Bim links ER stress and apoptosis in cells expressing mutant SOD1 associated with amyotrophic lateral sclerosis

Endoplasmic reticulum (ER) stress is an important pathway to cell death in amyotrophic lateral sclerosis (ALS). We previously demonstrated that ER stress is linked to neurotoxicity associated with formation of inclusions of mutant Cu,Zn-superoxide dismutase 1 (SOD1). Cells bearing mutant inclusions undergo mitochondrial apoptotic signalling. Here, we demonstrate that the BH3-only protein, Bim, is a direct link between ER stress and mitochondrial apoptosis. In the murine neuroblastoma cell line, Neuro2a, bearing mutant SOD1 inclusions, indicators of both ER stress and apoptosis are expressed. Bim knockdown by siRNA significantly reduced nuclear apoptotic features in these inclusion-bearing cells (but did not affect the proportion of cells overall that bear inclusions). Further, both Bax recruitment to mitochondria and cytochrome c redistribution were also decreased under Bim-depletion conditions. However, upregulation of CHOP, a marker of ER stress, was not reduced by Bim knockdown. Significantly, knockdown of CHOP by siRNA reduced the extent of apoptosis in cells bearing mutant SOD1 inclusions. These sequential links between ER stress, CHOP upregulation, and Bim activation of mitochondrial apoptotic signalling indicate a clear pathway to cell death mediated by mutant SOD1.     

Inducible Nitric Oxide Synthase Up-Regulation in a Transgenic Mouse Model of Familial Amyotrophic Lateral Sclerosis

Mutations in copper/zinc superoxide dismutase (SOD1) are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Here we show that, during the course of the disease, the spinal cord of transgenic mice expressing mutant SOD1 (mSOD1) is the site not only of a progressive loss of motor neurons, but also of a dramatic gliosis characterized by reactive astrocytes and activated microglial cells. These changes are absent from the spinal cord of age-matched transgenic mice expressing normal SOD1 and of wild-type mice. We also demonstrate that, during the course of the disease, the expression of inducible nitric oxide synthase (iNOS) increases. In both early symptomatic and end-stage transgenic mSOD1 mice, numerous cells with the appearance of glial cells are strongly iNOS-immunoreactive. In addition, iNOS mRNA level and catalytic activity are increased significantly in the spinal cord of these transgenic mSOD1 mice. None of these alterations are seen in the cerebellum of these animals, a region unaffected by mSOD1. Similarly, no up-regulation of iNOS is detected in the spinal cord of age-matched transgenic mice expressing normal SOD1 or of wild-type mice. The time course of the spinal cord gliosis and iNOS up-regulation parallels that of motor neuronal loss in transgenic mSOD1 mice. Neuronal nitric oxide synthase expression is only seen in neurons in the spinal cord of transgenic mSOD1 mice, regardless of the stage of the disease, and of age-matched transgenic mice expressing normal SOD1 and wild-type mice. Collectively, these data suggest that the observed alterations do not initiate the death of motor neurons, but may contribute to the propagation of the neurodegenerative process. Furthermore, the up-regulation of iNOS, which in turn may stimulate the production of nitric oxide, provides further support to the presumed deleterious role of nitric oxide in the pathogenesis of ALS. This observation also suggests that iNOS may represent a valuable target for the development of new therapeutic avenues for ALS.     

Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein-disulfide isomerase with superoxide dismutase 1

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.     

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.     

Interaction of amyotrophic lateral sclerosis (ALS)-related mutant copper-zinc superoxide dismutase with the dynein-dynactin complex contributes to inclusion formation

An important consequence of protein misfolding related to neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the formation of proteinaceous inclusions or aggregates within the central nervous system. We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interact and co-localize with the dynein-dynactin complex in cultured cells and affected tissues of ALS mice. In this study, we report that the interaction between mutant SOD1 and the dynein motor plays a critical role in the formation of large inclusions containing mutant SOD1. Disruption of the motor by overexpression of the p50 subunit of dynactin in neuronal and non-neuronal cell cultures abolished the association between aggregation-prone SOD1 mutants and the dynein-dynactin complex. The p50 overexpression also prevented mutant SOD1 inclusion formation and improved the survival of cells expressing A4V SOD1. Furthermore, we observed that two ALS-linked SOD1 mutants, H46R and H48Q, which showed a lower propensity to interact with the dynein motor, also produced less aggregation and fewer large inclusions. Overall, these data suggest that formation of large inclusions depends upon association of the abnormal SOD1s with the dynein motor. Whether the misfolded SOD1s directly perturb axonal transport or impair other functional properties of the dynein motor, this interaction could propagate a toxic effect that ultimately causes motor neuron death in ALS.     

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.     

SOD1 Mutations Causing Familial Amyotrophic Lateral Sclerosis Induce Toxicity in Astrocytes: Evidence for Bystander Effects in a Continuum of Astrogliosis

Astrocytes contribute to the death of motor neurons via non-cell autonomous mechanisms of injury in amyotrophic lateral sclerosis (ALS). Since mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) underlie the neuropathology of some forms of familial ALS, we explored how expression of mutant SOD1 protein A4V SOD1-EGFP affected the biology of secondary murine astrocytes. A4V SOD1-EGFP expressing astrocytes (72 h after transfection) displayed decreased mitochondrial activity (~45%) and l-glutamate transport (~25%), relative to cells expressing wild-type SOD1-EGFP. A4V SOD1-EGFP altered F-actin and Hoechst staining, indicative of cytoskeletal and nuclear changes, and altered GM130 labelling suggesting fragmentation of Golgi apparatus. SOD1 inclusion formation shifted from discrete to “punctate” over 72 h with A4V SOD1-EGFP more rapidly producing inclusions than G85R SOD1-EGFP, and forming more punctate aggregates. A4V, not wild-type SOD1-EGFP, exerted a substantial, time-dependent effect on GFAP expression, and ~60% of astrocytes became stellate and hypertrophic at 72 h. Spreading toxicity was inferred since at 72 h ~80% of bystander cells exhibited hypertrophy and stellation. This evidence favours mutant SOD1-containing astrocytes releasing destructive species that alter the biology of adjacent astrocytes. This panoply of mutant SOD1-induced destructive events favours recruitment of astrocytes to non-cell autonomous injury in ALS.     

Wild-type superoxide dismutase acquires binding and toxic properties of ALS-linked mutant forms through oxidation

Recent studies suggest that superoxide dismutase (SOD1) may represent a major target of oxidative damage in neurodegenerative diseases. To test the possibility that oxidized species of wild-type (WT) SOD1 might be involved in pathogenic processes, we analyzed the properties of the WT human SOD1 protein after its oxidation in vivo or in vitro by hydrogen peroxide (H2O2) treatment. Using transfected Neuro2a cells expressing WT or amyotrophic lateral sclerosis-linked SOD1 species, we show that exposure to H2O2 modifies the properties of WT SOD1. Western blot analysis of immunoprecipitates from cell lysates revealed that, like mutant SOD1, oxidized WT SOD1 can be conjugated with poly-ubiquitin and can interact with Hsp70. Chromogranin B, a neurosecretory protein that interacts with mutant SOD1 but not with WT SOD1, was co-immunoprecipitated with oxidized WT SOD1 from lysates of Neuro2a cells treated with H2O2. Treatment of microglial cells (line BV2) with either oxidized WT SOD1 or mutant SOD1 recombinant proteins induced tumor necrosis factor-alpha and inducible nitric oxide synthase. Furthermore, exposure of cultured motor neurons to oxidized WT SOD1 caused dose-dependent cell death like mutant SOD1 proteins. These results suggest that WT SOD1 may acquire binding and toxic properties of mutant forms of SOD1 through oxidative damage.     

Mitochondrial Ubiquitin Ligase MITOL Ubiquitinates Mutant SOD1 and Attenuates Mutant SOD1-induced Reactive Oxygen Species Generation

We have previously identified a novel mitochondrial ubiquitin ligase, MITOL, which is localized in the mitochondrial outer membrane and is involved in the control of mitochondrial dynamics. In this study, we examined whether MITOL eliminates misfolded proteins localized to mitochondria. Mutant superoxide dismutase1 (mSOD1), one of misfolded proteins, has been shown to localize in mitochondria and induce mitochondrial dysfunction, possibly involving in the onset and progression of amyotrophic lateral sclerosis. We found that in the mitochondria, MITOL interacted with and ubiquitinated mSOD1 but not wild-type SOD1. In vitro ubiquitination assay revealed that MITOL directly ubiquitinates mSOD1. Cycloheximide-chase assay in the Neuro2a cells indicated that MITOL overexpression promoted mSOD1 degradation and suppressed both the mitochondrial accumulation of mSOD1 and mSOD1-induced reactive oxygen species (ROS) generation. Conversely, the overexpression of MITOL CS mutant and MITOL knockdown by specific siRNAs resulted in increased accumulation of mSOD1 in mitochondria, which enhanced mSOD1-induced ROS generation and cell death. Thus, our findings indicate that MITOL plays a protective role against mitochondrial dysfunction caused by the mitochondrial accumulation of mSOD1 via the ubiquitin–proteasome pathway.     

Spy1, a unique cell cycle regulator,alters viability in ALS motor neurons and cell lines in response to mutant SOD1-induced DNA damage

Increasing evidence indicates that DNA damage and p53 activation play major roles in the pathological process of motor neuron death in amyotrophic lateral sclerosis (ALS). Human SpeedyA1 (Spy1), a member of the Speedy/Ringo family, enhances cell proliferation and promotes tumorigenesis. Further studies have demonstrated that Spy1 promotes cell survival and inhibits DNA damage-induced apoptosis. We showed that the Spy1 expression levels were substantially decreased in ALS motor neurons compared with wild-type controls both in vivo and in vitro by qRT-PCR, western blotting, and Immunoassay tests. In addition, we established that over-expression of human SOD1 mutant G93A led to a decreased expression of Spy1. Furthermore, DNA damage response was activated in SOD1^G93A-transfected cells (mSOD1 cells). Moreover, decreased Spy1 expression reduced cell viability and further activated the DNA damage response in mSOD1 cells. In contrast, increased Spy1 expression improved cell viability and inhibited the DNA damage response in mSOD1 cells. These results suggest that Spy1 plays a protective role in ALS motor neurons. Importantly, these findings provide a novel direction for therapeutic options for patients with ALS as well as for trial designs, such as investigating the role of oncogenic proteins in ALS.     

Mutant SOD1 inhibits ER‐Golgi transport in amyotrophic lateral sclerosis

Cu/Zn‐superoxide dismutase is misfolded in familial and sporadic amyotrophic lateral sclerosis, but it is not clear how this triggers endoplasmic reticulum (ER) stress or other pathogenic processes. Here, we demonstrate that mutant SOD1 (mSOD1) is predominantly found in the cytoplasm in neuronal cells. Furthermore, we show that mSOD1 inhibits secretory protein transport from the ER to Golgi apparatus. ER‐Golgi transport is linked to ER stress, Golgi fragmentation and axonal transport and we also show that inhibition of ER‐Golgi trafficking preceded ER stress, Golgi fragmentation, protein aggregation and apoptosis in cells expressing mSOD1. Restoration of ER‐Golgi transport by over‐expression of coatomer coat protein II subunit Sar1 protected against inclusion formation and apoptosis, thus linking dysfunction in ER‐Golgi transport to cellular pathology. These findings thus link several cellular events in amyotrophic lateral sclerosis into a single mechanism occurring early in mSOD1 expressing cells.

     

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.     

Replacement of microglial cells using Clodronate liposome and bone marrow transplantation in the central nervous system of SOD1(G93A) transgenic mice as an in vivo model of amyotrophic lateral sclerosis

Disease progression of amyotrophic lateral sclerosis (ALS) is partially mediated by the toxic microenvironment established by microglia. In the present study, we used SOD1G93A transgenic mice as an in vivo ALS model and replaced microglia expressing mutant SOD1 (mSOD1) with microglia expressing wild-type SOD1 (w/tSOD1) to modulate the toxic microenvironment. Stereotactic injection of Clodronate liposome, a selective toxin against the monocyte/macrophage system, into the fourth ventricle of the brains of 12-week-old asymptomatic ALS mice reduced the number of microglia effectively in the central nervous system. Subsequent bone marrow transplantation (BMT) with bone marrow cells (BMCs) expressing w/tSOD1 and GFP leads to replacement of the endogenous microglia of the ALS mice with microglia expressing w/tSOD1 and GFP. The expression of mSOD1 in the other neural cells was not influenced by the replacement procedures, and immunological side effects were not observed. The replacement of microglia significantly slowed disease progression and prolonged survival of the ALS mice compared with the ALS mice treated by stereotactic injection of PBS-liposome and BMT with BMCs expressing mSOD1 or w/tSOD1. These results suggest that replacement of microglia would improve the neural cell microenvironment, thereby slowing disease progression. The mechanisms and functional implications of this replacement require further elucidation.     

Exendin-4 ameliorates motor neuron degeneration in cellular and animal models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by a progressive loss of lower motor neurons in the spinal cord. The incretin hormone, glucagon-like peptide-1 (GLP-1), facilitates insulin signaling, and the long acting GLP-1 receptor agonist exendin-4 (Ex-4) is currently used as an anti-diabetic drug. GLP-1 receptors are widely expressed in the brain and spinal cord, and our prior studies have shown that Ex-4 is neuroprotective in several neurodegenerative disease rodent models, including stroke, Parkinson's disease and Alzheimer's disease. Here we hypothesized that Ex-4 may provide neuroprotective activity in ALS, and hence characterized Ex-4 actions in both cell culture (NSC-19 neuroblastoma cells) and in vivo (SOD1 G93A mutant mice) models of ALS. Ex-4 proved to be neurotrophic in NSC-19 cells, elevating choline acetyltransferase (ChAT) activity, as well as neuroprotective, protecting cells from hydrogen peroxide-induced oxidative stress and staurosporine-induced apoptosis. Additionally, in both wild-type SOD1 and mutant SOD1 (G37R) stably transfected NSC-19 cell lines, Ex-4 protected against trophic factor withdrawal-induced toxicity. To assess in vivo translation, SOD1 mutant mice were administered vehicle or Ex-4 at 6-weeks of age onwards to end-stage disease via subcutaneous osmotic pump to provide steady-state infusion. ALS mice treated with Ex-4 showed improved glucose tolerance and normalization of behavior, as assessed by running wheel, compared to control ALS mice. Furthermore, Ex-4 treatment attenuated neuronal cell death in the lumbar spinal cord; immunohistochemical analysis demonstrated the rescue of neuronal markers, such as ChAT, associated with motor neurons. Together, our results suggest that GLP-1 receptor agonists warrant further evaluation to assess whether their neuroprotective potential is of therapeutic relevance in ALS.     

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

Mitochondrial dysfunction and its role in motor neuron degeneration in ALS

Mitochondria play a pivotal role in many metabolic and apoptotic pathways that regulate the life and death of cells. Accumulating evidence suggests that mitochondrial dysfunction is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Mitochondrial dysfunction may cause motor neuron death by predisposing them to calcium-mediated excitotoxicity, by increasing generation of reactive oxygen species, and by initiating the intrinsic apoptotic pathway. Morphological and biochemical mitochondrial abnormalities have been described in sporadic human ALS cases, but the implications of these findings in terminally ill individuals or in post-mortem tissues are difficult to decipher. However, remarkable mitochondrial abnormalities have also been identified in transgenic mouse models of familial ALS expressing mutant Cu, Zn superoxide dismutase (SOD1). Detailed studies in these mouse models indicate that mitochondrial abnormalities begin prior to the clinical and pathological onset of the disease, suggesting that mitochondrial dysfunction may be causally involved in the pathogenesis of ALS. Although the mechanisms whereby mutant SOD1 damages mitochondria remain to be fully understood, the finding that a portion of mutant SOD1 is localized in mitochondria, where it forms aberrant aggregates and protein interactions, has opened a number of avenues of investigation. The future challenges are to devise models to better understand the effects of mutant SOD1 in mitochondria and the relative contribution of mitochondrial dysfunction to the pathogenesis of ALS, as well as to identify therapeutic approaches that target mitochondrial dysfunction and its consequences.     

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.     

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.     

The Copper Metabolism MURR1 Domain Protein 1 (COMMD1) Modulates the Aggregation of Misfolded Protein Species in a Client-Specific Manner

The Copper Metabolism MURR1 domain protein 1 (COMMD1) is a protein involved in multiple cellular pathways, including copper homeostasis, NF-κB and hypoxia signalling. Acting as a scaffold protein, COMMD1 mediates the levels, stability and proteolysis of its substrates (e.g. the copper-transporters ATP7B and ATP7A, RELA and HIF-1α). Recently, we established an interaction between the Cu/Zn superoxide dismutase 1 (SOD1) and COMMD1, resulting in a decreased maturation and activation of SOD1. Mutations in SOD1, associated with the progressive neurodegenerative disorder Amyotrophic Lateral Sclerosis (ALS), cause misfolding and aggregation of the mutant SOD1 (mSOD1) protein. Here, we identify COMMD1 as a novel regulator of misfolded protein aggregation as it enhances the formation of mSOD1 aggregates upon binding. Interestingly, COMMD1 co-localizes to the sites of mSOD1 inclusions and forms high molecular weight complexes in the presence of mSOD1. The effect of COMMD1 on protein aggregation is client-specific as, in contrast to mSOD1, COMMD1 decreases the abundance of mutant Parkin inclusions, associated with Parkinson’s disease. Aggregation of a polyglutamine-expanded Huntingtin, causative of Huntington’s disease, appears unaltered by COMMD1. Altogether, this study offers new research directions to expand our current knowledge on the mechanisms underlying aggregation disease pathologies.