Species-dependent neuropathology in transgenic SOD1 pigs

Mutations in the human copper/zinc superoxide dismutase 1 (hSOD1) gene cause familial amyotrophic lateral sclerosis (ALS). It remains unknown whether large animal models of ALS mimic more pathological events seen in ALS patients via novel mechanisms. Here, we report the generation of transgenic pigs expressing mutant G93A hSOD1 and showing hind limb motor defects, which are germline transmissible, and motor neuron degeneration in dose- and age-dependent manners. Importantly, in the early disease stage, mutant hSOD1 did not form cytoplasmic inclusions, but showed nuclear accumulation and ubiquitinated nuclear aggregates, as seen in some ALS patient brains, but not in transgenic ALS mouse models. Our findings revealed that SOD1 binds PCBP1, a nuclear poly(rC) binding protein, in pig brain, but not in mouse brain, suggesting that the SOD1-PCBP1 interaction accounts for nuclear SOD1 accumulation and that species-specific targets are key to ALS pathology in large mammals and in humans.     

Expression of SOD1 G93A or wild-type SOD1 in primary cultures of astrocytes down-regulates the glutamate transporter GLT-1: lack of involvement of oxidative stress

Glutamate excitotoxicity is implicated in the aetiology of amyotrophic lateral sclerosis (ALS) with impairment of glutamate transport into astrocytes a possible cause of glutamate-induced injury to motor neurons. It is possible that mutations of Cu/Zn superoxide dismutase (SOD1), responsible for about 20% of familial ALS, down-regulates glutamate transporters via oxidative stress. We transfected primary mouse astrocytes to investigate the effect of the FALS-linked mutant hSOD1(G93A) and wild-type SOD1 (hSOD1wt) on the glutamate uptake system. Using western blotting, immunocytochemistry and RT-PCR it was shown that expression of either hSOD1(G93A) or hSOD1wt in astrocytes produced down-regulation of the levels of a glutamate transporter GLT-1, without alterations in its mRNA level. hSOD1(G93A) or hSOD1wt expression caused a decrease of the monomeric form of GLT-1 without increasing oxidative multimers of GLT-1. The effects were selective to GLT-1, since another glutamate transporter GLAST protein and mRNA levels were not altered. Reflecting the decrease in GLT-1 protein, [3H]d-aspartate uptake was reduced in cultures expressing hSOD1(G93A) or hSOD1wt. The hSOD1-induced decline in GLT-1 protein and [3H]d-aspartate uptake was not blocked by the antioxidant Trolox nor potentiated by antioxidant depletion using catalase and glutathione peroxidase inhibitors. Measurement of 2',7'-dichlorofluorescein (DCF)-induced fluorescence revealed that expression of hSOD1(G93A) or hSOD1wt in astrocytes does not lead to detectable increase of intracellular reactive oxygen species. This study suggests that levels of GLT-1 protein in astrocytes are reduced rapidly by overexpression of hSOD1, and is due to a property shared between the wild-type and G93A mutant form, but does not involve the production of intracellular oxidative stress.     

Modulation of mutant superoxide dismutase 1 aggregation by co-expression of wild-type enzyme

Mutations in superoxide dismutase 1 (SOD1, EC 1.15.1.1) cause familial amyotrophic lateral sclerosis; with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop amyotrophic lateral sclerosis-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared with mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, hSOD1 or mouse SOD1, delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mouse SOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates the aggregation of mutant SOD1.     

Gene Expression Profiles of APP and BACE1 in Tg SOD1G93A Cortical Cells

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease defined by motor neuron loss. Transgenic mouse model (Tg SOD1G93A) shows pathological features that closely mimic those seen in ALS patients. An hypothetic link between AD and ALS was suggested by finding an higher amount of amyloid precursor protein (APP) in the spinal cord anterior horn neurons, and of Aβ peptides in ALS patients skin. In this work, we have investigated the expression of some genes involved in Alzheimer’s disease, as APP, β- and γ-secretase, in an animal model of ALS, to understand some possible common molecular mechanisms between these two pathologies. For gene expression analysis, we carried out a quantitative RT-PCR in ALS mice and in transgenic mice over-expressing human wild-type SOD1 (Tg hSOD1). We found that APP and BACE1 mRNA levels were increased 1.5-fold in cortical cells of Tg SOD1G93A mice respect to Tg hSOD1, whereas the expression of γ-secretase genes, as PSEN1, PSEN2, Nicastrin, and APH1a, showed no statistical differences between wild-type and ALS mice. Biochemical analysis carried out by immunostaining and western blotting, did not show any significant modulation of the protein expression compared to the genes, suggesting the existence of post-translational mechanisms that modify protein levels.     

Oxidation of the Tryptophan 32 Residue of Human Superoxide Dismutase 1 Caused by Its Bicarbonate-dependent Peroxidase Activity Triggers the Non-amyloid Aggregation of the Enzyme

The role of oxidative post-translational modifications of human superoxide dismutase 1 (hSOD1) in the amyotrophic lateral sclerosis (ALS) pathology is an attractive hypothesis to explore based on several lines of evidence. Among them, the remarkable stability of hSOD1^WT and several of its ALS-associated mutants suggests that hSOD1 oxidation may precede its conversion to the unfolded and aggregated forms found in ALS patients. The bicarbonate-dependent peroxidase activity of hSOD1 causes oxidation of its own solvent-exposed Trp³² residue. The resulting products are apparently different from those produced in the absence of bicarbonate and are most likely specific for simian SOD1s, which contain the Trp³² residue. The aims of this work were to examine whether the bicarbonate-dependent peroxidase activity of hSOD1 (hSOD1^WT and hSOD1^G93A mutant) triggers aggregation of the enzyme and to comprehend the role of the Trp³² residue in the process. The results showed that Trp³² residues of both enzymes are oxidized to a similar extent to hSOD1-derived tryptophanyl radicals. These radicals decayed to hSOD1-N-formylkynurenine and hSOD1-kynurenine or to a hSOD1 covalent dimer cross-linked by a ditryptophan bond, causing hSOD1 unfolding, oligomerization, and non-amyloid aggregation. The latter process was inhibited by tempol, which recombines with the hSOD1-derived tryptophanyl radical, and did not occur in the absence of bicarbonate or with enzymes that lack the Trp³² residue (bovine SOD1 and hSOD1^W32F mutant). The results support a role for the oxidation products of the hSOD1-Trp³² residue, particularly the covalent dimer, in triggering the non-amyloid aggregation of hSOD1.     

Amyotrophic lateral sclerosis-associated SOD1 mutant proteins bind and aggregate with Bcl-2 in spinal cord mitochondria

Familial amyotrophic lateral sclerosis (ALS)-linked mutations in the copper-zinc superoxide dismutase (SOD1) gene cause motor neuron death in about 3% of ALS cases. While the wild-type (wt) protein is anti-apoptotic, mutant SOD1 promotes apoptosis. We now demonstrate that both wt and mutant SOD1 bind the anti-apoptotic protein Bcl-2, providing evidence of a direct link between SOD1 and an apoptotic pathway. This interaction is evident in vitro and in vivo in mouse and human spinal cord. We also demonstrate that in mice and humans, Bcl-2 binds to high molecular weight SDS-resistant mutant SOD1 containing aggregates that are present in mitochondria from spinal cord but not liver. These findings provide new insights into the anti-apoptotic function of SOD1 and suggest that entrapment of Bcl-2 by large SOD1 aggregates may deplete motor neurons of this anti-apoptotic protein.     

Proteasomal inhibition by misfolded mutant superoxide dismutase 1 induces selective motor neuron death in familial amyotrophic lateral sclerosis

Accumulating evidence indicates that abnormal conformation of mutant superoxide dismutase 1 (SOD1) is an essential feature underlying the pathogenesis of mutant SOD1-linked familial amyotrophic lateral sclerosis (ALS). Here we investigated the role of ubiquitin-proteasome pathway in the mutant SOD1-related cell death and the effect of oxidative stress on the misfolding of mutant SOD1. Transient overexpression of ubiquitin with human SOD1 (wild-type, ala4val, gly85arg, gly93ala) in Neuro2A cells decreased the amount of mutant SOD1, but not of wild-type, while only mutants were co-immunoprecipitated with poly-ubiquitin. Proteasome inhibition by lactacystin augmented accumulation of mutant SOD1 in the non-ionic detergent-insoluble fraction. The spinal cord lysates from mutant SOD1 transgenic mice showed multiple carbonylated proteins, including mutant SOD1 with SDS-resistant dimer formation. Furthermore, the treatment of hSOD1-expressing cells with hydrogen peroxide promoted the oligomerization, and detergent-insolubility of mutant SOD1 alone, and the oxidized mutant SOD1 proteins were more heavily poly-ubiquitinated. In Neuro2A cells stably expressing human SOD1 protein, the proteasome function measured by chymotrypsin-like activity, was decreased over time without a quantitative alteration of the 20S proteasomal component. Finally, primary motor neurons from the mouse embryonic spinal cord were more vulnerable to lactacystin than non-motor neurons. These results indicate that the sustained expression of mutant SOD1 leads to proteasomal inhibition and motor neuronal death, which in part explains the pathogenesis of mutant SOD1-linked ALS.     

An immunological epitope selective for pathological monomer-misfolded SOD1 in ALS

Misfolding of Cu/Zn-superoxide dismutase (SOD1) is emerging as a mechanism underlying motor neuron degeneration in individuals with amyotrophic lateral sclerosis (ALS) who carry a mutant SOD1 gene (SOD1 ALS). Here we describe a structure-guided approach to developing an antibody that specifically recognizes monomer-misfolded forms of SOD1. We raised this antibody to an epitope that is normally buried in the SOD1 native homodimer interface. The SOD1 exposed dimer interface (SEDI) antibody recognizes only those SOD1 conformations in which the native dimer is disrupted or misfolded and thereby exposes the hydrophobic dimer interface. Using the SEDI antibody, we established the presence of monomer-misfolded SOD1 in three ALS mouse models, with G37R, G85R and G93A SOD1 mutations, and in a human individual with an A4V SOD1 mutation. Despite ubiquitous expression, misfolded SOD1 was found primarily within degenerating motor neurons. Misfolded SOD1 appeared before the onset of symptoms and decreased at the end stage of the disease, concomitant with motor neuron loss.     

SOD1 dimerization monitoring using a novel split NanoLuc,NanoBit

In the present study, we applied a highly sensitive NanoLuc‐based technology to understand the status of superoxide dismutase 1 (SOD1) within mammalian cells. Two fragments of NanoLuc (NanoBit), large N‐terminal and small C‐terminal regions, were fused with wild‐type (wt) and mutant human SOD1 (hSOD1) genes and transfected into cells. Luciferase activity through NanoBit assembly was only detected in NanoBit‐tagged wtSOD1‐expressing cells. Furthermore, the developed NanoLuc system was used to investigate the role of protein‐protein interactions in the pathogenesis of amyotrophic lateral sclerosis (ALS). In addition to SOD1, we also applied this NanoBit system for detecting the dimerization of wild‐type, M337V‐mutated human TAR‐binding protein 43 kDa (hTDP43) and its cleaved C‐terminal fragment (TDP25_M337V) as well as their interactions with SOD1. Luciferase activities of NanoBit‐tagged mutant SOD1, TDP43, or TDP25 were negligible. Finally, we found that a zinc chelator partially reduced the luciferase activity of NanoBit‐wtSOD1. Collectively, these results show that the present assay is sensitive and convenient to appreciate ALS and to develop useful agents for the modulation of SOD1 conformation.     

ALS-causing SOD1 mutations promote production of copper-deficient misfolded species

Point mutations scattered throughout the sequence of Cu,Zn superoxide dismutase (SOD1) cause a subset of amyotrophic lateral sclerosis (ALS) cases. SOD1 is a homodimer in which each subunit binds one copper atom and one zinc atom. Inclusions containing misfolded SOD1 are seen in motor neurons of SOD1-associated ALS cases. The mechanism by which these diverse mutations cause misfolding and converge on the same disease is still not well understood. Previously, we developed several time-resolved techniques to monitor structural changes in SOD1 as it unfolds in guanidine hydrochloride. By measuring the rates of Cu and Zn release using an absorbance-based assay, dimer dissociation through chemical cross-linking, and β-barrel conformation changes by tryptophan fluorescence, we established that wild-type SOD1 unfolds by a branched pathway involving a Zn-deficient monomer as the dominant intermediate of the major pathway, and with various metal-loaded and Cu-deficient dimers populated along the minor pathway. We have now compared the unfolding pathway of wild-type SOD1 with those of A4V, G37R, G85R, G93A, and I113T ALS-associated mutant SOD1. The kinetics of unfolding of the mutants were generally much faster than those of wild type. However, all of the mutants utilize the minority pathway to a greater extent than the wild-type protein, leading to greater populations of Cu-deficient intermediates and decreases in Zn-deficient intermediates relative to the wild-type protein. The greater propensity of the mutants to populate Cu-deficient states potentially implicates these species as a pathogenic form of SOD1 in SOD1-associated ALS and provides a novel target for therapeutic intervention.     

Features of wild-type human SOD1 limit interactions with misfolded aggregates of mouse G86R Sod1

Mutations in the gene encoding superoxide dismutase 1 (SOD1) account for about 20% of the cases of familial amyotrophic lateral sclerosis (fALS). It is well established that mutations in SOD1, associated with fALS, heighten the propensity of the protein to misfold and aggregate. Although aggregation appears to be a factor in the toxicity of mutant SOD1s, the precise nature of this toxicity has not been elucidated. A number of other studies have now firmly established that raising the levels of wild-type (WT) human SOD1 (hSOD1) proteins can in some manner augment the toxicity of mutant hSOD1 proteins. However, a recent study demonstrated that raising the levels of WT-hSOD1 did not affect disease in mice that harbor a mouse Sod1 gene (mSod1) encoding a well characterized fALS mutation (G86R). In the present study, we sought a potential explanation for the differing effects with WT-hSOD1 on the toxicity of mutant hSOD1 versus mutant mSod1. In the cell culture models used here, we observe poor interactions between WT-hSOD1 and misfolded G86R-mSod1, possibly explaining why over-expression of WT-hSOD1 does not synergize with mutant mSod1 to accelerate the course of the disease in mice.     

An Analysis of Interactions between Fluorescently-Tagged Mutant and Wild-Type SOD1 in Intracellular Inclusions

Background

By mechanisms yet to be discerned, the co-expression of high levels of wild-type human superoxide dismutase 1 (hSOD1) with variants of hSOD1 encoding mutations linked familial amyotrophic lateral sclerosis (fALS) hastens the onset of motor neuron degeneration in transgenic mice. Although it is known that spinal cords of paralyzed mice accumulate detergent insoluble forms of WT hSOD1 along with mutant hSOD1, it has been difficult to determine whether there is co-deposition of the proteins in inclusion structures.

Methodology/Principal Findings

In the present study, we use cell culture models of mutant SOD1 aggregation, focusing on the A4V, G37R, and G85R variants, to examine interactions between WT-hSOD1 and misfolded mutant SOD1. In these studies, we fuse WT and mutant proteins to either yellow or red fluorescent protein so that the two proteins can be distinguished within inclusions structures.

Conclusions/Significance

Although the interpretation of the data is not entirely straightforward because we have strong evidence that the nature of the fused fluorophores affects the organization of the inclusions that form, our data are most consistent with the idea that normal dimeric WT-hSOD1 does not readily interact with misfolded forms of mutant hSOD1. We also demonstrate the monomerization of WT-hSOD1 by experimental mutation does induce the protein to aggregate, although such monomerization may enable interactions with misfolded mutant SOD1. Our data suggest that WT-hSOD1 is not prone to become intimately associated with misfolded mutant hSOD1 within intracellular inclusions that can be generated in cultured cells.     

CHIP promotes proteasomal degradation of familial ALS-linked mutant SOD1 by ubiquitinating Hsp/Hsc70

Over 100 mutants in superoxide dismutase 1 (SOD1) are reported in familial amyotrophic lateral sclerosis (ALS). However, the precise mechanism by which they are degraded through a ubiquitin-proteasomal pathway (UPP) remains unclear. Here, we report that heat-shock protein (Hsp) or heat-shock cognate (Hsc)70, and the carboxyl terminus of the Hsc70-interacting protein (CHIP), are involved in proteasomal degradation of mutant SOD1. Only mutant SOD1 interacted with Hsp/Hsc70 in vivo, and in vitro experiments revealed that Hsp/Hsc70 preferentially interacted with apo-SOD1 or dithiothreitol (DTT)-treated holo-SOD1, compared with metallated or oxidized forms. CHIP, a binding partner of Hsp/Hsc70, interacted only with mutant SOD1 and promoted its degradation. Both Hsp70 and CHIP promoted polyubiquitination of mutant SOD1-associated molecules, but not of mutant SOD1, indicating that mutant SOD1 is not a substrate of CHIP. Moreover, mutant SOD1-associated Hsp/Hsc70, a known substrate of CHIP, was polyubiquitinated in vivo, and polyubiquitinated Hsc70 by CHIP interacted with the S5a subunit of the 26S proteasome in vitro. Furthermore, CHIP was predominantly expressed in spinal neurons, and ubiquitinated inclusions in the spinal motor neurons of hSOD1(G93A) transgenic mice were CHIP-immunoreactive. Taken together, we propose a novel pathway in which ubiquitinated Hsp/Hsc70 might deliver mutant SOD1 to, and facilitate its degradation, at the proteasome.     

Mitochondria-Targeted Catalase Reverts the Neurotoxicity of hSOD1G93A Astrocytes without Extending the Survival of ALS-Linked Mutant hSOD1 Mice

Dominant mutations in the Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons. The molecular mechanism underlying the toxic gain-of-function of mutant hSOD1s remains uncertain. Several lines of evidence suggest that toxicity to motor neurons requires damage to non-neuronal cells. In line with this observation, primary astrocytes isolated from mutant hSOD1 over-expressing rodents induce motor neuron death in co-culture. Mitochondrial alterations have been documented in both neuronal and glial cells from ALS patients as well as in ALS-animal models. In addition, mitochondrial dysfunction and increased oxidative stress have been linked to the toxicity of mutant hSOD1 in astrocytes and neurons. In mutant SOD1-linked ALS, mitochondrial alterations may be partially due to the increased association of mutant SOD1 with the outer membrane and intermembrane space of the mitochondria, where it can affect several critical aspects of mitochondrial function. We have previously shown that decreasing glutathione levels, which is crucial for peroxide detoxification in the mitochondria, significantly accelerates motor neuron death in hSOD1^G93A mice. Here we employed a catalase targeted to the mitochondria to investigate the effect of increased mitochondrial peroxide detoxification capacity in models of mutant hSOD1-mediated motor neuron death. The over-expression of mitochondria-targeted catalase improved mitochondrial antioxidant defenses and mitochondrial function in hSOD1^G93A astrocyte cultures. It also reverted the toxicity of hSOD1^G93A-expressing astrocytes towards co-cultured motor neurons, however ALS-animals did not develop the disease later or survive longer. Hence, while increased oxidative stress and mitochondrial dysfunction have been extensively documented in ALS, these results suggest that preventing peroxide-mediated mitochondrial damage alone is not sufficient to delay the disease.     

Mutation frequency is reduced in the cerebellum of Big Blue mice overexpressing a human wild type SOD1 gene

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder caused by motor neuron degeneration. A similar disease phenotype is observed in mice overexpressing a mutant human hSOD1 gene (G93A, 1Gurd(1)). Mice transgenic for lacI (Big Blue) and human mutant (1Gurd(1), Mut hSOD1) or wild type (2Gur, Wt hSOD1) SOD1 genes were used to examine spontaneous mutation, oxidative DNA damage, and neurodegeneration in vivo. The frequency and pattern of spontaneous mutation were determined for forebrain (90% glia), cerebellum (90% neurons) and thymus from 5-month-old male mice. Mutation frequency is not elevated significantly and mutation pattern is unaltered in Mut hSOD1 mice compared to control mice. Mutation frequency is reduced significantly in the cerebellum of Wt hSOD1 mice (1.6x10(-5); P=0.0093; Fisher's Exact Test) compared to mice without a human transgene (2.7x10(-5)). Mutation pattern is unaltered. This first report of an endogenous factor that can reduce in vivo, the frequency of spontaneous mutation suggests potential strategies for lowering mutagenesis related to aging, neurodegeneration, and carcinogenesis.     

Absence of Nrf2 or Its Selective Overexpression in Neurons and Muscle Does Not Affect Survival in ALS-Linked Mutant hSOD1 Mouse Models

The nuclear factor erythroid 2-related factor 2 (Nrf2) governs the expression of antioxidant and phase II detoxifying enzymes. Nrf2 activation can prevent or reduce cellular damage associated with several types of injury in many different tissues and organs. Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) cause familial forms of amyotrophic lateral sclerosis (ALS), a fatal disorder characterized by the progressive loss of motor neurons and subsequent muscular atrophy. We have previously shown that Nrf2 activation in astrocytes delays neurodegeneration in ALS mouse models. To further investigate the role of Nrf2 in ALS we determined the effect of absence of Nrf2 or its restricted overexpression in neurons or type II skeletal muscle fibers on symptoms onset and survival in mutant hSOD1 expressing mice. We did not observe any detrimental effect associated with the lack of Nrf2 in two different mutant hSOD1 animal models of ALS. However, restricted Nrf2 overexpression in neurons or type II skeletal muscle fibers delayed disease onset but failed to extend survival in hSOD1^G93A mice. These results highlight the concept that not only the pharmacological target but also the cell type targeted may be relevant when considering a Nrf2-mediated therapeutic approach for ALS.     

Analysis of Mutant SOD1 Electrophoretic Mobility by Blue Native Gel Electrophoresis; Evidence for Soluble Multimeric Assemblies

Mutations in superoxide dismutase 1 (SOD1) cause familial forms of amyotrophic lateral sclerosis (fALS). Disease causing mutations have diverse consequences on the activity and half-life of the protein, ranging from complete inactivity and short half-life to full activity and long-half-life. Uniformly, disease causing mutations induce the protein to misfold and aggregate and such aggregation tendencies are readily visualized by over-expression of the proteins in cultured cells. In the present study we have investigated the potential of using immunoblotting of proteins separated by Blue-Native gel electrophoresis (BNGE) as a means to identify soluble multimeric forms of mutant protein. We find that over-expressed wild-type human SOD1 (hSOD1) is generally not prone to form soluble high molecular weight entities that can be separated by BNGE. For ALS mutant SOD1, we observe that for all mutants examined (A4V, G37R, G85R, G93A, and L126Z), immunoblots of BN-gels separating protein solubilized by digitonin demonstrated varied amounts of high molecular weight immunoreactive entities. These entities lacked reactivity to ubiquitin and were partially dissociated by reducing agents. With the exception of the G93A mutant, these entities were not reactive to the C4F6 conformational antibody. Collectively, these data demonstrate that BNGE can be used to assess the formation of soluble multimeric assemblies of mutant SOD1.     

Mutated human SOD1 causes dysfunction of oxidative phosphorylation in mitochondria of transgenic mice

A growing body of evidence suggests that impaired mitochondrial energy production and increased oxidative radical damage to the mitochondria could be causally involved in motor neuron death in amyotrophic lateral sclerosis (ALS) and in familial ALS associated with mutations of Cu,Zn superoxide dismutase (SOD1). For example, morphologically abnormal mitochondria and impaired mitochondrial histoenzymatic respiratory chain activities have been described in motor neurons of patients with sporadic ALS. To investigate further the role of mitochondrial alterations in the pathogenesis of ALS, we studied mitochondria from transgenic mice expressing wild type and G93A mutated hSOD1. We found that a significant proportion of enzymatically active SOD1 was localized in the intermembrane space of mitochondria. Mitochondrial respiration, electron transfer chain, and ATP synthesis were severely defective in G93A mice at the time of onset of the disease. We also found evidence of oxidative damage to mitochondrial proteins and lipids. On the other hand, presymptomatic G93A transgenic mice and mice expressing the wild type form of hSOD1 did not show significant mitochondrial abnormalities. Our findings suggest that G93A-mutated hSOD1 in mitochondria may cause mitochondrial defects, which contribute to precipitating the neurodegenerative process in motor neurons.