ER stress and UPR in familial amyotrophic lateral sclerosis

The primary mechanism by which mutations in Cu, Zn-superoxide dismutase (SOD1) contribute to progressive motor neuron loss in familial amyotrophic lateral sclerosis (FALS) remains unknown. Misfolded protein aggregates, ubiquitin-proteasome system impairment and neuronal apoptosis mediated by death receptor or mitochondrial-dependent pathways are implicated in mutant SOD1-induced toxicity. Recent evidence from cellular and transgenic rodent models of FALS proposes activation of a third apoptotic pathway linked to sustained endoplasmic reticulum (ER) stress. Here, we review the emerging role of ER stress and the unfolded protein response (UPR) in the pathogenesis of mutant SOD1-linked FALS. The UPR observed in FALS rodents is described which encompasses induction of key ER-resident chaperones during presymptomatic disease, leading to activation of stress transducers and pro-apoptotic molecules by late stage disease. Importantly, mutant SOD1 co-aggregates with UPR components and recruits to the ER, suggesting a direct adverse effect on ER function. By contrast, the opposing neuroprotective effects of wild-type SOD1 overexpression on UPR signalling are also highlighted. In addition, the potential impact of neuronal Golgi apparatus (GA) fragmentation and subsequent disturbances in intracellular protein trafficking on motor neuron survival in FALS is also discussed. We propose that ER stress and UPR may be coupled to GA dysfunction in mutant SOD1-mediated toxicity, promoting ER-initiated cell death signalling in FALS.     

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.     

A novel Akt/PKB-interacting protein promotes cell adhesion and inhibits familial amyotrophic lateral sclerosis-linked mutant SOD1-induced neuronal death via inhibition of PP2A-mediated dephosphorylation of Akt/PKB

Akt/Protein Kinase B (PKB) family proteins (Akts), consisting of Akt1, 2, and 3, play a crucial role in multiple biological processes. We recently demonstrated that activation of Akt3 by the autosomal-recessive familial amyotrophic lateral sclerosis (ALS)-linked gene 2 (ALS2) product, alsinLF, led to the suppression of motoneuronal death induced by familial ALS-related mutant superoxide dismutase-1 (SOD1). To characterize the mechanism of neuroprotection mediated by Akt3 in detail, we performed a yeast two-hybrid system using Akt3 as a bait and identified BTBD10 as a novel Akt-interacting protein with a BTB/POZ domain. BTBD10 equally binds to any Akt. Overexpression of BTBD10 increased phosphorylation levels of Akts at both Thr(308) and Ser(473) while the reduction of the endogenous BTBD10 level resulted in a decrease in the phosphorylation levels of Akts. In vitro analysis indicated that BTBD10 bound to protein phosphatase 2A (PP2A) and inhibited dephosphorylation of Akts by PP2A. In agreement with BTBD10-mediated upregulation of the Akt phosphorylation levels, enforced expression of BTBD10 led to the suppression of mutant SOD1-induced neuronal death. Furthermore, overexpression of BTBD10 accelerated cell growth by enhancing cell adhesion. Given its ubiquitous expression, BTBD10 appears to behave as a suppressor of cell death including neuronal cell death related to ALS and an enhancer of cell growth via its positive regulation of Akt phosphorylation.     

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.     

Muscle cells and motoneurons differentially remove mutant SOD1 causing familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal motoneuronal disease which occurs in sporadic or familial forms, clinically indistinguishable. About 15% of familial ALS cases are linked to mutations of the superoxide dismutase 1 (SOD1) gene that may induce misfolding in the coded protein, exerting neurotoxicity to motoneurons. However, other cell types might be target of SOD1 toxicity, because muscle-restricted expression of mutant SOD1 correlates with muscle atrophy and motoneurons death. We analysed the molecular behaviour of mutant SOD1 in motoneuronal NSC34 and muscle C2C12 cells. We found that misfolded mutant SOD1 clearance is much more efficient in muscle C2C12 than in motoneuronal NSC34 cells. Mutant SOD1 forms aggregates and impairs the proteasome only in motoneuronal NSC34 cells. Interestingly, NSC34 cells expressing mutant SOD1 are more sensitive to a superoxide-induced oxidative stress. Moreover, in muscle C2C12 cells mutant SOD1 remains soluble even when proteasome is inhibited with MG132. The higher mutant SOD1 clearance in muscle cells correlates with a more efficient proteasome activity, combined with a robust autophagy activation. Therefore, muscle cells seem to better manage misfolded SOD1 species, not because of an intrinsic property of the mutant protein, but in function of the cell environment, indicating also that the SOD1 toxicity at muscle level may not directly depend on its aggregation rate.     

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.     

siRNA-based inhibition specific for mutant SOD1 with single nucleotide alternation in familial ALS, compared with ribozyme and DNA enzyme

In many of autosomal dominant diseases such as familial amyotrophic lateral sclerosis (ALS) with SOD1 mutation, a missense point mutation may induce the disease by its gain of adverse property. Reduction of such a mutant protein expression is expected to improve the disease phenotype. Duplex of 21-nt RNA, known as siRNA, has recently emerged as a powerful tool to silence gene, but the sequence specificity and efficacies have not been fully studied in comparison with ribozyme and DNA enzyme. We could make the siRNA which recognized even a single nucleotide alternation and selectively suppress G93A SOD1 expression leaving wild-type SOD1 intact. In mammalian cells, the siRNA much more efficiently suppressed the expression of mutant SOD1 than ribozyme or DNA enzyme. Furthermore, these siRNAs could suppress cell death of Neuro2a induced by over-expression of mutant SOD1s with stress of proteasome inhibition. Our results support the feasibility of utilizing siRNA-based gene therapy of familial ALS with mutant SOD1.     

Stress signaling from the endoplasmic reticulum: A central player in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the misfolding and aggregation of distinct proteins in affected tissues, however, the pathogenic cause of disease remains unknown. Recent evidence indicates that endoplasmic reticulum (ER) stress plays a central role in ALS pathogenesis. ER stress activates the unfolded protein response (UPR), a homeostatic response to misfolded proteins. The UPR is initially protective by up-regulation of specific ER stress-regulated genes and inhibition of general protein translation. However, long-term ER stress leads to cell death via apoptotic signaling, thus providing a link to neurodegeneration. Activation of the UPR is one of the earliest events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant superoxide dismutase 1 (SOD1). Recently, genetic manipulation of ER stress in several different SOD1 mouse models was shown to alter disease onset and progression, implicating an active role for the UPR in disease mechanisms. Furthermore, mutations to vesicle-associated membrane protein-associated protein B (VAPB), an ER transmembrane protein involved in ER stress regulation, also cause some cases of familial ALS. ER stress also occurs in spinal cord tissues of human sporadic ALS patients, and recent evidence suggests that perturbation of the ER could occur in ALS cases associated with TAR DNA binding protein 43 (TDP-43), fused in sarcoma (FUS) and valosin containing protein (VCP). Together these findings implicate ER stress as a potential upstream mechanism involved in both familial and sporadic forms of ALS.     

Disulfide bond mediates aggregation, toxicity, and ubiquitylation of familial amyotrophic lateral sclerosis-linked mutant SOD1

Mutations in the Cu/Zn-superoxide dismutase (SOD1) gene cause familial amyotrophic lateral sclerosis (ALS) through the gain of a toxic function; however, the nature of this toxic function remains largely unknown. Ubiquitylated aggregates of mutant SOD1 proteins in affected brain lesions are pathological hallmarks of the disease and are suggested to be involved in several proposed mechanisms of motor neuron death. Recent studies suggest that mutant SOD1 readily forms an incorrect disulfide bond upon mild oxidative stress in vitro, and the insoluble SOD1 aggregates in spinal cord of ALS model mice contain multimers cross-linked via intermolecular disulfide bonds. Here we show that a non-physiological intermolecular disulfide bond between cysteines at positions 6 and 111 of mutant SOD1 is important for high molecular weight aggregate formation, ubiquitylation, and neurotoxicity, all of which were dramatically reduced when the pertinent cysteines were replaced in mutant SOD1 expressed in Neuro-2a cells. Dorfin is a ubiquityl ligase that specifically binds familial ALS-linked mutant SOD1 and ubiquitylates it, thereby promoting its degradation. We found that Dorfin ubiquitylated mutant SOD1 by recognizing the Cys(6)- and Cys(111)-disulfide cross-linked form and targeted it for proteasomal degradation.     

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.     

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motorneurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signalling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motorneuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.     

Potential Effect of S-Nitrosylated Protein Disulfide Isomerase on Mutant SOD1 Aggregation and Neuronal Cell Death in Amyotrophic Lateral Sclerosis

Aggregation of misfolded protein and resultant intracellular inclusion body formation are common hallmarks of mutant superoxide dismutase (mSOD1)-linked familial amyotrophic lateral sclerosis (FALS) and have been associated with the selective neuronal death. Protein disulfide isomerase (PDI) represents a family of enzymatic chaperones that can fold nascent and aberrant proteins in the endoplasmic reticulum (ER) lumen. Recently, our group found that S-nitrosylated PDI could contribute to protein misfolding and subsequent neuronal cell death. However, the exact role of PDI in the pathogenesis of ALS remains unclear. In this study, we propose that PDI attenuates aggregation of mutant/misfolded SOD1 and resultant neurotoxicity associated with ER stress. ER stress resulting in PDI dysfunction therefore provides a mechanistic link between deficits in molecular chaperones, accumulation of misfolded proteins, and neuronal death in neurodegenerative diseases. In contrast, S-nitrosylation of PDI inhibits its activity, increases mSOD1 aggregation, and increases neuronal cell death. Specifically, our data show that S-nitrosylation abrogates PDI-mediated attenuation of neuronal cell death triggered by thapsigargin. Biotin switch assays demonstrate S-nitrosylated PDI both in the spinal cords of SOD1 (G93A) mice and human patients with sporadic ALS. Therefore, denitrosylation of PDI may have therapeutic implications. Taken together, our results suggest a novel strategy involving PDI as a therapy to prevent mSOD1 aggregation and neuronal degeneration. Moreover, the data demonstrate that inactivation of PDI by S-nitrosylation occurs in both mSOD1-linked and sporadic forms of ALS in humans as well as mice.     

Interaction between familial amyotrophic lateral sclerosis (ALS)-linked SOD1 mutants and the dynein complex

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive motor neuron death. More than 90 mutations in the copper-zinc superoxide dismutase (SOD1) gene cause a subset of familial ALS. Toxic properties have been proposed for the ALS-linked SOD1 mutants, but the nature of the toxicity has not been clearly specified. Cytoplasmic inclusion bodies containing mutant SOD1 and a number of other proteins are a pathological hallmark of mutant SOD1-mediated familial ALS, but whether such aggregates are toxic to motor neurons remains unclear. In this study, we identified a dynein subunit as a component of the mutant SOD1-containing high molecular weight complexes using proteomic techniques. We further demonstrated interaction and colocalization between dynein and mutant SOD1, but not normal SOD1, in cultured cells and also in G93A and G85R transgenic rodent tissues. Moreover, the interaction occurred early, prior to the onset of symptoms in the ALS animal models and increased over the disease progression. Motor neurons with long axons are particularly susceptible to defects in axonal transport. Our results demonstrate a direct "gain-of-interaction" between mutant SOD1 and dynein, which may provide insights into the mechanism by which mutant SOD1 could contribute to a defect in retrograde axonal transport or other dynein functions. The aberrant interaction is potentially critical to the formation of mutant SOD1 aggregates as well as the toxic cascades leading to motor neuron degeneration 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.     

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.     

ALS-Associated TDP-43 Induces Endoplasmic Reticulum Stress,Which Drives Cytoplasmic TDP-43 Accumulation and Stress Granule Formation

In amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration, TAR DNA binding protein 43 (TDP-43) accumulates in the cytoplasm of affected neurons and glia, where it associates with stress granules (SGs) and forms large inclusions. SGs form in response to cellular stress, including endoplasmic reticulum (ER) stress, which is induced in both familial and sporadic forms of ALS. Here we demonstrate that pharmacological induction of ER stress causes TDP-43 to accumulate in the cytoplasm, where TDP-43 also associates with SGs. Furthermore, treatment with salubrinal, an inhibitor of dephosphorylation of eukaryotic initiation factor 2-α, a key modulator of ER stress, potentiates ER stress-mediated SG formation. Inclusions of C-terminal fragment TDP-43, reminiscent of disease-pathology, form in close association with ER and Golgi compartments, further indicating the involvement of ER dysfunction in TDP-43-associated disease. Consistent with this notion, over-expression of ALS-linked mutant TDP-43, and to a lesser extent wildtype TDP-43, triggers several ER stress pathways in neuroblastoma cells. Similarly, we found an interaction between the ER chaperone protein disulphide isomerase and TDP-43 in transfected cell lysates and in the spinal cords of mutant A315T TDP-43 transgenic mice. This study provides evidence for ER stress as a pathogenic pathway in TDP-43-mediated disease.     

Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a chronic, adult-onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons, resulting in severe atrophy of muscles and death. Although the exact pathogenic mechanism of mutant superoxide dismutase 1 (SOD1) causing familial ALS is still elusive, toxic protein aggregation leading to insufficiency of chaperones is one of the main hypotheses. In this study, we investigated the effect of over-expressing one of these chaperones, heat shock protein 27 (Hsp27), in ALS. Mice over-expressing the human, mutant SOD1^G93A were crossed with mice that ubiquitously over-expressed human Hsp27. Even though the single transgenic hHsp27 mice showed protection against spinal cord ischemia, the double transgenic SOD1^G93A/hHsp27 mice did not live longer, and did not show a significant delay in the onset of disease compared to their SOD1^G93A littermates. There was no protective effect of hHsp27 over-expression on the motor neurons and on the mutant SOD1 aggregates in the double transgenic SOD1^G93A/hHsp27 mice. In conclusion, despite the protective action against acute motor neuron injury, Hsp27 alone is not sufficient to protect against the chronic motor neuron injury due to the presence of mutant SOD1.     

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.     

Alsin and SOD1(G93A) proteins regulate endosomal reactive oxygen species production by glial cells and proinflammatory pathways responsible for neurotoxicity

Recent studies have implicated enhanced Nox2-mediated reactive oxygen species (ROS) by microglia in the pathogenesis of motor neuron death observed in familial amyotrophic lateral sclerosis (ALS). In this context, ALS mutant forms of SOD1 enhance Rac1 activation, leading to increased Nox2-dependent microglial ROS production and neuron cell death in mice. It remains unclear if other genetic mutations that cause ALS also function through similar Nox-dependent pathways to enhance ROS-mediate motor neuron death. In the present study, we sought to understand whether alsin, which is mutated in an inherited juvenile form of ALS, functionally converges on Rac1-dependent pathways acted upon by SOD1(G93A) to regulate Nox-dependent ROS production. Our studies demonstrate that glial cell expression of SOD1(G93A) or wild type alsin induces ROS production, Rac1 activation, secretion of TNFα, and activation of NFκB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1(G93A) in glial cells attenuated these proinflammatory indicators and protected motor neurons in co-culture, although shRNAs against Nox1 and Nox4 had little effect. SOD1(G93A) expression dramatically enhanced TNFα-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1(G93A)-induced endosomal ROS and Rac1 activation. SOD1(G93A) expression enhanced recruitment of alsin to the endomembrane compartment in glial cells, suggesting that these two proteins act to modulate Nox2-dependent endosomal ROS and proinflammatory signals that modulate NFκB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS.