Mitochondrial proteomic analysis of a cell line model of familial amyotrophic lateral sclerosis

Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.     

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Recent findings indicate that nitric oxide (NO*) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R-SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO* bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO*. A decrease in the level of R-SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO*, the conditions for a closed, but continuous, loop of NO* biotransformation are present in the CSF of ALS patients.     

Effect of wild-type or mutant Parkin on oxidative damage,nitric oxide,antioxidant defenses,and the proteasome

Mutations in Parkin (a ubiquitin protein ligase) are involved in autosomal recessive juvenile parkinsonism, but it is not known how they cause nigral cell death. We examined the effect of Parkin overexpression on cellular levels of oxidative damage, antioxidant defenses, nitric oxide production, and proteasomal enzyme activity. Increasing expression of Parkin by gene transfection in NT-2 and SK-N-MC cells led to increased proteasomal activity, decreased levels of protein carbonyls, 3-nitrotyrosine-containing proteins, and a trend to a reduction in ubiquitinated protein levels. Transfection of these cells with DNA encoding three mutant Parkins associated with autosomal recessive juvenile parkinsonism (Del 3-5, T240R, and Q311X) gave smaller increases in proteasomal activity and led to elevated levels of protein carbonyls and lipid peroxidation. Turnover of the mutant proteins was slower than that of the wild-type protein, and both could be blocked by the proteasome inhibitor, lactacystin. A rise in levels of nitrated proteins and increased levels of NO2-/NO3- was also observed in cells transfected with mutant Parkins, apparently because of increased levels of neuronal nitric-oxide synthase. The presence of mutant Parkin in substantia nigra in juvenile parkinsonism may increase oxidative stress and nitric oxide production, sensitizing cells to death induced by other insults.     

Focal dysfunction of the proteasome: a pathogenic factor in a mouse model of amyotrophic lateral sclerosis

Mutations in the Cu/Zn-superoxide dismutase (SOD-1) gene are responsible for a familial form of amyotrophic lateral sclerosis (fALS). The present study demonstrated impaired proteasomal function in the lumbar spinal cord of transgenic mice expressing human SOD-1 with the ALS-causing mutation G93A (SOD-1(G93A)) compared to non-transgenic littermates (LM) and SOD-1(WT) transgenic mice. Chymotrypsin-like activity was decreased as early as 45 days of age. By 75 days, chymotrypsin-, trypsin-, and caspase-like activities of the proteasome were impaired, at about 50% of control activity in lumbar spinal cord, but unchanged in thoracic spinal cord and liver. Both total and specific activities of the proteasome were reduced to a similar extent, indicating that a change in proteasome function, rather than a decrease in proteasome levels, had occurred. Similar decreases of total and specific activities of the proteasome were observed in NIH 3T3 cell lines expressing fALS mutants SOD-1(G93A) and SOD-1(G41S), but not in SOD-1(WT) controls. Although overall levels of proteasome were maintained in spinal cord of SOD-1(G93A) transgenic mice, the level of 20S proteasome was substantially reduced in lumbar spinal motor neurons relative to the surrounding neuropil. It is concluded that impairment of the proteasome is an early event and contributes to ALS pathogenesis.     

Neuroprotective effects of the mitochondria-targeted antioxidant MitoQ in a model of inherited amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by motor neuron degeneration that ultimately results in progressive paralysis and death. Growing evidence indicates that mitochondrial dysfunction and oxidative stress contribute to motor neuron degeneration in ALS. To further explore the hypothesis that mitochondrial dysfunction and nitroxidative stress contribute to disease pathogenesis at the in vivo level, we assessed whether the mitochondria-targeted antioxidant [10-(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)decyl]triphenylphosphonium methane sulfonate (MitoQ) can modify disease progression in the SOD1^G93A mouse model of ALS. To do this, we administered MitoQ (500 µM) in the drinking water of SOD1^G93A mice from a time when early symptoms of neurodegeneration become evident at 90 days of age until death. This regime is a clinically plausible scenario and could be more easily translated to patients as this corresponds to initiating treatment of patients after they are first diagnosed with ALS. MitoQ was detected in all tested tissues by liquid chromatography/mass spectrometry after 20 days of administration. MitoQ treatment slowed the decline of mitochondrial function, in both the spinal cord and the quadriceps muscle, as measured by high-resolution respirometry. Importantly, nitroxidative markers and pathological signs in the spinal cord of MitoQ-treated animals were markedly reduced and neuromuscular junctions were recovered associated with a significant increase in hindlimb strength. Finally, MitoQ treatment significantly prolonged the life span of SOD1^G93A mice. Our results support a role for mitochondrial nitroxidative damage and dysfunction in the pathogenesis of ALS and suggest that mitochondria-targeted antioxidants may be of pharmacological use for ALS treatment.     

Bax and Bcl-2 interaction in a transgenic mouse model of familial amyotrophic lateral sclerosis

It has been proposed that mutations in copper/zinc-superoxide dismutase (SOD1), the only proven cause of amyotrophic lateral sclerosis (ALS), induce the disease by a toxic property that promotes apoptosis. Consistent with this, we have demonstrated that overexpression of Bcl-2, a protein that inhibits apoptosis, attenuates neurodegeneration produced by the familial ALS-linked SOD1 mutant G93A (mSOD1). Herein, we assessed the status of key members of the Bcl-2 family in the spinal cord of transgenic mSOD1 mice at different stages of the disease. In asymptomatic transgenic mSOD1 mice, expression of Bcl-2, Bcl-XL, Bad, and Bax does not differ from that in nontransgenic mice. In contrast, in symptomatic mice, expression of Bcl-2 and Bcl-XL, which inhibit apoptosis, is reduced, whereas expression of Bad and Bax, which stimulate apoptosis, is increased. These alterations are specific to affected brain regions and are caused by the mutant and not by the normal SOD1 enzyme. Relevant to the neuroprotective effects of Bcl-2 in transgenic mSOD1 mice, overexpression of Bcl-2 increases the formation of Bcl-2:Bax heterodimers, which abolish the Bax proapoptotic property. This study demonstrates significant alterations in the expression of key members of the Bcl-2 family associated with mSOD1 deleterious effects. That these changes contribute to the neurodegenerative process in this model of ALS is supported by our observations in double transgenic mSOD1/Bcl-2 mice in which the pernicious increase of Bax is tempered by an increase in formation of Bcl-2:Bax heterodimers. Based on these findings, it may be concluded that Bcl-2 family members appear as invaluable targets for the development of new neuroprotective therapies in ALS.     

Effect of overexpression of Bcl-2 on cellular oxidative damage, nitric oxide production, antioxidant defenses, and the proteasome

Bcl-2 is a gene family involved in the suppression of apoptosis in response to a wide range of cellular insults. Multiple papers have suggested a link between Bcl-2 and oxidative damage/antioxidant protection. We therefore examined parameters of antioxidant defense and oxidative damage in two different cell lines, NT-2/D1 (NT-2) and SK-N-MC, overexpressing Bcl-2 as compared with vector-only controls. Bcl-2 transfectants of both cell lines were more resistant to H₂O₂ and showed increases in GSH level and Cu/Zn-superoxide dismutase (SOD1) activity, but not in Mn-superoxide dismutase, glutathione peroxidase, or glutathione reductase activities. Catalase activity was increased in SK-N-MC cells. Overexpression of Bcl-2 did not significantly decrease levels of oxidative DNA damage (measured as 8-hydroxyguanine) or lipid peroxidation, but it decreased levels of 3-nitrotyrosine in both cell lines and protein carbonyls in SK-N-MC cells only. It also increased proteasome activity in both cell lines. We conclude that Bcl-2 raises cellular antioxidant defense status, but this is not necessarily reflected in decreased levels of oxidative damage to DNA and lipids. The ability of Bcl-2 overexpression to decrease 3-nitrotyrosine levels suggests that it may decrease formation of peroxynitrite or other reactive nitrogen species; this was confirmed as decreased production of NO₂⁻/NO₃⁻ in the transfected cells and a fall in the level of nNOS protein.     

Mitochondrial localization of mutant superoxide dismutase 1 triggers caspase-dependent cell death in a cellular model of familial amyotrophic lateral sclerosis

The mutations in superoxide dismutase 1 (SOD1) cause approximately 20% of familial amyotrophic lateral sclerosis cases. A toxic gain of function has been considered to be the cause of the disease, but its molecular mechanism remains uncertain. To determine whether the subcellular localization of mutant SOD1 is crucial to mutant SOD1-mediated cell death, we produced neuronal cell models with accumulation of SOD1 in each subcellular fraction/organelle, such as the cytosol, nucleus, endoplasmic reticulum, and mitochondria. We showed that the localization of mutant SOD1 in the mitochondria triggered the release of mitochondrial cytochrome c followed by the activation of caspase cascade and induced neuronal cell death without cytoplasmic mutant SOD1 aggregate formation. Nuclear and endoplasmic reticulum localization of mutant SOD1 did not induce cell death. These results suggest that the localization of mutant SOD1 in the mitochondria is critical in the pathogenesis of mutant SOD1-associated familial amyotrophic lateral sclerosis.     

Dorfin prevents cell death by reducing mitochondrial localizing mutant superoxide dismutase 1 in a neuronal cell model of familial amyotrophic lateral sclerosis

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.     

Protein nitration in a mouse model of familial amyotrophic lateral sclerosis: possible multifunctional role in the pathogenesis

Multiple mechanisms have been proposed to contribute to amyotrophic lateral sclerosis (ALS) pathogenesis, including oxidative stress. Early evidence of a role for oxidative damage was based on the finding, in patients and murine models, of high levels of markers, such as free nitrotyrosine (NT). However, no comprehensive study on the protein targets of nitration in ALS has been reported. We found an increased level of NT immunoreactivity in spinal cord protein extracts of a transgenic mouse model of familial ALS (FALS) at a presymptomatic stage of the disease compared with age-matched controls. NT immunoreactivity is increased in the soluble fraction of spinal cord homogenates and is found as a punctate staining in motor neuron perikarya of presymptomatic FALS mice. Using a proteome-based strategy, we identified proteins nitrated in vivo, under physiological or pathological conditions, and compared their level of specific nitration. alpha- and gamma-enolase, ATP synthase beta chain, and heat shock cognate 71-kDa protein and actin were overnitrated in presymptomatic FALS mice. We identified by matrix-assisted laser desorption/ionization mass spectrometry 16 sites of nitration in proteins oxidized in vivo. In particular, alpha-enolase nitration at Tyr(43), target also of phosphorylation, brings additional evidence on the possible interference of nitration with phosphorylation. In conclusion, we propose that protein nitration may have a role in ALS pathogenesis, acting directly by inhibiting the function of specific proteins and indirectly interfering with protein degradation pathways and phosphorylation cascades.     

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

Characterization of the properties of a novel mutation in VAPB in familial amyotrophic lateral sclerosis

Following the mutation screening of genes known to cause amyotrophic lateral sclerosis (ALS) in index cases from 107 familial ALS (FALS) kindred, a point mutation was identified in vesicle-associated membrane protein-associated protein B (VAPB), or VAMP-associated protein B, causing an amino acid change from threonine to isoleucine at codon 46 (T46I) in one FALS case but not in 257 controls. This is an important finding because it is only the second mutation identified in this gene that causes ALS. In order to investigate the pathogenic effects of this mutation, we have used a motor neuron cell line and tissue-specific expression of the mutant protein in Drosophila. We provide substantial evidence for the pathogenic effects of this mutation in abolishing the effect of wild type VAPB in the unfolded protein response, promoting ubiquitin aggregate formation, and activating neuronal cell death. We also report that expression of the mutant protein in the Drosophila motor system induces aggregate deposition, endoplasmic reticulum disorganization, and chaperone up-regulation both in neurons and in muscles. Our integrated analysis of the pathogenic effect of the T46I mutation and the previously identified P56S mutation indicate extensive commonalities in the disease mechanism for these two mutations. In summary, we show that this newly identified mutation in human FALS has a pathogenic effect, supporting and reinforcing the role of VAPB as a causative gene of ALS.     

Respiratory impairment in a mouse model of amyotrophic lateral sclerosis.

Amyothrophic lateral sclerosis (ALS) is a progressive, lethal neuromuscular disease that is associated with the degeneration of cortical and spinal motoneurons, leading to atrophy of limb, axial, and respiratory muscles. Patients with ALS invariably develop respiratory muscle weakness and most die from pulmonary complications. Overexpression of superoxide dismutase 1 (SOD1) gene mutations in mice recapitulates several of the clinical and pathological characteristics of ALS and is therefore a valuable tool to study this disease. The present study is intended to evaluate an age-dependent progression of respiratory complications in SOD1(G93A) mutant mice. In each animal, baseline measurements of breathing pattern [i.e., breathing frequency and tidal volume (VT)], minute ventilation (VE), and metabolism (i.e., oxygen consumption and carbon dioxide production) were repeatedly sampled at variable time points between 10 and 20 wk of age with the use of whole-body plethysmographic chambers. To further characterize the neurodegeneration of breathing, VE was also measured during 5-min challenges of hypercapnia (5% CO(2)) and hypoxia (10% O(2)). At baseline, breathing characteristics and metabolism remained relatively unchanged from 10 to 14 wk of age. From 14 to 18 wk of age, there were significant (P < 0.05) increases in baseline VT, VE, and the ventilatory equivalent (VE/oxygen consumption). After 18 wk of age, there was a rapid decline in VE due to significant (P < 0.05) reductions in both breathing frequency and VT. Whereas little change in hypoxic VE responses occurred between 10 and 18 wk, hypercapnic VE responses were significantly (P < 0.05) elevated at 18 wk due to an augmented VT response. Like baseline breathing characteristics, hypercapnic VE responses also declined rapidly after 18 wk of age. The phenotypic profile of SOD1(G93A) mutant mice was apparently unique because similar changes in respiration and metabolism were not observed in SOD1 controls. The present results outline the magnitude and time course of respiratory complications in SOD1(G93A) mutant mice as the progression of disease occurs in this mouse model of ALS.     

Galectin-1 is a component of neurofilamentous lesions in sporadic and familial amyotrophic lateral sclerosis

In amyotrophic lateral sclerosis (ALS), abnormal accumulation of neurofilaments induces pathological changes such as axonal spheroids, cord-like neurite swellings, and perikaryal conglomerate inclusions in degenerating motor neurons of the spinal cord, and the accumulation seems to cause motor neuron degeneration in this disease. Such ALS lesions were intensely labeled with HepSS-1, a monoclonal antibody to heparan sulfate. Since the identification of HepSS-1-immunoreactive substance seems to be an important step for understanding the molecular pathology of ALS, we purified the substance from human spinal cord tissue to homogeneity. Amino acid sequence of the protein was consistent with that of galectin-1. Immunohistochemistry using antibodies against recombinant human galectin-1 showed that galectin-1 was accumulated in these lesions in ALS. Although HepSS-1 was believed to be specific for heparan sulfate, it reacted with recombinant human galectin-1 which has no heparan sulfate moiety. The results show that galectin-1 is a component of the neurofilamentous lesions in ALS. Since galectin-1 has axonal regeneration-enhancing activity, the abnormal accumulation of galectin-1 to the lesions seems to be related to the pathological process of ALS.     

Informatics-assisted protein profiling in a transgenic mouse model of amyotrophic lateral sclerosis

One of the causes of amyotrophic lateral sclerosis (ALS) is due to mutations in Cu,Zn-superoxide dismutase (SOD1). The mutant protein exhibits a toxic gain of function that adversely affects the function of neurons in the spinal cord, brain stem, and motor cortex. A proteomic analysis of protein expression in a widely used mouse model of ALS was undertaken to identify differences in protein expression in the spinal cords of mice expressing a mutant protein with the G93A mutation found in human ALS. Protein profiling was done on soluble and particulate fractions of spinal cord extracts using high throughput two-dimensional liquid chromatography coupled to tandem mass spectrometry. An integrated proteomics-informatics platform was used to identify relevant differences in protein expression based upon the abundance of peptides identified by database searching of mass spectrometry data. Changes in the expression of proteins associated with mitochondria were particularly prevalent in spinal cord proteins from both mutant G93A-SOD1 and wild-type SOD1 transgenic mice. G93A-SOD1 mouse spinal cord also exhibited differences in proteins associated with metabolism, protein kinase regulation, antioxidant activity, and lysosomes. Using gene ontology analysis, we found an overlap of changes in mRNA expression in presymptomatic mice (from microarray analysis) in three different gene categories. These included selected protein kinase signaling systems, ATP-driven ion transport, and neurotransmission. Therefore, alterations in selected cellular processes are detectable before symptomatic onset in ALS mouse models. However, in late stage disease, mRNA expression analysis did not reveal significant changes in mitochondrial gene expression but did reveal concordant changes in lipid metabolism, lysosomes, and the regulation of neurotransmission. Thus, concordance of proteomic and mRNA expression data within multiple categories validates the use of gene ontology analysis to compare different types of "omic" data.     

DNA laddering and caspase 3-like activity in the spinal cord of a mouse model of familial amyotrophic lateral sclerosis.

Transgenic mice with several copies of a mutated human superoxide dismutase 1 (Gly93-Ala substitution) gene, i.e. a mutation responsible for the development of familial amyotrophic lateral sclerosis (ALS), integrated into the mouse genome, develop a slowly progressive paralysis of the hind-limbs accompanied by a corresponding degeneration of spinal cord neuronal tissue. We have used two different lines of these transgenic mice [a low (approximately 12 copies) or a high (approximately 24) copy number of the mutated human superoxide dismutase 1 gene] to find evidence of programmed cell death in affected spinal cord tissue at distinct age groups. Hallmarks of programmed cell death, i.e. DNA laddering and an increase in caspase 3-like activity, were found in the spinal cord of both lines of mice. Behavioural evaluation of the mice indicated that the hallmarks of programmed cell death were mainly, but not exclusively found in symptomatic animals just before or at end-stage. These data suggest that programmed cell death may play a role in the disease process of familial ALS particularly in its terminal phase.     

Parvalbumin overexpression alters immune-mediated increases in intracellular calcium, and delays disease onset in a transgenic model of familial amyotrophic lateral sclerosis.

Intracellular calcium is increased in vulnerable spinal motoneurons in immune-mediated as well as transgenic models of amyotrophic lateral sclerosis (ALS). To determine whether intracellular calcium levels are influenced by the calcium-binding protein parvalbumin, we developed transgenic mice overexpressing parvalbumin in spinal motoneurons. ALS immunoglobulins increased intracellular calcium and spontaneous transmitter release at motoneuron terminals in control animals, but not in parvalbumin overexpressing transgenic mice. Parvalbumin transgenic mice interbred with mutant SOD1 (mSOD1) transgenic mice, an animal model of familial ALS, had significantly reduced motoneuron loss, and had delayed disease onset (17%) and prolonged survival (11%) when compared with mice with only the mSOD1 transgene. These results affirm the importance of the calcium binding protein parvalbumin in altering calcium homeostasis in motoneurons. The increased motoneuron parvalbumin can significantly attenuate the immune-mediated increases in calcium and to a lesser extent compensate for the mSOD1-mediated 'toxic-gain-of-function' in transgenic mice.     

Hypoxia causes autophagic stress and derangement of metabolic adaptation in a cell model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease that affects motor neurons. The recruitment of autophagy (macroautophagy) and mitochondrial dysfunction are documented in amyotrophic lateral sclerosis patients and experimental models expressing mutant forms of Cu, Zn superoxide dismutase (SOD1) protein, but their impact in the disease remains unclear. Hypoxia is a stress closely related to the disease in patients and mutant SOD1 mice; in individual cells, hypoxia activates autophagy and regulates mitochondrial metabolism as fundamental adaptive mechanisms. Our aim was to examine whether mutant SOD1 changed this response. Hypoxia (1% O₂ for 22 h) caused greater loss of viability and more marked activation of caspase 3/7 in the motor neuronal NSC‐34 cell line stably transfected with the G93A mutant human SOD1 (G93A‐NSC) than in the one with the wild‐type SOD1 (WT‐NSC) or in untransfected NSC‐34. In the G93A‐NSC cells, there was a more marked accumulation of the LC3‐II autophagy protein, attributable to autophagic stress; 3‐methyladenine, which acts on initiation of autophagy, fully rescued G93A‐NSC viability and reduced the activation of caspase 3/7 indicating this was a secondary event; the metabolic handling of hypoxia was inappropriate possibly contributing to the autophagic stress. Our findings evidentiate that the G93A mutation of SOD1 profoundly altered the adaptive metabolic response to hypoxia and this could increase the cell susceptibility to this stress.

     

Superoxide dismutase 1: Identification of a novel mutation in a case of familial amyotrophic lateral sclerosis

Mutation analysis of the superoxide dismutase gene SOD1 in a familial case of amyotrophic lateral sclerosis revealed a T→C transition at codon 151 of exon 5. This mutation results in the substitution of an isoleucine for a threonine. It appears to affect formation of dimers of the protein and is the most C-terminal amino acid change in SOD1 described to date. Received: 5 February 1996 / Revised: 9 February 1996     

Structures of the G85R variant of SOD1 in familial amyotrophic lateral sclerosis

Mutations in the gene encoding human copper-zinc superoxide dismutase (SOD1) cause a dominant form of the progressive neurodegenerative disease amyotrophic lateral sclerosis. Transgenic mice expressing the human G85R SOD1 variant develop paralytic symptoms concomitant with the appearance of SOD1-enriched proteinaceous inclusions in their neural tissues. The process(es) through which misfolding or aggregation of G85R SOD1 induces motor neuron toxicity is not understood. Here we present structures of the human G85R SOD1 variant determined by single crystal x-ray diffraction. Alterations in structure of the metal-binding loop elements relative to the wild type enzyme suggest a molecular basis for the metal ion deficiency of the G85R SOD1 protein observed in the central nervous system of transgenic mice and in purified recombinant G85R SOD1. These findings support the notion that metal-deficient and/or disulfide-reduced mutant SOD1 species contribute to toxicity in SOD1-linked amyotrophic lateral sclerosis.