein, C.J. (1995) Dilated cardiomyopathy and neonatal lethality in mutant mice lacking manganese superoxide dismutase. Nat. Genet., 11, 376-381.

250. Reaume, A.G., Elliott, J.L., Hoffman, E.K., Kowall, N.W., Ferrante, R.J., Siwek, D.F., Wilcox, H.M., Flood, D.G., Beal, M.F., Brown, R.H.J., Scott, R.W., and Snider, W.D. (1996) Motor neurons in Cu/Zn superoxide dismutase-deficient mice develop normally but exhibit enhanced cell death after axonal injury. Nat. Genet., 13, 43-47.

251. Ho, Y.S., Gargano, M., Cao, J., Bronson, R.T., Heimler, I., and Hutz, R.J. (1998) Reduced fertility in female mice lacking copper-zinc superoxide dismutase. J. Biol. Chem., 273, 7765-7769.

252. Matzuk, M.M., Dionne, L., Guo, Q., Kumar, T.R., and Lebovitz, R.M. (1998) Ovarian function in superoxide dismutase 1 and 2 knockout mice. Endocrinology, 139, 4008-4011.

253. Shefner, J.M., Reaume, A.G., Flood, D.G., Scott, R.W., Kowall, N.W., Ferrante, R.J., Siwek, D.F., Upton-Rice, M., and Brown, R.H.J. (1999) Mice lacking cytosolic copper/zinc superoxide dismutase display a distinctive motor axonopathy. Neurology, 53, 1239-1246.


89

254. Astrom, E.M., Marklund, S.L., Karlsson, K., Brannstrom, T., and Behndig, A. (2005) In vitro glucose-induced cataract in copper-zinc superoxide dismutase null mice. Exp. Eye Res.

255. Keithley, E.M., Canto, C., Zheng, Q.Y., Wang, X., Fischel-Ghodsian, N., and Johnson, K.R. (2005) Cu/Zn superoxide dismutase and age-related hearing loss. Hear. Res.

256. Elchuri, S., Oberley, T.D., Qi, W., Eisenstein, R.S., Jackson, R.L., Van Remmen, H., Epstein, C.J., and Huang, T.T. (2005) CuZnSOD deficiency leads to persistent and widespread oxidative damage and hepatocarcinogenesis later in life. Oncogene, 24, 367-380.

257. Parge, H.E., Hallewell, R.A., and Tainer, J.A. (1992) Atomic structures of wild-type and thermostable mutant recombinant human Cu,Zn superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A, 89, 6109-6113.

258. Tainer, J.A., Getzoff, E.D., Beem, K.M., Richardson, J.S., and Richardson, D.C. (1982) Determination and analysis of the 2 A-structure of copper, zinc superoxide dismutase. J. Mol. Biol., 160, 181-217.

259. Tainer, J.A., Getzoff, E.D., Richardson, J.S., and Richardson, D.C. (1983) Structure and mechanism of copper, zinc superoxide dismutase. Nature, 306, 284-287.

260. Strange, R.W., Antonyuk, S., Hough, M.A., Doucette, P.A., Rodriguez, J.A., Hart, P.J., Hayward, L.J., Valentine, J.S., and Hasnain, S.S. (2003) The structure of holo and metal-deficient wild-type human Cu, Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis. J. Mol. Biol., 328, 877-891.

261. Getzoff, E.D., Tainer, J.A., Weiner, P.K., Kollman, P.A., Richardson, J.S., and Richardson, D.C. (1983) Electrostatic recognition between superoxide and copper, zinc superoxide dismutase. Nature, 306, 287-290.

262. Forman, H.J. and Fridovich, I. (1973) On the stability of bovine superoxide dismutase. The effects of metals. J. Biol. Chem., 248, 2645-2649.

263. Levanon, D., Lieman-Hurwitz, J., Dafni, N., Wigderson, M., Sherman, L., Bernstein, Y., Laver-Rudich, Z., Danciger, E., Stein, O., and Groner, Y. (1985) Architecture and anatomy of the chromosomal locus in human chromosome 21 encoding the Cu/Zn superoxide dismutase. EMBO J., 4, 77-84.

264. Kim, H.T., Kim, Y.H., Nam, J.W., Lee, H.J., Rho, H.M., and Jung, G. (1994) Study of 5'-flanking region of human Cu/Zn superoxide dismutase. Biochem. Biophys. Res. Commun., 201, 1526-1533.

265. Minc, E., de Coppet, P., Masson, P., Thiery, L., Dutertre, S., Amor-Gueret, M., and Jaulin, C. (1999) The human copper-zinc superoxide dismutase gene (SOD1) proximal promoter is regulated by Sp1, Egr-1, and WT1 via non-canonical binding sites. J. Biol. Chem., 274, 503-509.

266. Marklund, S.L., Westman, N.G., Lundgren, E., and Roos, G. (1982) Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues. Cancer Res., 42, 1955-1961.

267. Bergeron, C., Petrunka, C., and Weyer, L. (1996) Copper/zinc superoxide dismutase expression in the human central nervous system. Correlation with selective neuronal vulnerability. Am. J. Pathol., 148, 273-279.

P. Andreas Jonsson

90

268. Pardo, C.A., Xu, Z., Borchelt, D.R., Price, D.L., Sisodia, S.S., and Cleveland, D.W. (1995) Superoxide dismutase is an abundant component in cell bodies, dendrites, and axons of motor neurons and in a subset of other neurons. Proc. Natl. Acad. Sci. U. S. A., 92, 954-958.

269. Furuta, A., Price, D.L., Pardo, C.A., Troncoso, J.C., Xu, Z.S., Taniguchi, N., and Martin, L.J. (1995) Localization of superoxide dismutases in Alzheimer's disease and Down's syndrome neocortex and hippocampus. Am. J. Pathol., 146, 357-367.

270. Shaw, P.J., Chinnery, R.M., Thagesen, H., Borthwick, G.M., and Ince, P.G. (1997) Immunocytochemical study of the distribution of the free radical scavenging enzymes Cu/Zn superoxide dismutase (SOD1); MN superoxide dismutase (MN SOD) and catalase in the normal human spinal cord and in motor neuron disease. J. Neurol. Sci., 147, 115-125.

271. Pappolla, M.A., Omar, R.A., Kim, K.S., and Robakis, N.K. (1992) Immunohistochemical evidence of oxidative stress in Alzheimer's disease. Am. J. Pathol., 140, 621-628.

272. Higgins, C.M., Jung, C., Ding, H., and Xu, Z. (2002) Mutant Cu, Zn superoxide dismutase that causes motoneuron degeneration is present in mitochondria in the CNS. J. Neurosci., 22, RC215

273. Kira, Y., Sato, E.F., and Inoue, M. (2002) Association of Cu,Zn-type superoxide dismutase with mitochondria and peroxisomes. Arch. Biochem. Biophys., 399, 96-102.

274. Panchenko, L.F., Brusov, O.S., Gerasimov, A.M., and Loktaeva, T.D. (1975) Intramitochondrial localization and release of rat liver superoxide dismutase. FEBS Lett., 55, 84-87.

275. Okado-Matsumoto, A. and Fridovich, I. (2001) Subcellular distribution of superoxide dismutases (sod) in rat liver. cu,zn-sod in mitochondria. J. Biol. Chem., 276, 38388-38393.

276. Chang, L.Y., Slot, J.W., Geuze, H.J., and Crapo, J.D. (1988) Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes. J. Cell Biol., 107, 2169-2179.

277. Crapo, J.D., Oury, T., Rabouille, C., Slot, J.W., and Chang, L.Y. (1992) Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. Proc. Natl. Acad. Sci. U. S. A, 89, 10405-10409.

278. Dhaunsi, G.S., Gulati, S., Singh, A.K., Orak, J.K., Asayama, K., and Singh, I. (1992) Demonstration of Cu-Zn superoxide dismutase in rat liver peroxisomes. Biochemical and immunochemical evidence. J. Biol. Chem., 267, 6870-6873.

279. Geller, B.L. and Winge, D.R. (1982) Rat liver Cu,Zn-superoxide dismutase. Subcellular location in lysosomes. J. Biol. Chem., 257, 8945-8952.

280. Miyata-Asano, M., Ito, K., Ikeda, H., Sekiguchi, S., Arai, K., and Taniguchi, N. (1986) Purification of copper-zinc-superoxide dismutase and catalase from human erythrocytes by copper-chelate affinity chromatography. J. Chromatogr., 370, 501-507.

281. Marklund, S.L., Andersen, P.M., Forsgren, L., Nilsson, P., Ohlsson, P.I., Wikander, G., and Oberg, A. (1997) Normal binding and reactivity of copper in mutant superoxide dismutase isolated from amyotrophic lateral sclerosis patients. J. Neurochem., 69, 675-681.

282. DiGuiseppi, J. and Fridovich, I. (1984) The toxicology of molecular oxygen. Crit Rev. Toxicol., 12, 315-342.


91

283. Mei, G., Rosato, N., Silva, N., Jr., Rusch, R., Gratton, E., Savini, I., and Finazzi-Agro, A. (1992) Denaturation of human Cu/Zn superoxide dismutase by guanidine hydrochloride: a dynamic fluorescence study. Biochemistry, 31, 7224-7230.

284. Lindberg, M.J., Tibell, L., and Oliveberg, M. (2002) Common denominator of Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis: decreased stability of the apo state. Proc. Natl. Acad. Sci. U. S. A, 99, 16607-16612.

285. Doucette, P.A., Whitson, L.J., Cao, X., Schirf, V., Demeler, B., Valentine, J.S., Hansen, J.C., and Hart, P.J. (2004) Dissociation of human copper-zinc superoxide dismutase dimers using chaotrope and reductant. Insights into the molecular basis for dimer stability. J. Biol. Chem., 279, 54558-54566.

286. Lynch, S.M., Boswell, S.A., and Colon, W. (2004) Kinetic stability of Cu/Zn superoxide dismutase is dependent on its metal ligands: implications for ALS. Biochemistry, 43, 16525-16531.

287. Lindberg, M.J., Normark, J., Holmgren, A., and Oliveberg, M. (2004) Folding of human superoxide dismutase: disulfide reduction prevents dimerization and produces marginally stable monomers. Proc. Natl. Acad. Sci. U. S. A, 101, 15893-15898.

288. Furukawa, Y. and O'Halloran, T.V. (2005) Amyotrophic Lateral Sclerosis Mutations Have the Greatest Destabilizing Effect on the Apo- and Reduced Form of SOD1, Leading to Unfolding and Oxidative Aggregation. J. Biol. Chem., 280, 17266-17274.

289. Arnesano, F., Banci, L., Bertini, I., Martinelli, M., Furukawa, Y., and O'Halloran, T.V. (2004) The unusually stable quaternary structure of human Cu,Zn-superoxide dismutase 1 is controlled by both metal occupancy and disulfide status. J. Biol. Chem., 279, 47998-48003.

290. Ferraroni, M., Rypniewski, W., Wilson, K.S., Viezzoli, M.S., Banci, L., Bertini, I., and Mangani, S. (1999) The crystal structure of the monomeric human SOD mutant F50E/G51E/E133Q at atomic resolution. The enzyme mechanism revisited. J. Mol. Biol., 288, 413-426.

291. Hough, M.A. and Hasnain, S.S. (2003) Structure of fully reduced bovine copper zinc superoxide dismutase at 1.15 A. Structure. (Camb. ), 11, 937-946.

292. Rae, T.D., Schmidt, P.J., Pufahl, R.A., Culotta, V.C., and O'Halloran, T.V. (1999) Undetectable intracellular free copper: the requirement of a copper chaperone for superoxide dismutase. Science, 284, 805-808.

293. Culotta, V.C., Klomp, L.W., Strain, J., Casareno, R.L., Krems, B., and Gitlin, J.D. (1997) The copper chaperone for superoxide dismutase. J. Biol. Chem., 272, 23469-23472.

294. Schmidt, P.J., Rae, T.D., Pufahl, R.A., Hamma, T., Strain, J., O'Halloran, T.V., and Culotta, V.C. (1999) Multiple protein domains contribute to the action of the copper chaperone for superoxide dismutase. J. Biol. Chem., 274, 23719-23725.

295. Lamb, A.L., Wernimont, A.K., Pufahl, R.A., Culotta, V.C., O'Halloran, T.V., and Rosenzweig, A.C. (1999) Crystal structure of the copper chaperone for superoxide dismutase. Nat. Struct. Biol., 6, 724-729.

296. Lamb, A.L., Wernimont, A.K., Pufahl, R.A., O'Halloran, T.V., and Rosenzweig, A.C. (2000) Crystal structure of the second domain of the human copper chaperone for superoxide dismutase. Biochemistry, 39, 1589-1595.

P. Andreas Jonsson

92

297. Schmidt, P.J., Ramos-Gomez, M., and Culotta, V.C. (1999) A gain of superoxide dismutase (SOD) activity obtained with CCS, the copper metallochaperone for SOD1. J. Biol. Chem., 274, 36952-36956.

298. Lee, J., Prohaska, J.R., and Thiele, D.J. (2001) Essential role for mammalian copper transporter Ctr1 in copper homeostasis and embryonic development. Proc. Natl. Acad. Sci. U. S. A, 98, 6842-6847.

299. Puig, S. and Thiele, D.J. (2002) Molecular mechanisms of copper uptake and distribution. Curr. Opin. Chem. Biol., 6, 171-180.

300. Rae, T.D., Torres, A.S., Pufahl, R.A., and O'Halloran, T.V. (2001) Mechanism of Cu,Zn-Superoxide Dismutase Activation by the Human Metallochaperone hCCS. J. Biol. Chem., 276, 5166-5176.

301. Furukawa, Y., Torres, A.S., and O'Halloran, T.V. (2004) Oxygen-induced maturation of SOD1: a key role for disulfide formation by the copper chaperone CCS. EMBO J., 23, 2872-2881.

302. Lamb, A.L., Torres, A.S., O'Halloran, T.V., and Rosenzweig, A.C. (2000) Heterodimer formation between superoxide dismutase and its copper chaperone. Biochemistry, 39, 14720-14727.

303. Lamb, A.L., Torres, A.S., O'Halloran, T.V., and Rosenzweig, A.C. (2001) Heterodimeric structure of superoxide dismutase in complex with its metallochaperone. Nat. Struct. Biol., 8, 751-755.

304. Tegelstrom, H. (1975) Interspecific hybridisation of superoxide dismutase. Hereditas, 81 ,185-198.

305. Rothstein, J.D., Dykes-Hoberg, M., Corson, L.B., Becker, M., Cleveland, D.W., Price, D.L., Culotta, V.C., and Wong, P.C. (1999) The copper chaperone CCS is abundant in neurons and astrocytes in human and rodent brain. J. Neurochem., 72, 422-429.

306. Sturtz, L.A., Diekert, K., Jensen, L.T., Lill, R., and Culotta, V.C. (2001) A fraction of yeast cu,zn-superoxide dismutase and its metallochaperone, ccs, localize to the intermembrane space of mitochondria. a physiological role for sod1 in guarding against mitochondrial oxidative damage. J. Biol. Chem., 276, 38084-38089.

307. Okado-Matsumoto, A. and Fridovich, I. (2002) Amyotrophic lateral sclerosis: A proposed mechanism. Proc. Natl. Acad. Sci. U. S. A, 99, 9010-9014.

308. Field, L.S., Furukawa, Y., O'Halloran, T.V., and Culotta, V.C. (2003) Factors controlling the uptake of yeast Cu/Zn superoxide dismutase into mitochondria. J. Biol. Chem.

309. Wong, P.C., Waggoner, D., Subramaniam, J.R., Tessarollo, L., Bartnikas, T.B., Culotta, V.C., Price, D.L., Rothstein, J., and Gitlin, J.D. (2000) Copper chaperone for superoxide dismutase is essential to activate mammalian Cu/Zn superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A, :

310. Carroll, M.C., Girouard, J.B., Ulloa, J.L., Subramaniam, J.R., Wong, P.C., Valentine, J.S., and Culotta, V.C. (2004) Mechanisms for activating Cu- and Zn-containing superoxide dismutase in the absence of the CCS Cu chaperone. Proc. Natl. Acad. Sci. U. S. A, 101, 5964-5969.

311. Andersen, P.M., Sims, K.B., Xin, W.W., Kiely, R., O'Neill, G., Ravits, J., Pioro, E., Harati, Y., Brower, R.D., Levine, J.S., Heinicke, H.U., Seltzer, W., Boss, M., and Brown, R.H., Jr. (2003) Sixteen novel mutations in the Cu/Zn superoxide dismutase gene in amyotrophic lateral


93

sclerosis: a decade of discoveries, defects and disputes. Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 4, 62-73.

312. Watanabe, Y., Kato, S., Adachi, Y., and Nakashima, K. (2000) Frameshift, nonsense and non amino acid altering mutations in SOD1 in familial ALS: report of a Japanese pedigree and literature review. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 1, 251-258.

313. Valentine, J.S., Doucette, P.A., and Potter, S.Z. (2004) Copper-Zinc Superoxide Dismutase and Amyotrophic Lateral Sclerosis. Annu. Rev. Biochem.

314. Zu, J., Deng, H.X., Lo, T.P., Mitsumoto, H., Ahmed, M.S., Hung, W.Y., Cai, Z.J., Tainer, J.A., and Siddique, T. (1999) Exon 5 encoded domain is not required for the toxic function of mutant SOD1 but essential for the dismutase activity: identification and characterization of two new SOD1 mutations associated with familial amyotrophic lateral sclerosis. Neurogenetics, 1, 65-71.

315. Sapp, P.C., Rosen, D.R., Hosler, B.A., Esteban, J., McKenna-Yasek, D., O'Regan, J.P., Horvitz, H.R., and Brown, R.H.J. (1995) Identification of three novel mutations in the gene for Cu/Zn superoxide dismutase in patients with familial amyotrophic lateral sclerosis. Neuromuscul. Disord., 5, 353-357.

316. Cudkowicz, M.E., McKenna-Yasek, D., Sapp, P.E., Chin, W., Geller, B., Hayden, D.L., Schoenfeld, D.A., Hosler, B.A., Horvitz, H.R., and Brown, R.H. (1997) Epidemiology of mutations in superoxide dismutase in amyotrophic lateral sclerosis. Ann. Neurol., 41, 210-221.

317. Suthers, G., Laing, N., Wilton, S., Dorosz, S., and Waddy, H. (1994) "Sporadic" motoneuron disease due to familial SOD1 mutation with low penetrance. Lancet, 344, 1773

318. Deng, H.X., Tainer, J.A., Mitsumoto, H., Ohnishi, A., He, X., Hung, W.Y., Zhao, Y., Juneja, T., Hentati, A., and Siddique, T. (1995) Two novel SOD1 mutations in patients with familial amyotrophic lateral sclerosis. Hum. Mol. Genet., 4, 1113-1116.

319. Jackson, M., Al-Chalabi, A., Enayat, Z.E., Chioza, B., Leigh, P.N., and Morrison, K.E. (1997) Copper/zinc superoxide dismutase 1 and sporadic amyotrophic lateral sclerosis: analysis of 155 cases and identification of a novel insertion mutation. Ann. Neurol., 42, 803-807.

320. Jones, C.T., Brock, D.J., Chancellor, A.M., Warlow, C.P., and Swingler, R.J. (1993) Cu/Zn superoxide dismutase (SOD1) mutations and sporadic amyotrophic lateral sclerosis. Lancet, 342, 1050-1051.

321. Gellera, C. (2001) Genetics of ALS in Italian families. Amyotroph. Lateral Scler. Other Motor Neuron Disord., 2 Suppl 1, S43-S46.

322. Hayward, C., Swingler, R.J., Simpson, S.A., and Brock, D.J. (1996) A specific superoxide dismutase mutation is on the same genetic background in sporadic and familial cases of amyotrophic lateral sclerosis. Am. J. Hum. Genet., 59, 1165-1167.

323. Shaw, C.E., Enayat, Z.E., Chioza, B.A., Al-Chalabi, A., Radunovic, A., Powell, J.F., and Leigh, P.N. (1998) Mutations in all five exons of SOD-1 may cause ALS. Ann. Neurol., 43, 390-394.

324. Andersen, P.M., Nilsson, P., Keranen, M.L., Forsgren, L., Hagglund, J., Karlsborg, M., Ronnevi, L.O., Gredal, O., and Marklund, S.L. (1997) Phenotypic heterogeneity in motor neuron disease patients with CuZn- superoxide dismutase mutations in Scandinavia. Brain, 120, 1723-1737.

P. Andreas Jonsson

94

325. Orrell, R.W., Habgood, J.J., Malaspina, A., Mitchell, J., Greenwood, J., Lane, R.J., and deBelleroche, J.S. (1999) Clinical characteristics of SOD1 gene mutations in UK families with ALS. J. Neurol. Sci., 169, 56-60.

326. Boukaftane, Y., Khoris, J., Moulard, B., Salachas, F., Meininger, V., Malafosse, A., Camu, W., and Rouleau, G.A. (1998) Identification of six novel SOD1 gene mutations in familial amyotrophic lateral sclerosis. Can. J. Neurol. Sci., 25, 192-196.

327. Pramatarova, A., Figlewicz, D.A., Krizus, A., Han, F.Y., Ceballos-Picot, I., Nicole, A., Dib, M., Meininger, V., Brown, R.H., and Rouleau, G.A. (1995) Identification of new mutations in the Cu/Zn superoxide dismutase gene of patients with familial amyotrophic lateral sclerosis. Am. J. Hum. Genet., 56, 592-596.

328. Gellera, C., Castellotti, B., Riggio, M.C., Silani, V., Morandi, L., Testa, D., Casali, C., Taroni, F., Di Donato, S., Zeviani, M., and Mariotti, C. (2001) Superoxide dismutase gene mutations in Italian patients with familial and sporadic amyotrophic lateral sclerosis: identification of three novel missense mutations. Neuromuscul. Disord., 11, 404-410.

329. Jones, C.T., Swingler, R.J., Simpson, S.A., and Brock, D.J. (1995) Superoxide dismutase mutations in an unselected cohort of Scottish amyotrophic lateral sclerosis patients. J. Med. Genet., 32, 290-292.

330. Alexander, M.D., Traynor, B.J., Miller, N., Corr, B., Frost, E., McQuaid, S., Brett, F.M., Green, A., and Hardiman, O. (2002) "True" sporadic ALS associated with a novel SOD-1 mutation. Ann. Neurol., 52, 680-683.

331. Orrell, R.W., Habgood, J.J., Gardiner, I., King, A.W., Bowe, F.A., Hallewell, R.A., Marklund, S.L., Greenwood, J., Lane, R.J., and deBelleroche, J. (1997) Clinical and functional investigation of 10 missense mutations and a novel frameshift insertion mutation of the gene for copper-zinc superoxide dismutase in UK families with amyotrophic lateral sclerosis. Neurology, 48, 746-751.

332. Abe, K., Aoki, M., Ikeda, M., Watanabe, M., Hirai, S., and Itoyama, Y. (1996) Clinical characteristics of familial amyotrophic lateral sclerosis with Cu/Zn superoxide dismutase gene mutations. J. Neurol. Sci., 136, 108-116.

333. Radunovic, A. and Leigh, P.N. (1996) Cu/Zn superoxide dismutase gene mutations in amyotrophic lateral sclerosis: correlation between genotype and clinical features. J. Neurol. Neurosurg. Psychiatry, 61, 565-572.

334. Juneja, T., Pericak-Vance, M.A., Laing, N.G., Dave, S., and Siddique, T. (1997) Prognosis in familial amyotrophic lateral sclerosis: progression and survival in patients with glu100gly and ala4val mutations in Cu,Zn superoxide dismutase. Neurology, 48, 55-57.

335. Rosen, D.R., Bowling, A.C., Patterson, D., Usdin, T.B., Sapp, P., Mezey, E., McKenna-Yasek, D., O'Regan, J., Rahmani, Z., and Ferrante, R.J. (1994) A frequent ala 4 to val superoxide dismutase-1 mutation is associated with a rapidly progressive familial amyotrophic lateral sclerosis. Hum. Mol. Genet., 3, 981-987.

336. Cudkowicz, M.E., McKenna-Yasek, D., Chen, C., Hedley-Whyte, E.T., and Brown, R.H.J. (1998) Limited corticospinal tract involvement in amyotrophic lateral sclerosis subjects with the A4V mutation in the copper/zinc superoxide dismutase gene. Ann. Neurol., 43, 703-710.

337. Ratovitski, T., Corson, L.B., Strain, J., Wong, P., Cleveland, D.W., Culotta, V.C., and Borchelt, D.R. (1999) Variation in the biochemical/biophysical properties of mutant superoxide dismutase 1 enzymes and the rate of disease progression in familial amyotrophic lateral sclerosis kindreds. Hum. Mol. Genet., 8, 1451-1460.


95

338. Borchelt, D.R., Lee, M.K., Slunt, H.S., Guarnieri, M., Xu, Z.S., Wong, P.C., Brown, R.H., Jr., Price, D.L., Sisodia, S.S., and Cleveland, D.W. (1994) Superoxide dismutase 1 with mutations linked to familial amyotrophic lateral sclerosis possesses significant activity. Proc. Natl. Acad. Sci. U. S. A, 91, 8292-8296.

339. Nakano, R., Inuzuka, T., Kikugawa, K., Takahashi, H., Sakimura, K., Fujii, J., Taniguchi, N., and Tsuji, S. (1996) Instability of mutant Cu/Zn superoxide dismutase (Ala4Thr) associated with familial amyotrophic lateral sclerosis. Neurosci. Lett., 211, 129-131.

340. Hoffman, E.K., Wilcox, H.M., Scott, R.W., and Siman, R. (1996) Proteasome inhibition enhances the stability of mouse Cu/Zn superoxide dismutase with mutations linked to familial amyotrophic lateral sclerosis. J. Neurol. Sci., 139, 15-20.

341. Crow, J.P., Sampson, J.B., Zhuang, Y., Thompson, J.A., and Beckman, J.S. (1997) Decreased zinc affinity of amyotrophic lateral sclerosis-associated superoxide dismutase mutants leads to enhanced catalysis of tyrosine nitration by peroxynitrite. J. Neurochem., 69, 1936-1944.

342. Goto, J.J., Zhu, H., Sanchez, R.J., Nersissian, A., Gralla, E.B., Valentine, J.S., and Cabelli, D.E. (2000) Loss of in vitro metal ion binding specificity in mutant copper-zinc superoxide dismutases associated with familial amyotrophic lateral sclerosis. J. Biol. Chem., 275, 1007-1014.

343. Hayward, L.J., Rodriguez, J.A., Kim, J.W., Tiwari, A., Goto, J.J., Cabelli, D.E., Valentine, J.S., and Brown, R.H., Jr. (2002) Decreased metallation and activity in subsets of mutant superoxide dismutases associated with familial amyotrophic lateral sclerosis. J. Biol. Chem., 277, 15923-15931.

344. Tiwari, A. and Hayward, L.J. (2003) Familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase are susceptible to disulfide reduction. J. Biol. Chem., 278, 5984-5992.

345. Rodriguez, J.A., Valentine, J.S., Eggers, D.K., Roe, J.A., Tiwari, A., Brown, R.H., Jr., and Hayward, L.J. (2002) Familial amyotrophic lateral sclerosis-associated mutations decrease the thermal stability of distinctly metallated species of human copper/zinc superoxide dismutase. J. Biol. Chem., 277, 15932-15937.

346. Stathopulos, P.B., Rumfeldt, J.A., Scholz, G.A., Irani, R.A., Frey, H.E., Hallewell, R.A., Lepock, J.R., and Meiering, E.M. (2003) Cu/Zn superoxide dismutase mutants associated with amyotrophic lateral sclerosis show enhanced formation of aggregates in vitro. Proc. Natl. Acad. Sci. U. S. A, 100, 7021-7026.

347. Rodriguez, J.A., Shaw, B.F., Durazo, A., Sohn, S.H., Doucette, P.A., Nersissian, A.M., Faull, K.F., Eggers, D.K., Tiwari, A., Hayward, L.J., and Valentine, J.S. (2005) Destabilization of apoprotein is insufficient to explain Cu,Zn-superoxide dismutase-linked ALS pathogenesis. Proc. Natl. Acad. Sci. U. S. A, 102, 10516-10521.

348. DiDonato, M., Craig, L., Huff, M.E., Thayer, M.M., Cardoso, R.M., Kassmann, C.J., Lo, T.P., Bruns, C.K., Powers, E.T., Kelly, J.W., Getzoff, E.D., and Tainer, J.A. (2003) ALS mutants of human superoxide dismutase form fibrous aggregates via framework destabilization. J. Mol. Biol., 332, 601-615.

349. Lindberg, M.J., Bystrom, R., Boknas, N., Andersen, P.M., and Oliveberg, M. (2005) Systematically perturbed folding patterns of amyotrophic lateral sclerosis (ALS)-associated SOD1 mutants. Proc. Natl. Acad. Sci. U. S. A, 102, 9754-9759.

350. Rakhit, R., Cunningham, P., Furtos-Matei, A., Dahan, S., Qi, X.F., Crow, J.P., Cashman, N.R., Kondejewski, L.H., and Chakrabartty, A. (2002) Oxidation-induced misfolding and

P. Andreas Jonsson

96

aggregation of superoxide dismutase and its implications for amyotrophic lateral sclerosis. J. Biol. Chem., 277, 47551-47556.

351. Tiwari, A., Xu, Z., and Hayward, L.J. (2005) Aberrantly increased hydrophobicity shared by mutants of Cu,Zn-superoxide dismutase in familial amyotrophic lateral sclerosis. J. Biol. Chem., 280, 29771-29779.

352. Gurney, M.E., Pu, H., Chiu, A.Y., Dal Canto, M.C., Polchow, C.Y., Alexander, D.D., Caliendo, J., Hentati, A., Kwon, Y.W., and Deng, H.X. (1994) Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation. Science, 264, 1772-1775.

353. Wong, P.C., Pardo, C.A., Borchelt, D.R., Lee, M.K., Copeland, N.G., Jenkins, N.A., Sisodia, S.S., Cleveland, D.W., and Price, D.L. (1995) An adverse property of a familial ALS-linked SOD1 mutation causes motor neuron disease characterized by vacuolar degeneration of mitochondria. Neuron, 14, 1105-1116.

354. Wang, J., Xu, G., Slunt, H.H., Gonzales, V., Coonfield, M., Fromholt, D., Copeland, N.G., Jenkins, N.A., and Borchelt, D.R. (2005) Coincident thresholds of mutant protein for paralytic disease and protein aggregation caused by restrictively expressed superoxide dismutase cDNA. Neurobiol. Dis.

355. Pramatarova, A., Laganiere, J., Roussel, J., Brisebois, K., and Rouleau, G.A. (2001) Neuron-specific expression of mutant superoxide dismutase 1 in transgenic mice does not lead to motor impairment. J. Neurosci., 21, 3369-3374.

356. Chang-Hong, R., Wada, M., Koyama, S., Kimura, H., Arawaka, S., Kawanami, T., Kurita, K., Kadoya, T., Aoki, M., Itoyama, Y., and Kato, T. (2005) Neuroprotective effect of oxidized galectin-1 in a transgenic mouse model of amyotrophic lateral sclerosis. Exp. Neurol., 194, 203-211.

357. Nagai, M., Aoki, M., Miyoshi, I., Kato, M., Pasinelli, P., Kasai, N., Brown, R.H., Jr., and Itoyama, Y. (2001) Rats expressing human cytosolic copper-zinc superoxide dismutase transgenes with amyotrophic lateral sclerosis: associated mutations develop motor neuron disease. J. Neurosci., 21, 9246-9254.

358. Wang, J., Xu, G.L., Gonzales, V., Coonfield, M., Fromholt, D., Copeland, N.G., Jenkins, N.A., and Borchelt, D.R. (2002) Fibrillar inclusions and motor neuron degeneration in transgenic mice expressing superoxide dismutase 1 with a disrupted copper-binding site. Neurobiology of Disease, 10, 128-138.

359. Wang, J., Slunt, H., Gonzales, V., Fromholt, D., Coonfield, M., Copeland, N.G., Jenkins, N.A., and Borchelt, D.R. (2003) Copper-binding-site-null SOD1 causes ALS in transgenic mice: aggregates of non-native SOD1 delineate a common feature. Hum. Mol. Genet., 12, 2753-2764.

360. Bruijn, L.I., Becher, M.W., Lee, M.K., Anderson, K.L., Jenkins, N.A., Copeland, N.G., Sisodia, S.S., Rothstein, J.D., Borchelt, D.R., Price, D.L., and Cleveland, D.W. (1997) ALS-linked SOD1 mutant G85R mediates damage to astrocytes and promotes rapidly progressive disease with SOD1-containing inclusions. Neuron, 18, 327-338.

361. Lino, M.M., Schneider, C., and Caroni, P. (2002) Accumulation of SOD1 mutants in postnatal motoneurons does not cause motoneuron pathology or motoneuron disease. J. Neurosci., 22, 4825-4832.

362. Ripps, M.E., Huntley, G.W., Hof, P.R., Morrison, J.H., and Gordon, J.W. (1995) Transgenic mice expressing an altered murine superoxide dismutase gene provide an animal model of amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A., 92, 689-693.


97

363. Gong, Y.H., Parsadanian, A.S., Andreeva, A., Snider, W.D., and Elliott, J.L. (2000) Restricted expression of G86R Cu/Zn superoxide dismutase in astrocytes results in astrocytosis but does not cause motoneuron degeneration. J. Neurosci., 20, 660-665.

364. Howland, D.S., Liu, J., She, Y., Goad, B., Maragakis, N.J., Kim, B., Erickson, J., Kulik, J., DeVito, L., Psaltis, G., DeGennaro, L.J., Cleveland, D.W., and Rothstein, J.D. (2002) Focal loss of the glutamate transporter EAAT2 in a transgenic rat model of SOD1 mutant-mediated amyotrophic lateral sclerosis (ALS). Proc. Natl. Acad. Sci. U. S. A, 99, 1604-1609.

365. Wang, J., Xu, G., Li, H., Gonzales, V., Fromholt, D., Karch, C., Copeland, N.G., Jenkins, N.A., and Borchelt, D.R. (2005) Somatodendritic accumulation of misfolded SOD1-L126Z in motor neurons mediates degeneration: alphaB-crystallin modulates aggregation. Hum. Mol. Genet., 14, 2335-2347.

366. Watanabe, Y., Yasui, K., Nakano, T., Doi, K., Fukada, Y., Kitayama, M., Ishimoto, M., Kurihara, S., Kawashima, M., Fukuda, H., Adachi, Y., Inoue, T., and Nakashima, K. (2005) Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification. Brain Res. Mol. Brain Res., 135, 12-20.

367. Alexander, G.M., Erwin, K.L., Byers, N., Deitch, J.S., Augelli, B.J., Blankenhorn, E.P., and Heiman-Patterson, T.D. (2004) Effect of transgene copy number on survival in the G93A SOD1 transgenic mouse model of ALS. Brain Res. Mol. Brain Res., 130, 7-15.

368. Jaarsma, D., Haasdijk, E.D., Grashorn, J.A., Hawkins, R., van Duijn, W., Verspaget, H.W., London, J., and Holstege, J.C. (2000) Human Cu/Zn superoxide dismutase (SOD1) overexpression in mice causes mitochondrial vacuolization, axonal degeneration, and premature motoneuron death and accelerates motoneuron disease in mice expressing a familial amyotrophic lateral sclerosis mutant SOD1. Neurobiol. Dis., 7, 623-643.

369. Fukada, K., Nagano, S., Satoh, M., Tohyama, C., Nakanishi, T., Shimizu, A., Yanagihara, T., and Sakoda, S. (2001) Stabilization of mutant Cu/Zn superoxide dismutase (SOD1) protein by coexpressed wild SOD1 protein accelerates the disease progression in familial amyotrophic lateral sclerosis mice. Eur. J. Neurosci., 14, 2032-2036.

370. Bruijn, L.I., Houseweart, M.K., Kato, S., Anderson, K.L., Anderson, S.D., Ohama, E., Reaume, A.G., Scott, R.W., and Cleveland, D.W. (1998) Aggregation and motor neuron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Science, 281, 1851-1854.

371. Hayward, C., Brock, D.J., Minns, R.A., and Swingler, R.J. (1998) Homozygosity for Asn86Ser mutation in the CuZn-superoxide dismutase gene produces a severe clinical phenotype in a juvenile onset case of familial amyotrophic lateral sclerosis. J. Med. Genet., 35, 174

372. Ralph, G.S., Radcliffe, P.A., Day, D.M., Carthy, J.M., Leroux, M.A., Lee, D.C., Wong, L.F., Bilsland, L.G., Greensmith, L., Kingsman, S.M., Mitrophanous, K.A., Mazarakis, N.D., and Azzouz, M. (2005) Silencing mutant SOD1 using RNAi protects against neurodegeneration and extends survival in an ALS model. Nat. Med.

373. Raoul, C., Abbas-Terki, T., Bensadoun, J.C., Guillot, S., Haase, G., Szulc, J., Henderson, C.E., and Aebischer, P. (2005) Lentiviral-mediated silencing of SOD1 through RNA interference retards disease onset and progression in a mouse model of ALS. Nat. Med.

374. Clement, A.M., Nguyen, M.D., Roberts, E.A., Garcia, M.L., Boillee, S., Rule, M., McMahon, A.P., Doucette, W., Siwek, D., Ferrante, R.J., Brown, R.H., Jr., Julien, J.P., Goldstein, L.S., and Cleveland, D.W. (2003) Wild-type nonneuronal cells extend survival of SOD1 mutant motor neurons in ALS mice. Science, 302, 113-117.

P. Andreas Jonsson

98

375. Ferri, A., Nencini, M., Casciati, A., Cozzolino, M., Angelini, D.F., Longone, P., Spalloni, A., Rotilio, G., and Carri, M.T. (2004) Cell death in amyotrophic lateral sclerosis: interplay between neuronal and glial cells. FASEB J., 18, 1261-1263.

376. Liu, J., Lillo, C., Jonsson, P.A., Velde, C.V., Ward, C.M., Miller, T.M., Subramaniam, J.R., Rothstein, J.D., Marklund, S., Andersen, P.M., Brannstrom, T., Gredal, O., Wong, P.C., Williams, D.S., and Cleveland, D.W. (2004) Toxicity of Familial ALS-Linked SOD1 Mutants from Selective Recruitment to Spinal Mitochondria. Neuron, 43, 5-17.

377. Guegan, C. and Przedborski, S. (2003) Programmed cell death in amyotrophic lateral sclerosis. J. Clin. Invest, 111, 153-161.

378. Sathasivam, S. and Shaw, P.J. (2005) Apoptosis in amyotrophic lateral sclerosis-what is the evidence? Lancet Neurol., 4, 500-509.

379. Rabizadeh, S., Gralla, E.B., Borchelt, D.R., Gwinn, R., Valentine, J.S., Sisodia, S., Wong, P., Lee, M., Hahn, H., and Bredesen, D.E. (1995) Mutations associated with amyotrophic lateral sclerosis convert superoxide dismutase from an antiapoptotic gene to a proapoptotic gene: studies in yeast and neural cells. Proc. Natl. Acad. Sci. U. S. A., 92, 3024-3028.

380. Durham, H.D., Roy, J., Dong, L., and Figlewicz, D.A. (1997) Aggregation of mutant Cu/Zn superoxide dismutase proteins in a culture model of ALS. J. Neuropathol. Exp. Neurol., 56, 523-530.

381. Ghadge, G.D., Lee, J.P., Bindokas, V.P., Jordan, J., Ma, L., Miller, R.J., and Roos, R.P. (1997) Mutant superoxide dismutase-1-linked familial amyotrophic lateral sclerosis: molecular mechanisms of neuronal death and protection. J. Neurosci., 17, 8756-8766.

382. Patel, Y.J., Payne, S., de Belleroche, J., and Latchman, D.S. (2005) Hsp27 and Hsp70 administered in combination have a potent protective effect against FALS-associated SOD1-mutant-induced cell death in mammalian neuronal cells. Brain Res. Mol. Brain Res., 134, 256-274.

383. Pasinelli, P., Borchelt, D.R., Houseweart, M.K., Cleveland, D.W., and Brown, R.H.J. (1998) Caspase-1 is activated in neural cells and tissue with amyotrophic lateral sclerosis-associated mutations in copper-zinc superoxide dismutase. Proc. Natl. Acad. Sci. U. S. A., 95, 15763-15768.

384. Pasinelli, P., Houseweart, M.K., Brown, R.H., Jr., and Cleveland, D.W. (2000) Caspase-1 and -3 are sequentially activated in motor neuron death in Cu,Zn superoxide dismutase-mediated familial amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A, 97, 13901-13906.

385. Embacher, N., Kaufmann, W.A., Beer, R., Maier, H., Jellinger, K.A., Poewe, W., and Ransmayr, G. (2001) Apoptosis signals in sporadic amyotrophic lateral sclerosis: an immunocytochemical study. Acta Neuropathol. (Berl), 102, 426-434.

386. Migheli, A., Atzori, C., Piva, R., Tortarolo, M., Girelli, M., Schiffer, D., and Bendotti, C. (1999) Lack of apoptosis in mice with ALS. Nat. Med., 5, 966-967.

387. Kang, S.J., Sanchez, I., Jing, N., and Yuan, J. (2003) Dissociation between neurodegeneration and caspase-11-mediated activation of caspase-1 and caspase-3 in a mouse model of amyotrophic lateral sclerosis. J. Neurosci., 23, 5455-5460.

388. Li, M., Ona, V.O., Guegan, C., Chen, M., Jackson-Lewis, V., Andrews, L.J., Olszewski, A.J., Stieg, P.E., Lee, J.P., Przedborski, S., and Friedlander, R.M. (2000) Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model. Science, 288, 335-339.


99

389. Guegan, C., Vila, M., Rosoklija, G., Hays, A.P., and Przedborski, S. (2001) Recruitment of the mitochondrial-dependent apoptotic pathway in amyotrophic lateral sclerosis. J. Neurosci., 21, 6569-6576.

390. Vukosavic, S., Stefanis, L., Jackson-Lewis, V., Guegan, C., Romero, N., Chen, C., Dubois-Dauphin, M., and Przedborski, S. (2000) Delaying caspase activation by Bcl-2: A clue to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis. J. Neurosci., 20, 9119-9125.

391. Inoue, H., Tsukita, K., Iwasato, T., Suzuki, Y., Tomioka, M., Tateno, M., Nagao, M., Kawata, A., Saido, T.C., Miura, M., Misawa, H., Itohara, S., and Takahashi, R. (2003) The crucial role of caspase-9 in the disease progression of a transgenic ALS mouse model. EMBO J., 22, 6665-6674.

392. Friedlander, R.M., Brown, R.H., Gagliardini, V., Wang, J., and Yuan, J. (1997) Inhibition of ICE slows ALS in mice. Nature, 388, 31

393. Nguyen, M.D., Julien, J.P., and Rivest, S. (2001) Induction of proinflammatory molecules in mice with amyotrophic lateral sclerosis: no requirement for proapoptotic interleukin-1beta in neurodegeneration. Ann. Neurol., 50, 630-639.

394. Vukosavic, S., Dubois-Dauphin, M., Romero, N., and Przedborski, S. (1999) Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis. J. Neurochem., 73, 2460-2468.

395. Gonzalez de Aguilar, J.L., Gordon, J.W., Rene, F., de Tapia, M., Lutz-Bucher, B., Gaiddon, C., and Loeffler, J.P. (2000) Alteration of the Bcl-x/Bax Ratio in a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis: Evidence for the Implication of the p53 Signaling Pathway. Neurobiol. Dis., 7, 406-415.

396. Pasinelli, P., Belford, M.E., Lennon, N., Bacskai, B.J., Hyman, B.T., Trotti, D., and Brown, R.H., Jr. (2004) Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria. Neuron, 43, 19-30.

397. Kostic, V., Jackson-Lewis, V., de Bilbao, F., Dubois-Dauphin, M., and Przedborski, S. (1997) Bcl-2: prolonging life in a transgenic mouse model of familial amyotrophic lateral sclerosis. Science, 277, 559-562.

398. Martin, L.J. (1999) Neuronal death in amyotrophic lateral sclerosis is apoptosis: possible contribution of a programmed cell death mechanism. J. Neuropathol. Exp. Neurol., 58, 459-471.

399. Ekegren, T., Grundstrom, E., Lindholm, D., and Aquilonius, S.M. (1999) Upregulation of Bax protein and increased DNA degradation in ALS spinal cord motor neurons. Acta Neurol. Scand., 100, 317-321.

400. Mu, X., He, J., Anderson, D.W., Trojanowski, J.Q., and Springer, J.E. (1996) Altered expression of bcl-2 and bax mRNA in amyotrophic lateral sclerosis spinal cord motor neurons. Ann. Neurol., 40, 379-386.

401. Kong, J. and Xu, Z. (1998) Massive mitochondrial degeneration in motor neurons triggers the onset of amyotrophic lateral sclerosis in mice expressing a mutant SOD1. J. Neurosci., 18, 3241-3250.

402. Jaarsma, D., Rognoni, F., van Duijn, W., Verspaget, H.W., Haasdijk, E.D., and Holstege, J.C. (2001) CuZn superoxide dismutase (SOD1) accumulates in vacuolated mitochondria in

P. Andreas Jonsson

100

transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations. Acta Neuropathol. (Berl), 102, 293-305.

403. Higgins, C.M., Jung, C., and Xu, Z. (2003) ALS-associated mutant SOD1G93A causes mitochondrial vacuolation by expansion of the intermembrane space and by involvement of SOD1 aggregation and peroxisomes. BMC. Neurosci., 4, 16

404. Subramaniam, J.R., Lyons, W.E., Liu, J., Bartnikas, T.B., Rothstein, J., Price, D.L., Cleveland, D.W., Gitlin, J.D., and Wong, P.C. (2002) Mutant SOD1 causes motor neuron disease independent of copper chaperone-mediated copper loading. Nat. Neurosci., 5, 301-307.

405. Takeuchi, H., Kobayashi, Y., Ishigaki, S., Doyu, M., and Sobue, G. (2002) Mitochondrial localization of mutant superoxide dismutase 1 triggers caspase-dependent cell death in a cellular model of familial amyotrophic lateral sclerosis. J. Biol. Chem., 277, 50966-50972.

406. Kirkinezos, I.G., Bacman, S.R., Hernandez, D., Oca-Cossio, J., Arias, L.J., Perez-Pinzon, M.A., Bradley, W.G., and Moraes, C.T. (2005) Cytochrome c association with the inner mitochondrial membrane is impaired in the CNS of G93A-SOD1 mice. J. Neurosci., 25, 164-172.

407. Mattiazzi, M., D'Aurelio, M., Gajewski, C.D., Martushova, K., Kiaei, M., Beal, M.F., and Manfredi, G. (2002) Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice. J. Biol. Chem., 277, 29626-29633.

408. Jung, C., Higgins, C.M., and Xu, Z. (2002) Mitochondrial electron transport chain complex dysfunction in a transgenic mouse model for amyotrophic lateral sclerosis. J. Neurochem., 83, 535-545.

409. Browne, S.E., Bowling, A.C., Baik, M.J., Gurney, M., Brown-RH, J., and Beal, M.F. (1998) Metabolic dysfunction in familial, but not sporadic, amyotrophic lateral sclerosis. J. Neurochem., 71, 281-287.

410. Borthwick, G.M., Johnson, M.A., Ince, P.G., Shaw, P.J., and Turnbull, D.M. (1999) Mitochondrial enzyme activity in amyotrophic lateral sclerosis: implications for the role of mitochondria in neuronal cell death. Ann. Neurol., 46, 787-790.

411. Fujita, K., Yamauchi, M., Shibayama, K., Ando, M., Honda, M., and Nagata, Y. (1996) Decreased cytochrome c oxidase activity but unchanged superoxide dismutase and glutathione peroxidase activities in the spinal cords of patients with amyotrophic lateral sclerosis. J. Neurosci. Res., 45, 276-281.

412. Wiedemann, F.R., Manfredi, G., Mawrin, C., Beal, M.F., and Schon, E.A. (2002) Mitochondrial DNA and respiratory chain function in spinal cords of ALS patients. J. Neurochem., 80, 616-625.

413. Bowling, A.C., Schulz, J.B., Brown, R.H., Jr., and Beal, M.F. (1993) Superoxide dismutase activity, oxidative damage, and mitochondrial energy metabolism in familial and sporadic amyotrophic lateral sclerosis. J. Neurochem., 61, 2322-2325.

414. Klivenyi, P., Ferrante, R.J., Matthews, R.T., Bogdanov, M.B., Klein, A.M., Andreassen, O.A., Mueller, G., Wermer, M., Kaddurah-Daouk, R., and Beal, M.F. (1999) Neuroprotective effects of creatine in a transgenic animal model of amyotrophic lateral sclerosis. Nat. Med., 5, 347-350.

415. Zhu, S., Stavrovskaya, I.G., Drozda, M., Kim, B.Y., Ona, V., Li, M., Sarang, S., Liu, A.S., Hartley, D.M., Wu, d.C., Gullans, S., Ferrante, R.J., Przedborski, S., Kristal, B.S., and


101

Friedlander, R.M. (2002) Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice. Nature, 417, 74-78.

416. Kriz, J., Nguyen, M.D., and Julien, J.P. (2002) Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Neurobiol. Dis., 10, 268-278.

417. Van Den, B.L., Tilkin, P., Lemmens, G., and Robberecht, W. (2002) Minocycline delays disease onset and mortality in a transgenic model of ALS. Neuroreport, 13, 1067-1070.

418. Zhang, W., Narayanan, M., and Friedlander, R.M. (2003) Additive neuroprotective effects of minocycline with creatine in a mouse model of ALS. Ann. Neurol., 53, 267-270.

419. Groeneveld, G.J., Veldink, J.H., Van, D.T., I, Kalmijn, S., Beijer, C., De Visser, M., Wokke, J.H., Franssen, H., and Van den Berg, L.H. (2003) A randomized sequential trial of creatine in amyotrophic lateral sclerosis. Ann. Neurol., 53, 437-445.

420. Shefner, J.M., Cudkowicz, M.E., Schoenfeld, D., Conrad, T., Taft, J., Chilton, M., Urbinelli, L., Qureshi, M., Zhang, H., Pestronk, A., Caress, J., Donofrio, P., Sorenson, E., Bradley, W., Lomen-Hoerth, C., Pioro, E., Rezania, K., Ross, M., Pascuzzi, R., Heiman-Patterson, T., Tandan, R., Mitsumoto, H., Rothstein, J., Smith-Palmer, T., MacDonald, D., and Burke, D. (2004) A clinical trial of creatine in ALS. Neurology, 63, 1656-1661.

421. Gordon, P.H., Moore, D.H., Gelinas, D.F., Qualls, C., Meister, M.E., Werner, J., Mendoza, M., Mass, J., Kushner, G., and Miller, R.G. (2004) Placebo-controlled phase I/II studies of minocycline in amyotrophic lateral sclerosis. Neurology, 62, 1845-1847.

422. Sasaki, S., Ohsawa, Y., Yamane, K., Sakuma, H., Shibata, N., Nakano, R., Kikugawa, K., Mizutani, T., Tsuji, S., and Iwata, M. (1998) Familial amyotrophic lateral sclerosis with widespread vacuolation and hyaline inclusions. Neurology, 51, 871-873.

423. Beckman, J.S., Carson, M., Smith, C.D., and Koppenol, W.H. (1993) ALS, SOD and peroxynitrite. Nature, 364, 584

424. Ferrante, R.J., Shinobu, L.A., Schulz, J.B., Matthews, R.T., Thomas, C.E., Kowall, N.W., Gurney, M.E., and Beal, M.F. (1997) Increased 3-nitrotyrosine and oxidative damage in mice with a human copper/zinc superoxide dismutase mutation. Ann. Neurol., 42, 326-334.

425. Estevez, A.G., Crow, J.P., Sampson, J.B., Reiter, C., Zhuang, Y., Richardson, G.J., Tarpey, M.M., Barbeito, L., and Beckman, J.S. (1999) Induction of nitric oxide- dependent apoptosis in motor neurons by zinc- deficient superoxide dismutase. Science, 286, 2498-2500.

426. Bruijn, L.I., Beal, M.F., Becher, M.W., Schulz, J.B., Wong, P.C., Price, D.L., and Cleveland, D.W. (1997) Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)- like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant. Proc. Natl. Acad. Sci. U. S. A., 94, 7606-7611.

427. Beal, M.F., Ferrante, R.J., Browne, S.E., Matthews, R.T., Kowall, N.W., and Brown, R.H.J. (1997) Increased 3-nitrotyrosine in both sporadic and familial amyotrophic lateral sclerosis. Ann. Neurol., 42, 644-654.

428. Cha, C.I., Chung, Y.H., Shin, C., Shin, D.H., Kim, Y.S., Gurney, M.E., and Lee, K.W. (2000) Immunocytochemical study on the distribution of nitrotyrosine in the brain of the transgenic mice expressing a human Cu/Zn SOD mutation. Brain Res., 853, 156-161.

P. Andreas Jonsson

102

429. Abe, K., Pan, L.H., Watanabe, M., Kato, T., and Itoyama, Y. (1995) Induction of nitrotyrosine-like immunoreactivity in the lower motor neuron of amyotrophic lateral sclerosis. Neurosci. Lett., 199, 152-154.

430. Casoni, F., Basso, M., Massignan, T., Gianazza, E., Cheroni, C., Salmona, M., Bendotti, C., and Bonetto, V. (2005) Protein nitration in a mouse model of familial amyotrophic lateral sclerosis: possible multifunctional role in the pathogenesis. J. Biol. Chem., 280, 16295-16304.

431. Son, M., Fathallah-Shaykh, H.M., and Elliott, J.L. (2001) Survival in a transgenic model of FALS is independent of iNOS expression. Ann. Neurol., 50, 273

432. Facchinetti, F., Sasaki, M., Cutting, F.B., Zhai, P., MacDonald, J.E., Reif, D., Beal, M.F., Huang, P.L., Dawson, T.M., Gurney, M.E., and Dawson, V.L. (1999) Lack of involvement of neuronal nitric oxide synthase in the pathogenesis of a transgenic mouse model of familial amyotrophic lateral sclerosis. Neuroscience, 90, 1483-1492.

433. Wiedau, P.M., Goto, J.J., Rabizadeh, S., Gralla, E.B., Roe, J.A., Lee, M.K., Valentine, J.S., and Bredesen, D.E. (1996) Altered reactivity of superoxide dismutase in familial amyotrophic lateral sclerosis. Science, 271, 515-518.

434. Bogdanov, M.B., Ramos, L.E., Xu, Z., and Beal, M.F. (1998) Elevated "hydroxyl radical" generation in vivo in an animal model of amyotrophic lateral sclerosis. J. Neurochem., 71, 1321-1324.

435. Yim, H.S., Kang, J.H., Chock, P.B., Stadtman, E.R., and Yim, M.B. (1997) A familial amyotrophic lateral sclerosis-associated A4V Cu, Zn-superoxide dismutase mutant has a lower Km for hydrogen peroxide. Correlation between clinical severity and the Km value. J. Biol. Chem., 272, 8861-8863.

436. Kang, J.H. and Eum, W.S. (2000) Enhanced oxidative damage by the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants. Biochim. Biophys. Acta, 1524, 162-170.

437. Okado-Matsumoto, A., Myint, T., Fujii, J., and Taniguchi, N. (2000) Gain in functions of mutant Cu,Zn-superoxide dismutases as a causative factor in familial amyotrophic lateral sclerosis: less reactive oxidant formation but high spontaneous aggregation and precipitation. Free Radic. Res., 33, 65-73.

438. Singh, R.J., Karoui, H., Gunther, M.R., Beckman, J.S., Mason, R.P., and Kalyanaraman, B. (1998) Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2. Proc. Natl. Acad. Sci. U. S. A., 95, 6675-6680.

439. Sankarapandi, S. and Zweier, J.L. (1999) Evidence against the generation of free hydroxyl radicals from the interaction of Copper,Zinc-superoxide dismutase and hydrogen peroxide. J. Biol. Chem., 274, 34576-34583.

440. Shibata, N., Hirano, A., Kobayashi, M., Dal Canto, M.C., Gurney, M.E., Komori, T., Umahara, T., and Asayama, K. (1998) Presence of Cu/Zn superoxide dismutase (SOD) immunoreactivity in neuronal hyaline inclusions in spinal cords from mice carrying a transgene for Gly93Ala mutant human Cu/Zn SOD. Acta Neuropathol. (Berl. ), 95, 136-142.

441. Watanabe, M., Dykes-Hoberg, M., Culotta, V.C., Price, D.L., Wong, P.C., and Rothstein, J.D. (2001) Histological evidence of protein aggregation in mutant SOD1 transgenic mice and in amyotrophic lateral sclerosis neural tissues. Neurobiol. Dis., 8, 933-941.


103

442. Kato, S., Sumi-Akamaru, H., Fujimura, H., Sakoda, S., Kato, M., Hirano, A., Takikawa, M., and Ohama, E. (2001) Copper chaperone for superoxide dismutase co-aggregates with superoxide dismutase 1 (SOD1) in neuronal Lewy body-like hyaline inclusions: an immunohistochemical study on familial amyotrophic lateral sclerosis with SOD1 gene mutation. Acta Neuropathol. (Berl), 102, 233-238.

443. Takehisa, Y., Ujike, H., Ishizu, H., Terada, S., Haraguchi, T., Tanaka, Y., Nishinaka, T., Nobukuni, K., Ihara, Y., Namba, R., Yasuda, T., Nishibori, M., Hayabara, T., and Kuroda, S. (2001) Familial amyotrophic lateral sclerosis with a novel Leu126Ser mutation in the copper/zinc superoxide dismutase gene showing mild clinical features and lewy body-like hyaline inclusions. Arch. Neurol., 58, 736-740.

444. Maatkamp, A., Vlug, A., Haasdijk, E., Troost, D., French, P.J., and Jaarsma, D. (2004) Decrease of Hsp25 protein expression precedes degeneration of motoneurons in ALS-SOD1 mice. Eur. J. Neurosci., 20, 14-28.

445. Liu, J., Shinobu, L.A., Ward, C.M., Young, D., and Cleveland, D.W. (2005) Elevation of the Hsp70 chaperone does not effect toxicity in mouse models of familial amyotrophic lateral sclerosis. J. Neurochem., 93, 875-882.

446. Miyazaki, K., Fujita, T., Ozaki, T., Kato, C., Kurose, Y., Sakamoto, M., Kato, S., Goto, T., Itoyama, Y., Aoki, M., and Nakagawara, A. (2004) NEDL1, a novel ubiquitin-protein isopeptide ligase for dishevelled-1, targets mutant superoxide dismutase-1. J. Biol. Chem., 279, 11327-11335.

447. Niwa, J., Ishigaki, S., Hishikawa, N., Yamamoto, M., Doyu, M., Murata, S., Tanaka, K., Taniguchi, N., and Sobue, G. (2002) Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity. J. Biol. Chem., 277, 36793-36798.

448. Wang, J., Xu, G., and Borchelt, D.R. (2002) High molecular weight complexes of mutant superoxide dismutase 1: age-dependent and tissue-specific accumulation. Neurobiol. Dis., 9, 139-148.

449. Cheroni, C., Peviani, M., Cascio, P., Debiasi, S., Monti, C., and Bendotti, C. (2005) Accumulation of human SOD1 and ubiquitinated deposits in the spinal cord of SOD1G93A mice during motor neuron disease progression correlates with a decrease of proteasome. Neurobiol. Dis., 18, 509-522.

450. Shinder, G.A., Lacourse, M.C., Minotti, S., and Durham, H.D. (2001) Mutant Cu/Zn-Superoxide Dismutase Proteins Have Altered Solubility and Interact with Heat Shock/Stress Proteins in Models of Amyotrophic Lateral Sclerosis. J. Biol. Chem., 276, 12791-12796.

451. Johnston, J.A., Dalton, M.J., Gurney, M.E., and Kopito, R.R. (2000) Formation of high molecular weight complexes of mutant Cu,Zn-superoxide dismutase in a mouse model for familial amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A, 97, 12571-12576.

452. Ardley, H.C., Hung, C.C., and Robinson, P.A. (2005) The aggravating role of the ubiquitin-proteasome system in neurodegeneration. FEBS Lett., 579, 571-576.

453. Hatakeyama, S. and Nakayama, K.I. (2003) Ubiquitylation as a quality control system for intracellular proteins. J. Biochem. (Tokyo), 134, 1-8.

454. Zhou, P. (2005) Targeted protein degradation. Curr. Opin. Chem. Biol., 9, 51-55.

455. Roos-Mattjus, P. and Sistonen, L. (2004) The ubiquitin-proteasome pathway. Ann. Med., 36, 285-295.

P. Andreas Jonsson

104

456. Orlowski, M. and Wilk, S. (2003) Ubiquitin-independent proteolytic functions of the proteasome. Arch. Biochem. Biophys., 415, 1-5.

457. Zatz, M. and Starling, A. (2005) Calpains and disease. N. Engl. J. Med., 352, 2413-2423.

458. Yoshimori, T. (2004) Autophagy: a regulated bulk degradation process inside cells. Biochem. Biophys. Res. Commun., 313, 453-458.

459. Aquilano, K., Rotilio, G., and Ciriolo, M.R. (2003) Proteasome activation and nNOS down-regulation in neuroblastoma cells expressing a Cu,Zn superoxide dismutase mutant involved in familial ALS. J. Neurochem., 85, 1324-1335.

460. Urushitani, M., Kurisu, J., Tsukita, K., and Takahashi, R. (2002) Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis. J. Neurochem., 83, 1030-1042.

461. Hyun, D.H., Lee, M., Halliwell, B., and Jenner, P. (2003) Proteasomal inhibition causes the formation of protein aggregates containing a wide range of proteins, including nitrated proteins. J. Neurochem., 86, 363-373.

462. Hyun, D.H., Lee, M.H., Halliwell, B., and Jenner, P. (2002) Proteasomal dysfunction induced by 4-hydroxy-2,3-trans-nonenal, an end-product of lipid peroxidation: a mechanism contributing to neurodegeneration? J. Neurochem., 83, 360-370.

463. Rabouille, C., Strous, G.J., Crapo, J.D., Geuze, H.J., and Slot, J.W. (1993) The differential degradation of two cytosolic proteins as a tool to monitor autophagy in hepatocytes by immunocytochemistry. J. Cell Biol., 120, 897-908.

464. Muchowski, P.J. and Wacker, J.L. (2005) Modulation of neurodegeneration by molecular chaperones. Nat. Rev. Neurosci., 6, 11-22.

465. Mehta, T.A., Greenman, J., Ettelaie, C., Venkatasubramaniam, A., Chetter, I.C., and McCollum, P.T. (2005) Heat shock proteins in vascular disease--a review. Eur. J. Vasc. Endovasc. Surg., 29, 395-402.

466. Bruening, W., Roy, J., Giasson, B., Figlewicz, D.A., Mushynski, W.E., and Durham, H.D. (1999) Up-regulation of protein chaperones preserves viability of cells expressing toxic Cu/Zn-superoxide dismutase mutants associated with amyotrophic lateral sclerosis. J. Neurochem., 72, 693-699.

467. Tummala, H., Jung, C., Tiwari, A., Higgins, C.M., Hayward, L.J., and Xu, Z. (2005) Inhibition of chaperone activity is a shared property of several cu,zn-superoxide dismutase mutants that cause amyotrophic lateral sclerosis. J. Biol. Chem., 280, 17725-17731.

468. Batulan, Z., Shinder, G.A., Minotti, S., He, B.P., Doroudchi, M.M., Nalbantoglu, J., Strong, M.J., and Durham, H.D. (2003) High threshold for induction of the stress response in motor neurons is associated with failure to activate HSF1. J. Neurosci., 23, 5789-5798.

469. Vleminckx, V., Van Damme, P., Goffin, K., Delye, H., Van Den, B.L., and Robberecht, W. (2002) Upregulation of HSP27 in a transgenic model of ALS. J. Neuropathol. Exp. Neurol., 61, 968-974.

470. Takeuchi, H., Kobayashi, Y., Yoshihara, T., Niwa, J., Doyu, M., Ohtsuka, K., and Sobue, G. (2002) Hsp70 and Hsp40 improve neurite outgrowth and suppress intracytoplasmic aggregate formation in cultured neuronal cells expressing mutant SOD1. Brain Res., 949, 11-22.


105

471. Kieran, D., Kalmar, B., Dick, J.R., Riddoch-Contreras, J., Burnstock, G., and Greensmith, L. (2004) Treatment with arimoclomol, a coinducer of heat shock proteins, delays disease progression in ALS mice. Nat. Med., 10, 402-405.

472. Evgrafov, O.V., Mersiyanova, I., Irobi, J., Van Den, B.L., Dierick, I., Leung, C.L., Schagina, O., Verpoorten, N., Van Impe, K., Fedotov, V., Dadali, E., Auer-Grumbach, M., Windpassinger, C., Wagner, K., Mitrovic, Z., Hilton-Jones, D., Talbot, K., Martin, J.J., Vasserman, N., Tverskaya, S., Polyakov, A., Liem, R.K., Gettemans, J., Robberecht, W., De Jonghe, P., and Timmerman, V. (2004) Mutant small heat-shock protein 27 causes axonal Charcot-Marie-Tooth disease and distal hereditary motor neuropathy. Nat. Genet., 36, 602-606.

473. Irobi, J., Van Impe, K., Seeman, P., Jordanova, A., Dierick, I., Verpoorten, N., Michalik, A., De Vriendt, E., Jacobs, A., Van, G., V, Vennekens, K., Mazanec, R., Tournev, I., Hilton-Jones, D., Talbot, K., Kremensky, I., Van Den, B.L., Robberecht, W., Van Vandekerckhove, J., Broeckhoven, C., Gettemans, J., De Jonghe, P., and Timmerman, V. (2004) Hot-spot residue in small heat-shock protein 22 causes distal motor neuropathy. Nat. Genet., 36, 597-601.

474. Dobson, C.M. (2003) Protein folding and misfolding. Nature, 426, 884-890.

475. Rakhit, R., Crow, J.P., Lepock, J.R., Kondejewski, L.H., Cashman, N.R., and Chakrabartty, A. (2004) Monomeric Cu,Zn-superoxide dismutase is a common misfolding intermediate in the oxidation models of sporadic and familial amyotrophic lateral sclerosis. J. Biol. Chem., 279, 15499-15504.

476. Elam, J.S., Taylor, A.B., Strange, R., Antonyuk, S., Doucette, P.A., Rodriguez, J.A., Hasnain, S.S., Hayward, L.J., Valentine, J.S., Yeates, T.O., and Hart, P.J. (2003) Amyloid-like filaments and water-filled nanotubes formed by SOD1 mutant proteins linked to familial ALS. Nat. Struct. Biol., 10, 461-467.

477. Chung, J., Yang, H., de Beus, M.D., Ryu, C.Y., Cho, K., and Colon, W. (2003) Cu/Zn superoxide dismutase can form pore-like structures. Biochem. Biophys. Res. Commun., 312, 873-876.

478. Kato, S., Takikawa, M., Nakashima, K., Hirano, A., Cleveland, D.W., Kusaka, H., Shibata, N., Kato, M., Nakano, I., and Ohama, E. (2000) New consensus research on neuropathological aspects of familial amyotrophic lateral sclerosis with superoxide dismutase 1 (SOD1) gene mutations: inclusions containing SOD1 in neurons and astrocytes. Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 1, 163-184.

479. Stieber, A., Gonatas, J.O., and Gonatas, N.K. (2000) Aggregation of ubiquitin and a mutant ALS-linked SOD1 protein correlate with disease progression and fragmentation of the Golgi apparatus. J. Neurol. Sci., 173, 53-62.

480. Andersen, P.M., Nilsson, P., Ala-Hurula, V., Keranen, M.L., Tarvainen, I., Haltia, T., Nilsson, L., Binzer, M., Forsgren, L., and Marklund, S.L. (1995) Amyotrophic lateral sclerosis associated with homozygosity for an Asp90Ala mutation in CuZn-superoxide dismutase. Nat. Genet., 10, 61-66.

481. Andersen, P.M., Spitsyn, V.A., Makarov, S.V., Nilsson, L., Kravchuk, O.I., Bychkovskaya, L.S., and Marklund, S.L. (2001) The geographical and ethnic distribution of the D90A CuZn-SOD mutation in the Russian Federation. Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 2, 63-69.

482. Winter, S.M., Claus, A., Oberwittler, C., Volkel, H., Wenzler, S., and Ludolph, A.C. (2000) Recessively inherited amyotrophic lateral sclerosis: a Germany family with the D90A CuZn-SOD mutation. J. Neurol., 247, 783-786.

P. Andreas Jonsson

106

483. Khoris, J., Moulard, B., Briolotti, V., Hayer, M., Durieux, A., Clavelou, P., Malafosse, A., Rouleau, G.A., and Camu, W. (2000) Coexistence of dominant and recessive familial amyotrophic lateral sclerosis with the D90A Cu,Zn superoxide dismutase mutation within the same country. Eur. J. Neurol., 7, 207-211.

484. Mancuso, M., Filosto, M., Naini, A., Rocchi, A., Del Corona, A., Sartucci, F., Siciliano, G., and Murri, L. (2002) A screening for superoxide dismutase-1 D90A mutation in Italian patients with sporadic amyotrophic lateral sclerosis. Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 3, 215-218.

485. Skvortsova, V.I., Limborska, S.A., Slominsky, P.A., Levitskaya, N.I., Levitsky, G.N., Shadrina, M.I., and Kondratyeva, E.A. (2001) Sporadic ALS associated with the D90A Cu,Zn superoxide dismutase mutation in Russia. Eur. J. Neurol., 8, 167-172.

486. Mezei, M., Andersen, P.M., Stewart, H., Weber, M., and Eisen, A. (1999) Motor system abnormalities in heterozygous relatives of a D90A homozygous CuZn-SOD ALS patient of finnish extraction. J. Neurol. Sci., 169, 49-55.

487. Andersen, P.M. (2000) Genetic factors in the early diagnosis of ALS. ALS and other motor neuron disorders, 1 Suppl 1, S31-S42.

488. Andersen, P.M., Forsgren, L., Binzer, M., Nilsson, P., Ala-Hurula, V., Keranen, M.L., Bergmark, L., Saarinen, A., Haltia, T., Tarvainen, I., Kinnunen, E., Udd, B., and Marklund, S.L. (1996) Autosomal recessive adult-onset amyotrophic lateral sclerosis associated with homozygosity for Asp90Ala CuZn-superoxide dismutase mutation. A clinical and genealogical study of 36 patients. Brain, 119, 1153-1172.

489. Robberecht, W., Aguirre, T., Van den Bosch, L., Tilkin, P., Cassiman, J.J., and Matthijs, G. (1996) D90A heterozygosity in the SOD1 gene is associated with familial and apparently sporadic amyotrophic lateral sclerosis. Neurology, 47, 1336-1339.

490. Al Chalabi, A., Andersen, P.M., Chioza, B., Shaw, C., Sham, P.C., Robberecht, W., Matthijs, G., Camu, W., Marklund, S.L., Forsgren, L., Rouleau, G., Laing, N.G., Hurse, P.V., Siddique, T., Leigh, P.N., and Powell, J.F. (1998) Recessive amyotrophic lateral sclerosis families with the D90A SOD1 mutation share a common founder: evidence for a linked protective factor. Hum. Mol. Genet., 7, 2045-2050.

491. Parton, M.J., Broom, W., Andersen, P.M., Al Chalabi, A., Nigel, L.P., Powell, J.F., and Shaw, C.E. (2002) D90A-SOD1 mediated amyotrophic lateral sclerosis: a single founder for all cases with evidence for a Cis-acting disease modifier in the recessive haplotype. Hum. Mutat., 20, 473

492. Hansen, C., Gredal, O., Werdelin, L., Andersen, P.M., Marklund, S.L., Nilsson, P., Petersen, M.B., and Brøndum-Nielsen, K. (1999) Novel 4-bp insertion in exon 5 of the CuZn-superoxide dismutase (SOD1) gene associated with familial amyotrophic lateral sclerosis. Mutation Notes, S327-S328.

493. Mockett, R.J., Radyuk, S.N., Benes, J.J., Orr, W.C., and Sohal, R.S. (2003) Phenotypic effects of familial amyotrophic lateral sclerosis mutant Sod alleles in transgenic Drosophila. Proc. Natl. Acad. Sci. U. S. A, 100, 301-306.

494. Elia, A.J., Parkes, T.L., Kirby, K., St, Boulianne, G.L., Phillips, J.P., and Hilliker, A.J. (1999) Expression of human FALS SOD in motorneurons of Drosophila. Free Radic. Biol. Med., 26, 1332-1338.


107

495. Oeda, T., Shimohama, S., Kitagawa, N., Kohno, R., Imura, T., Shibasaki, H., and Ishii, N. (2001) Oxidative stress causes abnormal accumulation of familial amyotrophic lateral sclerosis-related mutant SOD1 in transgenic Caenorhabditis elegans. Hum. Mol. Genet., 10, 2013-2023.

496. Ghadge, G.D., Wang, L., Sharma, K., Monti, A.L., Bindokas, V., Stevens, F.J., and Roos, R.P. (2005) Truncated wild-type SOD1 and FALS-linked mutant SOD1 cause neural cell death in the chick embryo spinal cord. Neurobiol. Dis.

497. Williamson, T.L. and Cleveland, D.W. (1999) Slowing of axonal transport is a very early event in the toxicity of ALS-linked SOD1 mutants to motorneurons. Nat. Neurosci., 2, 50-56.

498. Heiman-Patterson, T.D., Deitch, J.S., Blankenhorn, E.P., Erwin, K.L., Perreault, M.J., Alexander, B.K., Byers, N., Toman, I., and Alexander, G.M. (2005) Background and gender effects on survival in the TgN(SOD1-G93A)1Gur mouse model of ALS. J. Neurol. Sci., 236, 1-7.

499. Kunst, C.B., Messer, L., Gordon, J., Haines, J., and Patterson, D. (2000) Genetic mapping of a mouse modifier gene that can prevent ALS onset. Genomics, 70, 181-189.

500. Behndig, A., Karlsson, K., Reaume, A.G., Sentman, M., and Marklund, S.L. (2001) In vitro photochemical cataract in mice lacking copper-zinc superoxide dismutase. Free Radic. Biol. Med., 31, 738-744.

501. Marklund, S. (1976) Spectrophotometric study of spontaneous disproportionation of superoxide anion radical and sensitive direct assay for superoxide dismutase. J. Biol. Chem., 251, 7504-7507.

502. Marklund, S.L. (1985) Direct assay with potassium superoxide. In: Handbook of methods for oxygen radical research, 249-255, Greenwald, R.A. (ed.), CRC press, Boca Raton.

503. Andersen, P.M., Nilsson, P., Forsgren, L., and Marklund, S.L. (1998) CuZn-superoxide dismutase, extracellular superoxide dismutase, and glutathione peroxidase in blood from individuals homozygous for Asp90Ala CuZu-superoxide dismutase mutation. J. Neurochem., 70, 715-720.

504. Tibell, L., Hjalmarsson, K., Edlund, T., Skogman, G., Engstrom, A., and Marklund, S.L. (1987) Expression of human extracellular superoxide dismutase in Chinese hamster ovary cells and characterization of the product. Proc. Natl. Acad. Sci. U. S. A, 84, 6634-6638.

505. Marklund, S.L. (1985) Pyrogallol autooxidation. In: Handbook of methods for oxygen radical research, 243-247, Greenwald, R.A. (ed.), CRC press, Boca Raton.

506. Marklund, S. and Marklund, G. (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem., 47, 469-474.

507. Beauchamp, C. and Fridovich, I. (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem., 44, 276-287.

508. Behndig, A., Svensson, B., Marklund, S.L., and Karlsson, K. (1998) Superoxide dismutase isoenzymes in the human eye. Invest Ophthalmol. Vis. Sci., 39, 471-475.

509. Karlsson, K. and Marklund, S.L. (1988) Plasma clearance of human extracellular-superoxide dismutase C in rabbits. J. Clin. Invest., 82, 762-766.

P. Andreas Jonsson

108

510. Kisselev, A.F. and Goldberg, A.L. (2001) Proteasome inhibitors: from research tools to drug candidates. Chem. Biol., 8, 739-758.

511. Birch-Machin, M.A., Briggs, H.L., Saborido, A.A., Bindoff, L.A., and Turnbull, D.M. (1994) An evaluation of the measurement of the activities of complexes I-IV in the respiratory chain of human skeletal muscle mitochondria. Biochem. Med. Metab Biol., 51, 35-42.

512. Ahl, I.M., Lindberg, M.J., and Tibell, L.A. (2004) Coexpression of yeast copper chaperone (yCCS) and CuZn-superoxide dismutases in Escherichia coli yields protein with high copper contents. Protein Expr. Purif., 37, 311-319.

513. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248-254.

514. Choi, J., Rees, H.D., Weintraub, S.T., Levey, A.I., Chin, L.S., and Li, L. (2005) Oxidative modifications and aggregation of Cu,Zn-superoxide dismutase associated with Alzheimer and Parkinson diseases. J. Biol. Chem., 280, 11648-11655.

515. Wenisch, E., Vorauer, K., Jungbauer, A., Katinger, H., and Righetti, P.G. (1994) Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes. Electrophoresis, 15, 647-653.

516. Hartman, J.R., Geller, T., Yavin, Z., Bartfeld, D., Kanner, D., Aviv, H., and Gorecki, M. (1986) High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli. Proc. Natl. Acad. Sci. U. S. A, 83, 7142-7146.

517. Poon, H.F., Hensley, K., Thongboonkerd, V., Merchant, M.L., Lynn, B.C., Pierce, W.M., Klein, J.B., Calabrese, V., and Butterfield, D.A. (2005) Redox proteomics analysis of oxidatively modified proteins in G93A-SOD1 transgenic mice--a model of familial amyotrophic lateral sclerosis. Free Radic. Biol. Med., 39, 453-462.

518. Csar, X.F., Wilson, N.J., Strike, P., Sparrow, L., McMahon, K.A., Ward, A.C., and Hamilton, J.A. (2001) Copper/zinc superoxide dismutase is phosphorylated and modulated specifically by granulocyte-colony stimulating factor in myeloid cells. Proteomics., 1, 435-443.

519. Canet-Aviles, R.M., Wilson, M.A., Miller, D.W., Ahmad, R., McLendon, C., Bandyopadhyay, S., Baptista, M.J., Ringe, D., Petsko, G.A., and Cookson, M.R. (2004) The Parkinson's disease protein DJ-1 is neuroprotective due to cysteine-sulfinic acid-driven mitochondrial localization. Proc. Natl. Acad. Sci. U. S. A, 101, 9103-9108.

520. Schinina, M.E., Carlini, P., Polticelli, F., Zappacosta, F., Bossa, F., and Calabrese, L. (1996) Amino acid sequence of chicken Cu, Zn-containing superoxide dismutase and identification of glutathionyl adducts at exposed cysteine residues. Eur. J. Biochem., 237, 433-439.

521. Harris, H., Hopkinson, D.A., and Robson, E.B. (1974) The incidence of rare alleles determining electrophoretic variants: data on 43 enzyme loci in man. Ann. Hum. Genet., 37, 237-253.

522. Blennow, K. and Hampel, H. (2003) CSF markers for incipient Alzheimer's disease. Lancet Neurol., 2, 605-613.

523. Bracco, F., Scarpa, M., Rigo, A., and Battistin, L. (1991) Determination of superoxide dismutase activity by the polarographic method of catalytic currents in the cerebrospinal fluid of aging brain and neurologic degenerative diseases. Proc. Soc. Exp. Biol. Med., 196, 36-41.


109

524. Ilzecka, J. (2004) Cerebrospinal fluid vascular endothelial growth factor in patients with amyotrophic lateral sclerosis. Clin. Neurol. Neurosurg., 106, 289-293.

525. Sussmuth, S.D., Tumani, H., Ecker, D., and Ludolph, A.C. (2003) Amyotrophic lateral sclerosis: disease stage related changes of tau protein and S100 beta in cerebrospinal fluid and creatine kinase in serum. Neurosci. Lett., 353, 57-60.

526. Jimenez-Jimenez, F.J., Hernanz, A., Medina-Acebron, S., de Bustos, F., Zurdo, J.M., Alonso, H., Puertas, I., Barcenilla, B., Sayed, Y., and Cabrera-Valdivia, F. (2005) Tau protein concentrations in cerebrospinal fluid of patients with amyotrophic lateral sclerosis. Acta Neurol. Scand., 111, 114-117.

527. Rosengren, L.E., Karlsson, J.E., Karlsson, J.O., Persson, L.I., and Wikkelso, C. (1996) Patients with amyotrophic lateral sclerosis and other neurodegenerative diseases have increased levels of neurofilament protein in CSF. J. Neurochem., 67, 2013-2018.

528. Kuncl, R.W., Bilak, M.M., Bilak, S.R., Corse, A.M., Royal, W., and Becerra, S.P. (2002) Pigment epithelium-derived factor is elevated in CSF of patients with amyotrophic lateral sclerosis. J. Neurochem., 81, 178-184.

529. Sjogren, M., Davidsson, P., Wallin, A., Granerus, A.K., Grundstrom, E., Askmark, H., Vanmechelen, E., and Blennow, K. (2002) Decreased CSF-beta-amyloid 42 in Alzheimer's disease and amyotrophic lateral sclerosis may reflect mismetabolism of beta-amyloid induced by disparate mechanisms. Dement. Geriatr. Cogn Disord., 13, 112-118.

530. Strand, T. and Marklund, S.L. (1992) Release of superoxide dismutase into cerebrospinal fluid as a marker of brain lesion in acute cerebral infarction. Stroke, 23, 515-518.

531. Yoshida, E., Mokuno, K., Aoki, S., Takahashi, A., Riku, S., Murayama, T., Yanagi, T., and Kato, K. (1994) Cerebrospinal fluid levels of superoxide dismutases in neurological diseases detected by sensitive enzyme immunoassays. J. Neurol. Sci., 124, 25-31.

532. Iwasaki, Y., Ikeda, K., and Kinoshita, M. (1993) Decreased cerebrospinal-fluid superoxide dismutase in amyotrophic lateral sclerosis. Lancet, 342, 1118

533. Boll, M.C., Alcaraz-Zubeldia, M., Montes, S., Murillo-Bonilla, L., and Rios, C. (2003) Raised nitrate concentration and low SOD activity in the CSF of sporadic ALS patients. Neurochem. Res., 28, 699-703.

534. Di Noto, L., Whitson, L.J., Cao, X., Hart, P.J., and Levine, R.L. (2005) Proteasomal degradation of mutant superoxide dismutases linked to amyotrophic lateral sclerosis. J. Biol. Chem.

535. Corson, L.B., Strain, J.J., Culotta, V.C., and Cleveland, D.W. (1998) Chaperone-facilitated copper binding is a property common to several classes of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants. Proc. Natl. Acad. Sci. U. S. A., 95, 6361-6366.

536. Nishida, C.R., Gralla, E.B., and Valentine, J.S. (1994) Characterization of three yeast copper-zinc superoxide dismutase mutants analogous to those coded for in familial amyotrophic lateral sclerosis. Proc. Natl. Acad. Sci. U. S. A., 91, 9906-9910.

537. Fujii, J., Myint, T., Seo, H.G., Kayanoki, Y., Ikeda, Y., and Taniguchi, N. (1995) Characterization of wild-type and amyotrophic lateral sclerosis-related mutant Cu,Zn-superoxide dismutases overproduced in baculovirus-infected insect cells. J. Neurochem., 64, 1456-1461.

P. Andreas Jonsson

110

538. Battistoni, A., Mazzetti, A.P., and Rotilio, G. (1999) In vivo formation of Cu,Zn superoxide dismutase disulfide bond in Escherichia coli. FEBS Lett., 443, 313-316.

539. Vijayvergiya, C., Beal, M.F., Buck, J., and Manfredi, G. (2005) Mutant superoxide dismutase 1 forms aggregates in the brain mitochondrial matrix of amyotrophic lateral sclerosis mice. J. Neurosci., 25, 2463-2470.

540. Borchelt, D.R., Wong, P.C., Becher, M.W., Pardo, C.A., Lee, M.K., Xu, Z.S., Thinakaran, G., Jenkins, N.A., Copeland, N.G., Sisodia, S.S., Cleveland, D.W., Price, D.L., and Hoffman, P.N. (1998) Axonal transport of mutant superoxide dismutase 1 and focal axonal abnormalities in the proximal axons of transgenic mice. Neurobiol. Dis., 5, 27-35.

541. Zhang, B., Tu, P., Abtahian, F., Trojanowski, J.Q., and Lee, V.M. (1997) Neurofilaments and orthograde transport are reduced in ventral root axons of transgenic mice that express human SOD1 with a G93A mutation. J. Cell Biol., 139, 1307-1315.

542. Murakami, T., Nagano, I., Hayashi, T., Manabe, Y., Shoji, M., Setoguchi, Y., and Abe, K. (2001) Impaired retrograde axonal transport of adenovirus-mediated E. coli LacZ gene in the mice carrying mutant SOD1 gene. Neurosci. Lett., 308, 149-152.

543. Vlug, A.S. and Jaarsma, D. (2004) Long term proteasome inhibition does not preferentially afflict motor neurons in organotypical spinal cord cultures. Amyotroph. Lateral. Scler. Other Motor Neuron Disord., 5, 16-21.

544. Lee, J.P., Gerin, C., Bindokas, V.P., Miller, R., Ghadge, G., and Roos, R.P. (2002) No correlation between aggregates of Cu/Zn superoxide dismutase and cell death in familial amyotrophic lateral sclerosis. J. Neurochem., 82, 1229-1238.

545. Kopito, R.R. (2000) Aggresomes, inclusion bodies and protein aggregation. Trends Cell Biol., 10, 524-530.

546. Kirkitadze, M.D., Bitan, G., and Teplow, D.B. (2002) Paradigm shifts in Alzheimer's disease and other neurodegenerative disorders: the emerging role of oligomeric assemblies. J. Neurosci. Res., 69, 567-577.


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Superoxide Dismutase 1 and Amyotrophic Lateral Sclerosis

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