Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Umeå University Medical Dissertations, New Series No 1285 Mutant Superoxide Dismutase-1-caused pathogenesis in Amyotrophic Lateral Sclerosis Daniel Bergemalm Department of Medical Biosciences, Clinical Chemistry and Pathology Department of Pharmacology and Clinical Neurosciences Umeå 2010 Responsible publisher under Swedish law: the Dean of the Medical Faculty Copyright © 2010 Daniel Bergemalm ISBN: 978-91-7264-838-8 ISSN: 0346-6612 Cover: Two hands with atrophy of intrinsic muscles reaching towards the diseasecausing SOD1 molecule. Painted by Per-Olof Bergemalm. E-version available at http://umu.diva-portal.org/ Printed by: Print och Media Umeå, Sweden 2010 To my beloved children Hanna and Ludvig Man's conquest of Nature turns out, in the moment of its consummation, to be Nature's conquest of Man - C.S Lewis Abstract Amyotrophic lateral sclerosis (ALS) is a devastating disease that affects people in their late mid-life, with fatal outcome usually within a few years. The progressive degeneration of neurons responsible for muscle movement (motor neurons) throughout the central nervous system (CNS) leads to muscle wasting and paralysis, and eventually affects respiratory function. Most cases have no familial background (sporadic) whereas about 10% of cases have relatives affected by the disease. A substantial number of familial cases are caused by mutations in the gene encoding superoxide dismutase-1 (SOD1). Since the initial discovery of this relationship about 17 years ago, numerous workers have tried to identify the pathogenicity of mutant SOD1 but without any final agreement or consensus regarding mechanism. The experiments in this thesis have been aimed at finding common pathogenic mechanisms by analyzing transgenic mouse models expressing mutant SOD1s with widely different properties. Mitochondrial pathology and dysfunction have been reported in both ALS patients and murine models. We used density gradient ultracentrifugation for comparison of mitochondrial partitioning of SOD1 in our transgenic models. It was found that models with high levels of mutant protein, overloaded mitochondria with high levels of SOD1protein whereas models with wild type-like levels of mutant protein did not. No significant association of the truncation mutant G127X with mitochondria was found. Thus, if mitochondrial dysfunction and pathology are fundamental for ALS pathogenesis this is unlikely to be caused by physical association of mutant SOD1 with mitochondria. Density gradient ultracentrifugation was used to study SOD1 inclusions in tissues from an ALS patient with a mutant SOD1 (G127X). We found large amounts in the ventral horns of the spinal cord but also in the liver and kidney, although at lower levels. This showed that such signs of the disease can also be found outside the CNS. This method was used further to characterize SOD1 inclusions with regard to the properties of mutant SOD1 and the presence of other proteins. The inclusions were found to be complex detergent-sensitive structures with mutant SOD1 reduced at disulfide C57C146 being the major inclusion protein, constituting at least 50% of the protein content. Ten co-aggregating proteins were isolated, some of which were already known to be present in cellular inclusions. Of great interest was the presence of several proteins that normally reside in the endoplasmic reticulum (ER), which is in accordance with recent data suggesting that the unfolded protein response (UPR) has a role in ALS. To obtain unbiased information on the pathogenesis of mutant SOD1, we performed a total proteome study on spinal cords from ALS transgenic mice. By multivariate analysis of the 1,800 protein spots detected, 420 (23%) were found to significantly contribute to the difference between transgenic and control mice. From 53 proteins finally identified, we found pathways such as mitochondrial function, oxidative stress, and protein degradation to be affected by the disease. We also identified a previously uncharacterized covalent SOD1 dimer. In conclusion, the work described in this thesis suggests that mutant SOD1 affects the function of mitochondria, but not mainly through direct accumulation of SOD1 protein. It also suggests that SOD1 inclusions, present in both the CNS and peripheral tissues, mainly consist of SOD1 but they also trap proteins involved in the UPR. This might be deleterious as motor neurons, unable to renew themselves, are dependent on proper protein folding and degradation. Keywords: ALS, SOD1, mitochondria, proteome, transgenic mice, inclusion. Table of contents Abbreviations ....................................................................................... 1 Preface .................................................................................................. 2 Introduction .......................................................................................... 3 Amyotrophic lateral sclerosis ........................................................... 3 Epidemiology ............................................................................... 3 Pathology...................................................................................... 4 Etiology ............................................................................................ 6 Genetics of ALS ........................................................................... 6 Genetic risk factors..................................................................... 10 Other risk factors ........................................................................ 13 Excitotoxicity ............................................................................. 13 Superoxide dismutase-1 ................................................................. 15 The protein ................................................................................. 15 SOD1 in ALS ............................................................................. 17 How do mutant SOD1s cause disease? ...................................... 19 The “–omics” in ALS/SOD1 models ......................................... 28 Aims ................................................................................................... 31 Methods .............................................................................................. 32 SOD1 transgenic mice ................................................................ 32 G127X patient ............................................................................ 32 Homogenization of tissue ........................................................... 32 Density gradient separation ........................................................ 33 Marker enzyme assays ............................................................... 34 Dot-blot assay............................................................................. 35 Antibody-based assays ............................................................... 35 Size-exclusion chromatography ................................................. 37 Proteomic techniques ................................................................. 37 Network analysis ........................................................................ 39 Statistics ..................................................................................... 40 Results and Discussion ....................................................................... 41 Paper I ............................................................................................ 41 Paper II ........................................................................................... 44 Paper III .......................................................................................... 45 Paper IV.......................................................................................... 47 Conclusions ........................................................................................ 51 Acknowledgements ............................................................................ 53 References .......................................................................................... 55 Abbreviations ALS CCS CHAPS CNS DIGE DTT EDTA EGTA ER FALS GWAS HEPES hSOD1 HSP ir IMS IPKB LBHI LC-MS LMN MALDI-TOF MND MOWSE mRNA mSOD1 NRF2 NP-40 OPLS-DA PAGE PLS PMA ROS SALS SDH SDS SLI SMA SOD1 SOD2 SOD3 UI UMN UPR UPS wt Amyotrophic lateral sclerosis Copper chaperone for SOD1 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate (1) Central nervous system Differential in-gel electrophoresis Dithiothreitol Ethylenediaminetetraacetic acid Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′ tetraacetic acid Endoplasmic reticulum Familial amyotrophic lateral sclerosis Genome-wide association study 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid Human superoxide dismutase-1 Heat shock protein immunoreactive Intermembrane space (mitochondria) Ingenuity pathway knowledge base Lewy body-like hyaline inclusion Liquid chromatography mass spectrometry Lower motor neuron Matrix-assisted laser desorption ionization time of flight Motor neuron disease Molecular weight search Messenger RNA Mouse superoxide dismutase-1 Nuclear factor erythroid 2-related factor 2 Nonidet P-40 (octyl phenoxylpolyethoxylethanol) Orthogonal projections to latent structures discriminant analysis Polyacrylamide gel electrophoresis Primary lateral sclerosis Progressive muscular atrophy Reactive oxygen species Sporadic amyotrophic lateral sclerosis Succinate dehydrogenase Sodium dodecylsulfate Skein-like inclusion Spinal muscular atrophy CuZn superoxide dismutase Mn superoxide dismutase Extracellular superoxide dismutase Ubiquitinated inclusion Upper motor neuron Unfolded protein response Ubiquitin proteasome system Wild type 1 Preface In this thesis, I have tried to target the pathogenic events taking place when mutant superoxide dismutase-1 (SOD1) causes amyotrophic lateral sclerosis (ALS). Although more than 17 years have passed since the original report on this subject, no consensus on the disease mechanism has been reached. In the Introduction I will review the main characteristics of the disease and the most common theories of pathogenesis. The focus is, however, on previous and current work relevant to the experiments described here, including mitochondrial toxicity, SOD1 aggregates, and the use of proteomic methods in ALS. The four papers that constitute this thesis are listed below. I. Daniel Bergemalm, P. Andreas Jonsson, Karin S. Graffmo, Peter M. Andersen, Thomas Brännström, Anna Rehnmark, Stefan L. Marklund. Overloading of stable and exclusion of unstable human superoxide dismutase-1 variants in mitochondria of murine amyotrophic lateral sclerosis models. Journal of Neuroscience. 2006 Apr 19;26:4147-54. II. P. Andreas Jonsson, Daniel Bergemalm, Peter M. Andersen, Ole Gredal, Thomas Brännström, Stefan L. Marklund. Inclusions of amyotrophic lateral sclerosis-linked superoxide dismutase in ventral horns, liver, and kidney. Annals of Neurology. 2008 May;63:671-5. III. Daniel Bergemalm, Karin Forsberg, Vaibhav Srivastava, Karin S Graffmo, Peter M. Andersen, Thomas Brännström, Gunnar Wingsle and Stefan L. Marklund. Superoxide dismutase-1 and other proteins in inclusions of transgenic amyotrophic lateral sclerosis model mice. (Submitted) IV. Daniel Bergemalm, Karin Forsberg, P. Andreas Jonsson, Karin S. Graffmo, Thomas Brännström, Peter M. Andersen, Henrik Antti, Stefan L. Marklund. Changes in the spinal cord proteome of an amyotrophic lateral sclerosis murine model determined by differential in-gel electrophoresis. Molecular and Cellular Proteomics. 2009 Jun;8:1306-17 2 Introduction Amyotrophic lateral sclerosis Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease that is invariably fatal. The progressive loss of motor neurons in the spinal cord, the brain stem, and the motor cortex leads to muscle wasting, paralysis, and eventually respiratory failure. As originally described in the 19th century by Charcot and others, the symptoms are derived from loss of upper and lower motor neurons (UMNs and LMNs) (1;2). Modern criteria are still based on signs of degeneration from both of these compartments in combination with progression and spread of symptoms (the so-called El Escorial criteria) (3;4). The control of muscle movement involves both UMNs and LMNs, and symptoms reflect their functional characteristics. UMNs are involved in voluntary movements and regulation of reflexes. Degeneration of UMNs and the corticospinal tract (descending nerve fibers from UMNs in the spinal cord) therefore leads to weakness in groups of coordinated muscles, symptoms of increased muscular tonus (spasticity), hyperreflexia and repetitive reflexes (clonus). LMNs are responsible for the direct innervations of individual muscle fibers. Depending on the requirements of fine motor ability, one LMN can innervate from a few up to hundreds of muscle fibers. Degeneration of LMNs therefore leads to loss of reflexes, muscle paralysis, spontaneous muscle contractions (fasciculations), and muscle atrophy due to loss of signaling. Despite the fact that there are clearly defined diagnostic criteria, the clinical situation is often more complex and establishing an ALS diagnosis takes a lot of time and resources; it is sometimes never established (5). This complexity is partly due to motor neuronal diseases that affect only UMNs (primary lateral sclerosis, PLS) or LMNs (progressive muscular atrophy, PMA) but which sometimes eventually develop the criteria of ALS (6;7). As the long-term prognosis is different, for example, for PLS, PMA, and classical ALS, clinical studies have recently been carried out to establish the criteria for and prognosis of these diseases and similar intermediate syndromes with accentuation of UMN or LMN symptoms (6;8-10). Epidemiology The incidence, prevalence, and mortality of ALS have been shown to have increased slightly over the past 50 years (11). Most studies have been performed in Europe and the United States, where the incidences have been quite similar with a rough mean of about 2 per 100,000 person-years (variation depending on study ~0.6–3.2 per 100,000) (12-16). In Sweden, the incidence has been shown to have increased from 2.3 per 100,000 personyears in 1991 to 3 per 100,000 person-years in 2005 (17). This corresponds to an approximate lifetime risk of developing ALS of 1 in 400 for the 3 population studied. Cronin and colleagues performed a review of global epidemiology and concluded that few studies have been performed outwith Caucasian populations but that the incidence is most probably lower in other ethnic groups (18). The mortality rates follow the incidence with marginally lower counts, probably due to under-reporting of ALS on death certificates (19). Historically, it has been shown that males have an increased risk of developing ALS. In the 1960s, a male-to-female risk ratio of about 2:1 was reported whereas this ratio was reported to average 1.3 in the 1990s (11). The decrease in male preponderance (or increase in incidence in women) now appears to have stabilized and recent reports have shown similar numbers (13;15;17). However, a small study recently reported a higher incidence in women than in men in an Italian population (14). It has been speculated that the gender difference may be attributed to protective female hormones and increased exposure to potential risk factors in males, such as smoking (20;21). Age of onset and survival Most ALS patients are diagnosed at ages ranging between 55 and 85, with a peak incidence at 68 years of age (17;19;20). The variation is, however, great and in a recent report the patients included were between 20 and 90 years of age—which is typical of studies in large populations (15). Though juvenile forms exist, with very early onset of disease, it is nevertheless unusual to be diagnosed before the age of 30 (22). The mean survival time of ALS patients has been estimated to be 32.6 months (range 26.3–43) (11) and this is partly dependent on the site of onset, with the bulbar form being more aggressive (23). Between 5% and 10% of patients have a disease duration of more than 10 years (24;25). Familial ALS (FALS, see below) shows a significantly lower age of onset and higher variability of disease duration (26;27). Pathology The main characteristics of ALS pathology are due to selective loss of motor neurons in affected areas of the motor cortex, the brain stem, and the anterior horns of the spinal cord (28;29). This affects the fibers that are involved in connecting these different cellular populations, the corticospinal tract, and efferent axons in the anterior roots. Macroscopically, atrophy of the spinal cord and anterior roots is often quite obvious, while atrophy of the precentral gyrus (motor cortex) can only be seen in patients with a very long disease duration (28). Microscopically, the loss of motor neurons is followed by astrocytic gliosis and shrinkage of the few remaining motor neurons. Few of these findings are, however, specific for ALS even though their simultaneous presence clearly strengthens the diagnosis. As a further aid to diagnosis, some characteristic cellular inclusions are visible with routine staining, and these are more or less specific for ALS. 4 Inclusions Neurodegenerative diseases are often characterized by the presence of intracellular and extracellular inclusions in the CNS. In Alzheimer’s disease and Parkinson’s disease, inclusions such as senile plaques, neurofibrillary tangles, and Lewy bodies are fundamental for the pathological diagnosis and they have been studied extensively. The mechanisms that give rise to the different types of inclusions are as yet unknown. Studies in cell lines have involved modeling of different entities that arise due to, for example, high expression of mutant proteins or inhibition of protein degradation (e.g. the ubiquitin-proteasome pathway). Examples of such entities are aggresomes, JUNQ/IPOD, and ERAC (30-32). A series of different inclusions are known in ALS; some are more common in SALS or FALS, but there is often overlap (29). Bunina bodies. Bunina bodies are almost pathognomonic for ALS and are generally found in the cytoplasm of surviving motor neurons. They are round or irregular in shape, with a diameter ranging from 1 to 6 µm. When stained with hematoxylin and eosin (H&E), they are eosinophilic hyaline inclusions occasionally surrounded by a thin halo. Bunina bodies stain with antibodies to cystatin C and transferrin, and they have been suggested to be derived from the endoplasmic reticulum or Golgi apparatus (28;29;33). Ubiquitinated inclusions (UIs). UIs are very common in ALS patients (close to 100%) and can be further subdivided according to morphology into Skein-like inclusions (SLIs) and Lewy-body like hyaline inclusions (LBHIs). It has recently been shown that one of the major proteins in UIs from SALS and non-SOD1 FALS patients is TAR DNA binding protein 43 (TDP-43) (34) (discussed further below). SLIs range from 5 to 25 µm and have a filamentous morphology of aggregated fibrils. Similar to SLIs, LBHIs are composed of filaments and are coated with small granules. LBHIs are seen with routine H&E staining and are rounder and denser than SLIs. LBHIs have been found in both astrocytes and neurons from SALS and mutant SOD1-associated FALS, and are the most frequently found inclusions in these patients as well as in SOD1 transgenic mouse models. In both SOD1 FALS and SOD1 transgenic mice, the LBHIs stain intensively for the SOD1 protein (35;36). A range of other proteins have been reported to be present in LBHIs such as neurofilaments, peripherin, and ER chaperones (28;29;37). Spheroids. Spheroids are mainly composed of accumulated phosphorylated neurofilaments, found in the proximal part of motor neuronal axons. They are variable in size, eosinophilic on H&E staining, and are clearly seen in silver impregnations. Spheroids are non-specific and can be found in tissue from both controls and patients with other neurological diseases, but the numbers are far greater in ALS patients (28;29). 5 Other inclusions. Several other inclusions have been described in ALS patients. One is hyaline conglomerate (neurofilament) inclusions (HCIs), which appear to be rather specific for FALS rather than SALS but have been found in other neurodegenerative diseases (20). Another is basophilic inclusions, which are found in juvenile ALS (28). Etiology Genetics of ALS Most cases of ALS develop without any familial history whereas about 5– 10% are regarded as inherited (FALS) (38). The majority of these families have an autosomal dominant disease with variable penetrance, but recessive inheritance also exists (39;40). To be regarded as “familial”, the proband must have at least one or two first- or second-degree relatives that are also affected (41). As the clinical and pathological presentations between SALS and FALS are often similar (42), it is obviously of great importance to characterize familial forms and identify the gene or genes that are involved. So far, mutations in ten genes have been coupled to typical or atypical ALS, and there are six more loci whose genes are as yet unidentified (Table 1). Table 1. Loci and genes identified in familial ALS Disease Type ALS1 ALS2 ALS4 ALS6 ALS8 LMND ALS ALS ALS-FTD ALS-FTD Gene SOD1 ALSIN SETX FUS VAPB DCTN1 Angiogenin TARDP CHMP2B PRGN Locus 21q22.11 2q33 9q34 16q21 20q13.3 2p13 14q11.2 1p36 3p11.2 17q21.32 Inheritance AD (AR) AR AD AD AD AD AD AD AD AD Clinical Pattern Classical Young onset Young onset Classical Varied LMND Classical Classical FTD and ALS FTD and ALS ALS3 ALS5 ALS7 ALS-X ALS-FTD ALS-FTD ? ? ? ? ? ? 18q21 15q15 20p13 Xp11-q12 9q21-q22 9p21.3 AD AR AD XD AD AD Classical Young onset Classical Classical ALS and FTD ALS and FTD AD, autosomal dominant; AR, autosomal recessive; XD, X-linked dominant; FTD, frontotemporal dementia; LMND, lower motor neuron disease; SETX, senataxin; DCTN1, dynactin. ALS1/SOD1. The identification of mutations in the gene for superoxide dismutase-1 (SOD1) in 1993 was a real breakthrough in our understanding 6 of ALS pathogenesis (43). It had been preceded by a publication of a disease-linked locus on chromosome 21, found in families with autosomal dominant ALS (44). The numbers of SOD1 mutations detected in ALS are still increasing, and currently more than 150 different mutations are known (38) (alsod.iop.kcl.ac.uk/als/summary /summary.aspx). As SOD1 toxicity is the main subject of this thesis, a more detailed description follows. ALS2/Alsin. In 2001, it was shown that mutations in the gene encoding a 184-kDa protein called alsin could cause a slowly progressing atypical ALS mainly with UMN involvement (45;46). Mutations were also found in cases of recessively inherited juvenile ALS. Alsin contains three guanine nucleotide exchange factor (GEF) domains and is involved in the cycling of GDP/GTP in GTPases (46). GTPases are involved in important intracellular signaling such as the RAS pathway, and alsin has been shown to interact with Rab5, a small GTPase involved in endosomal dynamics (47). There are long and short splice products of the alsin gene transcript. Somewhat surprisingly, Kanekura and colleagues showed that overexpression of the long form of alsin was able to rescue cells from the toxicity of mutant SOD1. The long form of alsin could interact physically with mutant SOD1 but not wild-type (wt) SOD1 (48). Subsequently, alsin has been coupled to glutamate signaling and excitotoxicity (49), and shown to be involved in the survival of motorneurons through its interaction with Rac1 (50;51). As most ALS patients with alsin mutations are homozygous and most mutations are predicted to truncate the protein, it has been suggested that ALS is caused by a loss of function (42). There are, however, no clear signs of motor neuron disease in the four different alsin knock-out models that have been presented (reviewed in (52)). ALS4/Senataxin. In 2004, mutations in the senataxin gene were shown to be involved in juvenile-onset, autosomal dominant ALS (53). In the same year, it was shown that the autosomal recessive disease ataxia-ocular apraxia 2 was due to mutations in the same gene (54). The phenotype of senataxincaused ALS is quite atypical, with juvenile onset, distal weakness, and muscle atrophy but normal lifespan (53). From sequence homology, the protein has been suggested to function as a DNA/RNA helicase, and it has been shown to be distributed in both the cytosol and the nucleus in multiple cell types of the CNS (with highest density in the cerebellum and the hippocampus) (55). ALS6/FUS. Recently, mutations in the gene encoding the protein fused in sarcoma (FUS) were shown to be responsible for the familial ALS variant previously called ALS6 (56;57). A total of 16 mutations were described. FUS is mainly a nuclear protein involved in transcriptional regulation and splicing, with functions that partly overlap with those of TDP-43 (see below) (58). In patients with FUS mutations, similar to TDP-43, the mutant protein was shown to aberrantly accumulate and aggregate in the cytoplasm. 7 ALS8/VAPB. In 2004, Nishimura et al. published the identification of a locus (20q13.3, called ALS8) associated with an atypical form of autosomal dominant ALS with slow progression (59). Later that same year, they reported that a mutation in a gene called VAPB is responsible for ALS8 (60). They found the same mutation in several families with different phenotypes including classical rapid-progression ALS, atypical ALS, and late-onset spinal muscular atrophy (SMA)—another motor neuron disease. Since then, screens for mutations other than the P56S mutation originally described have been fruitless (61;62). These studies found polymorphisms present in both ALS patients (e.g. the deletion mutant Delta S160) and healthy controls. From this, it was speculated that the P56S mutation confers a specific toxicity that is not conferred by other mutations (61;63). The normal function of VAPB was initially suggested to involve regulation of the unfolded protein response (UPR) in the endoplasmic reticulum, a function that is lost with the P56S mutation (64). Furthermore, it has been shown that the mutant protein interacts with wt subunits and causes them to coaggregate (64-66). This results in a dominant negative effect with decrease in UPR even in the presence of wt protein. A reduction in the level of VAPB protein has also been found in both ALS patients and SOD1 transgenic mice without VAPB mutations, indicating that there may be a functional link between general ALS pathogenesis, SOD1 toxicity, and VAPB protein (66;67). Apart from actions involving the UPR, Tsuda et al. showed that a domain of VAPB, including the P56S mutation, can be secreted and can act as a trophic factor at Eph receptors. The aggregation of VAPB abolished secretion and resulted in depletion of this non-cell autonomous function (68). Dynactin. Dynactin is a multiprotein complex, required for functioning of the microtubule motor protein dynein. In 2003, a G59S mutation in the largest subunit, p150(glued), was found to be associated with familial ALS (69). Mutational screening of p150(glued) has later revealed four other mutations in SALS, FALS, and ALS that are coupled to frontotemporal dementia (ALS-FTD) (70;71). In 2008, Laird and colleagues showed that expression of mutant p150(glued) protein, but not the wt protein, resulted in a motor neuron disease phenotype in transgenic mouse models (72). The model showed defects in vesicular transport and motor neuron pathology with axonal swelling. Another mouse model, aimed at impairing dynactin/dynein function (through means other than point mutation), was published the same year (73). Although showing neuronal pathology, the mice never developed symptoms of motor neuron disease. Interestingly, the authors showed that inhibition of dynactin/dynein function in mutant SOD1 transgenic mice resulted in extended survival, indicating that there was a functional link. Earlier, two mouse strains, loa and cra1, with chemicallyinduced mutations shown to be located in the dynein gene, had been found to show motor neuron disease (74). Co-expression of different mutant SOD1 proteins in these model mice has recently been shown to be beneficial (75- 8 77). The result was, however, not uniform for all the SOD1 mutant models tested (77). Furthermore, mutant SOD1 has been shown to interact with the dynactin/dynein complex, which is important for inclusion formation by mutant SOD1 proteins (78). These authors also showed that overexpression of a dynactin subunit can prevent the deposition of large SOD1 inclusions. Angiogenin. Since the original finding of ALS-associated mutations in the angiogenin gene in 2004 (79), several more have been identified in SALS, FALS, and in ALS-FTD (80-86). Angiogenin belongs to the ribonuclease A superfamily and has been shown to induce angiogenesis through four major pathways: degradation of RNA, basement membrane degradation, signal transduction, and nuclear translocation (87). Recent studies have indicated that angiogenin mutations lead to loss of function, affecting several pathways mentioned above, with consequences for neurons dealing with hypoxia (82;88-92). TARDP. Since the discovery of ubiquitinated TDP-43 protein in inclusions from both FTD and ALS patients in 2006 (34), there has been an explosion of reports regarding this protein and the relationship between the two diseases. The gene TARDP, encoding the TDP-43 protein, is located on chromosome 1 and is ubiquitously expressed in multiple splice variants (reviewed in (93)). TDP-43 binds RNA/DNA and is involved in diverse functions such as gene transcription, splicing, and mRNA stability. The initial histopathological studies indicated that anti-TDP-43 staining of ubiquitinated inclusions (UIs) was located in both the nucleus and the cytoplasm of neuronal and glial cells, but that TDP-43 was excluded from the nuclei and accumulated in the cytoplasm of affected cells (34;94). TDP43 was also shown to be cleaved, producing a 25-kDa C-terminal fragment, and to be phosphorylated in extracts from patients but not from controls (34). TDP-43 positive staining of neuronal and glial cell UIs was later shown to be present in SALS patients, FALS patients without SOD1-mutations, and ALS-FTD patients but not in ALS patients with SOD1 mutations (95;96) or in SOD1 transgenic mouse models (97). This has, however, been challenged by a more recent report based on transgenic mice (98). During the spring of 2008, multiple—almost simultaneous—reports appeared on mutations in the TARDB gene in SALS and in autosomal dominant FALS (99-103). Since then, reports on findings of new ALSassociated mutations have cointinued to appear; today, 30 mutations in 22 unrelated families and 29 sporadic cases have been found (reviewed in (58)). Although there are similarities in pathology between FTD and ALS regarding TDP-43 positive ubiquitinated inclusions, only one ALS patient with a TDP-43 mutation has so far been reported to develop cognitive impairment (of Alzheimer type) (104). As to the mechanism(s) of toxicity of mutant TDP-43 in ALS, there is still considerable confusion. Some recent reports have come with possible explanations. Mutated TDP-43 and also its 9 truncated C-terminal fragment have been shown to sequester the wt protein into cytoplasmic inclusions and thereby deprive the cell of TDP-43 activity (105;106). In contrast, expression of different TDP-43 fragments in a cell line showed that the C-terminal fragment alone was responsible for toxicity without affecting the activity of full-length TDP-43 (107). An antibody specific for the cleaved C-terminal fragment was also shown to selectively stain neuronal cytoplasmic inclusions in tissues from ALS patients (107). Furthermore, TDP-43 has been shown to stabilize neurofilament-light (NFL) mRNA through direct interaction, and it might therefore be involved in the formation of neurofilament inclusions in ALS (108). Iguchi and colleagues studied the effect of siRNA knock-down of TDP43 in Neuro-2a cells (109). This resulted in reduced viability, which was at least partly due to loss of regulation of Rho-family GTPases. As judged by the enormous amount of work invested in such a short time, we are only at the beginning of understanding these mechanisms, and the field will probably become clearer in the near future. CHMP2B. In 2005, mutations in charged multivesicular body protein 2b (CHMP2B) were reported in families with FTD (110). Later, mutations were found in one patient with PMA and in another with ALS-FTD (111). CHMP2B is involved in the endosomal sorting complex (ESCRT) and mutations were suggested to disturb endosome dynamics, as with alsin and dynactin (111). Recently, it has been suggested that mutations in CHMP2B inhibit autophagy, which is an important function for clearance of aggregated proteins (112;113). Progranulin. Mutations in progranulin (PRGN) were initially found in cases of FTD and linked to ALS through one family with a history of both diseases (114). Progranulin is a secreted growth factor with multiple functions and has recently been shown to be involved in caspase-dependent cleavage of TDP-43 (115). In a mutational screen of a spectrum of ALS variants, PRGN mutations were found in one SALS patient and one patient with ALS-FTD (116). In another study in Belgian and Dutch ALS patients, mutations were found but sequence variants were also present in control subjects (117). The authors instead suggested that allelic PRGN variants modify the disease progression and might therefore be more correctly regarded as a genetic risk factor. Genetic risk factors The distinction between a genetic risk factor and a causative genetic alteration is not always simple, as many findings have been in single SALS patients and not all inheritable mutations have complete penetrance. The number of genes that have been associated with ALS and suggested to modulate the risk in one way or another are numerous, and beyond the scope 10 of this review (recently reviewed in (118). I will briefly mention a few of the more interesting findings. Neurofilament-heavy subunit and peripherin. Neurofilaments (NFs) have been implicated in ALS from pathological findings of accumulated NFs in inclusion bodies (discussed above) and from NF overexpression and knockout models in mice that in some cases develop motor neuron deficits (119). Different genetic alterations in the C-terminal domain of the gene encoding the heavy variant of neurofilaments (NF-H) have been found in ALS patients (120-122). Another intermediate filament, peripherin, generates a motor neuron disease phenotype when overexpressed in mice (123). In mutational screening of the peripherin (PRPH) gene in SALS and FALS patients, 20 polymorphisms were found. One gave a truncated protein and was found only in one SALS patient (124). Another homozygous mutation has been found in one patient (125). Although there is not a strong correlation between ALS and neurofilament genes, these proteins appear to be important in the pathogenesis in several ways, including aggregation and axonal transport for example. VEGF. Oosthuyse et al. demonstrated that neuronal knock-down of expression of the gene for vascular endothelial growth factor (VEGF) through deletion of the hypoxia-response element resulted in an adult-onset motor neuronal disease in mice (126). This was followed by the finding that polymorphisms in the promoter sequence of VEGF, causing lower levels of circulating protein, increased the risk of ALS by 80% (127). Later studies have not been able to demonstrate this strong correlation in other populations, but they have found differences in risk attributed to gender (128;129). Like angiogenin, VEGF is involved in response to hypoxia and neovascularisation. The association of both of these molecules with ALS, although not clearly ruled out, indicates that these mechanisms are fundamental for motor neuron survival. Administration of VEGF through injection of viral vectors or as a purified protein has also shown to modify disease in a positive way in ALS transgenic mice (130;131). SMN1/2. The protein survival of motor neuron (SMN) is found in the cytoplasm and nucleus, and is involved in RNA processing through assembly of small ribonucleoproteins into functional complexes (132). Homozygous mutations in SMN1 are responsible for most cases of spinal muscular atrophy (SMA), a motor neuronal disease with juvenile onset with variable survival (but adult forms exist). Another copy of the gene, SMN2, produces a homologous protein that can modulate disease depending on copy number, with more copies being favorable for survival and resulting in less aggressive disease. As SMA is caused by the selective death of motor neurons, similar to ALS, the role of SMN in ALS has been addressed by several studies. Although the results are divergent, there are indications that SMN genetic variants that produce less SMN protein are a risk factor for 11 ALS (133-135). In co-transfection models with simultaneous overexpression of SMN and mutant SOD1, SMN has been shown to upregulate chaperone activity and to rescue cells from SOD1 toxicity (136). Recently, transgenic mice expressing mutant SOD1 (G93A) were produced in an SMN heterozygote deletion background (SMN +/-) (137). SMN -/- is embryonically lethal but SMN +/- mice show no sign of motor neuron disease. Co-expression of G93A/SMN +/- modulated disease, resulting in a more aggressive ALS phenotype. APOE. Patients with late-onset Alzheimer’s disease have an overrepresentation of the epsilon 4 allele of the apoliporotein E (APOE4) gene (138). Several correlation studies in various populations of ALS patients regarding APOE genotype have been conducted (139-145). All such studies have shown that there is no increase in risk of ALS associated with APOE genotype. However, APOE4 carriers have been suggested to have an younger age of onset and a worse prognosis (139;142) while the opposite appears to be true of APOE2/3 (140;145). Promoter deletions in SOD1. In sporadic ALS cases, the homozygous deletion of a 50-bp region of the SOD1 promoter has been shown to be correlated with increased age of onset in cases from British and Irish populations (146). The deletion resulted in a 50% lower promoter activity when expressed in various cell lines, but a reduction in SOD1 protein levels could not be confirmed in brain extracts of SALS patients. Genome-wide studies The recent advances in genetics have revealed new methods (e.g. microchip technology) for screening of genetic material, which makes it possible to find DNA sequence polymorphisms that are associated with disease in large patient material. Genome-wide association studies (GWAS) use dense maps of single nucleotide polymorphisms (SNPs) to scan the human genome for disease-common alleles (147). This approach has been successful in common diseases such as diabetes and breast cancer where the patient material is huge, and during the last few years they have been performed on different ALS materials (reviewed in (118;148)). The first GWAS performed on ALS in 2007 was conducted in a material of 276 patients. Thirty-four SNPs were found that were potentially associated with disease, but none survived correction for multiple testing (Bonferroni correction) (149). Later that year, a GWAS in a total of 1,337 European patients showed association with a genetic variant in the inositol 1,4,5-triphosphate receptor 2 gene (ITPR2), a protein involved in regulation of intracellular calcium concentration (150). These and other studies in different populations has been conflicting and unable to corroborate each other (151-156). Single studies have reported an association with dipeptidyl-peptidase 6 (DPP6) (154;155) and FGGY (153). These inconclusive results have been suggested 12 to be due to the problem of sample size in ALS, which will need to be overcome by the use of global multicenter studies in the future (118;148). In a recent, comparatively large study involving 1,821 SALS patients, Landers and colleagues found an association between survival and alleles in the KIFAP3 gene (157). Homozygosity for the allele was associated with a long survival time but not with risk, site of onset, or age of onset. With a slightly different genetic approach using microsatellite markers, another recent study presented an association between a component of RNA polymerase II (ELP3) and SALS (158). This is interesting in the context of other RNA processing enzymes that have been implicated in ALS pathogenesis, but the association must be confirmed in other studies. Other risk factors Besides genetic factors, age, and gender, the only risk factor that has been found to be somewhat associated with ALS is smoking (21). This association has been strengthened by a recent prospective study (159). As smoking among women has increased during the last fifty years, this has also been suggested to partly explain the shift in gender ratio towards equality. During the last decade, an unforeseen clustering of ALS cases in professional soccer players has drawn new attention to a possible link between extensive physical activity and motor neuron disease (reviewed in (160)). The studies performed do, however, suffer from small sample sizes and the possible confounding effects of other common exogenous or lifestyle risk factors. In a recent report comparing cohorts of professional soccer players, basketball players, and road cyclists, a higher than expected incidence of ALS was found only in soccer players, indicating that high physical activity per se may not be a risk factor (161). Excitotoxicity Glutamate is the dominant excitatory neurotransmitter in synapses of the brain and spinal cord. Upon release, glutamate interacts with various presynaptic and postsynaptic receptors (NMDA, AMPA, and KA receptors) and is finally taken up by astrocytes where it is turned into inactive glutamine. This uptake is mediated through glutamate transporters known as EAAT1 to -5, where glial EAAT2 is the predominant species in the CNS and is responsible for removal of glutamate from the synapses. Excessive glutamate results in toxic effects that are mediated by, e.g. Ca2+ influx and increased oxidative stress (162). This pathway has been implicated in ALS for a number of reasons. (I) Motor neurons of the ventral horn have been shown to be especially vulnerable to excessive glutamate levels and signaling through AMPA receptors (163-166). It has also been shown that a subset of AMPA receptors, lacking a GluR2 subunit that makes them permeable to Ca2+, might be specially toxic for motor neurons (167;168). Activation of AMPA receptors by administration of agonists in vivo has also 13 resulted in motor deficits in mice (165). (II) An early finding was that deficits in glutamate transport were specific to ALS patients in affected regions of the spinal cord and motor cortex (169). This was shown to be due to a loss of EAAT2 protein in ALS patients (170). Although controversial, this has been suggested to be caused by aberrant EAAT2 mRNAs (171). These species have also been found in controls, however (172). More recently, high levels of glutamate have been found in the plasma of ALS patients (173), as well as reduced glutamate uptake in platelets (174), suggesting that alterations in glutamate levels are not confined to the CNS. (III) In experiments on cultured cells from the cerebral cortex and spinal cord, cerebrospinal fluid (CSF) from ALS patients has been shown to mediate toxic effects (175-177). One measurable effect was an increase in cellular Ca2+ levels (175) and these studies also showed that administration of glutamate receptor inhibitors was protective. (IV) The finding that riluzole, an antiglutamate agent, had an effect on disease progression was a great breakthrough since no approved drugs existed (178). This effect was also confirmed in later studies (179;180). How this antiglutamate effect is produced is not fully known, but it has been suggested that levels of glutamate in serum decrease after intake of riluzole (181), a finding that could not be reproduced in a recent study (182). In cell culture experiments, it has been shown that riluzole significantly increases the activity of glutamate transporters such as EAAT2, with increased clearance of extracellular glutamate (183). It has also been suggested that riluzole regulates glutamate release and postsynaptic receptor activation, and inhibits voltage-sensitive channels (162). The riluzole effect appears to be the strongest evidence for the involvement of glutamate metabolism in ALS pathogenesis. Whether excitotoxicity is a primary toxic event or just secondary due to the neurodegenerative disease process requires further study. How this correlates to other etiological pathways such as mutant SOD1 toxicity also requires investigation. 14 Superoxide dismutase-1 The protein Activity and subcellular distribution. Seventy years ago, Mann and Keilin described a protein that was responsible for binding most of the Cu2+ in bovine erythrocytes, which they called haemocuprein (184). It took another thirty years before McCord and Fridovich discovered and reported the function of this copper protein and renamed it superoxide dismutase (SOD, later SOD1) (185). The function described, dismutation of superoxide, is a copper-dependent reaction that dismutes two molecules of superoxide (O2·-) into hydrogen peroxide (H2O2) in a two-step reaction: O2·- + SOD-Cu2+ + - 2H + O2· + SOD-Cu O2 + SOD-Cu+ + H2O2 + SOD-Cu (Step 1) 2+ (Step 2) Oxygen, a molecule crucial for all human cells, is used as substrate for energy production mainly in the mitochondrial respiratory chain (MRC). During these reactions, O2 is reduced to water in multistep reactions (186). The superoxide anion is a free radical that can be formed when oxygen picks up one electron in reactions involving the complexes of the MRC. Another major source of superoxide is the group of membrane-bound enzymes known as the nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases (several other cellular oxidases that produce superoxide are known). These enzymes are highly expressed by cells of the immune system, such as neutrophils, microglia, and macrophages, and are activated during inflammation for example. Depending on the type of NADPH oxidase (several Nox genes are known), superoxide can be formed both extracellularly and intracellulary. It is of particular interest that recent experiments have shown that massive activation of these enzymes takes place both in SALS and in murine models of ALS (187), and Nox2 knockout (the predominant species in microglia) has been shown to slow disease progression in ALS transgenic mice (187;188). The activation of NOX2 and increased amounts of superoxide have also been shown to be positively regulated by different mutant SOD1s when expressed in cellular models (189). Superoxide is considered to be a moderately reactive molecule but it reacts with FeS clusters, which occur in several enzymes. It can also be highly toxic through its very rapid reaction with NO to form peroxynitrite (ONOO-). Peroxynitrite reacts quickly with CO2 to form nitrosoperoxycarbonate (NOOCO2-) a highly toxic species that can react with virtually all macromolecules in the cell (e.g. through tyrosine nitration). NO is produced by three different nitric oxide synthetases (NOS): endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS) (190). Endothelial NOS produces the NO that is essential for 15 vascular homeostasis whereas nNOS is expressed in neurons (as well as in other cells), where NO is involved in diverse processes, e.g. neurogenesis and memory. Superoxide can also cause adverse effects by interfering with these normal physiological functions of NO. The activities of eNOS and nNOS are regarded as essentially constitutive whereas the level of iNOS depends on different cellular stimuli such as infection, inflammation, and ischemia. A number of pathological conditions have been shown to cause a significant increase in iNOS, one of them being ALS (191;192), where high NO levels have been speculated to contribute to pathogenesis. OH·, by far the most reactive free radical, can be formed from O2·- and H2O2 through reactions involving transition metals such as Fe2+ (or Cu2+); this is known as the Haber-Weiss reaction. O2·- + Fe3+ O2 + Fe2+ H2O2 + Fe2+ OH· + OH- + Fe3+ (Fenton) The second reaction is also called the Fenton reaction. Fe3+ and Cu2+ can also be reduced by other reductants such as ascorbate. As O2·- and H2O2 are involved in reactions resulting in the formation of OH·, the cell needs efficient systems for handling of these molecules. The oxidation of H2O2 into water is catalyzed by several cellular enzymes (e.g. peroxyredoxins, glutathione peroxidases, thioredoxin, and catalase) and is extremely rapid. Superoxide can be scavenged by ascorbate but is mainly taken care of through dismutation via SOD. The importance of this activity is highlighted by the existence of two other SOD enzymes besides SOD1, manganese-SOD (SOD2) (193;194) and EC-SOD (SOD3) (195). These three enzymes are located in different compartments, with SOD1 being responsible for the cytosol (194), the nucleus (193;194;196;197) and the intermembrane space (IMS) of mitochondria (193;198), whereas SOD2 is located in the mitochondrial matrix (194) and SOD3 is secreted into the extracellular space (195;199). The subcellular distribution of SOD1 is, however, controversial and the subject of discussions involving other compartments such as peroxisomes (196;200;201) and the endoplasmic reticulum (ER) (198;202;203). As the mitochondrion is one of the main sites for superoxide production (204) it is not surprising that the matrix has its own enzyme in combination with protection from SOD1 in the IMS. Deletion of SOD2 in mice has also been shown to be lethal during the first days of life (205;206). SOD1 knock-out mice, although not completely healthy (e.g. reduced fertility, axonal repair deficiencies, increased oxidative stress, cataract), do not display any severe phenotype (207-212). Similar to knock-out of SOD1, knock-out of SOD3 has no obvious spontaneous phenotype (212;213). This is apparently caused by adaptions in the germ-line SOD3 knock-outs. Partial conditional knock-out of SOD3 in the lungs of adult mice leads to death from lung failure in just a few days (214). The relatively mild phenotype of 16 germ-line SOD1 knock-out mice might also be explained by compensatory adaptions. Structure and properties. SOD1 is composed of two equivalent 153-aa long subunits, each containing one Cu2+ ion and one Zn2+ ion, with a molecular weight of roughly 16 kDa (whereby the enzyme is also known as Cu, Zn-SOD). The gene is located on chromosome 21q22.11. SOD1 is widely expressed, with the highest amounts in the liver and kidneys (215). The core of the SOD1 structure is eight β-strands connected through seven loops and arranged as a β-barrel (216;217). As mentioned, the Cu2+ ion is involved in the dismutase reaction but is also important for structure/folding, whereas the Zn2+ ion functions as a stabilizer (218). Four histidines (His46, His48, His63, and His120) are the ligands for the binding of copper and His63, His71, His80, and Asp83 are the ligands for Zn (219). Many ligands are present in the Zn-binding loop IV (His48-Asp83). Apart from the binding of the metal ions, the tertiary structure of the monomer is stabilized through an intrasubunit disulfide bond between Cys57 and Cys146. The presence of a structural disulfide bond is an unusual property of a protein located in the reducing environment of the cytosol, and it may be an Achilles heel of SOD1. There is a specific copper chaperone (copper chaperone for SOD1, CCS), responsible for Cu loading of SOD1 in eukaryotes (220). CCS knock-out mice were shown to display reduced SOD1 activity and a phenotype similar to that seen in SOD1 deficiency (e.g. reduced fertility) (221), but other experiments have shown that human SOD1 can be Cu-loaded to some extent by other means (222). SOD1 is one of the most stable proteins known, and it retains most of its activity in 10 M urea, 4% SDS, and heating to 80°C (218;223). This stability is totally lost in the apo state, when the protein is deprived of metals and upon reduction of the intrasubunit disulfide bond between cysteines 57 and 146. After loss of metals and reduction of the disulfide bond, SOD1 is unable to dimerize and the monomeric form is favored (224-226). SOD1 in ALS As discussed above, the number of SOD1 mutations found in ALS cases is steadily increasing and today mutations are known in approximately half of the codons (74 out of 153). Most cases are missense mutations, but insertion and deletion mutations exist and for the purposes of this thesis, the truncation mutation G127insTGGG (G127X) serves as an example (discussed below). The mutations confer widely different effects on the SOD1 molecule, with some—such as G127X—causing a severely truncated protein without SOD activity and loss of Cu/Zn binding and normal folding, whereas the common D90A mutant is fully active and properly folded. Despite such diversity, these mutations result in a similar disease phenotype, indicating the existence of an ALS-causing common toxic SOD1 species. 17 When caused by mutated SOD1, the disease is generally dominantly inherited with varying penetrance. However, the most common of the mutations, D90A, causes mostly recessively inherited ALS (39). Interestingly, this mutation has been found to be inherited as a dominant trait among FALS and SALS cases in populations where the mutation is rare (227). About 20% of FALS has been reported to be caused by mutations in SOD1, which represents about 2% of all cases (223;228). In a study of a mixed population of ALS patients, the frequency of SOD1 mutations was found to be 7.2% (38). This is in accordance with other findings of rather frequent SOD1 mutations in apparently sporadic ALS (229-231). The phenotype of mutant SOD1 ALS is generally that of typical ALS, although some mutations are known to be associated with atypical features such as long-term survival and bladder control disturbances (e.g. D90A) (231). The similarities between mutant SOD1 ALS and typical ALS have drawn attention to the wild-type SOD1 protein, and recent experiments suggest that SOD1 may be involved in the majority of cases (232-238). Transgenic murine SOD1 models Since taking CNS tissue samples from living ALS patients is associated with great risks, the need for model systems has been obvious. Different cell lines expressing various mutant SOD1s are routinely used, but since the spinal cord is a very complex tissue with multiple cell types, animal models are often needed. Mice have a highly developed CNS that is in many ways similar to ours, and genetic techniques have been used successfully for many years in this species. Since the discovery of mutant SOD1-induced ALS, various transgenic murine models have emerged. By far the most frequently used one is the G93A mouse, which was first described in 1994 (239). The widespread use of this model is mainly due to the conveniently short survival time (about 125 days, depending on the copy number) and the predictable course of the disease. These characteristics have made it the model of choice for pharmacological trials and virtually all potential therapies have been tested in G93A. The poor reproducibility between preclinical G93A trials and human drug trials, including riluzole, has recently pulled into question the suitability of the model and the relevance of positive drug reports to date (240). From a pathological point of view, there are similarities with human disease, e.g. ubiquitin-staining inclusion bodies, motor neuron loss, and reactive gliosis. During disease progression, the G93A mouse develops overt vacuolar pathology of the CNS (241). These are speculated to be remnants of damaged mitochondria and are common to other “high-level” models (discussed below), but are rarely found in human ALS subjects. The G93A mutation has also been transgenically expressed in rats (Rattus norvegicus), a model similar in most ways to the mouse strains used (242). The next mutant mouse model to be published was that with the G85R mutation in 1997 (243). This model develops an ALS-like disease 18 within approximately one year, despite having a mutant SOD1 level no greater than the endogenous mSOD1 (i.e. low-level, as discussed below). Histopathologically these mice differ from the G93A strains, as they lack vacuoles and show SOD1 inclusions in astrocytes as well as in neurons. The G127X mouse was generated in our laboratory and the mice are now bred as homozygotes (35). This mutation was found in a Danish family and is an insertion of four novel nucleotides after the glycine 127 codon (Gly127insTGGG) (231). The insertion leads to five novel amino acids followed by a premature stop codon. This protein is present at very low levels, about half that in the wt mSOD1 situation. G127X mice live approximately 216 days and succumb to motor neuron disease after an extremely rapid disease progression (of less than one week). D90A mice were also generated in our laboratory and, similarly to G93A, produce high levels of mutant protein (234). Multiple mutant SOD1 transgenic mouse models exist, but they will not be discussed further here. Several lines of mice expressing wt hSOD1 have been generated (239;244;245). None of them have displayed an ALS phenotype, but pathologically some similar characteristics are apparent such as loss of neurons in the ventral horn, vacuolization of mitochondria, and late accumulation of aggregated SOD1 (234;246;247). There are several other animal models that express mutant SOD1s, e.g. Drosophila melanogaster (248), Caenorhabditis elegans (249), zebrafish (250), and pigs (unpublished). Although not extensively studied, these might prove to be important in new ways. Recently, canine degenerative myelopathy, a disease in dogs similar to human ALS, was found to be caused by a point mutation in the SOD1 gene (251). Pathological examination also revealed neuronal SOD1 inclusions similar to those seen in human mutant SOD1 ALS cases. How do mutant SOD1s cause disease? Loss of function It soon became obvious from multiple sources that loss of SOD1 function does not cause ALS. (i) SOD1 knock-out mice do not present an ALS phenotype. (ii) Transgenic overexpression of mutant SOD1 leads to ALS despite there being normal levels of endogenous wild-type enzyme. (iii) Transgenic overexpression of most mutations results in several times more functional SOD1 enzyme. (iv) Disease is almost always dominantly inherited, and some mutations do not reduce the total SOD activity (e.g. D90A). 19 Thus, mutations in SOD1 confer a toxic property to the protein, which is most likely of the same basic character for all mutants. A non cell-autonomous toxicity Several reports have suggested that the toxic effect of mutant SOD1 is noncell autonomous. This conclusion is based on the following findings: (I) Neuron-specific expression of mutant SOD1 does not generate an MND phenotype (252;253). (This was challenged recently; see below). (II) Astrocyte-specific expression of mutant SOD1 does not generate an MND phenotype (254). (III) Chimeric mice that have been generated from mice expressing wt and mutant SOD1 (mixed cell-types), develop motor neuron pathology but do not develop a symptomatic MND. In these mice, motor neuron pathology is dependent on a mutant SOD1 context (255). Another recent report has shown that selective expression of mutant SOD1 in motor neurons and oligodendrocytes surrounded by wild-type neurons results in a delayed onset of disease (256). (IV) Transplantation of wild-type myeloid cells into G93A mice was shown to slow down disease progression (257) and microglia expressing mutant SOD1 were shown to have higher production of ROS and nitric oxide than those expressing WTSOD1 (258). (V) During disease progression, an increasing amount of lymphocytes migrating into the CNS has been reported (259) and the embryonic knock-out of T-lymphocytes in G93A mice has shown an accelerated disease progression (259;260). (VI) Extracellular mutant SOD1 has been shown to be toxic to motor neuronal cells only when co-cultured with microglia (192) (VII) Removal of mutant SOD1 expression in neurons through the Cre/lox system resulted in delayed onset and extended survival but did not abolish MND (261). Using the same technique, mutant SOD1 expression was removed from microglia (261) and astrocytes (262), which slowed disease progression but did not alter the time of onset. (VIII) Somewhat contradictory, knock-down of mutant SOD1 expression in Schwann cells has been shown to enhance disease progression (263). 20 (IX) Deletion of mutant SOD1 expression in muscle does not alter the phenotype, suggesting that muscle cells is not involved in the disease (264). (X) The expression of mutant SOD1 in glial-type cells has been shown to induce apoptotic mechansims in co-cultured neuroblastoma cells and embryonal spinal motor neurons from mutant SOD1 transgenic mice (265). Co-culture of wt SOD1 motor neurons and G93A glial cells from differentiated embryonic stem cells has resulted in motor neuron damage, indicating a soluble factor (266-268). It has also been shown that mutant SOD1 can interact with chromogranins and thereby be secreted to the extracellular compartment (269). Recently, it has been shown that neuron-specific expression of high levels of SOD1 is indeed able to produce an MND phenotype in transgenic mice (270). Similar findings have been reported elsewhere (271). These results argue against the initial findings (I) and they probably depend on the level of expression. In combination, these results argue that multiple cell types present in motor areas modulate disease through different parameters such as disease onset and progression. Although neuron-specific expression of mutant SOD1 might be sufficient, the natural form of the disease is probably dependent in different ways on interactions between most cells of the CNS. Aberrant redox chemistry and peroxidase activity During the first decade of mutant SOD1 studies, it was speculated whether mutant SOD1 could cause ALS through altered redox chemistry. It was suggested that the active site copper became increasingly exposed and could therefore participate in reactions with peroxynitrite and induce damage to proteins by tyrosine nitration (272-274). Another similar theory suggested that mutant SOD1 could catalyze copper-mediated conversion of hydrogen peroxide to hydroxyl radicals, which—like peroxynitrite—is one of the most reactive ROS (275). Other data contradict these theories. Several SOD1 mutations have either been shown or predicted to be deficient in copper binding and to lack activity (35;243;276;277). An artificial mutant lacking all four copper-binding histidines can still cause disease in transgenic mice (278). In line with this, the knock-out of CCS did not ameliorate the MND phenotype in mutant SOD1 mice despite substantial loss of SOD1 copper binding (279). This theory has not been completely discarded, however, and recently suggestions have been made that metal-deficient SOD1 can bind copper or other transition metals in the zinc site, and thereby take part in aberrant redox reactions (223;280). Recent studies have also suggested that the excessive nitration of proteins, found in the G93A mouse model, may contribute to disease (281). 21 SOD1 and mitochondria As already mentioned, SOD1 was shown in the early 1970s to be localized not only in the cytosol, but also in the intermembrane space (IMS) of mitochondria (193). This partioning was due to an unknown mechanism, as SOD1 does not contain a mitochondrial localization sequence. It was later shown that SOD1 is translated in the cytosol and imported in a disulfidereduced state, without any metals bound (the apo state) (282;283). It was also shown that CCS is present in the IMS to aid apo-SOD1 turn into mature holo-SOD1, and thereby to get trapped in this compartment (282;284). The mitochondrion is the major cellular site for energy production through oxidative phosphorylation, and therefore also a potential site of generation of oxidative free radicals (204). As mentioned, Mn-SOD is present in the mitochondrial matrix (193) for scavenging of free radicals and it was therefore not surprising that SOD1 was found to be localized in the IMS, probably for the same purpose. It has been shown that knock-out of SOD1 leads to increased carbonyl levels in mitochondria, which can be used as a marker of increased oxidative stress (284). The first reports on mitochondrial pathology in ALS came from studies on autopsy specimens and from functional measurements on peripheral tissues of patients (reviewed in (285)). Later, similar studies carried out on CNS tissue have revealed aggregated, swollen mitochondria and functional deficits (286-289). This has, however, not been a uniform feature of several other reports (290-292). Autopsy studies are also subject to several drawbacks, such as the issue of post-mortem time, tissue preservation, and the fact that only end-stage disease can be studied (285). The real take-off in the subject of mitochondrial pathogenesis came with the first reports of severe vacuolization of mitochondria present in the early lines of mutant SOD1 transgenic mice (241;244). These vacuoles were later shown by electron microscopy to be derived from expansion of the IMS evident before the onset of symptoms (293-296). The process of vacuolization was described by Higgins et al. to be distinct from earlier reported vacuole processes (mitochondrial permeability transition (MPT) or autophagy-derived), and it was referred to as Mitochondrial Vacuolation by Intermembrane Space Expansion (293). It was also found that mutant SOD1 accumulates in vacuolated mitochondria and co-localizes with cytochrome C, suggesting a direct toxicity through interaction (293;294). This idea is, however, challenged by the findings that mice overexpressing wt hSOD1 also show vacuolar pathology and accumulation of wt hSOD1 protein without any signs of ALS disease (234;241;244;246;247;294). Other work has shown that not all SOD1 mouse models, e.g. the G85R and H46R (243;297), display this pathology and that it is rarely found in autopsy samples (298). In line with the mitochondrial pathology, several reports have indicated that expression of mutant SOD1 in both cells and transgenic mice 22 results in alterations in mitochondrial function (299-302) similar to what was originally found in human subjects. After these initial findings of mitochondrial pathology, dysfunction, and direct association with SOD1, the obvious question was how mutant SOD1 might be toxic to the organelle. Several somewhat divergent mechanisms of toxicity have been suggested. These are not mutually exclusive but are more or less linked. (I) Mitochondrial accumulation of SOD1 protein aggregates. Several studies have shown that mutant SOD1, and under certain circumstances wt SOD1, can oligomerize and form protein aggregates on the outer surface of the outer mitochondrial membrane or in the IMS (293;303-308). Another report suggested that mutant SOD1 could aggregate in the mitochondrial matrix (309). Although not a well-defined mechanism, the idea of SOD1 that accumulates in mitochondria being toxic was supported by the report that overexpression of CCS along with mutant SOD1 in transgenic mice induced an even heavier mitochondrial load of SOD1 followed by a remarkable decrease in survival time (310). These findings are the subject of some debate due to findings by us (extensively discussed in paper 1) and others showing that mutant SOD1s appear to accumulate (to a relative level higher than mSOD1) in mitochondria in vivo only if the total cellular level of mutant protein is extremely high (e.g. the G93Amouse model) (297). (II) Mitochondrial release of apoptotic molecules. As mitochondria are important in the regulation of apoptotic pathways, these have been studied in several reports. Cytochrome C is a mitochondrial IMS protein that, upon mitochondrial release, induces apoptosis through activation of cellular caspases. The discovery of mutant SOD1 and cytochrome C co-localization, (293;294) was followed by reports on its cytosolic release (311-313). Cozzolino et al. showed that mutant SOD1 expression caused apoptosis through a pathway involving Apaf1, a scaffold protein of the apoptosome (a protein complex involved in activation of caspases) (311). When Apaf1 was knocked out, apoptosis and mitochondrial pathology was abolished despite similar SOD1 mitochondrial accumulation. Mutant SOD1 has also been shown to interact directly with the anti-apoptotic protein Bcl-2 and, through co-aggregation, to deplete cells of this important function (304). This finding has not, however, been reproduced by another group (314). These and other findings of increase in apoptotic mediators have been partly challenged by experiments in transgenic mice expressing mutant SOD1 in which different important apoptotic mediators have been knocked out: caspase-11 (315) and BAX (314). Both of these mouse models develop an ALS phenotype despite the inhibition of apoptotic pathways. Remarkably, BAX knock-out mice were found to develop motor neuronal disease without loss of motor neuronal cell bodies in the spinal cord. If apoptosis is involved in motor neuronal death in ALS, clearly apoptotic routes other than these must be involved. 23 (III) Increased production of reactive oxygen species (ROS). There have been several reports on increase in oxidative stress and ROS produced from mitochondria following expression of mutant SOD1s (300-302;306;316318). The precise source of ROS has not been established, but mutant SOD1 expression appears to inhibit the normal function of the electron transport chain, either through direct interaction/aggregation or through secondary effects, generating a higher leakage of free electrons and thereby more ROS (discussed above). Increase in ROS triggers several pathways such as cytochrome C release/apoptosis, induces damage to mitochondrial DNA, causes protein modifications/alterations, and stimulates nuclear transcription of genes involved in protection and inflammation (285;287;318-323). (IV) Mitochondrial damage through cytosolic/non-cell autonomous events. It has been suggested that binding of mutant SOD1 to proteins in the cytosol, such as heat shock proteins (HSPs), causes depletion of and toxicity to mitochondria (283;285;324). SOD1 has been shown to interact with a lysyl-tRNA synthetase (mitoKARS) in the cytosol and to co-aggregate at the cytoplasmic face of the mitochondrial outer membrane, thus contributing to mitochondrial dysfunction (325). Recently, it has been shown that selective expression of mutant SOD1 in astrocytes leads to mitochondrial pathology and dysfunction in motor neurons, indicating that the toxicity can be transferred from one cell to another (non-cell autonomous), possibly through oxidative stress (326;327). Cassina et al. also showed that inhibition of mitochondrial function in astrocytes caused by means other than mutant SOD1 resulted in similar toxicity to motor neurons. (V) Excitotoxicity. Excitotoxicity through extracellular glutamate signaling (discussed above) is a well-studied pathogenic mechanism in both in vivo and in vitro models (285). Mitochondrial dysfunction may enhance the sensitivity to this assault even more in motor neurons than in other cells (285;328). Mitochondria are the main storage site of intracellular Ca2+, and dysfunction of Ca2+ regulation seems to be important for excitotoxicity (329). Defects in intracellular Ca2+ regulation have been found in SOD1 mutant mice (330) and cell models (299;331). (VI) Impairment of axonal transport. In motor neurons, adequate anterograde and retrograde transport of mitochondria is necessary for cellular function and energy metabolism all through their extremely long axons. This is achieved through interaction with motor proteins such as kinesin/dynein and movement along microtubules (332). There is recent evidence to suggest that these movements are inhibited either by mitochondrial dysfunction or through dysfunction of the transport systems (reviewed in (332)). Disturbance of mitochondrial transport has been reported in SOD1 transgenic mice (333;334) as well as in SALS patients (289). This also suggests functional links between SOD1 toxicity and mutations in dynactin (as discussed above). Furthermore, mutant SOD1 has 24 recently been shown to co-aggregate with and possibly cause depletion of components of the anterograde transport system (335). SOD1 aggregation and inclusions As mentioned above, CNS tissues from FALS patients with mutant SOD1 and transgenic murine models show inclusions with SOD1 immunoreactivity (36;336). The formation of cellular inclusions could be toxic as they may, for example, trap essential proteins and block or interfere with cellular functions (283;335). There is no consensus on how mutant SOD1 ends up in inclusions, but most of the evidence points to impaired degradation, misfolding, and aggregation (32;337;338). SOD1 degradation. Although the routes of protein degradation in cells of the CNS have not been fully worked out, proteasomal degradation and autophagic/lysosomal degradation appear to be the two main pathways (339). These systems are partly complementary, but the ubiquitinproteasome system (UPS) is mainly responsible for degradation of ubiquitintagged soluble cytosolic proteins and the lysosome for endocytosed or autophagocytosed proteins (339). Wild-type and mutant SOD1 has been shown to be degraded by the UPS (337;340-343). Important mediators in this degradation have been found to be the chaperones HSP70/HSC70 and the ubiquitin E3-ligases CHIP, dorfin, and NEDL1 (342;344-347). Inhibition of the UPS accelerates aggregation of mutant SOD1 and formation of cellular SOD1 immunoreactive (ir) inclusions in cell models and tissue cultures with similarities to those found in transgenic mice and FALS patients (337;343;348-350). Further on, SOD1ir inclusions co-stain for ubiquitin, chaperones, and proteasomal components (336;342), and SOD1 has been shown to be oligo-ubiquitinated in cell models (351) and in vivo (35;352). Apart from the constitutive proteasome, there is an inducible form called the immunoproteasome (353). The activities of these two entities have been measured in tissues from mutant SOD1 transgenic mice, and it appears that at least the constitutive proteasome is inhibited (354-356). Reduced proteasomal activity has also been found in cells expressing mutant SOD1 (351) as well as in mice overexpressing wild-type protein (357). Although the proteasomal route to SOD1 degradation seems important, its involvement in ALS pathogenesis is not clear. Transgenic mice expressing G93A SOD1 have been crossed with mice that are deficient in the immunoproteasome (LMP2 -/-) (358). This model did not show a significant alteration in phenotype. Furthermore, inhibition of proteasomal function in organo-typical spinal cord cultures showed no difference in survival of motor neurons between those expressing mutant SOD1 and those from normal tissue (359). 25 Our knowledge of the role of autophagy in degradation of proteins of the CNS was given a remarkable turnabout when mice deficient in this pathway (atg7, atg5 -/-) were found to show severe neurodegeneration (360;361). The disease was associated with neuronal pathology at several levels, with protein aggregates and inclusion bodies. It was later shown that SOD1 is partly degraded by macroautophagy (362) and that this route is altered in G93A mice (363;364). Ligation of ubiquitin monomers to proteins targeted for autophagic degradation is mediated by lysine 63 (K63) on ubiquitin. When a ubiquitin K63 mutant was co-expressed with mutant SOD1, it resulted in generation of more cellular inclusions (365). Misfolding/aggregation. The formation of cellular inclusions and SOD1 aggregates could thus be caused by deficiencies in degradation and cellular overloading. Other theories and complementary ones suggest that SOD1 aggregation is caused by misfolding of mutant SOD1 monomers either in combination with deficient degradation, loss of folding of chaperones, or by intrinsic aggregation—through aberrant disulfide coupling, for example. In vitro studies of wt apo-SOD1 have shown that it can aggregate even under physiological conditions (366;367), although the wt holo protein is regarded as an extremely stable protein. These propensities to misfold may be aggravated in mutant SOD1 (368-370). For nucleation of aggregation, the least common denominator is disulfide-reduced, non-metallated SOD1 (367;367;367;371;372). Similar species have been identified in vivo, in a series of transgenic mouse models (247;373). Although intermolecular disulfide bonding exists in SOD1 aggregates both in vitro and in vivo (247;366;374;375), resulting in SOD1 oligomers, mutation of all four cysteines does not prevent SOD1 aggregation (371;376). C57-C146 disulfide-reduced SOD1 has also recently been shown to be the predominant species in detergent-resistant aggregates (375). This highlights the importance of this in vivo phenomenon and indicates that nucleation and building of SOD1 aggregates derives from the constant pool of soluble, disulfide-reduced apo-SOD1 (discussed in paper III). The composition of SOD1ir inclusions has mainly been explored by histochemical approaches in transgenic mice and FALS patients. Co-staining of inclusions has been found for a number of proteins such as CCS, heat shock proteins, ER chaperones, neurofilaments, kinesin-associated protein 3, p38MAPK (202;298;335;336;377;378), and parts of the UPS mentioned above (discussed in paper III). Analysis of detergent-resistant aggregates has revealed similar proteins such as different heat shock proteins (278;281;324;379). Proteomic approaches have recently emerged as a promising tool for identification of aggregate composition, and they will be discussed below. Although it is clear that SOD1 aggregates/inclusions are present in most murine models and ALS cases carrying SOD1 mutations, it is not clear whether they are a toxic prerequisite. In G93A mice, overexpression of CCS 26 has resulted in a more aggressive phenotype without any signs of SOD1ir inclusions (310). Other experiments, on inhibition of proteasomal function in mutant SOD1 cell models for example, have induced SOD1 aggregation but have not resulted in more pronounced cell death than in controls (380). SOD1-interacting heat shock proteins. Apart from the proteins found to be co-aggregated with SOD1 in inclusions, a number of proteins have been found to interact with SOD1, through co-immunoprecipitation experiments for example. These are mainly proteins from the family of heat shock proteins (HSPs), a group of proteins involved in stress responses and protein folding. The most frequently reported SOD1 interaction partner has been HSP70/HSC70 (283;324;344;345). As mentioned above, HSP70 has been proposed to bind misfolded SOD1 and mediate degradation or possibly refolding. The initial reports stated that this interaction was indeed confined to mutant SOD1, but later experiments have shown that oxidized wild-type enzyme acquires mutant-like properties and binds HSP70 (232). Other HSPs found to interact with SOD1 are HSP105, HSP90, HSP40, HSP25, and αβcrystallin (283;324;344;381). The heat shock response is crucial for the cellular response to misfolded proteins, and it has been reported that these pathways may be deficient in SOD1 transgenic mice (382). Furthermore, the interaction of HSPs and SOD1 has been suggested to be the reason for cellular depletion of chaperones, as they may be trapped in inclusions (283). In this way, the initially beneficial SOD1/HSP interaction eventually leads to loss of important functions and a vicious circle arises through generation of more misfolded proteins. From the above-mentioned experiments, it was suggested that induction, overexpression, or exogenous delivery of HSPs could be beneficial in ALS. Overexpression of HSP70 in cultured cells has been shown to reduce the amount of insoluble SOD1 aggregates (349) but not to ameliorate lifespan in SOD1 transgenic mice (383). Administration of recombinant HSP70 intraperitoneally in SOD1 transgenic mice has been shown to be beneficial when treatment is started before the onset of disease (384). In 2004, Kieran and colleagues reported that treatment with arimoclomol, a co-inducer of heat shock proteins, significantly ameliorated the disease in SOD1 transgenic mice (385). Although arimoclomol induces expression of several HSPs, increases in HSP70 and 90 were the most pronounced. This treatment has also been shown to reduce the number of ubiquitin-positive inclusions (386). To be effective in survival, treatment has to be started before or very early after onset of disease, which of course is difficult to achieve in human ALS subjects (385;386). Still, this is one of the most promising therapies and human trials are ongoing (387). 27 The “–omics” in ALS/SOD1 models The expanding field of proteomic and genomic experimental methods has introduced new ways of exploring pathogenic events without input of prior knowledge. In genomics, microarrays confer the possibility of exploring gene expression patterns in practically the whole genome—during progression of a disease, for example. Proteomic techniques are still more limited, but they may be more reliable since mRNA levels do not always reflect the amount of protein finally produced. Genomics also misses the whole area of post-translational modification of proteins. This and other technical discrepancies have led to partly conflicting results between genomic and proteomic findings. These issues will probably be solved some time in the future and the different approaches shown to complement each other. Here, will follow a brief review of “global” genomic and proteomic experiments performed in the context of ALS, i.e. those involving total changes in a tissue or cell. Genomics In recent years, several gene-expression studies using the microarray technique have been performed both in transgenic mice (321;388-392) and on autopsy material from human ALS cases (393-397). One serious drawback of autopsy material is the difficulties associated with post-mortem time and major cellular/mRNA alterations that take place after death. While the early studies were performed on whole spinal cord tissue or parts thereof (motor cortex in (396;397)) with a wide range of cell types, more recent genomic studies have been specifically performed on motor neurons collected by laser-capture techniques. As all of these studies have been highly individual in terms of the techniques and models used, the findings have been quite diverse regarding alterations in individual genes. The focus here must be to find similarities in expressional regulation of pathways and functional classes of genes. Most studies have found alterations in mitochondria, in oxidative stress, in the citric acid cycle, and in neurotransmission (GABA and glutamate metabolism). The effect on energy metabolism appears to be a common denominator. Proteomics A series of global proteomic studies have been performed in slightly different murine models of ALS. In a study on pooled homogenates from terminal G93A mice, Strey et al. found seven proteins to be differentially regulated compared to non-transgenic mice and mice expressing wt hSOD1 (398). These protein alterations were not present in asymptomatic mice. Atkin et al. performed a study at disease onset in the G93A rat model (202). Out of 15 proteins that were altered, 12 were identified of which some were ER chaperones involved in the unfolded protein response (UPR). In a study 28 comparing G93A mice to transgenic mice expressing wt hSOD1, Massignan et al. reported a mutant-specific change in 15 proteins in presymptomatic mice (399). Similarly to the study by Atkin et al., they found upregulation of the ER chaperone protein disulfide isomerase (PDI). However, most alterations were in proteins that are involved in metabolism, particularly from mitochondria. Lukas et al. performed a similar study on presymptomatic and terminal G93A mice but also included non-transgenic mice as controls (400). Using fractionation of spinal cord homogenates and LC-MS/MS, they compared the proteome from different fractions on the basis of functional categories (gene ontology analysis). This was preferred, since LC-MS generates semi-quantitative data and the alterations in individual proteins become less reliable. Interestingly, they performed a set of microarray experiments for comparison of proteome and gene expression data in the same model. As in other reports, the most obvious alterations were found in mitochondrial pathways, results that were not, however, corroborated by gene expression analysis. A study looking for differentially expressed proteins in the hippocampus of mice overexpressing wt hSOD1 found changes in 41 out of 157 proteins identified (401). This model has a high steady-state level of SOD1, much like the G93A model, and does develop loss of motor neurons but not overt symptoms. Several of the differentially expressed proteins involved mitochondria and the cytoskeleton. Using a cell model expressing different mutant and wt hSOD1s, with proteomic techniques Di Poto et al. found 17 proteins to be differentially regulated (402). Most proteins were involved in cellular metabolism and antioxidant defense. The most serious drawback of studies with cell lines may be the lack of a diverse cellular context, which in recent years has been shown to be important for motor neuronal disease (403) (discussed above). Proteomic methods have also been used for screening of potential biomarkers in cerebrospinal fluid and plasma (404-409). Although no consensus biomarker has been found, cystatin C may be a candidate as its potential as a biomarker has been able to replicate in the CSF of ALS patients. Proteomics on SOD1-interacting proteins Different methods have been used to try to identify SOD1 interaction partners. Proteomic methods have recently given new insights in this area. With the addition of a chemical cross-linker in lysates from mutant SOD1 expressing cells, Zhang et al. were able to immunoprecipitate and—by proteomic methods (LC-MS/MS)—identify SOD1 interaction partners (410). Interestingly, apart from known partners such as CCS and HSP70, the heavy chain of dynein was implicated, thus linking mutant SOD1 with dysfunction of motor proteins as suggested previously. Using a similar strategy, Watanabe et al. used transgenic mice expressing FLAG-tagged truncated 29 SOD1 for immunoprecipitation and identification of SOD1 interaction partners by LC-MS/MS (411). Thirty proteins were found to co-precipitate with SOD1 in mutant mice, whereas only a few were present in homogenates from wild-type expressing or non-transgenic mice. Wang and colleagues immunoprecipitated soluble and insoluble fractions containing mutant SOD1 (G85R coupled to yellow fluorescent protein) from murine spinal cord (379). The soluble fractions contained CCS and various HSPs, whereas the insoluble fractions consisted mainly of cytoskeletal components. In a similar study on spinal cords from terminal G93A mice, proteins in detergentresistant (insoluble) pellets were analyzed with 2-dimensional gels and MALDI-TOF (281). They found 42 protein spots selectively accumulated in insoluble pellets from G93A mice, as compared to a strain expressing wt hSOD1. Shaw et al. performed a study on detergent-insoluble aggregates and immunoprecipitated SOD1 aggregates for identification of co-aggregation proteins in mutant SOD1 transgenic mice with different characteristics (412). The only other protein found with reasonable consistency was vimentin, suggesting that SOD1 was the main constituent of these species. Metabolomics In the search for possible biomarkers of ALS disease, the expanding field of metabolomics may be more promising than genomic and proteomic methods. These new methods deal with quantification and analysis of small molecules (of 60–1,000 Da), such as peptides and degradation products, derived from accessible extracellular fluids. Work in identifying biomarkers in ALS is in progress (413). 30 Aims • To determine whether ALS can be ascribed to toxic effects of mutant SOD1 accumulating in mitochondria. Are different mutant SOD1 species, like the truncated G127X protein, uniformly present in mitochondria? (paper I) • To relate tissue vulnerability to distribution of the defective, truncated G127X mutant SOD1. Are the toxic properties of mutant SOD1 expression confined to the CNS in human ALS? (paper II) • To determine the composition of SOD1 inclusions in the spinal cord of mutant SOD1 transgenic mice and the properties of SOD1 protein trapped in them (paper III) • To gain insight into ALS pathogenesis from analysis of changes in individual protein levels in the spinal cord of SOD1 transgenic mice at onset of disease. (paper IV). 31 Methods SOD1 transgenic mice A variety of transgenic mouse models of ALS were used in the different experiments discussed here. The high-expressing G93A mutant line (G93AGur) was obtained from the Jackson laboratory, as were the mice expressing wt hSOD1. The mouse strain expressing the G85R mutant was obtained from Dr D.W. Cleveland. G127X and D90A mice were generated in our laboratory (discussed above). Mice were deemed terminally ill when, owing to limb weakness, they could no longer reach for food in the cages. This occurred for the D90A, G85R, G127X, and G93A mice at an average age of 407, 345, 216, and 124 days, respectively. The presymptomatic D90A, G85R, and G127X mice used were around 100 days old, whereas the G93A mice were around 50 days old. For the experiments in paper IV, mice were collected at their peak body weight. This occurred at day 195 ± 14 for the G127X mice and at day 325 ± 22 for G85R mice. As controls, we used non-transgenic littermates from the G93A and D90A breeding program, or occasionally pure C57BL/6JBomTac (B6) mice that were used for all breeding. G127X patient With a post-mortem delay of only 10 h, CNS and peripheral tissue from a Danish patient carrying the G127X mutation were quick-frozen at –80°C or stored in formalin after the post-mortem examination. This patient had showed rapidly progressing disease with truncal-thoracic onset and a survival time of 6 months. Homogenization of tissue Generally, tissues were homogenized in 25 volumes of appropriate buffer. In all experiments on organelles and inclusions with density gradient separations, an iso-osmotic buffer was used (mitochondrial buffer, consisting of 0.21 M mannitol, 0.11 M sucrose, 10 mM K HEPES, pH 7.2, 1 mM EGTA, and the Complete antiproteolytic cocktail without EDTA (Roche Diagnostics, Basel, Switzerland)). Tissues for immunoblot were supplied with 25 volumes of PBS (10 mM K phosphate, pH 7.0, 0.15 M NaCl and EDTA-free Complete antiproteolytic cocktail). For discrimination between disulfide-reduced and oxidized SOD1, the homogenization buffer was 32 supplied with 40 mM iodoacetamide. For 2-D gels, the spinal cords were homogenized in 10 volumes of lysis buffer (1 M Tris-HCl, pH 8.5, 2 M thiourea, 7 M urea, and 4% CHAPS). For 2-D gels and immunoblot, tissue was homogenized using an Ultraturrax (IKA, Stufen, Germany) followed by sonication using a Sonifier Cell Disruptor (Branson, Danbury, CT) for 30 s. For the preservation of organelles and separation of inclusions, a more gentle approach using dounce glass homogenizers was used. Density gradient separation Separation of organelles from tissue extracts can be performed in different ways, although density gradient centrifugation is the most commonly used method. Other methods involve gel filtration and cell sorting/flow cytometry. For density gradient separations, sucrose solutions have been the medium that is traditionally used. The advantage has mainly been low cost and availability, but for the separation of organelles the hypertonic characteristics of sucrose gradients have been a great disadvantage. This causes organelles to shrink due to osmotic water loss and affects their density (414;415). As organelles such as lysosomes, mitochondria, and peroxisomes exhibit very similar densities, hypertonic gradients make them inseparable. Furthermore, the high viscosity of sucrose solutions requires long centrifugation times at high speed. The introduction of non-ionic, iodinated, high-density substances developed as contrast media for X-ray purposes, such as metrizoic acid (415;416), brought a great improvement to organelle separation. The latest addition, iodixanol (5,5’-[(2-hydroxy-1-3propanediyl)-bis (acetylamino)] bis [N,N’bis (2,3-dihydroxypropyl)-2,4,6triiodo-1,3-benzenecarboxamide]) has a molecular weight of 1,550 Da and a density of 2.08 g/ml combined with a relatively low viscosity and low osmolarity (417). These characteristics preserve organelle morphology and increase the resolution of particles of similar density. Also, iodixanol is compatible with antibody-based and spectrophotometric assays. Density gradients are generally used as discontinuous gradients or continuous gradients. Discontinuous gradients are made with a series of different concentrations (at least two) of the medium that are put on top of each other. In this way, particles of similar density are trapped at the interfaces between gradient steps and can be easily identified. The major drawback in organelle separation is the overlap in density distribution between different organelles or other subcellular particles. A well-known example is the similarity in buoyant density between mitochondria and lysosomes from liver tissue. The problem can be partially avoided by the use of continuous gradients. In this case, the gradients are premade, more or less linearly, depending on the method used. We used the “two-chamber” technique, which is simple and highly reproducible (418). The two chambers are loaded with iodixanol diluted to the two appropriate densities representing the higher and lower 33 ends of the density range; here we used 12% and 38%. The chambers are connected at the bottom, which allows passage of the denser medium into the chamber of the lighter. A peristaltic pump draws the mixed medium from the chamber of the lighter medium and delivers it to the bottom of a centrifuge tube. During this process, the denser medium is mixed consecutively with the lighter medium, bringing a denser mix to underlay the medium already present (Figure 1). Figure 1. Two-chamber set-up used for making linear gradients. The preformed gradient is close to linear and the centrifugation will not affect the slope in any significant way, at least during the short centrifugation times used here. Although vertical and fixed-angle rotors are efficient for gradient separation, swinging-bucket rotors are traditionally used as the effect on the preformed gradient is minimal. These also minimize the risk of material adhering to the walls of the tube, and they were therefore used for all gradient separations. For our separations, a 9-ml gradient was produced with a linear density covering 1.07–1.21 g/ml using 12–38% iodixanol in mitochondrial buffer. Homogenates were passed though a nylon filter of 5-µm pore size by centrifugation at 20 g for 5–10 min. Filtered homogenates were supplied with iodixanol to a final concentration of 8% to minimize the formation of a thick lipid layer at the homogenate/gradient interface, and loaded on top of the gradient. Centrifugation was carried out at 100,000 g for 90 min at 4°C and fractions were collected from the bottom of the tube. To separate mitochondria from peroxisomes, the homogenate was supplied with 6 mM CaCl2, 5 mM succinate, and 5 mM K2HPO4 prior to separation in order to cause permeability transition pore opening and swelling of the mitochondria. Marker enzyme assays For determination of organelle separation in density gradients, enzymatic activities or protein content (by immunoblot) of certain compartmentspecific markers were measured. For mitochondria, the activity of succinate 34 dehydrogenase (SDH) was determined. Samples were incubated with 100 mM Na HEPES, pH 7.4, 1 mM EDTA, 10 mM succinic acid, and 0.08% 3(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) for 90 min. To stop the reaction, three volumes of 2-propanol were added, followed by centrifugation at 14,000 g for 10 min. The absorbance of the supernatants at 550 nm was then measured. For detection of lysosomes, alkaline phosphatase activity was determined with p-nitrophenyl phosphate as substrate. The activity of lactate dehydrogenase (LD) was measured using the CytoTox96 kit (Promega, Madison, WI) and used as a cytosolic marker. The distribution of other organelles was determined by immunoblotting. Dot-blot assay A filter-trap assay for aggregated proteins was used in papers I and III, somewhat modified from that originally described by Wang et al. (419). Fractions from separations were filtered through 0.15- or 0.2-µm cellulose acetate membranes (Schleicher and Schuell, Dassel, Germany) in a dot-blot apparatus. In paper I, the homogenates and separated fractions were supplied with 0.1% SDS and gently sonicated before centrifugation and filtration. This increased the sensitivity of the assay 25-fold. After filtration, the membranes were subjected to immunoblotting. In paper III, the fractions from density separations were subjected to filtration without any prior treatment. Individual wells with trapped aggregates were then punched out and incubated in 1 × SDS-PAGE sample buffer for immunoblotting or DeStreak rehydration buffer (GE Healthcare) for 2-dimensional (2-D) electrophoresis. Antibody-based assays Immunoblotting Immunoblotting (western blots) were performed according to standard procedures. Samples were diluted with the same amount of 2 × SDS-PAGE sample buffer (100 mM Tris-HCl, pH 6.8, 10% β-mercaptoethanol, 20% glycerol, 4% SDS, and bromophenol blue) and separated on SDS-PAGE (Criterion™ Cell, Bio-Rad). When discrimination between C57-C146 disulfide-reduced and oxidized SOD1 was required (paper III), βmercaptoethanol was omitted from the sample buffer. Gels were blotted onto polyvinylidine difluoride (PVDF) membranes and probed with primary antibody. All secondary antibodies were conjugated with horseradish peroxidase and chemiluminescence was generated with the addition of ECL Advance substrate (GE Healthcare). Separated bands were visualized using film and digital imaging (Chemidoc XRS, Bio-Rad). For quantification of 35 hSOD1, wt hSOD1 with its concentration determined by quantitative amino acid analysis was used as original standard (420). Histopathology Tissues were fixed with 4% paraformaldehyde in phosphate-buffered salt solution (10 mM K phosphate, pH 7.4, 0.15 M NaCl), dehydrated and embedded in paraffin. Transverse paraffin sections were prepared for immunohistochemistry or stained with hematoxylin/eosin. In paper II, liver and kidney sections were stained with periodic acid-Schiff, van Gieson, and reticulin stains and liver also with periodic acid-Schiff diastase, Fe, and Faucet’s stain. For immunohistochemistry, enhancement using microwaveheating for 20 min in citrate buffer, pH 6.0, was performed and the sections were blocked for 30 min in goat sera prior to the staining procedures. Single immunohistochemistry was performed on the sections using the Ventana AEC (Ventana Medical Systems, Tucson, AZ) and alkaline phosphatase red immunohistochemistry system, according to the protocol of the manufacturer. Antibodies SOD1 antibodies. The primary antibodies used for detection of human and murine SOD1 were mainly polyclonal rabbit antibodies raised against keyhole limpet hemocyanin-coupled peptides corresponding to amino acids (aa) 24–39 (hSOD1-specific) or 24–36 (mSOD1-specific) (35). For analysis of the G127X protein in human tissue, an antibody raised against the novel C-terminus of the truncated protein was used (ADDLGGGQRWK, with new amino acids highlighted) (35). Commercial antibodies. The antibodies to lamin A/C, Tim23, and β-COP (Golgi β-coatomer protein) were obtained from BD Biosciences (Franklin Lakes, NJ), anti-PMP70 (70-kDa peroxisomal membrane protein) was from Affinity BioReagents (Golden, CO) or Zymed (San Francisco, CA), and the anti-synaptotagmin antibody was from Stressgen (Victoria, British Columbia, Canada). Antibodies to HSC70, isocitrate dehydrogenase, UCHL1, isopeptide, and CNP were from Abcam (Cambridge, UK), antibodies to Cop9s8 were from PTGlab (Chicago, IL), antibodies to FABP were from R&D Systems (Minneapolis, MN), and antibodies to FIS1 were from Biosite (Täby, Sweden). Anti-rabbit or anti-mouse IgG secondary antibodies were from GE Healthcare (Uppsala, Sweden), anti-goat IgG was from Santa Cruz (Santa Cruz, CA), anti-sheep IgG was from DakoCytomation (Glostrup, Denmark), and anti-rat IgG secondary antibodies were from Abcam (Cambridge, UK). 36 Size-exclusion chromatography Tissues were homogenized after the addition of 20 mM iodoacetamide using an Ultraturrax and sonication as described above. The iodoacetamide was added to alkylate free thiol groups in proteins and low-molecular-weight compounds. After centrifugation at 20,000 g for 30 min, the supernatants were subjected to chromatography in 1 cm × 30 cm Superdex 75 columns (GE Healthcare, Uppsala, Sweden) using PBS with 5 mM iodoacetamide. The elution rate was 45 ml/h, and the eluate was collected in 0.3-ml fractions. The SOD activities (421) and hSOD1 protein content of the fractions were determined. Proteomic techniques Differential in-gel electrophoresis (DIGE) DIGE uses the properties of different protein-binding fluorophores with similar mobilities on SDS-PAGE. At present, three different fluors are available, which makes it possible to separate two different samples along with an internal standard on every gel in a 2-D gel experiment. This minimizes the number of gels needed and the internal standard makes it possible to compare a great number of different samples from different gels. The procedure was performed according to personal communications and the handbook of the manufacturer (GE Healthcare). For each new DIGE experiment, the internal standard was generated by mixing the same amount of protein from all the animals included. Quantified homogenates were mixed with Cy2 (internal standard), Cy3, or Cy5 (0.25 mM in dimethyl formamide) and incubated on ice for 30 min in the dark. Samples from transgenic or control mice were labeled with either Cy3 or Cy5 in a random way, as suggested by the manufacturer. Reactions were quenched by addition of 1 µl of 10 mM lysine (Riedel-De Haën AG, Seelze, Germany) and incubated on ice. Cy2-, Cy3-, and Cy5-labeled samples were mixed, to be run on a single gel, and incubated with the same amount of 2 × sample buffer for DIGE (1 M Tris-HCl, pH 8.5, 2 M thiourea, 7 M urea, 4% CHAPS, and 40 mM DTT). 2-D electrophoresis DIGE samples were run on 24-cm pI 3-11NL or pI 4-7 strips (GE Healthcare, Uppsala, Sweden). The pooled samples were supplied with IPG buffer (GE Healthcare) and DeStreak rehydration buffer (GE Healthcare), and rehydrated into IPG strips for 12–16 h. Isoelectric focusing (IEF) was performed on a Protean IEF cell (Bio-Rad) at 20°C and maximum current was set to 50 µA/strip, with increase of the voltage stepwise until 35,000 or 45,000 Vh was reached (3-11NL and 4-7, respectively). Following IEF, the 37 strips were equilibrated for 15 min in equilibration buffer (6 M urea, 50 mM Tris-HCl, pH 8.8, 30% (v/v) glycerol, 2% SDS, and 0.002% bromophenol blue) containing 1% DTT and then for 15 min with 2.5% iodoacetamide in the same buffer. The second dimension was run using the Ettan DALTsix electrophoresis system (GE Healthcare) with low-fluorescence glass plates. In paper III, unlabeled samples were run on 11-cm pI 3-11NL strips and the Criterion™ Cell electrophoresis system (Bio-Rad) was used for the second dimension. For analysis, gels were post-stained with a silver stain (422). Briefly, the gels were fixed for 30 min in 40% methanol/10% acetic acid, sensitized (with 30% methanol, 0.1% sodium thiosulfate, and 6.8% sodium acetate), stained with silver nitrate (0.25% silver nitrate and 0.015% formaldehyde), developed (2.5% sodium carbonate and 0.03% formaldehyde), and the reaction was stopped in 1.46% disodium EDTA. For Coomassie staining, the gels were fixed in 10% methanol/7% acetic acid for 1 h. Staining was performed overnight with G-250 staining solution (0.8% phosphoric acid, 8% ammonium sulfate, 20% methanol, and 0.08% CBBG250), briefly washed with 25% methanol, and stored in deionised water. Computer analysis CyDye-labeled DIGE gels were scanned in a Typhoon 9410 imager (GE Healthcare) using excitation and emission filters specified by the manufacturer (Cy2, Cy3, and Cy5; excitation 480 nm, 540 nm, and 620 nm, and emission 530 nm, 590 nm, and 680 nm, respectively). Further analysis was done using DeCyder 6.5 software. Spot detection was done using the differential in-gel analysis (DIA) module and data from this application were then entered into the biological variation analysis (BVA) software for spot matching between gels and variation analysis. Statistical significance was calculated using Student’s t-test. MALDI-TOF Selected spots were picked manually and dehydrated with acetonitrile, dried in a Speed-vac (Savant; GMI, Ramsey, MN) and finally incubated with trypsin (Promega, Madison, WI). The protein digest was applied to a MALDI-TOF target plate (Applied Biosystems) with α-cyana-4 hydroxycinnamic acid solution (Agilent Technologies, Santa Clara, CA). Calibrant was spotted onto the plate and used for external calibration. The plate was put in a Voyager DE-STR mass spectrometer (Applied Biosystems) and the peptide spectra recorded. Peptide peak lists were generated using Data Explorer software version 4 (Applied Biosystems). Proteins were identified by searching the Swissprot and IPI databases using an in-house Mascot server (Matrix Science Inc., Boston, MA). To obtain a Mowse score for the G127X dimer, this sequence was added to the Swissprot database searched. Search parameters were set to allow mass deviations of ± 50 ppm and one or two missed cleavage sites, as well as fixed modifications such as 38 carbamidomethylation of cysteine and oxidation of methionine. Identifications that were statistically significant (with a Mowse score in the range of at least 60–66, p-value < 0.05), and that showed a gel position close to the theoretical mass and isoelectric point, were regarded as positive. LC-ESI MS/MS and data analysis Samples were incubated in 10 mM dithiothreitol (DTT) and 6 M guanidine hydrochloride with Tris, pH 9.0. Iodoacetamide was added and the samples were kept at 37°C in the dark. The reaction mixture was filtered and washed twice with 0.2 M ammonium bicarbonate and trypsin added. The resulting peptides were collected by centrifugation, lyophilized, and resuspended in 0.1% formic acid. Peptide analysis was done by reverse-phase liquid chromatography electrospray ionization-tandem mass spectrometry as described by Srivastava et al. (423) using a capillary HPLC system coupled to a quadrupole time-of-flight mass spectrometer (CapLC Q-TOF UltimaTM, Waters Corporation). ProteinLynx Global Server software version 2.2.5 was used to convert raw data to peak lists for database searching. Proteins were identified by a local version of the MASCOT search program and the Mascot Daemon application (version 2.1.6) using the Swissprot protein sequence database with mouse taxonomy. Peptides with Mascot ion scores exceeding the threshold for statistical significance (p < 0.05) were selected. Proteins were classified as having been identified either if at least two peptides with a Mascot score above the statistically relevant threshold (p < 0.05) were found or if only one peptide achieved the required Mascot score with at least four consecutive y- or b-ions and with a significant signal-tobackground ratio. Network analysis Proteomic studies generate huge amounts of data. To find common themes among altered proteins, network analysis can be used. Ingenuity Pathways Knowledge Base (IPKB) uses published information on interactions between proteins and clusters the data into networks, and identifies cellular pathways that may be significantly altered (www.ingenuity.com). The data set containing protein identifiers (Swissprot) and corresponding expression values was uploaded to the IPKB. Networks of the identified proteins were then generated algorithmically along with identification of functional and canonical pathways, and diseases in which the uploaded proteins are known to be involved. Fisher’s exact test was used by the software to calculate a pvalue for biological functions and diseases. The significance of canonical pathways was measured in 2 ways: (1) a ratio of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway, and (2) Fisher’s exact test. 39 Statistics Means and standard deviations were calculated using Microsoft Excel (paper I). In paper IV, DeCyder 6.5 used Student’s t-test for calculation of p-values. To minimize the risk of false positives due to multiple comparisons in paper IV, orthogonal projections to latent structures discriminant analysis (OPLSDA) was used for multivariate statistical evaluation in the SIMCA-P_ software version 11.5 (Umetrics AB, Umeå, Sweden). 40 Results and Discussion Paper I Experiments in our laboratory had shown that the extremely high level of SOD1 protein present in G93A, D90A, and wthSOD1 transgenic mouse models of ALS was associated with incomplete Cu2+-charging of 30–50% of the hSOD1. This was probably caused by CCS insufficiency (247). In contrast to these models (called high-level models), other models expressed SOD1 at similar rates (in terms of mRNA levels) but accumulated mutant protein at levels that were similar to the normal level of mSOD1 or even less (G85R, G127X) (low-level models). This is probably due to rapid recognition for degradation, as these two mutant SOD1s are highly destabilized by their mutations. At that point in time, one line of evidence had suggested that mutant SOD1 preferentially localizes to mitochondria, where it interferes with normal function and may be responsible for vacuole pathology (293;294;303;304;309) (discussed above). It had also been shown that SOD1 is imported to the mitochondrial IMS in the apo state, where it is loaded with metals and disulfide-oxidized—at least partly due to activities of CCS (282;283). As high-level models accumulate comparable high levels of metal-deficient, C57-C146 disulfide-reduced subunits (247), we hypothesized that this might lead to a propensity for excessive mitochondrial accumulation. Furthermore, we wanted to explore whether destabilized mutants like G85R and G127X would be retained inside the mitochondrial IMS as they, at least in the G127X case, cannot be disulfide-oxidized and are unlikely to bind the prosthetic metals. Ultracentrifugation in density gradients is the method of choice for separation of organelles (as discussed in Methods). We chose to work with continuous iodixanol gradients, as this system offers the advantages of high resolution and iso-osmotic properties. From these gradients, we were able to look at patterns of segregation rather than accumulation of similar-density particles at interfaces. As also shown by others, we found an extremely high association of mutant SOD1s with mitochondria in the high-level models, and they reached levels 100-fold higher than that of endogenous mSOD1. The quantity of this association was similar for wild-type enzyme and mutant enzymes, with only slightly higher levels for mutants, and there was no difference between spinal cord and brain—or between time points (presymptomatic stage and terminal stage). This showed that the mitochondrial association is due to an intrinsic property of SOD1 that is not qualitively different between the wt protein and mutant proteins. In the low-level G85R model, the relative levels that appeared to co-fractionate with mitochondria were similar to what was 41 found for the wild-type murine enzyme in control mice (~2%). This contrasted with the relative levels found in high-level models (> 9% of all mutant SOD1) and suggested that the level of SOD1 protein and also the disulfide-reduced/oxidized state are major determinants of mitochondrial association. In presymptomatic G127X mice, very low levels were found at the position of mitochondria (0.6%) and the pattern of gradient distribution suggested that there was no co-fractionation. When the cytosolic marker lactate dehydrogenase (LD) was measured, the contamination from this compartment was at a similar level. In terminal G127X mice, the levels of aggregated SOD1 distributed all over the gradient (showed by a filter-trap assay) made it impossible to quantify any mitochondrial association but there were no signs of apparent co-fractionation. These results did indeed indicate that the truncated G127X protein does not accumulate in mitochondria, probably due to its inability to achieve a holo state (i.e. to bind the metals and form the disulfide bridge). We also explored the association between murine wt SOD1 and mitochondria. In the controls and low-level models, this was quantified to be approximately 2%, with no difference between tissue (brain and spinal cord) and time point (presymptomatic or terminal). In the high-level models, however, the relative associations were similar to that of the mutant human enzyme: ~8–9%. In the study by Jonsson et al., it was shown that the Cu2+ and CCS deficiency caused by overexpression of mutant enzyme cause a similar amount of murine SOD1 to be trapped in the apo state (at least disulfide-reduced) (247). These results suggested that the accumulation of disulfide-reduced protein in these models is responsible for the high association between SOD1s and mitochondria. These findings were somewhat different to those in the report by Liu and colleagues, who studied a similar set of transgenic animals (303). They found preferential association of mutant SOD1 (but not wt hSOD1) with mitochondria and an age-dependent accumulation and aggregation at the cytoplasmic surface of the mitochondrial outer membrane. The discrepancies are mainly due to different methods of separation. Liu et al. used discontinuous gradients, which collect particles of similar density at interfaces, in combination with differential centrifugations that are likely to co-pellet heavy aggregates and mitochondria, resulting in artificial association. As shown by us in this work, and as explored further in paper III, all of these models accumulate high levels of aggregated SOD1 material with age. These aggregates are trapped in inclusions that show overlapping densities with all organelles when subjected to gradient separation in the absence of detergent (paper III). In a recent study by the same group, these authors tried to overcome the issue of aggregate co-fractionation by flotation of mitochondria through loading of the sample at the bottom of the tube (305). This is unlikely to overcome the problem, however, since such SOD1 inclusions behave like organelles, in terms of density, when not exposed to detergents. A weakness of our study is that, despite gentle treatment, a 42 significant amount of mitochondria are trapped at the top of the gradient, probably due to the sticky lipid materials released from CNS tissue. This loss of separation might, however, be due to swollen or broken mitochondria with low density. The accumulation of mutant SOD1 in such organelles cannot be ruled out. A recent study has shown that physiological association of wt SOD1 with mitochondria is dependent on intramolecular disulfide bonding and interaction with CCS, but that this regulation is absent in mutant SOD1s— causing aggregation and intramitochondrial accumulation (308). Another similar study has indicated the importance of misfolded/aggregated SOD1 in mitochondrial toxicity and that the aggregation is partly dependent on SOD1 cysteines (424). As the association between mitochondria and aggregated SOD1 is impossible to explore in terminal mice by density separation methods, we cannot rule out the possibility that such interaction takes place even in the context of G127X. In recent unpublished experiments, we have been able to concentrate mitochondria by a filter-trap assay but we are still unable to find any mitochondrial association in presymptomatic G127X. Is the mitochondrial pathogenic hypothesis, then, a simple artifact in transgenic mouse models? This is unlikely, since there is overwhelming evidence from patients and transgenic mouse models that there are signs of dysfunction and pathology in mitochondria (discussed above). In paper IV, we also found evidence of mitochondrial dysfunction in the G127X mice. Similarly to our own findings, in a recent immunohistochemical study on H46R mice—another model with low-levels of SOD1 devoid of mitochondrial vacuolization (but with other mitochondrial pathology)—the authors were unable to find any association between SOD1 and mitochondria (297). This strengthens the conclusion that models with extreme levels of mutant protein are those that display mitochondrial vacuoles. From my point of view, mitochondria are, according to these findings, most probably involved in the pathogenic process of ALS but might also suffer from unspecific damage due to high accumulation of mutant SOD1. The main damage is not related to direct interaction/accumulation of mutant SOD1 and mitochondria, but is due to other mechanisms (as discussed above). In the G93A model where extreme amounts of mutant protein accumulate in the IMS, this probably causes overt pathology (e.g. vacuoles) and modulates disease with an effect on the survival of motor neurons. Depletion of mitochondrial S-nitrosothiol, caused exclusively by mutant SOD1s with intact copper binding, has been suggested to be responsible for vacuole pathology in these kinds of models (425). The toxic effect of G93A SOD1 overloading was recently shown to be a disease modulator by simultaneous overexpression of CCS and G93A in transgenic mice, leading to even higher loads of mitochondrial SOD1 and a marked shortening of survival (310). This could be the explanation for why certain 43 therapeutic interventions in this model have had an effect on the course of disease in mice but lack any effect in human trials. Paper II As discussed above, the G127X mutation was first described in a Danish pedigree in 1997 (231). The insertion of TGGG after codon 127 leads to a terminal stop codon preceded by five novel amino acids. This allows generation of mutant-specific antibodies and analysis of mutant SOD1 in the presence of wild-type SOD1 from the non-mutated allele. In control humans, SOD1 levels are by far the highest in the liver and the kidneys (215). When mutant SOD1 levels are measured from different tissues in low-level transgenic mice such as G127X, the highest protein levels accumulate in the spinal cord (247). As the spinal cord is the main tissue affected by ALS, the vulnerability may be caused by inadequate recognition and degradation of misfolded SOD1. From studies of CNS tissues from a G127X patient, it had previously been shown that mutant protein accumulates at very low levels and is mainly seen in inclusions (35). In paper II, we studied both CNS tissue and peripheral tissues from a second G127X patient who succumbed to ALS. Similar to what has been found in G127X mice, we found the highest levels of mutant protein to be in the CNS. By far the highest levels were found in the vulnerable ventral horns. The amount was estimated to be about 8% of the total amount of SOD1 protein. The wild-type SOD1 levels were highest in liver and kidney, as expected. To explore the properties of mutant and wild-type protein further, tissue homogenates were subjected to density gradient ultracentrifugation as described in paper I. As in brain tissue of terminal G127X mice, we did not find detectable levels of dense SOD1 material in tissue from the frontal lobe. From ventral horns, about 50% of the mutant protein entered the gradient with a pattern identical to that seen in spinal cord of terminal G127X mice. Interestingly, similar to spinal cord, a substantial amount of the G127X mutant protein from homogenates of liver and kidney entered the gradient. Thus, aggregated mutant SOD1 can also appear in peripheral tissues. From histopathological examinations using the G127X-specific antibody, we found SOD1 inclusions in motor areas of the ventral horn as well as in hepatocytes and kidney tubular epithelium. These results suggest that degradation of G127X protein is subject to difficulties, not only in the CNS but in peripheral tissues. In quantitative terms, this accumulation is by far the most abundant in the ventral horns of the spinal cord. Whether aggregated/inclusion SOD1 reflects the disease process or actually causes it is, however, unclear (discussed above). If defined as a marker of disease, the ALS pathogenesis might also involve peripheral tissues but at a much slower rate and obvious organ dysfunction might not be prominent during the lifespan of a human. This particular 44 subject had signs of chronic liver disease, for which we found no definite explanation. Future studies in peripheral tissues will be able to tell us whether ALS is a disease that is restricted to the CNS alone or if some signs of damage appear elsewhere. Paper III In the density gradient ultracentrifugation studies of spinal cord homogenates from terminal transgenic mice, we had found that dense SOD1 material was distributed all over the gradient (paper I). This material was not present in presymptomatic mice and was aggregated, as it could be captured in a filter-trap assay. Aggregated SOD1 is known to accumulate in large inclusions in both ALS transgenic mice and human ALS patients carrying SOD1 mutations and it has been suggested that this process could be deleterious, e.g. through depletion of essential proteins. Pure protein aggregates have a very high density and had been shown to accumulate in the bottom of the tubes when the homogenate was subjected to detergent (paper I). The composition and properties of such detergent-resistant aggregates have been extensively analyzed before (discussed in introduction) and the difference between such entities and what we refer to as “inclusions” are described in figure 2. Firstly, we wanted to address the physical character of SOD1 inclusions from murine models expressing mutant SOD1 of widely different character (G93A, D90A, G85R, and G127X). The inclusions were shown to be distributed all over the gradient at densities lower than expected from pure protein aggregates, and to be disrupted upon addition of weak detergent. SOD1 protein in inclusions was not accessible to antibodies without the addition of detergent, but it was sensitive to digestion by a proteinase. This suggested that the SOD1 protein is not present on the surface of inclusions and that it is not completely covered by a continuous lipid membrane, for example. In presymptomatic high-level models G93A and D90A, large amounts of SOD1 protein enter the gradient. This material was shown in paper I to accumulate mainly in mitochondria. To characterize the properties of SOD1 in the gradient of these high-level models in greater detail, non-reducing immunoblotting was used to discriminate between SOD1 species with the C57-C146 disulfide bond oxidized and reduced. It was shown that SOD1 with the disulfide bond intact closely followed the distribution of mitochondria. In presymptomatic G85R and G127X models, only minute amounts entered the gradient. In gradients from terminally ill mice of all models, large amounts of mutant protein entered the gradient, some at higher densities than in gradients from presymptomatic animals. When subjected to weak detergent for disruption of organelles and filter-trapping followed by non-reducing 45 immunoblotting, only disulfide-reduced SOD1 and, in some cases, another SOD1-species with a non-native disulfide bond, were trapped, showing that these are the predominant species in aggregates. A minute portion of the aggregated SOD1 was found to be ubiquitinated and about 30% was shown to oligomerize or bind to other proteins through disulfide coupling. Figure 2. Biochemical isolation of inclusions. There are different routes for isolation of aggregated materials. The standard procedure involves different detergents and sequential centrifugations producing a detergent-resistant pellet (middle). In contrast to simple centrifugations (left), more or less stringent protocols produce a reproducible pellet containing detergentresistant material free from detergent-soluble cellular components. In contrast, density gradient centrifugations on homogenates free from detergents (right), enables the separation of in vivo particles containing both soluble and detergent-resistant materials with much less contamination than though a pure centrifugation protocol. 46 In G85R and G127X mice, a proportion of the inclusions were denser than most organelles. These dense SOD1 inclusions gave us the opportunity to study their composition without any major contamination from organelles and cytosolic proteins. When separated on 2-D gels, apart from SOD1, 10 proteins not present in the controls could subsequently be identified using MALDI-TOF MS. The most abundant was mutant SOD1, representing at least 50% of the total protein. Two were cytoplasmic chaperones, 4 were cytoskeletal proteins, and 4 were proteins that normally reside in the endoplasmic reticulum. Most of these proteins had been found to interact or co-aggregate with SOD1 before, but the ER chaperone calreticulin had not previously been found to associate with SOD1. When tissue sections are stained for mutant SOD1s, inclusions are clearly visible in motor neurons, astrocytes, and in the neuropil. The entities isolated here most probably represent a mixture of such inclusions. The set of proteins found to co-fractionate with SOD1 also shows that the inclusions are not only derived from motor neurons, but also from glial cells. The fact that the pathology induced by mutant SOD1s involves not only motor neurons but also glial cells is well established (non-cell autonomous), and the presence of inclusions in other cell types has been reported before (35;243;336). It is possible that there may be different inclusions, with high densities that overlap, formed in the terminal phase of the disease—some containing SOD1 and some not. Although they might possibly be different cellular entities, they might all include aggregated material that could deplete the cell of important functions. Several immunohistochemical studies have explored the composition of ALS inclusions. The most frequently described inclusions from SOD1 transgenic mice have been ubiquitinated inclusions (UIs, described in introduction) in motor neurons and astrocytic hyaline inclusions (AST-His). These are also present in both sporadic and familial ALS patients. In mice, UIs are frequently SOD1-positive but they also stain for proteins such as CCS, heat shock proteins (HSPs), GRP78, neurofilaments, NEDL1, dorfin, proteasome 20s, EAAC1, and p38MAPK with varying frequency (336;342;347;377;378). AST-HIs contain similar proteins, but with the astrocytic markers αβ-crystallin and GFAP at high frequency (243;426). The relationship between such histological entities and the biochemical isolates from these experiments is not easy to determine, however. Paper IV Proteomic techniques allow screening for pathogenic alterations with minimal input of prior knowledge. Use of the latest techniques makes it possible to look at thousands of proteins at the same time. Differential in-gel electrophoresis (DIGE) enables the separation of two different samples and an internal standard on the same 2-D gel using pre-labeling with 47 fluorophores of similar molecular weight (discussed in more detail in Methods). We hypothesized that expression of mutant SOD1 in transgenic mice affects several different pathways in affected cells. One way of exploring these events simultaneously is to subject tissue homogenates from suitable time points to DIGE. We chose to work with G127X mice, as their mutant protein level is only half the level of the endogenous mSOD1, thus reducing the risk of overexpression artifacts (discussed in paper I). As time point, we choose their peak body weight, as this has been shown to reflect the onset of symptoms where the systemic effect of motor neuron disease is still small (383). The DIGE gels revealed about 1,800 protein spots that could be processed further. From multivariate analysis, we found that about 420 protein spots contributed significantly to the difference between controls and transgenics. This is more than expected and suggests that 20–25% of proteins are altered by pathogenesis in affected tissue. We can only speculate that the majority of these alterations probably reflect compensatory actions taken for the survival of the cell. From another angle, this suggests that protein alterations are taking place in most cells of the spinal cord and not only in motor neurons. This is in line with recent reports of SOD1 pathogenesis being non-cell autonomous. Owing to limitations of 2-D gels and MALDI-TOF, 53 significantly altered protein spots could be reliably identified. As it is inevitable to encounter false-positive hits in multiple comparisons such as this, we used stringent exclusion criteria. The DIGE handbook suggests that differences down to 10% can be viewed as reliable, but we chose 25%. The use of stringent statistics with a univariate p-value of 0.01 and a multivariate correlation of p(corr)p > 0.07 reduced the risk of false positives significantly. In fact, when the different samples were assigned randomly (controls and trangenics mixed), virtually no significant spots appeared using these criteria. For interpretation of the 53 protein alterations identified, the material was subjected to analysis using Ingenuity pathway knowledge base (IPKB, described in Methods). One of the main findings was the representation of altered mitochondrial proteins. We had shown in paper I that the G127X protein does not associate with mitochondria (a finding strengthened in paper III), but still mitochondrial functions are clearly altered in this model. This suggests that the mitochondrial involvement may be secondary to primary noxious action of mutant SOD1s elsewhere. Furthermore, we found alterations in the NRF2-mediated stress response, a pathway involved in protection from oxidative stress. Regulation of proteins involved in oxidative stress has been reported previously (427;428). It is particularly interesting that all proteins found in this pathway were upregulated. A previous geneexpression study on NSC34 cells expressing mutant G93A found NRF2 and some downstream targets to be downregulated (429). This was also reported 48 in a study on isolated motor neurons of G93A rats (430). As our study only detected downstream targets of this oxidative response, we can only speculate that (I) this response might be heavily upregulated in other cells of the spinal cord but still be downregulated in motor neurons, or that (II) the isolated system of motor neuronal/NSC34 cells does not adequately reflect the in vivo situation, or that (III) the upregulation of the proteins in this study is due to stimulation of another pathway similar to NRF2. In a study by Kraft et al. (431), double transgenic mice expressing mutant SOD1 with a reporter for NRF2 activation were generated. When these authors followed NRF2 activation in different cell types during the course of the disease, they found a clear activation of this antioxidant response in several cell types of the spinal cord. This correlates well with our finding, and might indicate that the NRF2-mediated response activation in transgenic mice is dependent on an accurate cellular context. One consequence of increased oxidative stress is protein oxidation, which has been shown to be elevated in G93A mice (432). Protein degradation was another pathway that appeared to be affected by mutant SOD1 expression. Proper degradation of protein is essential to cells, particularly for neurons as they lack the ability to regenerate. The UPS is one of the major degradation pathways (discussed in introduction), and here it was found to be differentially regulated. Oxidation and altered expression of UCH-L1 has been found in brains from both Alzheimer’s and Parkinson’s disease cases by proteomic methods (433). The UCH-L1 upregulation found here is supported by an earlier report from our group showing intense immunostaining in motor neurons and astrocytes of a G127X patient (35). The COP9 signalosome complex is a multifunctional complex involved in apoptosis, protein degradation, and hypoxia (reviewed in(434)). An important function of the signalosome is to inhibit ubiquitination through deneddylation of cullin-type E3 ubiquitin ligases. As subunits 4 and 8, which are scaffold proteins in this complex, were found to be upregulated, this could indicate inhibition of ubiquitin ligase activity and of proper protein degradation. In immunoblots of samples from G127X patients and transgenic mice, a SOD1 band is present at double the molecular weight of the monomeric species (35). A similar band has been seen in other SOD1 transgenic mouse models, and also in ventral horn extracts from SALS cases (233;247). This species was identified in the DIGE gels, and MALDI-TOF analysis revealed a spectrum including only G127X SOD1 peptides. This indicates that the 32kDa band seen in immunoblots is a covalently-coupled SOD1 dimer, previously uncharacterized. In conclusion, we found alterations in cellular pathways such as oxidative stress, mitochondrial function, and protein degradation. This is in agreement with earlier proteomic studies on the G93A model, although few specific proteins overlap (399;400). We also found evidence for a novel dimeric 49 SOD1-species that might be common in ALS regardless of genetic background. 50 Conclusions Paper I • High-level mutant SOD1 models accumulate more than 100 times more SOD1 protein in mitochondria than nontransgenic mice, probably due to high levels of metaldeficient, disulfide-reduced SOD1 species. There is no obvious quantitative difference between brain and spinal cord or between presymptomatic stage and terminal stage. • The truncated G127X protein is not retained in the mitochondria of presymptomatic animals. • The difference in mitochondrial accumulation of SOD1 between different mouse models correlates with differences in mitochondrial pathology (such as vacuoles) and indicates that these may represent overexpression artifacts. • If mitochondrial deficits are universally present in mutant SOD1 ALS, this is apparently not caused by mitochondrial accumulation of SOD1 protein. Paper II • Aggregation of mutant SOD1 is not confined to the CNS of a human ALS patient, but is also evident in peripheral tissues such as the liver and kidney. • Defects in recognition and degradation of misfolded SOD1 might explain the vulnerability of motor areas. Paper III • SOD1 inclusions are complex entities containing both detergent-resistant and detergent-susceptible protein aggregates, and they are not enclosed by a continuous membrane. • Disulfide-reduced SOD1 protein is the major species in SOD1 inclusions from the spinal cord of transgenic mice. • Besides cytoskeletal proteins, the main constituents of SOD1 inclusions are protein chaperones, normally residing in the ER. This is in line with recent reports on the 51 involvement of the ER-controlled response in the pathogenesis of ALS. unfolded protein Paper IV • Expression of the G127X mutant of SOD1 in transgenic mice affects the expression of up to 25% of other proteins in the spinal cord. Alterations in important mitochondrial pathways, response to oxidative stress, and protein degradation are most prominent. • The G127X protein gives rise to a previously uncharacterized, covalently-coupled dimer. This species may be formed by other SOD1 mutants and wild-type SOD1 and might be a common modification in both ALS patients and transgenic mouse models. 52 Acknowledgements To my supervisor, Stefan Marklund. I feel favored to have been guided by someone with such genuine knowledge and devotion. Your leadership is a true model for how to handle confused students and make them feel as if they have actually achieved something. To Peter Andersen and Thomas Brännström, my co-supervisors. Thank you for ideas, fruitful discussions, and inspiration. Per Zetterström, my room-mate and travel companion for many years. Thank you for the discussions about work and other important areas of life. Looking forward to the next big party you need to accomplish in a short time. Andreas Jonsson, my unofficial supervisor. You are a brilliant scientist and your extensive teaching in practical and theoretical science has made a great difference. Karin Graffmo, for handling all the mouse issues and for being an excellent travel companion across the world. Karin Forsberg, for all your help with histochemistry and for just being such a great fellow Ph.D. student. I know that all your work will soon pay off and that you will finally get your reward. Eva Bern and Agneta Öberg, the two number-one western-blotters of the world. You have been so important for this work that to try to quantitate your efforts would look silly. Thanks also for the very nice atmosphere you have created in the SOD lab. Micael Granström, for being an excellent room-mate – where are you? Former lab companions Anna Rehnmark andLena Larsson, for productive collaboration. Heather Stewart and Monica Vestling, for interesting discussions. All former and present collaborators in the SOD lab. The atmosphere mentioned above can also be attributed to you. Jörgen Andersson, Anna Birve, Caroline Dahlberg, Sabine Eriksson, Ingalis Fransson, Johan Bergh, Karin Hjertqvist, Ann-Charlotte Nilsson, Eva Olofsson and Ulla-Stina Spetz. 53 The Mikael Oliveberg group in Stockholm and Robert Byström in Umeå: thank you for making protein folding an issue for those who don’t understand it. To all the wonderful administrators at the department of Medical Biosciences whom I have worked with during these years. Birgitta Berglund, Karin Bodén, Åsa Lundstén, Terry Persson, Iris Svanholm, and Monica Sörman. Your helpful attitude and knowledge are some of this department’s most valuable treasures. To all the technical stuff who handle our numerous mice. Elisabeth Nilsson, Karin Wallgren, and all the others at UTCF and GDL. Kjell Granqvist, Parviz Behnam-Motlagh, Johan Hultdin, and the others at 6M, 2 tr. My collaborators: Henrik Antti – for brilliant statistics. Thomas Kieselbach, Gunnar Wingsle, and Vaibhav Srivastava for proteomic expertise and helpful discussions. All my friends at Umeå Vineyard, especially families Skog, Rydin, and Petersson. You underestimate your own importance. To my family in Örebro: Pappa, Mamma, Jacob, Anna, Johannes, and Ellinor. To Runar, Christina, Ann-Christine, Bosse, AnnaCarin, and Klas. And to all my friends back home in Örebro. For more than ten years I have longed to say: “we are coming home”. To my heavenly Father, without whom the quest would never end. Most of all to my wife, Jenny, and my two wonderful children, Hanna and Ludvig. You know that this would never have been possible without your loving support. 54 References 1. Aran, F. A. (1850) Recherches sur une maladie non encore décrite du système musculaire (atrophie musculaire progressive) Arch. Gen. Med. 24, 5;172-35;214 2. Charcot, J. M. and Joffroy, A. (1869) Deux cas d'atrophie musculaire progressive avec lésions de la substance grise et des faisceaux antéro-latéraux de la moelle épinière Arch. Physiol. Neurol. Path. 2, 744-760 3. 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 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, 96107 5. 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 6. Gordon, P. H., Cheng, B., Katz, I. B., Pinto, M., Hays, A. P., Mitsumoto, H., and Rowland, L. P. (2006) The natural history of primary lateral sclerosis Neurology. 66, 647-653 7. Rosenfeld, J. and Swash, M. (2006) What's in a name? Lumping or splitting ALS, PLS, PMA, and the other motor neuron diseases Neurology 66, 624-625 8. Gordon, P. H., Cheng, B., Katz, I. B., Mitsumoto, H., and Rowland, L. P. (2009) Clinical features that distinguish PLS, upper motor neuron-dominant ALS, and typical ALS Neurology. 72, 1948-1952 9. De Carvalho M., Scotto, M., and Swash, M. (2007) Clinical patterns in progressive muscular atrophy (PMA): a prospective study Amyotroph. Lateral. Scler. 8, 296299 10. Van den Berg-Vos RM, Visser, J., Kalmijn, S., Fischer, K., de, V. M., de, J., V, de Haan, R. J., Franssen, H., Wokke, J. H., and van den Berg, L. H. (2009) A longterm prospective study of the natural course of sporadic adult-onset lower motor neuron syndromes Arch. Neurol. 66, 751-757 11. Worms, P. M. (2001) The epidemiology of motor neuron diseases: a review of recent studies J. Neurol. Sci. 191, 3-9 12. Mortara, P., Chio, A., Rosso, M. G., Leone, M., and Schiffer, D. (1984) Motor neuron disease in the province of Turin, Italy, 1966-1980. Survival analysis in an unselected population J. Neurol. Sci. 66, 165-173 13. Abhinav, K., Stanton, B., Johnston, C., Hardstaff, J., Orrell, R. W., Howard, R., Clarke, J., Sakel, M., Ampong, M. A., Shaw, C. E., Leigh, P. N., and Al-Chalabi, A. (2007) Amyotrophic lateral sclerosis in South-East England: a population-based study. The South-East England register for amyotrophic lateral sclerosis (SEALS Registry) Neuroepidemiology. 29, 44-48 55 14. Cima, V., Logroscino, G., D'Ascenzo, C., Palmieri, A., Volpe, M., Briani, C., Pegoraro, E., Angelini, C., and Soraru, G. (2009) Epidemiology of ALS in Padova district, Italy, from 1992 to 2005 Eur. J. Neurol. 16, 920-924 15. Chio, A., Mora, G., Calvo, A., Mazzini, L., Bottacchi, E., and Mutani, R. (2009) Epidemiology of ALS in Italy: a 10-year prospective population-based study Neurology. 72, 725-731 16. Preux, P. M., Druet-Cabanac, M., Couratier, P., Debrock, C., Truong, T., Marcharia, W., Vallat, J. M., Dumas, M., and Boutros-Toni, F. (2000) Estimation of the amyotrophic lateral sclerosis incidence by capture-recapture method in the Limousin region of France J. Clin. Epidemiol. 53, 1025-1029 17. Fang, F., Valdimarsdottir, U., Bellocco, R., Ronnevi, L. O., Sparen, P., Fall, K., and Ye, W. (2009) Amyotrophic lateral sclerosis in Sweden, 1991-2005 Arch. Neurol. 66, 515-519 18. Cronin, S., Hardiman, O., and Traynor, B. J. (2007) Ethnic variation in the incidence of ALS: a systematic review Neurology. 68, 1002-1007 19. McGuire, V. and Nelson, L. M. (2006) Amyotrophic lateral sclerosis. Mitsumoto, H., Przedborski, S., and Gordon, P. H., editors. Taylor & Francis Group, New York 20. Wijesekera, L. C. and Leigh, P. N. (2009) Amyotrophic lateral sclerosis Orphanet. J. Rare. Dis. 4., 3 21. Armon, C. (2003) An evidence-based medicine approach to the evaluation of the role of exogenous risk factors in sporadic amyotrophic lateral sclerosis Neuroepidemiology 22, 217-228 22. Gouveia, L. O. and De, C. M. (2007) Young-onset sporadic amyotrophic lateral sclerosis: a distinct nosological entity? Amyotroph. Lateral. Scler. 8, 323-327 23. Chio, A., Mora, G., Leone, M., Mazzini, L., Cocito, D., Giordana, M. T., Bottacchi, E., and Mutani, R. (2002) Early symptom progression rate is related to ALS outcome: a prospective population-based study Neurology 59, 99-103 24. Chio, A., Logroscino, G., Hardiman, O., Swingler, R., Mitchell, D., Beghi, E., and Traynor, B. G. (2008) Prognostic factors in ALS: A critical review Amyotroph. Lateral. Scler. 10, 310-323 25. 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 26. Chio, A., Brignolio, F., Meineri, P., and Schiffer, D. (1987) Phenotypic and genotypic heterogeneity of dominantly inherited amyotrophic lateral sclerosis Acta Neurol. Scand. 75, 277-282 27. 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 28. Kato, S., Shaw, P. J., Wood-Allum, C. A., Leigh, N., and Shaw, C. E. (2003) Motor neuron disorders. Dickson, D. W., editor. In Neurodegeneration: The molecular pathology of dementia and movement disorders, ISN Neuropath Press, Basel 29. Hays, A. P. (2006) Pathology of amyotrophic lateral sclerosis. Mitsumoto, H., Przedborski, S., and Gordon, P. H., editors. In Amyotrophic lateral sclerosis, Taylor & Francis group, New York 56 30. Huyer, G., Longsworth, G. L., Mason, D. L., Mallampalli, M. P., McCaffery, J. M., Wright, R. L., and Michaelis, S. (2004) A striking quality control subcompartment in Saccharomyces cerevisiae: the endoplasmic reticulum-associated compartment Mol. Biol. Cell 15, 908-921 31. Kaganovich, D., Kopito, R., and Frydman, J. (2008) Misfolded proteins partition between two distinct quality control compartments Nature 454, 1088-1095 32. Kopito, R. R. (2000) Aggresomes, inclusion bodies and protein aggregation Trends Cell Biol. 10, 524-530 33. Okamoto, K., Mizuno, Y., and Fujita, Y. (2008) Bunina bodies in amyotrophic lateral sclerosis Neuropathology. 28, 109-115 34. Neumann, M., Sampathu, D. M., Kwong, L. K., Truax, A. C., Micsenyi, M. C., Chou, T. T., Bruce, J., Schuck, T., Grossman, M., Clark, C. M., McCluskey, L. F., Miller, B. L., Masliah, E., Mackenzie, I. R., Feldman, H., Feiden, W., Kretzschmar, H. A., Trojanowski, J. Q., and Lee, V. M. (2006) Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis Science 314, 130-133 35. Jonsson, P. A., Ernhill, K., Andersen, P. M., Bergemalm, D., Brannstrom, T., Gredal, O., Nilsson, P., and Marklund, S. L. (2004) Minute quantities of misfolded mutant superoxide dismutase-1 cause amyotrophic lateral sclerosis Brain 127, 7388 36. 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 37. Yamagishi, S., Koyama, Y., Katayama, T., Taniguchi, M., Hitomi, J., Kato, M., Aoki, M., Itoyama, Y., Kato, S., and Tohyama, M. (2007) An in vitro model for Lewy body-like hyaline inclusion/astrocytic hyaline inclusion: induction by ER stress with an ALS-linked SOD1 mutation PLoS ONE 2, e1030 38. 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. L., Heinicke, H. U., Seltzer, W., Boss, M., and Brown, R. H. J. (2003) Sixteen novel mutations in the Cu/Zn superoxide dismutase gene in amyotrophic lateral sclerosis: a decade of discoveries, defects and disputes Als and other motor neuron disorders 4, 62-73 39. 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 CuZnsuperoxide dismutase Nat. Genet. 10, 61-66 40. Stewart, H. G. and Andersen, P. M. (2004) Neurophysiology of hereditary amyotrophic lateral sclerosis. Eisen, A., editor. In Clinical neurophysiology of motor neuron diseases, Elsevier B.V., Amsterdam 41. Valdmanis, P. N. and Rouleau, G. A. (2008) Genetics of familial amyotrophic lateral sclerosis Neurology 70, 144-152 42. Pasinelli, P. and Brown, R. H. (2006) Molecular biology of amyotrophic lateral sclerosis: insights from genetics Nat. Rev. Neurosci. 7, 710-723 57 43. 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., Helperin, J. J., Herzfeldt, B., Van den Bergh, R., Hung, W. Y., Bird, T., Deng, G., Mulder, D. W., Swyth, C., Laing, N. S., Sorlano, E., Pericak-Vance, M. A., Haines, J., Rouleau, G. A., Gusella, J. S., Horvitz, H. R., and Brown Jr, R. H. (1993) Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis Nature 362, 59-62 44. Siddique, T., Figlewicz, D. A., Pericak-Vance, M. A., Haines, J. L., Rouleau, G., Jeffers, A. J., Sapp, P., Hung, W. Y., Bebout, J., Kenna-Yasek, D., and . (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 45. 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., BenHamida, 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 46. 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 47. 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 48. Kanekura, K., Hashimoto, Y., Niikura, T., Aiso, S., Matsuoka, M., and Nishimoto, I. (2004) Alsin, the product of ALS2 gene, suppresses SOD1 mutant neurotoxicity through RhoGEF domain by interacting with SOD1 mutants J. Biol. Chem. 279, 19247-19256 49. Lai, C., Xie, C., McCormack, S. G., Chiang, H. C., Michalak, M. K., Lin, X., Chandran, J., Shim, H., Shimoji, M., Cookson, M. R., Huganir, R. L., Rothstein, J. D., Price, D. L., Wong, P. C., Martin, L. J., Zhu, J. J., and Cai, H. (2006) Amyotrophic lateral sclerosis 2-deficiency leads to neuronal degeneration in amyotrophic lateral sclerosis through altered AMPA receptor trafficking J. Neurosci. 26, 11798-11806 50. Jacquier, A., Buhler, E., Schafer, M. K., Bohl, D., Blanchard, S., Beclin, C., and Haase, G. (2006) Alsin/Rac1 signaling controls survival and growth of spinal motoneurons Ann. Neurol. 60, 105-117 51. Tudor, E. L., Perkinton, M. S., Schmidt, A., Ackerley, S., Brownlees, J., Jacobsen, N. J., Byers, H. L., Ward, M., Hall, A., Leigh, P. N., Shaw, C. E., McLoughlin, D. M., and Miller, C. C. (2005) ALS2/Alsin regulates Rac-PAK signaling and neurite outgrowth J. Biol. Chem. 280, 34735-34740 52. Cai, H., Shim, H., Lai, C., Xie, C., Lin, X., Yang, W. J., and Chandran, J. (2008) ALS2/alsin knockout mice and motor neuron diseases Neurodegener. Dis. 5, 359366 58 53. 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., uer-Grumbach, M., Wagner, K., De, J. 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 54. 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 55. Chen, Y. Z., Hashemi, S. H., Anderson, S. K., Huang, Y., Moreira, M. C., Lynch, D. R., Glass, I. A., Chance, P. F., and Bennett, C. L. (2006) Senataxin, the yeast Sen1p orthologue: characterization of a unique protein in which recessive mutations cause ataxia and dominant mutations cause motor neuron disease Neurobiol. Dis. 23, 97-108 56. Kwiatkowski, T. J., Jr., Bosco, D. A., Leclerc, A. L., Tamrazian, E., Vanderburg, C. R., Russ, C., Davis, A., Gilchrist, J., Kasarskis, E. J., Munsat, T., Valdmanis, P., Rouleau, G. A., Hosler, B. A., Cortelli, P., de Jong, P. J., Yoshinaga, Y., Haines, J. L., Pericak-Vance, M. A., Yan, J., Ticozzi, N., Siddique, T., Kenna-Yasek, D., Sapp, P. C., Horvitz, H. R., Landers, J. E., and Brown, R. H., Jr. (2009) Mutations in the FUS/TLS gene on chromosome 16 cause familial amyotrophic lateral sclerosis Science. 323, 1205-1208 57. Vance, C., Rogelj, B., Hortobagyi, T., De Vos, K. J., Nishimura, A. L., Sreedharan, J., Hu, X., Smith, B., Ruddy, D., Wright, P., Ganesalingam, J., Williams, K. L., Tripathi, V., Al-Saraj, S., Al-Chalabi, A., Leigh, P. N., Blair, I. P., Nicholson, G., de, B. J., Gallo, J. M., Miller, C. C., and Shaw, C. E. (2009) Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6 Science. 323, 1208-1211 58. Lagier-Tourenne, C. and Cleveland, D. W. (2009) Rethinking ALS: the FUS about TDP-43 Cell 136, 1001-1004 59. 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 60. 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 61. Landers, J. E., Leclerc, A. L., Shi, L., Virkud, A., Cho, T., Maxwell, M. M., Henry, A. F., Polak, M., Glass, J. D., Kwiatkowski, T. J., Al-Chalabi, A., Shaw, C. E., Leigh, P. N., Rodriguez-Leyza, I., Kenna-Yasek, D., Sapp, P. C., and Brown, R. H., Jr. (2008) New VAPB deletion variant and exclusion of VAPB mutations in familial ALS Neurology. 70, 1179-1185 62. Kirby, J., Hewamadduma, C. A., Hartley, J. A., Nixon, H. C., Evans, H., Wadhwa, R. R., Kershaw, C., Ince, P. G., and Shaw, P. J. (2007) Mutations in VAPB are not associated with sporadic ALS Neurology. 68, 1951-1953 59 63. Hirano, M. (2008) VAPB: new genetic clues to the pathogenesis of ALS Neurology. 70, 1161-1162 64. Kanekura, K., Nishimoto, I., Aiso, S., and Matsuoka, M. (2006) Characterization of amyotrophic lateral sclerosis-linked P56S mutation of vesicle-associated membrane protein-associated protein B (VAPB/ALS8) J. Biol. Chem. 281, 30223-30233 65. Suzuki, H., Kanekura, K., Levine, T. P., Kohno, K., Olkkonen, V. M., Aiso, S., and Matsuoka, M. (2009) ALS-linked P56S-VAPB, an aggregated loss-of-function mutant of VAPB, predisposes motor neurons to ER stress-related death by inducing aggregation of co-expressed wild-type VAPB J. Neurochem. 108, 973-985 66. Teuling, E., Ahmed, S., Haasdijk, E., Demmers, J., Steinmetz, M. O., Akhmanova, A., Jaarsma, D., and Hoogenraad, C. C. (2007) Motor neuron disease-associated mutant vesicle-associated membrane protein-associated protein (VAP) B recruits wild-type VAPs into endoplasmic reticulum-derived tubular aggregates J. Neurosci. 27, 9801-9815 67. Anagnostou, G., Akbar, M. T., Paul, P., Angelinetta, C., Steiner, T. J., and de, B. J. (2008) Vesicle associated membrane protein B (VAPB) is decreased in ALS spinal cord Neurobiol. Aging. Aug 12 (Epub), 68. Tsuda, H., Han, S. M., Yang, Y., Tong, C., Lin, Y. Q., Mohan, K., Haueter, C., Zoghbi, A., Harati, Y., Kwan, J., Miller, M. A., and Bellen, H. J. (2008) The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors Cell. 133, 963-977 69. 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., Jr., Ludlow, C. L., and Fischbeck, K. H. (2003) Mutant dynactin in motor neuron disease Nat. Genet. 33, 455-456 70. 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 71. Munch, C., Rosenbohm, A., Sperfeld, A. D., Uttner, I., Reske, S., Krause, B. J., Sedlmeier, R., Meyer, T., Hanemann, C. O., Stumm, G., and Ludolph, A. C. (2005) Heterozygous R1101K mutation of the DCTN1 gene in a family with ALS and FTD Ann. Neurol. 58, 777-780 72. Laird, F. M., Farah, M. H., Ackerley, S., Hoke, A., Maragakis, N., Rothstein, J. D., Griffin, J., Price, D. L., Martin, L. J., and Wong, P. C. (2008) Motor neuron disease occurring in a mutant dynactin mouse model is characterized by defects in vesicular trafficking J. Neurosci. 28, 1997-2005 73. Teuling, E., van, D., V, Wulf, P. S., Haasdijk, E. D., Akhmanova, A., Hoogenraad, C. C., and Jaarsma, D. (2008) A novel mouse model with impaired dynein/dynactin function develops amyotrophic lateral sclerosis (ALS)-like features in motor neurons and improves lifespan in SOD1-ALS mice Hum. Mol. Genet. 17, 28492862 74. Hafezparast, M., Klocke, R., Ruhrberg, C., Marquardt, A., hmad-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., Hrabe de, A. M., Schiavo, G., Shima, D. T., Russ, A. P., 60 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 75. Kieran, D., Hafezparast, M., Bohnert, S., Dick, J. R., Martin, J., Schiavo, G., Fisher, E. M., and Greensmith, L. (2005) A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice J. Cell Biol. 169, 561-567 76. Teuchert, M., Fischer, D., Schwalenstoecker, B., Habisch, H. J., Bockers, T. M., and Ludolph, A. C. (2006) A dynein mutation attenuates motor neuron degeneration in SOD1(G93A) mice Exp. Neurol. 198, 271-274 77. Ilieva, H. S., Yamanaka, K., Malkmus, S., Kakinohana, O., Yaksh, T., Marsala, M., and Cleveland, D. W. (2008) Mutant dynein (Loa) triggers proprioceptive axon loss that extends survival only in the SOD1 ALS model with highest motor neuron death Proc. Natl. Acad. Sci. U. S. A. 105, 12599-12604 78. Strom, A. L., Shi, P., Zhang, F., Gal, J., Kilty, R., Hayward, L. J., and Zhu, H. (2008) Interaction of amyotrophic lateral sclerosis (ALS)-related mutant copperzinc superoxide dismutase with the dynein-dynactin complex contributes to inclusion formation J. Biol. Chem. 283, 22795-22805 79. Greenway, M. J., Alexander, M. D., Ennis, S., Traynor, B. J., Corr, B., Frost, E., Green, A., and Hardiman, O. (2004) A novel candidate region for ALS on chromosome 14q11.2 Neurology. 63, 1936-1938 80. Greenway, M. J., Andersen, P. M., Russ, C., Ennis, S., Cashman, S., Donaghy, C., Patterson, V., Swingler, R., Kieran, D., Prehn, J., Morrison, K. E., Green, A., Acharya, K. R., Brown, R. H., and Hardiman, O. (2006) ANG mutations segregate with familial and 'sporadic' amyotrophic lateral sclerosis Nat. Genet. 38, 411-413 81. Conforti, F. L., Sprovieri, T., Mazzei, R., Ungaro, C., La, B., V, Tessitore, A., Patitucci, A., Magariello, A., Gabriele, A. L., Tedeschi, G., Simone, I. L., Majorana, G., Valentino, P., Condino, F., Bono, F., Monsurro, M. R., Muglia, M., and Quattrone, A. (2008) A novel Angiogenin gene mutation in a sporadic patient with amyotrophic lateral sclerosis from southern Italy Neuromuscul. Disord. 18, 68-70 82. Wu, D., Yu, W., Kishikawa, H., Folkerth, R. D., Iafrate, A. J., Shen, Y., Xin, W., Sims, K., and Hu, G. F. (2007) Angiogenin loss-of-function mutations in amyotrophic lateral sclerosis Ann. Neurol. 62, 609-617 83. Gellera, C., Colombrita, C., Ticozzi, N., Castellotti, B., Bragato, C., Ratti, A., Taroni, F., and Silani, V. (2008) Identification of new ANG gene mutations in a large cohort of Italian patients with amyotrophic lateral sclerosis Neurogenetics. 9, 33-40 84. Paubel, A., Violette, J., Amy, M., Praline, J., Meininger, V., Camu, W., Corcia, P., Andres, C. R., and Vourc'h, P. (2008) Mutations of the ANG gene in French patients with sporadic amyotrophic lateral sclerosis Arch. Neurol. 65, 1333-1336 85. van Es, M. A., Diekstra, F. P., Veldink, J. H., Baas, F., Bourque, P. R., Schelhaas, H. J., Strengman, E., Hennekam, E. A., Lindhout, D., Ophoff, R. A., and van den Berg, L. H. (2009) A case of ALS-FTD in a large FALS pedigree with a K17I ANG mutation Neurology. 72, 287-288 86. Fernandez-Santiago, R., Hoenig, S., Lichtner, P., Sperfeld, A. D., Sharma, M., Berg, D., Weichenrieder, O., Illig, T., Eger, K., Meyer, T., Anneser, J., Munch, C., Zierz, S., Gasser, T., and Ludolph, A. (2009) Identification of novel Angiogenin (ANG) gene missense variants in German patients with amyotrophic lateral sclerosis J. Neurol. 256, 1337-1342 61 87. Gao, X. and Xu, Z. (2008) Mechanisms of action of angiogenin Acta Biochim. Biophys. Sin. (Shanghai). 40, 619-624 88. Crabtree, B., Thiyagarajan, N., Prior, S. H., Wilson, P., Iyer, S., Ferns, T., Shapiro, R., Brew, K., Subramanian, V., and Acharya, K. R. (2007) Characterization of human angiogenin variants implicated in amyotrophic lateral sclerosis Biochemistry. 46, 11810-11818 89. Subramanian, V., Crabtree, B., and Acharya, K. R. (2008) Human angiogenin is a neuroprotective factor and amyotrophic lateral sclerosis associated angiogenin variants affect neurite extension/pathfinding and survival of motor neurons Hum. Mol. Genet. 17, 130-149 90. Kieran, D., Sebastia, J., Greenway, M. J., King, M. A., Connaughton, D., Concannon, C. G., Fenner, B., Hardiman, O., and Prehn, J. H. (2008) Control of motoneuron survival by angiogenin J. Neurosci. 28, 14056-14061 91. Sebastia, J., Kieran, D., Breen, B., King, M. A., Netteland, D. F., Joyce, D., Fitzpatrick, S. F., Taylor, C. T., and Prehn, J. H. (2009) Angiogenin protects motoneurons against hypoxic injury Cell Death. Differ. 16, 1238-1247 92. Subramanian, V. and Feng, Y. (2007) A new role for angiogenin in neurite growth and pathfinding: implications for amyotrophic lateral sclerosis Hum. Mol. Genet. 16, 1445-1453 93. Buratti, E. and Baralle, F. E. (2008) Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease Front Biosci. 13, 867-878 94. Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., and Oda, T. (2006) TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis Biochem. Biophys. Res. Commun. 351, 602-611 95. Tan, C. F., Eguchi, H., Tagawa, A., Onodera, O., Iwasaki, T., Tsujino, A., Nishizawa, M., Kakita, A., and Takahashi, H. (2007) TDP-43 immunoreactivity in neuronal inclusions in familial amyotrophic lateral sclerosis with or without SOD1 gene mutation Acta Neuropathol. 113, 535-542 96. Mackenzie, I. R., Bigio, E. H., Ince, P. G., Geser, F., Neumann, M., Cairns, N. J., Kwong, L. K., Forman, M. S., Ravits, J., Stewart, H., Eisen, A., McClusky, L., Kretzschmar, H. A., Monoranu, C. M., Highley, J. R., Kirby, J., Siddique, T., Shaw, P. J., Lee, V. M., and Trojanowski, J. Q. (2007) Pathological TDP-43 distinguishes sporadic amyotrophic lateral sclerosis from amyotrophic lateral sclerosis with SOD1 mutations Ann. Neurol. 61, 427-434 97. Robertson, J., Sanelli, T., Xiao, S., Yang, W., Horne, P., Hammond, R., Pioro, E. P., and Strong, M. J. (2007) Lack of TDP-43 abnormalities in mutant SOD1 transgenic mice shows disparity with ALS Neurosci. Lett. 420, 128-132 98. Shan, X., Vocadlo, D., and Krieger, C. (2009) Mislocalization of TDP-43 in the G93A mutant SOD1 transgenic mouse model of ALS Neurosci. Lett. 458, 70-74 99. Sreedharan, J., Blair, I. P., Tripathi, V. B., Hu, X., Vance, C., Rogelj, B., Ackerley, S., Durnall, J. C., Williams, K. L., Buratti, E., Baralle, F., de, B. J., Mitchell, J. D., Leigh, P. N., Al-Chalabi, A., Miller, C. C., Nicholson, G., and Shaw, C. E. (2008) TDP-43 mutations in familial and sporadic amyotrophic lateral sclerosis Science. 319, 1668-1672 62 100. Kabashi, E., Valdmanis, P. N., Dion, P., Spiegelman, D., McConkey, B. J., Vande, V. C., Bouchard, J. P., Lacomblez, L., Pochigaeva, K., Salachas, F., Pradat, P. F., Camu, W., Meininger, V., Dupre, N., and Rouleau, G. A. (2008) TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis Nat. Genet. 40, 572-574 101. Yokoseki, A., Shiga, A., Tan, C. F., Tagawa, A., Kaneko, H., Koyama, A., Eguchi, H., Tsujino, A., Ikeuchi, T., Kakita, A., Okamoto, K., Nishizawa, M., Takahashi, H., and Onodera, O. (2008) TDP-43 mutation in familial amyotrophic lateral sclerosis Ann. Neurol. 63, 538-542 102. Van Deerlin, V., Leverenz, J. B., Bekris, L. M., Bird, T. D., Yuan, W., Elman, L. B., Clay, D., Wood, E. M., Chen-Plotkin, A. S., Martinez-Lage, M., Steinbart, E., McCluskey, L., Grossman, M., Neumann, M., Wu, I. L., Yang, W. S., Kalb, R., Galasko, D. R., Montine, T. J., Trojanowski, J. Q., Lee, V. M., Schellenberg, G. D., and Yu, C. E. (2008) TARDBP mutations in amyotrophic lateral sclerosis with TDP-43 neuropathology: a genetic and histopathological analysis Lancet Neurol. 7, 409-416 103. Gitcho, M. A., Baloh, R. H., Chakraverty, S., Mayo, K., Norton, J. B., Levitch, D., Hatanpaa, K. J., White, C. L., III, Bigio, E. H., Caselli, R., Baker, M., Al-Lozi, M. T., Morris, J. C., Pestronk, A., Rademakers, R., Goate, A. M., and Cairns, N. J. (2008) TDP-43 A315T mutation in familial motor neuron disease Ann. Neurol. 63, 535-538 104. Corrado, L., Ratti, A., Gellera, C., Buratti, E., Castellotti, B., Carlomagno, Y., Ticozzi, N., Mazzini, L., Testa, L., Taroni, F., Baralle, F. E., Silani, V., and D'Alfonso, S. (2009) High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis Hum. Mutat. 30, 688-694 105. Winton, M. J., Van, D., V, Kwong, L. K., Yuan, W., Wood, E. M., Yu, C. E., Schellenberg, G. D., Rademakers, R., Caselli, R., Karydas, A., Trojanowski, J. Q., Miller, B. L., and Lee, V. M. (2008) A90V TDP-43 variant results in the aberrant localization of TDP-43 in vitro FEBS Lett. 582, 2252-2256 106. Nonaka, T., Kametani, F., Arai, T., Akiyama, H., and Hasegawa, M. (2009) Truncation and pathogenic mutations facilitate the formation of intracellular aggregates of TDP-43 Hum. Mol. Genet. 18, 3353-3364 107. Zhang, Y. J., Xu, Y. F., Cook, C., Gendron, T. F., Roettges, P., Link, C. D., Lin, W. L., Tong, J., Castanedes-Casey, M., Ash, P., Gass, J., Rangachari, V., Buratti, E., Baralle, F., Golde, T. E., Dickson, D. W., and Petrucelli, L. (2009) Aberrant cleavage of TDP-43 enhances aggregation and cellular toxicity Proc. Natl. Acad. Sci. U. S. A 106, 7607-7612 108. Strong, M. J., Volkening, K., Hammond, R., Yang, W., Strong, W., Leystra-Lantz, C., and Shoesmith, C. (2007) TDP43 is a human low molecular weight neurofilament (hNFL) mRNA-binding protein Mol. Cell Neurosci. 35, 320-327 109. Iguchi, Y., Katsuno, M., Niwa, J. I., Yamada, S. I., Sone, J., Waza, M., Adachi, H., Tanaka, F., Nagata, K. I., Arimura, N., Watanabe, T., Kaibuchi, K., and Sobue, G. (2009) TDP-43 depletion induces neuronal cell damage through dysregulation of Rho family GTPases J. Biol. Chem. 284, 22059-22066 110. Skibinski, G., Parkinson, N. J., Brown, J. M., Chakrabarti, L., Lloyd, S. L., Hummerich, H., Nielsen, J. E., Hodges, J. R., Spillantini, M. G., Thusgaard, T., Brandner, S., Brun, A., Rossor, M. N., Gade, A., Johannsen, P., Sorensen, S. A., Gydesen, S., Fisher, E. M., and Collinge, J. (2005) Mutations in the endosomal 63 ESCRTIII-complex subunit CHMP2B in frontotemporal dementia Nat. Genet. 37, 806-808 111. Parkinson, N., Ince, P. G., Smith, M. O., Highley, R., Skibinski, G., Andersen, P. M., Morrison, K. E., Pall, H. S., Hardiman, O., Collinge, J., Shaw, P. J., and Fisher, E. M. (2006) ALS phenotypes with mutations in CHMP2B (charged multivesicular body protein 2B) Neurology 67, 1074-1077 112. Filimonenko, M., Stuffers, S., Raiborg, C., Yamamoto, A., Malerod, L., Fisher, E. M., Isaacs, A., Brech, A., Stenmark, H., and Simonsen, A. (2007) Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease J. Cell Biol. 179, 485-500 113. Rusten, T. E., Filimonenko, M., Rodahl, L. M., Stenmark, H., and Simonsen, A. (2007) ESCRTing autophagic clearance of aggregating proteins Autophagy. 4, 114. Spina, S., Murrell, J. R., Huey, E. D., Wassermann, E. M., Pietrini, P., Baraibar, M. A., Barbeito, A. G., Troncoso, J. C., Vidal, R., Ghetti, B., and Grafman, J. (2007) Clinicopathologic features of frontotemporal dementia with progranulin sequence variation Neurology. 68, 820-827 115. Zhang, Y. J., Xu, Y. F., Dickey, C. A., Buratti, E., Baralle, F., Bailey, R., PickeringBrown, S., Dickson, D., and Petrucelli, L. (2007) Progranulin mediates caspasedependent cleavage of TAR DNA binding protein-43 J. Neurosci. 27, 10530-10534 116. Schymick, J. C., Yang, Y., Andersen, P. M., Vonsattel, J. P., Greenway, M., Momeni, P., Elder, J., Chio, A., Restagno, G., Robberecht, W., Dahlberg, C., Mukherjee, O., Goate, A., Graff-Radford, N., Caselli, R. J., Hutton, M., Gass, J., Cannon, A., Rademakers, R., Singleton, A. B., Hardiman, O., Rothstein, J., Hardy, J., and Traynor, B. J. (2007) Progranulin mutations and amyotrophic lateral sclerosis or amyotrophic lateral sclerosis-frontotemporal dementia phenotypes J. Neurol. Neurosurg. Psychiatry. 78, 754-756 117. Sleegers, K., Brouwers, N., Maurer-Stroh, S., van Es, M. A., Van, D. P., van Vught, P. W., van der, Z. J., Serneels, S., De, P. T., Van den, B. M., Cruts, M., Schymkowitz, J., De, J. P., Rousseau, F., van den Berg, L. H., Robberecht, W., and Van, B. C. (2008) Progranulin genetic variability contributes to amyotrophic lateral sclerosis Neurology. 71, 253-259 118. Beleza-Meireles, A. and Al-Chalabi, A. (2009) Genetic studies of amyotrophic lateral sclerosis: controversies and perspectives Amyotroph. Lateral. Scler. 10, 1-14 119. Lariviere, R. C. and Julien, J. P. (2004) Functions of intermediate filaments in neuronal development and disease J. Neurobiol. 58, 131-148 120. 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 121. Al-Chalabi, A., Andersen, P. M., Nilsson, P., Chioza, B., Andersson, J. L., Russ, C., Shaw, C. E., Powell, J. F., and Leigh, P. N. (1999) Deletions of the heavy neurofilament subunit tail in amyotrophic lateral sclerosis Hum. Mol. Genet. 8, 157164 122. 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 64 123. Beaulieu, J. M., Nguyen, M. D., and Julien, J. P. (1999) Late onset of motor neurons in mice overexpressing wild-type peripherin J. Cell Biol. 147, 531-544 124. 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 125. 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, 290-296 126. Oosthuyse, B., Moons, L., Storkebaum, E., Beck, H., Nuyens, D., Brusselmans, K., Van, D. J., Hellings, P., Gorselink, M., Heymans, S., Theilmeier, G., Dewerchin, M., Laudenbach, V., Vermylen, P., Raat, H., Acker, T., Vleminckx, V., Van Den, B. L., 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, 131-138 127. 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., LangLazdunski, 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, 383-394 128. Fernandez-Santiago, R., Sharma, M., Mueller, J. C., Gohlke, H., Illig, T., Anneser, J., Munch, C., Ludolph, A., Kamm, C., and Gasser, T. (2006) Possible genderdependent association of vascular endothelial growth factor (VEGF) gene and ALS Neurology. 66, 1929-1931 129. Lambrechts, D., Poesen, K., Fernandez-Santiago, R., Al-Chalabi, A., Del, B. R., van Vught, P. W., Khan, S., Marklund, S., Brockington, A., Van, M., I, Anneser, J., Shaw, C., Ludolph, A., Leigh, N., Comi, G., Gasser, T., Shaw, P. J., Morrison, K., Andersen, P., van den Berg, L. H., Thijs, V., Siddique, T., Robberecht, W., and Carmeliet, P. (2008) Meta-analysis of VEGF variations in ALS: increased susceptibility in male carriers of the -2578AA genotype J. Med. Genet. 46, 840-846 130. Azzouz, M., Ralph, G. S., Storkebaum, E., Walmsley, L. E., Mitrophanous, K. A., Kingsman, S. M., Carmeliet, P., and Mazarakis, N. D. (2004) VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model Nature 429, 413-417 131. Storkebaum, E., Lambrechts, D., Dewerchin, M., Moreno-Murciano, M. P., Appelmans, S., Oh, H., Van Damme, P., Rutten, B., Man, W. Y., De Mol, M., Wyns, S., Manka, D., Vermeulen, K., Van Den, B. L., Mertens, N., Schmitz, C., Robberecht, W., Conway, E. M., Collen, D., Moons, L., and Carmeliet, P. (2005) Treatment of motoneuron degeneration by intracerebroventricular delivery of VEGF in a rat model of ALS Nat. Neurosci. 8, 85-92 132. Burghes, A. H. and Beattie, C. E. (2009) Spinal muscular atrophy: why do low levels of survival motor neuron protein make motor neurons sick? Nat. Rev. Neurosci. 10, 597-609 65 133. Veldink, J. H., Kalmijn, S., Van der Hout, A. H., Lemmink, H. H., Groeneveld, G. J., Lummen, C., Scheffer, H., Wokke, J. H., and van den Berg, L. H. (2005) SMN genotypes producing less SMN protein increase susceptibility to and severity of sporadic ALS Neurology. 65, 820-825 134. 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, 749-752 135. Corcia, P., Camu, W., Halimi, J. M., Vourc'h, P., Antar, C., Vedrine, S., Giraudeau, B., de, T. B., and Andres, C. R. (2006) SMN1 gene, but not SMN2, is a risk factor for sporadic ALS Neurology. 67, 1147-1150 136. Zou, T., Ilangovan, R., Yu, F., Xu, Z., and Zhou, J. (2007) SMN protects cells against mutant SOD1 toxicity by increasing chaperone activity Biochem. Biophys. Res. Commun. 364, 850-855 137. Turner, B. J., Parkinson, N. J., Davies, K. E., and Talbot, K. (2009) Survival motor neuron deficiency enhances progression in an amyotrophic lateral sclerosis mouse model Neurobiol. Dis. 34, 511-517 138. Bu, G. (2009) Apolipoprotein E and its receptors in Alzheimer's disease: pathways, pathogenesis and therapy Nat. Rev. Neurosci. 10, 333-344 139. Zetterberg, H., Jacobsson, J., Rosengren, L., Blennow, K., and Andersen, P. M. (2008) Association of APOE with age at onset of sporadic amyotrophic lateral sclerosis J. Neurol. Sci. 273, 67-69 140. Li, Y. J., Pericak-Vance, M. A., Haines, J. L., Siddique, N., Kenna-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, 209213 141. Lacomblez, L., Doppler, V., Beucler, I., Costes, G., Salachas, F., Raisonnier, A., Le, F. N., Pradat, P. F., Bruckert, E., and Meininger, V. (2002) APOE: a potential marker of disease progression in ALS Neurology. 58, 1112-1114 142. Drory, V. E., Birnbaum, M., Korczyn, A. D., and Chapman, J. (2001) Association of APOE epsilon4 allele with survival in amyotrophic lateral sclerosis J. Neurol. Sci. 190, 17-20 143. Siddique, T., Pericak-Vance, M. A., Caliendo, J., Hong, S. T., Hung, W. Y., Kaplan, J., Kenna-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, 213-216 144. Mui, S., Rebeck, G. W., Kenna-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, 460-463 145. 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., 34-37 146. Broom, W. J., Greenway, M., Sadri-Vakili, G., Russ, C., Auwarter, K. E., Glajch, K. E., Dupre, N., Swingler, R. J., Purcell, S., Hayward, C., Sapp, P. C., Kenna- 66 Yasek, D., Valdmanis, P. N., Bouchard, J. P., Meininger, V., Hosler, B. A., Glass, J. D., Polack, M., Rouleau, G. A., Cha, J. H., Hardiman, O., and Brown Jr, R. H. (2008) 50bp deletion in the promoter for superoxide dismutase 1 (SOD1) reduces SOD1 expression in vitro and may correlate with increased age of onset of sporadic amyotrophic lateral sclerosis Amyotroph. Lateral. Scler. 9, 229-237 147. Kruglyak, L. (2008) The road to genome-wide association studies Nat. Rev. Genet. 9, 314-318 148. Dupre, N. and Valdmanis, P. (2009) Genome-wide association studies in amyotrophic lateral sclerosis Eur. J. Hum. Genet. 17, 137-138 149. Schymick, J. C., Scholz, S. W., Fung, H. C., Britton, A., Arepalli, S., Gibbs, J. R., Lombardo, F., Matarin, M., Kasperaviciute, D., Hernandez, D. G., Crews, C., Bruijn, L., Rothstein, J., Mora, G., Restagno, G., Chio, A., Singleton, A., Hardy, J., and Traynor, B. J. (2007) Genome-wide genotyping in amyotrophic lateral sclerosis and neurologically normal controls: first stage analysis and public release of data Lancet Neurol. 6, 322-328 150. van Es, M. A., van Vught, P. W., Blauw, H. M., Franke, L., Saris, C. G., Andersen, P. M., Van Den, B. L., de Jong, S. W., van 't, S. R., Birve, A., Lemmens, R., de, J., V, Baas, F., Schelhaas, H. J., Sleegers, K., Van, B. C., Wokke, J. H., Wijmenga, C., Robberecht, W., Veldink, J. H., Ophoff, R. A., and van den Berg, L. H. (2007) ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genomewide association study Lancet Neurol. 6, 869-877 151. Chio, A., Schymick, J. C., Restagno, G., Scholz, S. W., Lombardo, F., Lai, S. L., Mora, G., Fung, H. C., Britton, A., Arepalli, S., Gibbs, J. R., Nalls, M., Berger, S., Kwee, L. C., Oddone, E. Z., Ding, J., Crews, C., Rafferty, I., Washecka, N., Hernandez, D., Ferrucci, L., Bandinelli, S., Guralnik, J., Macciardi, F., Torri, F., Lupoli, S., Chanock, S. J., Thomas, G., Hunter, D. J., Gieger, C., Wichmann, H. E., Calvo, A., Mutani, R., Battistini, S., Giannini, F., Caponnetto, C., Mancardi, G. L., La, B., V, Valentino, F., Monsurro, M. R., Tedeschi, G., Marinou, K., Sabatelli, M., Conte, A., Mandrioli, J., Sola, P., Salvi, F., Bartolomei, I., Siciliano, G., Carlesi, C., Orrell, R. W., Talbot, K., Simmons, Z., Connor, J., Pioro, E. P., Dunkley, T., Stephan, D. A., Kasperaviciute, D., Fisher, E. M., Jabonka, S., Sendtner, M., Beck, M., Bruijn, L., Rothstein, J., Schmidt, S., Singleton, A., Hardy, J., and Traynor, B. J. (2009) A two-stage genome-wide association study of sporadic amyotrophic lateral sclerosis Hum. Mol. Genet. 18, 1524-1532 152. van Es, M. A., van Vught, P. W., Veldink, J. H., Andersen, P. M., Birve, A., Lemmens, R., Cronin, S., Van Der Kooi, A. J., Visser, M. D., Schelhaas, H. J., Hardiman, O., Ragoussis, I., Lambrechts, D., Robberecht, W., Wokke, J. H., Ophoff, R. A., and van den Berg, L. H. (2009) Analysis of FGGY as a risk factor for sporadic amyotrophic lateral sclerosis Amyotroph. Lateral. Scler. 10, 441-447 153. Dunckley, T., Huentelman, M. J., Craig, D. W., Pearson, J. V., Szelinger, S., Joshipura, K., Halperin, R. F., Stamper, C., Jensen, K. R., Letizia, D., Hesterlee, S. E., Pestronk, A., Levine, T., Bertorini, T., Graves, M. C., Mozaffar, T., Jackson, C. E., Bosch, P., McVey, A., Dick, A., Barohn, R., Lomen-Hoerth, C., Rosenfeld, J., O'connor, D. T., Zhang, K., Crook, R., Ryberg, H., Hutton, M., Katz, J., Simpson, E. P., Mitsumoto, H., Bowser, R., Miller, R. G., Appel, S. H., and Stephan, D. A. (2007) Whole-genome analysis of sporadic amyotrophic lateral sclerosis N. Engl. J. Med. 357, 775-788 154. van Es, M. A., van Vught, P. W., Blauw, H. M., Franke, L., Saris, C. G., Van Den, B. L., de Jong, S. W., de, J., V, Baas, F., Van't, S. R., Lemmens, R., Schelhaas, H. J., Birve, A., Sleegers, K., Van, B. C., Schymick, J. C., Traynor, B. J., Wokke, J. H., 67 Wijmenga, C., Robberecht, W., Andersen, P. M., Veldink, J. H., Ophoff, R. A., and van den Berg, L. H. (2008) Genetic variation in DPP6 is associated with susceptibility to amyotrophic lateral sclerosis Nat. Genet. 40, 29-31 155. Cronin, S., Berger, S., Ding, J., Schymick, J. C., Washecka, N., Hernandez, D. G., Greenway, M. J., Bradley, D. G., Traynor, B. J., and Hardiman, O. (2008) A genome-wide association study of sporadic ALS in a homogenous Irish population Hum. Mol. Genet. 17, 768-774 156. Fogh, I., D'Alfonso, S., Gellera, C., Ratti, A., Cereda, C., Penco, S., Corrado, L., Soraru, G., Castellotti, B., Tiloca, C., Gagliardi, S., Cozzi, L., Lupton, M. K., Ticozzi, N., Mazzini, L., Shaw, C. E., Al-Chalabi, A., Powell, J., and Silani, V. (2009) No association of DPP6 with amyotrophic lateral sclerosis in an Italian population Neurobiol. Aging. Jun 12 (Epub), 157. Landers, J. E., Melki, J., Meininger, V., Glass, J. D., van den Berg, L. H., van Es, M. A., Sapp, P. C., van Vught, P. W., Kenna-Yasek, D. M., Blauw, H. M., Cho, T. J., Polak, M., Shi, L., Wills, A. M., Broom, W. J., Ticozzi, N., Silani, V., Ozoguz, A., Rodriguez-Leyva, I., Veldink, J. H., Ivinson, A. J., Saris, C. G., Hosler, B. A., Barnes-Nessa, A., Couture, N., Wokke, J. H., Kwiatkowski, T. J., Jr., Ophoff, R. A., Cronin, S., Hardiman, O., Diekstra, F. P., Leigh, P. N., Shaw, C. E., Simpson, C. L., Hansen, V. K., Powell, J. F., Corcia, P., Salachas, F., Heath, S., Galan, P., Georges, F., Horvitz, H. R., Lathrop, M., Purcell, S., Al-Chalabi, A., and Brown, R. H., Jr. (2009) Reduced expression of the Kinesin-Associated Protein 3 (KIFAP3) gene increases survival in sporadic amyotrophic lateral sclerosis Proc. Natl. Acad. Sci. U. S. A. 106, 9004-9009 158. Simpson, C. L., Lemmens, R., Miskiewicz, K., Broom, W. J., Hansen, V. K., van Vught, P. W., Landers, J. E., Sapp, P., Van Den, B. L., Knight, J., Neale, B. M., Turner, M. R., Veldink, J. H., Ophoff, R. A., Tripathi, V. B., Beleza, A., Shah, M. N., Proitsi, P., Van, H. A., Carmeliet, P., Horvitz, H. R., Leigh, P. N., Shaw, C. E., van den Berg, L. H., Sham, P. C., Powell, J. F., Verstreken, P., Brown, R. H., Jr., Robberecht, W., and Al-Chalabi, A. (2009) Variants of the elongator protein 3 (ELP3) gene are associated with motor neuron degeneration Hum. Mol. Genet. 18, 472-481 159. Gallo, V., Bueno-de-Mesquita, H. B., Vermeulen, R., Andersen, P. M., Kyrozis, A., Linseisen, J., Kaaks, R., Allen, N. E., Roddam, A. W., Boshuizen, H. C., Peeters, P. H., Palli, D., Mattiello, A., Sieri, S., Tumino, R., Jimenez-Martin, J. M., Diaz, M. J., Suarez, L. R., Trichopoulou, A., Agudo, A., Arriola, L., Barricante-Gurrea, A., Bingham, S., Khaw, K. T., Manjer, J., Lindkvist, B., Overvad, K., Bach, F. W., Tjonneland, A., Olsen, A., Bergmann, M. M., Boeing, H., Clavel-Chapelon, F., Lund, E., Hallmans, G., Middleton, L., Vineis, P., and Riboli, E. (2009) Smoking and risk for amyotrophic lateral sclerosis: analysis of the EPIC cohort Ann. Neurol. 65, 378-385 160. Harwood, C. A., McDermott, C. J., and Shaw, P. J. (2009) Physical activity as an exogenous risk factor in motor neuron disease (MND): a review of the evidence Amyotroph. Lateral. Scler. 10, 191-204 161. Chio, A., Calvo, A., Dossena, M., Ghiglione, P., Mutani, R., and Mora, G. (2009) ALS in Italian professional soccer players: the risk is still present and could be soccer-specific Amyotroph. Lateral. Scler. 10, 205-209 162. Foran, E. and Trotti, D. (2009) Glutamate transporters and the excitotoxic path to motor neuron degeneration in amyotrophic lateral sclerosis Antioxid. Redox. Signal. 11, 1587-1602 68 163. Vandenberghe, W., Ihle, E. C., Patneau, D. K., Robberecht, W., and Brorson, J. R. (2000) AMPA receptor current density, not desensitization, predicts selective motoneuron vulnerability J. Neurosci. 20, 7158-7166 164. Vandenberghe, W., Robberecht, W., and Brorson, J. R. (2000) AMPA receptor calcium permeability, GluR2 expression, and selective motoneuron vulnerability J. Neurosci. 20, 123-132 165. Nakamura, R., Kamakura, K., and Kwak, S. (1994) Late-onset selective neuronal damage in the rat spinal cord induced by continuous intrathecal administration of AMPA Brain Res. 654, 279-285 166. Rothstein, J. D., Jin, L., Dykes-Hoberg, M., and Kuncl, R. W. (1993) Chronic inhibition of glutamate uptake produces a model of slow neurotoxicity Proc. Natl. Acad. Sci. U. S. A. 90, 6591-6595 167. Van Den Bosch L., Vandenberghe, W., Klaassen, H., Van, H. E., and Robberecht, W. (2000) Ca(2+)-permeable AMPA receptors and selective vulnerability of motor neurons J. Neurol. Sci. 180, 29-34 168. Van Den Bosch L. and Robberecht, W. (2000) Different receptors mediate motor neuron death induced by short and long exposures to excitotoxicity Brain Res. Bull. 53, 383-388 169. 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 170. Rothstein, J. D., Van, K. 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, 73-84 171. 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, 589-602 172. 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, 643-649 173. Andreadou, E., Kapaki, E., Kokotis, P., Paraskevas, G. P., Katsaros, N., Libitaki, G., Petropoulou, O., Zis, V., Sfagos, C., and Vassilopoulos, D. (2008) Plasma glutamate and glycine levels in patients with amyotrophic lateral sclerosis In Vivo. 22, 137-141 174. Ferrarese, C., Sala, G., Riva, R., Begni, B., Zoia, C., Tremolizzo, L., Galimberti, G., Millul, A., Bastone, A., Mennini, T., Balzarini, C., Frattola, L., and Beghi, E. (2001) Decreased platelet glutamate uptake in patients with amyotrophic lateral sclerosis Neurology. 56, 270-272 175. Sen, I., Nalini, A., Joshi, N. B., and Joshi, P. G. (2005) Cerebrospinal fluid from amyotrophic lateral sclerosis patients preferentially elevates intracellular calcium and toxicity in motor neurons via AMPA/kainate receptor J. Neurol. Sci. 235, 45-54 176. Cid, C., varez-Cermeno, J. C., Regidor, I., Salinas, M., and Alcazar, A. (2003) Low concentrations of glutamate induce apoptosis in cultured neurons: implications for amyotrophic lateral sclerosis J. Neurol. Sci. 206, 91-95 69 177. Tikka, T. M., Vartiainen, N. E., Goldsteins, G., Oja, S. S., Andersen, P. M., Marklund, S. L., and Koistinaho, J. (2002) Minocycline prevents neurotoxicity induced by cerebrospinal fluid from patients with motor neurone disease Brain. 125, 722-731 178. Bensimon, G., Lacomblez, L., and Meininger, V. (1994) A controlled trial of riluzole in amyotrophic lateral sclerosis. ALS/Riluzole Study Group N. Engl. J. Med. 330, 585-591 179. Lacomblez, L., Bensimon, G., Leigh, P. N., Guillet, P., Powe, L., Durrleman, S., Delumeau, J. C., and Meininger, V. (1996) A confirmatory dose-ranging study of riluzole in ALS. ALS/Riluzole Study Group-II Neurology. 47, S242-S250 180. Lacomblez, L., Bensimon, G., Leigh, P. N., Guillet, P., and Meininger, V. (1996) Dose-ranging study of riluzole in amyotrophic lateral sclerosis. Amyotrophic Lateral Sclerosis/Riluzole Study Group II Lancet. 347, 1425-1431 181. Niebroj-Dobosz, I., Janik, P., and Kwiecinski, H. (2002) Effect of Riluzole on serum amino acids in patients with amyotrophic lateral sclerosis Acta Neurol. Scand. 106, 39-43 182. Andreadou, E., Kapaki, E., Kokotis, P., Paraskevas, G. P., Katsaros, N., Libitaki, G., Zis, V., Sfagos, C., and Vassilopoulos, D. (2008) Plasma glutamate and glycine levels in patients with amyotrophic lateral sclerosis: the effect of riluzole treatment Clin. Neurol. Neurosurg. 110, 222-226 183. Fumagalli, E., Funicello, M., Rauen, T., Gobbi, M., and Mennini, T. (2008) Riluzole enhances the activity of glutamate transporters GLAST, GLT1 and EAAC1 Eur. J. Pharmacol. 578, 171-176 184. Mann, T. and Keilin, D. (1938) Haemocuprein and hepatocuprein, copper-protein compounds of blood and liver in animals Proc. R. Soc. Lond. B 126, 303-315 185. McCord, J. M. and Fridovich, I. (1969) Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein) J. Biol. Chem. 244, 6049-6055 186. Marklund, S. L. (2005) Endogenous free radicals and antioxidants in the brain. Beal, M. F., Lang, A. E., and Ludolph, A., editors. In Neurodegenerative Diseases, Cambridge University Press, Cambridge, UK 187. Wu, D. C., Re, D. B., Nagai, M., Ischiropoulos, H., and Przedborski, S. (2006) The inflammatory NADPH oxidase enzyme modulates motor neuron degeneration in amyotrophic lateral sclerosis mice Proc. Natl. Acad. Sci. U. S. A 103, 12132-12137 188. Marden, J. J., Harraz, M. M., Williams, A. J., Nelson, K., Luo, M., Paulson, H., and Engelhardt, J. F. (2007) Redox modifier genes in amyotrophic lateral sclerosis in mice J. Clin. Invest 117, 2913-2919 189. Harraz, M. M., Marden, J. J., Zhou, W., Zhang, Y., Williams, A., Sharov, V. S., Nelson, K., Luo, M., Paulson, H., Schoneich, C., and Engelhardt, J. F. (2008) SOD1 mutations disrupt redox-sensitive Rac regulation of NADPH oxidase in a familial ALS model J. Clin. Invest. 118, 659-670 190. Zhou, L. and Zhu, D. Y. (2009) Neuronal nitric oxide synthase: structure, subcellular localization, regulation, and clinical implications Nitric. Oxide. 20, 223230 191. Barbeito, L. H., Pehar, M., Cassina, P., Vargas, M. R., Peluffo, H., Viera, L., Estevez, A. G., and Beckman, J. S. (2004) A role for astrocytes in motor neuron loss in amyotrophic lateral sclerosis Brain Res. Brain Res. Rev. 47, 263-274 70 192. Zhao, W., Beers, D. R., Henkel, J. S., Zhang, W., Urushitani, M., Julien, J. P., and Appel, S. H. (2010) Extracellular mutant SOD1 induces microglial-mediated motoneuron injury Glia. 58, 231-243 193. Weisiger, R. A. and Fridovich, I. (1973) Mitochondrial superoxide dismutase. Site of synthesis and intramitochondrial localization J. Biol. Chem. 248, 4793-4796 194. Weisiger, R. A. and Fridovich, I. (1973) Superoxide dismutase. Organelle specificity J. Biol. Chem. 248, 3582-3592 195. Marklund, S. L. (1982) Human copper-containing superoxide dismutase of high molecular weight Proc. Natl. Acad. Sci. U. S. A 79, 7634-7638 196. 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 197. 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 198. 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 199. 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 200. Kira, Y., Sato, E. F., and Inoue, M. (2002) Association of Cu,Zn-type superoxide dismutase with mitochondria and peroxisomes Arch. Biochem. Biophys. 399, 96102 201. 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 202. Atkin, J. D., Farg, M. A., Turner, B. J., Tomas, D., Lysaght, J. A., Nunan, J., Rembach, A., Nagley, P., Beart, P. M., Cheema, S. S., and Horne, M. K. (2006) Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein disulfide isomerase with superoxide dismutase 1 J. Biol. Chem. 281, 30152-30165 203. Urushitani, M., Ezzi, S. A., Matsuo, A., Tooyama, I., and Julien, J. P. (2008) The endoplasmic reticulum-Golgi pathway is a target for translocation and aggregation of mutant superoxide dismutase linked to ALS FASEB J. 22, 2476-2487 204. Starkov, A. A. (2008) The role of mitochondria in reactive oxygen species metabolism and signaling Ann. N. Y. Acad. Sci. 1147, 37-52 205. Lebovitz, R. M., Zhang, H., Vogel, H., Cartwright, J., Jr., 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 206. 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 71 207. 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., Jr., 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, 43-47 208. 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 209. 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 210. Olofsson, 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. 81, 639-646 211. Olofsson, E. M., Marklund, S. L., and Behndig, A. (2007) Glucose-induced cataract in CuZn-SOD null lenses: an effect of nitric oxide? Free Radic. Biol. Med. 42, 1098-1105 212. Sentman, M. L., Granstrom, M., Jakobson, H., Reaume, A., Basu, S., and Marklund, S. L. (2006) Phenotypes of mice lacking extracellular superoxide dismutase and copper- and zinc-containing superoxide dismutase J. Biol. Chem. 281, 6904-6909 213. Carlsson, L. M., Jonsson, J., Edlund, T., and Marklund, S. L. (1995) Mice lacking extracellular superoxide dismutase are more sensitive to hyperoxia Proc. Natl. Acad. Sci. U. S. A. 92, 6264-6268 214. Gongora, M. C., Lob, H. E., Landmesser, U., Guzik, T. J., Martin, W. D., Ozumi, K., Wall, S. M., Wilson, D. S., Murthy, N., Gravanis, M., Fukai, T., and Harrison, D. G. (2008) Loss of extracellular superoxide dismutase leads to acute lung damage in the presence of ambient air: a potential mechanism underlying adult respiratory distress syndrome Am. J. Pathol. 173, 915-926 215. 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 216. Parge, H. E., Hallewell, R. A., and Tainer, J. A. (1992) Atomic structures of wildtype and thermostable mutant recombinant human Cu,Zn superoxide dismutase Proc. Natl. Acad. Sci. U. S. A 89, 6109-6113 217. 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, 877-891 218. Forman, H. J. and Fridovich, I. (1973) On the stability of bovine superoxide dismutase. The effects of metals J. Biol. Chem. 248, 2645-2649 219. Shaw, B. F. and Valentine, J. S. (2007) How do ALS-associated mutations in superoxide dismutase 1 promote aggregation of the protein? Trends Biochem. Sci. 32, 78-85 72 220. 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 221. 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 97, 2886-2891 222. 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 Zncontaining superoxide dismutase in the absence of the CCS Cu chaperone Proc. Natl. Acad. Sci. U. S. A 101, 5964-5969 223. Beckman, J. S. and Estevez, A. G. (2006) Superoxide dismutase, oxidative stress, and ALS. Mitsumoto, H., Przedborski, S., and Gordon, P. H., editors. In Amyotrophic lateral sclerosis, Taylor & Francis group, New York 224. 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 225. Assfalg, M., Banci, L., Bertini, I., Turano, P., and Vasos, P. R. (2003) Superoxide Dismutase Folding/Unfolding Pathway: Role of the Metal Ions in Modulating Structural and Dynamical Features J. Mol. Biol. 330, 145-158 226. Hornberg, A., Logan, D. T., Marklund, S. L., and Oliveberg, M. (2006) The Coupling between Disulphide Status, Metallation and Dimer Interface Strength in Cu/Zn Superoxide Dismutase J. Mol. Biol. 365, 333-342 227. Robberecht, W., Aguirre, T., Van Den, B. 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, 13361339 228. Cudkowicz, M. E., Kenna-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, 210-221 229. 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, 290-292 230. 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, 803-807 231. 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 232. Ezzi, S. A., Urushitani, M., and Julien, J. P. (2007) Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation J. Neurochem. 102, 170-178 73 233. Gruzman, A., Wood, W. L., Alpert, E., Prasad, M. D., Miller, R. G., Rothstein, J. D., Bowser, R., Hamilton, R., Wood, T. D., Cleveland, D. W., Lingappa, V. R., and Liu, J. (2007) Common molecular signature in SOD1 for both sporadic and familial amyotrophic lateral sclerosis Proc. Natl. Acad. Sci. U. S. A 104, 12524-12529 234. Jonsson, P. A., Graffmo, K. S., Brannstrom, T., Nilsson, P., Andersen, P. M., and Marklund, S. L. (2006) Motor neuron disease in mice expressing the wild type-like D90A mutant superoxide dismutase-1 J. Neuropathol. Exp. Neurol. 65, 1126-1136 235. Jonsson, P. A., Graffmo, K. S., Andersen, P. M., Marklund, S. L., and Brannstrom, T. (2009) Superoxide dismutase in amyotrophic lateral sclerosis patients homozygous for the D90A mutation Neurobiol. Dis. 36, 421-424 236. Kabashi, E., Valdmanis, P. N., Dion, P., and Rouleau, G. A. (2007) Oxidized/misfolded superoxide dismutase-1: the cause of all amyotrophic lateral sclerosis? Ann. Neurol. 62, 553-559 237. Wang, L., Deng, H. X., Grisotti, G., Zhai, H., Siddique, T., and Roos, R. P. (2009) Wild-type SOD1 overexpression accelerates disease onset of a G85R SOD1 mouse Hum. Mol. Genet. 18, 1642-1651 238. 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 ALS J. Biol. Chem. 279, 15499-15504 239. 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., Deng, H. X., Chen, W., Zhai, P., Sufit, R. L., and Siddique, T. (1994) Motor neuron degeneration in mice that express a human Cu,Zn superoxide dismutase mutation Science 264, 1772-1775 240. Scott, S., Kranz, J. E., Cole, J., Lincecum, J. M., Thompson, K., Kelly, N., Bostrom, A., Theodoss, J., Al-Nakhala, B. M., Vieira, F. G., Ramasubbu, J., and Heywood, J. A. (2008) Design, power, and interpretation of studies in the standard murine model of ALS Amyotroph. Lateral. Scler. 9, 4-15 241. Dal Canto, M. C. and Gurney, M. E. (1995) Neuropathological changes in two lines of mice carrying a transgene for mutant human Cu,Zn SOD, and in mice overexpressing wild type human SOD: a model of familial amyotrophic lateral sclerosis (FALS) Brain Res. 676, 25-40 242. 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 243. 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, 327-338 244. 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 245. Epstein, C. J., Avraham, K. B., Lovett, M., Smith, S., Elroy-Stein, O., Rotman, G., Bry, C., and Groner, Y. (1987) Transgenic mice with increased Cu/Zn-superoxide 74 dismutase activity: animal model of dosage effects in Down syndrome Proc. Natl. Acad. Sci. U. S. A. 84, 8044-8048 246. 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, 623-643 247. Jonsson, P. A., Graffmo, K. S., Andersen, P. M., Brannstrom, T., Lindberg, M., Oliveberg, M., and Marklund, S. L. (2006) Disulphide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models Brain 129, 451-464 248. Phillips, J. P., Tainer, J. A., Getzoff, E. D., Boulianne, G. L., Kirby, K., and Hilliker, A. J. (1995) Subunit-destabilizing mutations in Drosophila copper/zinc superoxide dismutase: neuropathology and a model of dimer dysequilibrium Proc. Natl. Acad. Sci. U. S. A. 92, 8574-8578 249. 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 250. Lemmens, R., Van, H. A., Hersmus, N., Geelen, V., D'Hollander, I., Thijs, V., Van Den, B. L., Carmeliet, P., and Robberecht, W. (2007) Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish Hum. Mol. Genet. 16, 2359-2365 251. Awano, T., Johnson, G. S., Wade, C. M., Katz, M. L., Johnson, G. C., Taylor, J. F., Perloski, M., Biagi, T., Baranowska, I., Long, S., March, P. A., Olby, N. J., Shelton, G. D., Khan, S., O'Brien, D. P., Lindblad-Toh, K., and Coates, J. R. (2009) Genome-wide association analysis reveals a SOD1 mutation in canine degenerative myelopathy that resembles amyotrophic lateral sclerosis Proc. Natl. Acad. Sci. U. S. A. 106, 2794-2799 252. 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 253. 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 254. 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, 660-665 255. 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, 113-117 256. Yamanaka, K., Boillee, S., Roberts, E. A., Garcia, M. L., onis-Downes, M., Mikse, O. R., Cleveland, D. W., and Goldstein, L. S. (2008) Mutant SOD1 in cell types 75 other than motor neurons and oligodendrocytes accelerates onset of disease in ALS mice Proc. Natl. Acad. Sci. U. S. A. 105, 7594-7599 257. Beers, D. R., Henkel, J. S., Xiao, Q., Zhao, W., Wang, J., Yen, A. A., Siklos, L., McKercher, S. R., and Appel, S. H. (2006) Wild-type microglia extend survival in PU.1 knockout mice with familial amyotrophic lateral sclerosis Proc. Natl. Acad. Sci. U. S. A 103, 16021-16026 258. Xiao, Q., Zhao, W., Beers, D. R., Yen, A. A., Xie, W., Henkel, J. S., and Appel, S. H. (2007) Mutant SOD1(G93A) microglia are more neurotoxic relative to wild-type microglia J. Neurochem. 102, 2008-2019 259. Chiu, I. M., Chen, A., Zheng, Y., Kosaras, B., Tsiftsoglou, S. A., Vartanian, T. K., Brown, R. H., Jr., and Carroll, M. C. (2008) T lymphocytes potentiate endogenous neuroprotective inflammation in a mouse model of ALS Proc. Natl. Acad. Sci. U. S. A 105, 17913-17918 260. Beers, D. R., Henkel, J. S., Zhao, W., Wang, J., and Appel, S. H. (2008) CD4+ T cells support glial neuroprotection, slow disease progression, and modify glial morphology in an animal model of inherited ALS Proc. Natl. Acad. Sci. U. S. A. 105, 15558-15563 261. Boillee, S., Yamanaka, K., Lobsiger, C. S., Copeland, N. G., Jenkins, N. A., Kassiotis, G., Kollias, G., and Cleveland, D. W. (2006) Onset and Progression in Inherited ALS Determined by Motor Neurons and Microglia Science 312, 13891392 262. Yamanaka, K., Chun, S. J., Boillee, S., Fujimori-Tonou, N., Yamashita, H., Gutmann, D. H., Takahashi, R., Misawa, H., and Cleveland, D. W. (2008) Astrocytes as determinants of disease progression in inherited amyotrophic lateral sclerosis Nat. Neurosci. 11, 251-253 263. Lobsiger, C. S., Boillee, S., onis-Downes, M., Khan, A. M., Feltri, M. L., Yamanaka, K., and Cleveland, D. W. (2009) Schwann cells expressing dismutase active mutant SOD1 unexpectedly slow disease progression in ALS mice Proc. Natl. Acad. Sci. U. S. A. 106, 4465-4470 264. Miller, T. M., Kim, S. H., Yamanaka, K., Hester, M., Umapathi, P., Arnson, H., Rizo, L., Mendell, J. R., Gage, F. H., Cleveland, D. W., and Kaspar, B. K. (2006) Gene transfer demonstrates that muscle is not a primary target for non-cellautonomous toxicity in familial amyotrophic lateral sclerosis Proc. Natl. Acad. Sci. U. S. A. 103, 19546-19551 265. 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 266. Nagai, M., Re, D. B., Nagata, T., Chalazonitis, A., Jessell, T. M., Wichterle, H., and Przedborski, S. (2007) Astrocytes expressing ALS-linked mutated SOD1 release factors selectively toxic to motor neurons Nat. Neurosci. 10, 615-622 267. Di Giorgio, F. P., Carrasco, M. A., Siao, M. C., Maniatis, T., and Eggan, K. (2007) Non-cell autonomous effect of glia on motor neurons in an embryonic stem cellbased ALS model Nat. Neurosci. 10, 608-614 268. Di Giorgio, F. P., Boulting, G. L., Bobrowicz, S., and Eggan, K. C. (2008) Human embryonic stem cell-derived motor neurons are sensitive to the toxic effect of glial cells carrying an ALS-causing mutation Cell Stem Cell. 3, 637-648 76 269. Urushitani, M., Sik, A., Sakurai, T., Nukina, N., Takahashi, R., and Julien, J. P. (2006) Chromogranin-mediated secretion of mutant superoxide dismutase proteins linked to amyotrophic lateral sclerosis Nat. Neurosci. 9, 108-118 270. Jaarsma, D., Teuling, E., Haasdijk, E. D., De Zeeuw, C. I., and Hoogenraad, C. C. (2008) Neuron-specific expression of mutant superoxide dismutase is sufficient to induce amyotrophic lateral sclerosis in transgenic mice J. Neurosci. 28, 2075-2088 271. Wang, L., Sharma, K., Deng, H. X., Siddique, T., Grisotti, G., Liu, E., and Roos, R. P. (2008) Restricted expression of mutant SOD1 in spinal motor neurons and interneurons induces motor neuron pathology Neurobiol. Dis. 29, 400-408 272. 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 273. Beckman, J. S., Carson, M., Smith, C. D., and Koppenol, W. H. (1993) ALS, SOD and peroxynitrite Nature. 364, 584 274. 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 oxidedependent apoptosis in motor neurons by zinc-deficient superoxide dismutase Science. 286, 2498-2500 275. Wiedau-Pazos, 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, 515-518 276. 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 277. 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 278. 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 279. 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, 301-307 280. Trumbull, K. A. and Beckman, J. S. (2009) A role for copper in the toxicity of zincdeficient superoxide dismutase to motor neurons in Amyotrophic Lateral Sclerosis Antioxid. Redox. Signal. 11, 1627-1639 281. Basso, M., Samengo, G., Nardo, G., Massignan, T., D'Alessandro, G., Tartari, S., Cantoni, L., Marino, M., Cheroni, C., De, B. S., Giordana, M. T., Strong, M. J., Estevez, A. G., Salmona, M., Bendotti, C., and Bonetto, V. (2009) Characterization of detergent-insoluble proteins in ALS indicates a causal link between nitrative stress and aggregation in pathogenesis PLoS ONE 4, e8130 77 282. 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. 278, 28052-28059 283. Okado-Matsumoto, A. and Fridovich, I. (2002) Amyotrophic lateral sclerosis: a proposed mechanism Proc. Natl. Acad. Sci. U. S. A 99, 9010-9014 284. 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 285. Xu, Z. and Kong, J. (2006) Role of mitochondria in motor neuron degeneration in ALS. Mitsumoto, H., Przedborski, S., and Gordon, P. H., editors. In Amyotrophic lateral sclerosis, Taylor & Francis group, New York 286. Sasaki, S. and Iwata, M. (1996) Ultrastructural study of synapses in the anterior horn neurons of patients with amyotrophic lateral sclerosis Neurosci. Lett. 204, 5356 287. 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, 616-625 288. Sasaki, S., Horie, Y., and Iwata, M. (2007) Mitochondrial alterations in dorsal root ganglion cells in sporadic amyotrophic lateral sclerosis Acta Neuropathol. 114, 633639 289. Sasaki, S. and Iwata, M. (2007) Mitochondrial alterations in the spinal cord of patients with sporadic amyotrophic lateral sclerosis J. Neuropathol. Exp. Neurol. 66, 10-16 290. Takahashi, H., Makifuchi, T., Nakano, R., Sato, S., Inuzuka, T., Sakimura, K., Mishina, M., Honma, Y., Tsuji, S., and Ikuta, F. (1994) Familial amyotrophic lateral sclerosis with a mutation in the Cu/Zn superoxide dismutase gene Acta Neuropathol. 88, 185-188 291. Kato, S., Shimoda, M., Watanabe, Y., Nakashima, K., Takahashi, K., and Ohama, E. (1996) Familial amyotrophic lateral sclerosis with a two base pair deletion in superoxide dismutase 1: gene multisystem degeneration with intracytoplasmic hyaline inclusions in astrocytes J. Neuropathol. Exp. Neurol. 55, 1089-1101 292. Ince, P. G., Shaw, P. J., Slade, J. Y., Jones, C., and Hudgson, P. (1996) Familial amyotrophic lateral sclerosis with a mutation in exon 4 of the Cu/Zn superoxide dismutase gene: pathological and immunocytochemical changes Acta Neuropathol. 92, 395-403 293. 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, 16 294. 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 transgenic mice expressing amyotrophic lateral sclerosis-linked SOD1 mutations Acta Neuropathol. (Berl) 102, 293-305 295. 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 78 296. Bendotti, C., Calvaresi, N., Chiveri, L., Prelle, A., Moggio, M., Braga, M., Silani, V., and De, B. S. (2001) Early vacuolization and mitochondrial damage in motor neurons of FALS mice are not associated with apoptosis or with changes in cytochrome oxidase histochemical reactivity J. Neurol. Sci. 191, 25-33 297. Sasaki, S., Aoki, M., Nagai, M., Kobayashi, M., and Itoyama, Y. (2009) Mitochondrial alterations in transgenic mice with an H46R mutant Cu/Zn superoxide dismutase gene J. Neuropathol. Exp. Neurol. 68, 365-373 298. 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, 871-873 299. Carri, M. T., Ferri, A., Battistoni, A., Famhy, L., Gabbianelli, R., Poccia, F., and Rotilio, G. (1997) Expression of a Cu,Zn superoxide dismutase typical of familial amyotrophic lateral sclerosis induces mitochondrial alteration and increase of cytosolic Ca2+ concentration in transfected neuroblastoma SH-SY5Y cells FEBS Lett. 414, 365-368 300. Fujita, K., Shibayama, K., Yamauchi, M., Kato, T., Ando, M., Takahashi, H., Iritani, K., Yoshimoto, N., and Nagata, Y. (1998) Alteration of enzymatic activities implicating neuronal degeneration in the spinal cord of the motor neuron degeneration mouse during postnatal development Neurochem. Res. 23, 557-562 301. 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, 535-545 302. 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, 2962629633 303. 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, 5-17 304. 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, 19-30 305. Vande Velde C., Miller, T. M., Cashman, N. R., and Cleveland, D. W. (2008) Selective association of misfolded ALS-linked mutant SOD1 with the cytoplasmic face of mitochondria Proc. Natl. Acad. Sci. U. S. A 105, 4022-4027 306. Ferri, A., Cozzolino, M., Crosio, C., Nencini, M., Casciati, A., Gralla, E. B., Rotilio, G., Valentine, J. S., and Carri, M. T. (2006) Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials Proc. Natl. Acad. Sci. U. S. A 103, 13860-13865 307. Deng, H. X., Shi, Y., Furukawa, Y., Zhai, H., Fu, R., Liu, E., Gorrie, G. H., Khan, M. S., Hung, W. Y., Bigio, E. H., Lukas, T., Dal Canto, M. C., O'Halloran, T. V., and Siddique, T. (2006) Conversion to the amyotrophic lateral sclerosis phenotype is associated with intermolecular linked insoluble aggregates of SOD1 in mitochondria Proc. Natl. Acad. Sci. U. S. A 103, 7142-7147 79 308. Kawamata, H. and Manfredi, G. (2008) Different regulation of wild-type and mutant Cu,Zn superoxide dismutase localization in mammalian mitochondria Hum. Mol. Genet. 17, 3303-3317 309. 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 310. Son, M., Puttaparthi, K., Kawamata, H., Rajendran, B., Boyer, P. J., Manfredi, G., and Elliott, J. L. (2007) Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology Proc. Natl. Acad. Sci. U. S. A. 104, 6072-6077 311. Cozzolino, M., Ferri, A., Ferraro, E., Rotilio, G., Cecconi, F., and Carri, M. T. (2006) Apaf1 mediates apoptosis and mitochondrial damage induced by mutant human SOD1s typical of familial amyotrophic lateral sclerosis Neurobiol. Dis. 21, 69-79 312. Takeuchi, H., Kobayashi, Y., Ishigaki, S., Doyu, M., and Sobue, G. (2002) Mitochondrial localization of mutant superoxide dismutase 1 triggers caspasedependent cell death in a cellular model of familial amyotrophic lateral sclerosis J. Biol. Chem. 277, 50966-50972 313. 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 Friedlander, R. M. (2002) Minocycline inhibits cytochrome c release and delays progression of amyotrophic lateral sclerosis in mice Nature. 417, 74-78 314. Gould, T. W., Buss, R. R., Vinsant, S., Prevette, D., Sun, W., Knudson, C. M., Milligan, C. E., and Oppenheim, R. W. (2006) Complete dissociation of motor neuron death from motor dysfunction by Bax deletion in a mouse model of ALS J. Neurosci. 26, 8774-8786 315. 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 316. Ahtoniemi, T., Jaronen, M., Keksa-Goldsteine, V., Goldsteins, G., and Koistinaho, J. (2008) Mutant SOD1 from spinal cord of G93A rats is destabilized and binds to inner mitochondrial membrane Neurobiol. Dis. 32, 479-485 317. Zimmerman, M. C., Oberley, L. W., and Flanagan, S. W. (2007) Mutant SOD1induced neuronal toxicity is mediated by increased mitochondrial superoxide levels J. Neurochem. 102, 609-618 318. Martin, L. J., Liu, Z., Chen, K., Price, A. C., Pan, Y., Swaby, J. A., and Golden, W. C. (2007) Motor neuron degeneration in amyotrophic lateral sclerosis mutant superoxide dismutase-1 transgenic mice: mechanisms of mitochondriopathy and cell death J. Comp Neurol. 500, 20-46 319. Wood-Allum, C. A., Barber, S. C., Kirby, J., Heath, P., Holden, H., Mead, R., Higginbottom, A., Allen, S., Beaujeux, T., Alexson, S. E., Ince, P. G., and Shaw, P. J. (2006) Impairment of mitochondrial anti-oxidant defence in SOD1-related motor neuron injury and amelioration by ebselen Brain 129, 1693-1709 320. Martin, L. J., Gertz, B., Pan, Y., Price, A. C., Molkentin, J. D., and Chang, Q. (2009) The mitochondrial permeability transition pore in motor neurons: Involvement in the pathobiology of ALS mice Exp. Neurol. 218, 333-346 80 321. Yoshihara, T., Ishigaki, S., Yamamoto, M., Liang, Y., Niwa, J., Takeuchi, H., Doyu, M., and Sobue, G. (2002) Differential expression of inflammation- and apoptosis-related genes in spinal cords of a mutant SOD1 transgenic mouse model of familial amyotrophic lateral sclerosis J. Neurochem. 80, 158-167 322. Casciati, A., Ferri, A., Cozzolino, M., Celsi, F., Nencini, M., Rotilio, G., and Carri, M. T. (2002) Oxidative modulation of nuclear factor-kappaB in human cells expressing mutant fALS-typical superoxide dismutases J. Neurochem. 83, 10191029 323. Zhao, K., Zhao, G. M., Wu, D., Soong, Y., Birk, A. V., Schiller, P. W., and Szeto, H. H. (2004) Cell-permeable peptide antioxidants targeted to inner mitochondrial membrane inhibit mitochondrial swelling, oxidative cell death, and reperfusion injury J. Biol. Chem. 279, 34682-34690 324. 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 325. Kawamata, H., Magrane, J., Kunst, C., King, M. P., and Manfredi, G. (2008) LysyltRNA synthetase is a target for mutant SOD1 toxicity in mitochondria J. Biol. Chem. 283, 28321-28328 326. Bilsland, L. G., Nirmalananthan, N., Yip, J., Greensmith, L., and Duchen, M. R. (2008) Expression of mutant SOD1 in astrocytes induces functional deficits in motoneuron mitochondria J. Neurochem. 107, 1271-1283 327. Cassina, P., Cassina, A., Pehar, M., Castellanos, R., Gandelman, M., de, L. A., Robinson, K. M., Mason, R. P., Beckman, J. S., Barbeito, L., and Radi, R. (2008) Mitochondrial dysfunction in SOD1G93A-bearing astrocytes promotes motor neuron degeneration: prevention by mitochondrial-targeted antioxidants J. Neurosci. 28, 4115-4122 328. Kanki, R., Nakamizo, T., Yamashita, H., Kihara, T., Sawada, H., Uemura, K., Kawamata, J., Shibasaki, H., Akaike, A., and Shimohama, S. (2004) Effects of mitochondrial dysfunction on glutamate receptor-mediated neurotoxicity in cultured rat spinal motor neurons Brain Res. 1015, 73-81 329. Stout, A. K., Raphael, H. M., Kanterewicz, B. I., Klann, E., and Reynolds, I. J. (1998) Glutamate-induced neuron death requires mitochondrial calcium uptake Nat. Neurosci. 1, 366-373 330. Damiano, M., Starkov, A. A., Petri, S., Kipiani, K., Kiaei, M., Mattiazzi, M., Flint, B. M., and Manfredi, G. (2006) Neural mitochondrial Ca2+ capacity impairment precedes the onset of motor symptoms in G93A Cu/Zn-superoxide dismutase mutant mice J. Neurochem. 96, 1349-1361 331. Jaiswal, M. K., Zech, W. D., Goos, M., Leutbecher, C., Ferri, A., Zippelius, A., Carri, M. T., Nau, R., and Keller, B. U. (2009) Impairment of mitochondrial calcium handling in a mtSOD1 cell culture model of motoneuron disease BMC. Neurosci. 10, 64 332. Magrane, J. and Manfredi, G. (2009) Mitochondrial function, morphology, and axonal transport in amyotrophic lateral sclerosis Antioxid. Redox. Signal. 11, 16151626 333. De Vos, K. J., Chapman, A. L., Tennant, M. E., Manser, C., Tudor, E. L., Lau, K. F., Brownlees, J., Ackerley, S., Shaw, P. J., McLoughlin, D. M., Shaw, C. E., 81 Leigh, P. N., Miller, C. C., and Grierson, A. J. (2007) Familial amyotrophic lateral sclerosis-linked SOD1 mutants perturb fast axonal transport to reduce axonal mitochondria content Hum. Mol. Genet. 16, 2720-2728 334. Sotelo-Silveira, J. R., Lepanto, P., Elizondo, M. V., Horjales, S., Palacios, F., Martinez, P. L., Marin, M., Beckman, J. S., and Barbeito, L. (2009) Axonal mitochondrial clusters containing mutant SOD1 in transgenic models of ALS Antioxid. Redox. Signal. 11, 1535-1545 335. Tateno, M., Kato, S., Sakurai, T., Nukina, N., Takahashi, R., and Araki, T. (2009) Mutant SOD1 impairs axonal transport of choline acetyltransferase and acetylcholine release by sequestering KAP3 Hum. Mol. Genet. 18, 942-955 336. 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, 933-941 337. 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 338. Rajan, R. S., Illing, M. E., Bence, N. F., and Kopito, R. R. (2001) Specificity in intracellular protein aggregation and inclusion body formation Proc. Natl. Acad. Sci. U. S. A 98, 13060-13065 339. Tai, H. C. and Schuman, E. M. (2008) Ubiquitin, the proteasome and protein degradation in neuronal function and dysfunction Nat. Rev. Neurosci. 9, 826-838 340. Di, N. 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. 280, 39907-39913 341. 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, 15-20 342. 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, 3679336798 343. Puttaparthi, K., Wojcik, C., Rajendran, B., DeMartino, G. N., and Elliott, J. L. (2003) Aggregate formation in the spinal cord of mutant SOD1 transgenic mice is reversible and mediated by proteasomes J. Neurochem. 87, 851-860 344. Choi, J. S., Cho, S., Park, S. G., Park, B. C., and Lee, D. H. (2004) Co-chaperone CHIP associates with mutant Cu/Zn-superoxide dismutase proteins linked to familial amyotrophic lateral sclerosis and promotes their degradation by proteasomes Biochem. Biophys. Res. Commun. 321, 574-583 345. Urushitani, M., Kurisu, J., Tateno, M., Hatakeyama, S., Nakayama, K., Kato, S., and Takahashi, R. (2004) CHIP promotes proteasomal degradation of familial ALSlinked mutant SOD1 by ubiquitinating Hsp/Hsc70 J. Neurochem. 90, 231-244 346. Ishigaki, S., Niwa, J., Yamada, S., Takahashi, M., Ito, T., Sone, J., Doyu, M., Urano, F., and Sobue, G. (2007) Dorfin-CHIP chimeric proteins potently ubiquitylate and degrade familial ALS-related mutant SOD1 proteins and reduce their cellular toxicity Neurobiol. Dis. 25, 331-341 82 347. 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 348. 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, 363-373 349. Koyama, S., Arawaka, S., Chang-Hong, R., Wada, M., Kawanami, T., Kurita, K., Kato, M., Nagai, M., Aoki, M., Itoyama, Y., Sobue, G., Chan, P. H., and Kato, T. (2006) Alteration of familial ALS-linked mutant SOD1 solubility with disease progression: its modulation by the proteasome and Hsp70 Biochem. Biophys. Res. Commun. 343, 719-730 350. Turner, B. J., Lopes, E. C., and Cheema, S. S. (2004) Inducible superoxide dismutase 1 aggregation in transgenic amyotrophic lateral sclerosis mouse fibroblasts J. Cell Biochem. 91, 1074-1084 351. 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 352. Basso, M., Massignan, T., Samengo, G., Cheroni, C., De, B. S., Salmona, M., Bendotti, C., and Bonetto, V. (2006) Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice J. Biol. Chem. 281, 3332533335 353. Kabashi, E. and Durham, H. D. (2006) Failure of protein quality control in amyotrophic lateral sclerosis Biochim. Biophys. Acta 1762, 1038-1050 354. 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, 509-522 355. Cheroni, C., Marino, M., Tortarolo, M., Veglianese, P., De, B. S., Fontana, E., Zuccarello, L. V., Maynard, C. J., Dantuma, N. P., and Bendotti, C. (2009) Functional alterations of the ubiquitin-proteasome system in motor neurons of a mouse model of familial amyotrophic lateral sclerosis Hum. Mol. Genet. 18, 82-96 356. Kabashi, E., Agar, J. N., Taylor, D. M., Minotti, S., and Durham, H. D. (2004) Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis J. Neurochem. 89, 1325-1335 357. Le, P. M., Bourdon, E., Paly, E., Farout, L., Friguet, B., and London, J. (2005) Oxidized SOD1 alters proteasome activities in vitro and in the cortex of SOD1 overexpressing mice FEBS Lett. 579, 3613-3618 358. Puttaparthi, K., Van, K. L., and Elliott, J. L. (2007) Assessing the role of immunoproteasomes in a mouse model of familial ALS Exp. Neurol. 206, 53-58 359. 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, 16-21 360. Hara, T., Nakamura, K., Matsui, M., Yamamoto, A., Nakahara, Y., SuzukiMigishima, R., Yokoyama, M., Mishima, K., Saito, I., Okano, H., and Mizushima, N. (2006) Suppression of basal autophagy in neural cells causes neurodegenerative disease in mice Nature 441, 885-889 83 361. Komatsu, M., Waguri, S., Chiba, T., Murata, S., Iwata, J., Tanida, I., Ueno, T., Koike, M., Uchiyama, Y., Kominami, E., and Tanaka, K. (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice Nature 441, 880884 362. Kabuta, T., Suzuki, Y., and Wada, K. (2006) Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-superoxide dismutase proteins by macroautophagy and the proteasome J. Biol. Chem. 281, 30524-30533 363. Morimoto, N., Nagai, M., Ohta, Y., Miyazaki, K., Kurata, T., Morimoto, M., Murakami, T., Takehisa, Y., Ikeda, Y., Kamiya, T., and Abe, K. (2007) Increased autophagy in transgenic mice with a G93A mutant SOD1 gene Brain Res. 1167, 112-117 364. Li, L., Zhang, X., and Le, W. (2008) Altered macroautophagy in the spinal cord of SOD1 mutant mice Autophagy. 4, 290-293 365. Tan, J. M., Wong, E. S., Kirkpatrick, D. S., Pletnikova, O., Ko, H. S., Tay, S. P., Ho, M. W., Troncoso, J., Gygi, S. P., Lee, M. K., Dawson, V. L., Dawson, T. M., and Lim, K. L. (2008) Lysine 63-linked ubiquitination promotes the formation and autophagic clearance of protein inclusions associated with neurodegenerative diseases Hum. Mol. Genet. 17, 431-439 366. Banci, L., Bertini, I., Durazo, A., Girotto, S., Gralla, E. B., Martinelli, M., Valentine, J. S., Vieru, M., and Whitelegge, J. P. (2007) Metal-free superoxide dismutase forms soluble oligomers under physiological conditions: a possible general mechanism for familial ALS Proc. Natl. Acad. Sci. U. S. A 104, 1126311267 367. Chattopadhyay, M., Durazo, A., Sohn, S. H., Strong, C. D., Gralla, E. B., Whitelegge, J. P., and Valentine, J. S. (2008) Initiation and elongation in fibrillation of ALS-linked superoxide dismutase Proc. Natl. Acad. Sci. U. S. A 105, 1866318668 368. Sandelin, E., Nordlund, A., Andersen, P. M., Marklund, S. S., and Oliveberg, M. (2007) Amyotrophic lateral sclerosis-associated copper/zinc superoxide dismutase mutations preferentially reduce the repulsive charge of the proteins J. Biol. Chem. 282, 21230-21236 369. Prudencio, M., Hart, P. J., Borchelt, D. R., and Andersen, P. M. (2009) Variation in aggregation propensities among ALS-associated variants of SOD1: Correlation to human disease Hum. Mol. Genet. 18, 3217-3226 370. Banci, L., Bertini, I., Boca, M., Girotto, S., Martinelli, M., Valentine, J. S., and Vieru, M. (2008) SOD1 and amyotrophic lateral sclerosis: mutations and oligomerization PLoS One. 3, e1677 371. Furukawa, Y., Kaneko, K., Yamanaka, K., O'Halloran, T. V., and Nukina, N. (2008) Complete loss of post-translational modifications triggers fibrillar aggregation of SOD1 in familial form of ALS J. Biol. Chem. 283, 24167-24176 372. 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 373. Zetterstrom, P., Stewart, H. G., Bergemalm, D., Jonsson, P. A., Graffmo, K. S., Andersen, P. M., Brannstrom, T., Oliveberg, M., and Marklund, S. L. (2007) Soluble misfolded subfractions of mutant superoxide dismutase-1s are enriched in 84 spinal cords throughout life in murine ALS models Proc. Natl. Acad. Sci. U. S. A 104, 14157-14162 374. Furukawa, Y., Fu, R., Deng, H. X., Siddique, T., and O'Halloran, T. V. (2006) Disulfide cross-linked protein represents a significant fraction of ALS-associated Cu, Zn-superoxide dismutase aggregates in spinal cords of model mice Proc. Natl. Acad. Sci. U. S. A 103, 7148-7153 375. Karch, C. M., Prudencio, M., Winkler, D. D., Hart, P. J., and Borchelt, D. R. (2009) Role of mutant SOD1 disulfide oxidation and aggregation in the pathogenesis of familial ALS Proc. Natl. Acad. Sci. U. S. A 106, 7774-7779 376. Karch, C. M. and Borchelt, D. R. (2008) A limited role for disulfide cross-linking in the aggregation of mutant SOD1 linked to familial amyotrophic lateral sclerosis J. Biol. Chem. 283, 13528-13537 377. Wate, R., Ito, H., Zhang, J. H., Ohnishi, S., Nakano, S., and Kusaka, H. (2005) Expression of an endoplasmic reticulum-resident chaperone, glucose-regulated stress protein 78, in the spinal cord of a mouse model of amyotrophic lateral sclerosis Acta Neuropathol. 110, 557-562 378. Bendotti, C., Atzori, C., Piva, R., Tortarolo, M., Strong, M. J., Debiasi, S., and Migheli, A. (2004) Activated p38MAPK is a novel component of the intracellular inclusions found in human amyotrophic lateral sclerosis and mutant SOD1 transgenic mice J. Neuropathol. Exp. Neurol. 63, 113-119 379. Wang, J., Farr, G. W., Zeiss, C. J., Rodriguez-Gil, D. J., Wilson, J. H., Furtak, K., Rutkowski, D. T., Kaufman, R. J., Ruse, C. I., Yates, J. R., III, Perrin, S., Feany, M. B., and Horwich, A. L. (2009) Progressive aggregation despite chaperone associations of a mutant SOD1-YFP in transgenic mice that develop ALS Proc. Natl. Acad. Sci. U. S. A 106, 1392-1397 380. 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 381. Yamashita, H., Kawamata, J., Okawa, K., Kanki, R., Nakamizo, T., Hatayama, T., Yamanaka, K., Takahashi, R., and Shimohama, S. (2007) Heat-shock protein 105 interacts with and suppresses aggregation of mutant Cu/Zn superoxide dismutase: clues to a possible strategy for treating ALS J. Neurochem. 102, 1497-1505 382. 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, 693-699 383. 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, 875-882 384. Gifondorwa, D. J., Robinson, M. B., Hayes, C. D., Taylor, A. R., Prevette, D. M., Oppenheim, R. W., Caress, J., and Milligan, C. E. (2007) Exogenous delivery of heat shock protein 70 increases lifespan in a mouse model of amyotrophic lateral sclerosis J. Neurosci. 27, 13173-13180 385. 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 85 386. Kalmar, B., Novoselov, S., Gray, A., Cheetham, M. E., Margulis, B., and Greensmith, L. (2008) Late stage treatment with arimoclomol delays disease progression and prevents protein aggregation in the SOD1 mouse model of ALS J. Neurochem. 107, 339-350 387. Cudkowicz, M. E., Shefner, J. M., Simpson, E., Grasso, D., Yu, H., Zhang, H., Shui, A., Schoenfeld, D., Brown, R. H., Wieland, S., and Barber, J. R. (2008) Arimoclomol at dosages up to 300 mg/day is well tolerated and safe in amyotrophic lateral sclerosis Muscle Nerve. 38, 837-844 388. Lobsiger, C. S., Boillee, S., and Cleveland, D. W. (2007) Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons Proc. Natl. Acad. Sci. U. S. A 104, 7319-7326 389. Perrin, F. E., Boisset, G., Docquier, M., Schaad, O., Descombes, P., and Kato, A. C. (2005) No widespread induction of cell death genes occurs in pure motoneurons in an amyotrophic lateral sclerosis mouse model Hum. Mol. Genet. 14, 3309-3320 390. Ferraiuolo, L., Heath, P. R., Holden, H., Kasher, P., Kirby, J., and Shaw, P. J. (2007) Microarray analysis of the cellular pathways involved in the adaptation to and progression of motor neuron injury in the SOD1 G93A mouse model of familial ALS J. Neurosci. 27, 9201-9219 391. Fukada, Y., Yasui, K., Kitayama, M., Doi, K., Nakano, T., Watanabe, Y., and Nakashima, K. (2007) Gene expression analysis of the murine model of amyotrophic lateral sclerosis: studies of the Leu126delTT mutation in SOD1 Brain Res. 1160, 1-10 392. Olsen, M. K., Roberds, S. L., Ellerbrock, B. R., Fleck, T. J., McKinley, D. K., and Gurney, M. E. (2001) Disease mechanisms revealed by transcription profiling in SOD1-G93A transgenic mouse spinal cord Ann. Neurol. 50, 730-740 393. Ishigaki, S., Niwa, J., Ando, Y., Yoshihara, T., Sawada, K., Doyu, M., Yamamoto, M., Kato, K., Yotsumoto, Y., and Sobue, G. (2002) Differentially expressed genes in sporadic amyotrophic lateral sclerosis spinal cords--screening by molecular indexing and subsequent cDNA microarray analysis FEBS Lett. 531, 354-358 394. Dangond, F., Hwang, D., Camelo, S., Pasinelli, P., Frosch, M. P., Stephanopoulos, G., Stephanopoulos, G., Brown Jr, R. H., and Gullans, S. R. (2003) The molecular signature of late-stage human ALS revealed by expression profiling of post-mortem spinal cord gray matter Physiol Genomics 16, 229-239 395. Jiang, Y. M., Yamamoto, M., Kobayashi, Y., Yoshihara, T., Liang, Y., Terao, S., Takeuchi, H., Ishigaki, S., Katsuno, M., Adachi, H., Niwa, J. I., Tanaka, F., Doyu, M., Yoshida, M., Hashizume, Y., and Sobue, G. (2005) Gene expression profile of spinal motor neurons in sporadic amyotrophic lateral sclerosis Ann. Neurol. 57, 236-251 396. Wang, X. S., Simmons, Z., Liu, W., Boyer, P. J., and Connor, J. R. (2006) Differential expression of genes in amyotrophic lateral sclerosis revealed by profiling the post mortem cortex Amyotroph. Lateral. Scler. 7, 201-210 397. Lederer, C. W., Torrisi, A., Pantelidou, M., Santama, N., and Cavallaro, S. (2007) Pathways and genes differentially expressed in the motor cortex of patients with sporadic amyotrophic lateral sclerosis BMC. Genomics 8, 26 398. Strey, C. W., Spellman, D., Stieber, A., Gonatas, J. O., Wang, X., Lambris, J. D., and Gonatas, N. K. (2004) Dysregulation of stathmin, a microtubule-destabilizing protein, and up-regulation of Hsp25, Hsp27, and the antioxidant peroxiredoxin 6 in 86 a mouse model of familial amyotrophic lateral sclerosis Am. J. Pathol. 165, 17011718 399. Massignan, T., Casoni, F., Basso, M., Stefanazzi, P., Biasini, E., Tortarolo, M., Salmona, M., Gianazza, E., Bendotti, C., and Bonetto, V. (2007) Proteomic analysis of spinal cord of presymptomatic amyotrophic lateral sclerosis G93A SOD1 mouse Biochem. Biophys. Res. Commun. 353, 719-725 400. Lukas, T. J., Luo, W. W., Mao, H., Cole, N., and Siddique, T. (2006) Informaticsassisted protein profiling in a transgenic mouse model of amyotrophic lateral sclerosis Mol. Cell Proteomics. 5, 1233-1244 401. Shin, J. H., London, J., Le, P. M., Weitzdoerfer, R., Hoeger, H., and Lubec, G. (2005) Proteome analysis in hippocampus of mice overexpressing human Cu/Znsuperoxide dismutase 1 Neurochem. Int. 46, 641-653 402. Di, P. C., Iadarola, P., Bardoni, A. M., Passadore, I., Giorgetti, S., Cereda, C., Carri, M. T., Ceroni, M., and Salvini, R. (2007) 2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis Electrophoresis 28, 4320-4329 403. Boillee, S., Vande, V. C., and Cleveland, D. W. (2006) ALS: a disease of motor neurons and their nonneuronal neighbors Neuron 52, 39-59 404. Pasinetti, G. M., Ungar, L. H., Lange, D. J., Yemul, S., Deng, H., Yuan, X., Brown, R. H., Cudkowicz, M. E., Newhall, K., Peskind, E., Marcus, S., and Ho, L. (2006) Identification of potential CSF biomarkers in ALS Neurology 66, 1218-1222 405. Ranganathan, S., Nicholl, G. C., Henry, S., Lutka, F., Sathanoori, R., Lacomis, D., and Bowser, R. (2007) Comparative proteomic profiling of cerebrospinal fluid between living and post mortem ALS and control subjects Amyotroph. Lateral. Scler. 8, 373-379 406. Ranganathan, S., Williams, E., Ganchev, P., Gopalakrishnan, V., Lacomis, D., Urbinelli, L., Newhall, K., Cudkowicz, M. E., Brown, R. H., Jr., and Bowser, R. (2005) Proteomic profiling of cerebrospinal fluid identifies biomarkers for amyotrophic lateral sclerosis J. Neurochem. 95, 1461-1471 407. Ramstrom, M., Ivonin, I., Johansson, A., Askmark, H., Markides, K. E., Zubarev, R., Hakansson, P., Aquilonius, S. M., and Bergquist, J. (2004) Cerebrospinal fluid protein patterns in neurodegenerative disease revealed by liquid chromatographyFourier transform ion cyclotron resonance mass spectrometry Proteomics. 4, 40104018 408. Goldknopf, I. L., Sheta, E. A., Bryson, J., Folsom, B., Wilson, C., Duty, J., Yen, A. A., and Appel, S. H. (2006) Complement C3c and related protein biomarkers in amyotrophic lateral sclerosis and Parkinson's disease Biochem. Biophys. Res. Commun. 342, 1034-1039 409. Palma, A. S., De, C. M., Grammel, N., Pinto, S., Barata, N., Conradt, H. S., and Costa, J. (2008) Proteomic analysis of plasma from Portuguese patients with familial amyotrophic lateral sclerosis Amyotroph. Lateral. Scler. 9, 339-349 410. Zhang, F., Strom, A. L., Fukada, K., Lee, S., Hayward, L. J., and Zhu, H. (2007) Interaction between familial amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants and the dynein complex J. Biol. Chem. 282, 16691-16699 411. Watanabe, Y., Morita, E., Fukada, Y., Doi, K., Yasui, K., Kitayama, M., Nakano, T., and Nakashima, K. (2008) Adherent monomer-misfolded SOD1 PLoS ONE 3, e3497 87 412. Shaw, B. F., Lelie, H. L., Durazo, A., Nersissian, A. M., Xu, G., Chan, P. K., Gralla, E. B., Tiwari, A. J., Hayward, L. J., Borchelt, D. R., Valentine, J. S., and Whitelegge, J. P. (2008) Detergent insoluble aggregates associated with amyotrophic lateral sclerosis in transgenic mice contain primarily full-length, unmodified superoxide dismutase-1 J. Biol. Chem. 283, 8340-8350 413. Wuolikainen, A., Hedenstrom, M., Moritz, T., Marklund, S. L., Antti, H., and Andersen, P. M. (2009) Optimization of procedures for collecting and storing of CSF for studying the metabolome in ALS Amyotroph. Lateral. Scler. 10, 229-236 414. Beaufay, H., Jacques, P., Baudhuin, P., Sellinger, O. Z., Berthet, J., and De, D. C. (1964) Tissue fractionation studies. 18. Resolution of mitochondrial fractions from rat liver into three distinct populations of cytoplasmic particles by means of density equilibration in various gradients Biochem. J. 92, 184-205 415. Wattiaux, R., Wattiaux-De, C. S., Ronveaux-dupal, M. F., and Dubois, F. (1978) Isolation of rat liver lysosomes by isopycnic centrifugation in a metrizamide gradient J. Cell Biol. 78, 349-368 416. Rickwood, D., Hell, A., and Birnie, G. D. (1973) Isopycnic centrifugation of sheared chromatin in metrizamide gradients FEBS Lett. 33, 221-224 417. Ford, T., Graham, J., and Rickwood, D. (1994) Iodixanol: a nonionic iso-osmotic centrifugation medium for the formation of self-generated gradients Anal. Biochem. 220, 360-366 418. Patel, D., Ford, T. C., and Rickwood, D. (1999) Cell separation, 1 Ed., Oxford University Press, USA, 419. 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, 139-148 420. 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, 675-681 421. 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 422. Heukeshoven, J. and Dernick, R. (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining Electrophoresis 6, 103-112 423. Srivastava, V., Srivastava, M. K., Chibani, K., Nilsson, R., Rouhier, N., Melzer, M., and Wingsle, G. (2009) Alternative splicing studies of the reactive oxygen species gene network in Populus reveal two isoforms of high-isoelectric-point superoxide dismutase Plant Physiol. 149, 1848-1859 424. Cozzolino, M., Pesaresi, M. G., Amori, I., Crosio, C., Ferri, A., Nencini, M., and Carri, M. T. (2009) Oligomerization of mutant SOD1 in mitochondria of motoneuronal cells drives mitochondrial damage and cell toxicity Antioxid. Redox. Signal. 11, 1547-1558 425. Schonhoff, C. M., Matsuoka, M., Tummala, H., Johnson, M. A., Estevez, A. G., Wu, R., Kamaid, A., Ricart, K. C., Hashimoto, Y., Gaston, B., Macdonald, T. L., Xu, Z., and Mannick, J. B. (2006) S-nitrosothiol depletion in amyotrophic lateral sclerosis Proc. Natl. Acad. Sci. U. S. A 103, 2404-2409 88 426. Kato, S., Hayashi, H., Nakashima, K., Nanba, E., Kato, M., Hirano, A., Nakano, I., Asayama, K., and Ohama, E. (1997) Pathological characterization of astrocytic hyaline inclusions in familial amyotrophic lateral sclerosis Am. J. Pathol. 151, 611620 427. Di, P. C., Iadarola, P., Bardoni, A. M., Passadore, I., Giorgetti, S., Cereda, C., Carri, M. T., Ceroni, M., and Salvini, R. (2007) 2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis Electrophoresis 28, 4320-4329 428. Barber, S. C., Mead, R. J., and Shaw, P. J. (2006) Oxidative stress in ALS: a mechanism of neurodegeneration and a therapeutic target Biochim. Biophys. Acta 1762, 1051-1067 429. Kirby, J., Halligan, E., Baptista, M. J., Allen, S., Heath, P. R., Holden, H., Barber, S. C., Loynes, C. A., Wood-Allum, C. A., Lunec, J., and Shaw, P. J. (2005) Mutant SOD1 alters the motor neuronal transcriptome: implications for familial ALS Brain 128, 1686-1706 430. Pehar, M., Vargas, M. R., Robinson, K. M., Cassina, P., az-Amarilla, P. J., Hagen, T. M., Radi, R., Barbeito, L., and Beckman, J. S. (2007) Mitochondrial superoxide production and nuclear factor erythroid 2-related factor 2 activation in p75 neurotrophin receptor-induced motor neuron apoptosis J. Neurosci. 27, 7777-7785 431. Kraft, A. D., Resch, J. M., Johnson, D. A., and Johnson, J. A. (2007) Activation of the Nrf2-ARE pathway in muscle and spinal cord during ALS-like pathology in mice expressing mutant SOD1 Exp. Neurol. 207, 107-117 432. 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, 453-462 433. Choi, J., Levey, A. I., Weintraub, S. T., Rees, H. D., Gearing, M., Chin, L. S., and Li, L. (2004) Oxidative modifications and down-regulation of ubiquitin carboxylterminal hydrolase L1 associated with idiopathic Parkinson's and Alzheimer's diseases J. Biol. Chem. 279, 13256-13264 434. Mikus, P. and Zundel, W. (2005) COPing with hypoxia Semin. Cell Dev. Biol. 16, 462-473 89