Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.     

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.     

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.     

Inhibition of chaperone activity is a shared property of several Cu,Zn-superoxide dismutase mutants that cause amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive motor neuron degeneration, paralysis, and death. Mutant Cu,Zn-superoxide dismutase (SOD1) causes a subset of ALS by an unidentified toxic property. Increasing evidence suggests that chaperone dysfunction plays a role in motor neuron degeneration in ALS. To investigate the relationship between mutant SOD1 expression and chaperone dysfunction, we measured chaperone function in central nervous system tissue lysates from normal mice and transgenic mice expressing human SOD1 variants. We observed a significant decrease in chaperone activity in tissues from mice expressing ALS-linked mutant SOD1 but not control mice expressing human wild type SOD1. This decrease was detected only in the spinal cord, became apparent by 60 days of age (before the onset of muscle weakness and significant motor neuron loss), and persisted throughout the late stages. In addition, this impairment of chaperone activity occurred only in cytosolic but not in mitochondrial and nuclear fractions. Furthermore, multiple recombinant human SOD1 mutants with differing biochemical and biophysical properties inhibited chaperone function in a cell-free extract of normal mouse spinal cords. Thus, mutant SOD1 proteins may impair chaperone function independent of gene expression in vivo, and this inhibition may be a shared property of ALS-linked mutant SOD1 proteins.     

Increased mitochondrial antioxidative activity or decreased oxygen free radical propagation prevent mutant SOD1-mediated motor neuron cell death and increase amyotrophic lateral sclerosis-like transgenic mouse survival

The molecular mechanisms of selective motor neuron degeneration in human amyotrophic lateral sclerosis (ALS) disease remain largely unknown and effective therapies are not currently available. Mitochondrial dysfunction is an early event of motor neuron degeneration in transgenic mice overexpressing mutant superoxide dismutase (SOD)1 gene and mitochondrial abnormality is observed in human ALS patients. In an in vitro cell culture system, we demonstrated that infection of mouse NSC-34 motor neuron-like cells with adenovirus containing mutant G93A-SOD1 gene increased cellular oxidative stress, mitochondrial dysfunction, cytochrome c release and motor neuron cell death. Cells pretreated with highly oxidizable polyunsaturated fatty acid elevated lipid peroxidation and synergistically exacerbated motor neuron-like cell death with mutant G93A-SOD1 but not with wild-type SOD1. Similarly, overexpression of mitochondrial antioxidative genes, MnSOD and GPX4 by stable transfection significantly increased NSC-34 motor neuron-like cell resistance to mutant SOD1. Pre-incubation of cells with spin trapping molecule, 5',5'-dimethylpryrroline-N-oxide (DMPO), prevented mutant SOD1-mediated mitochondrial dysfunction and cell death. Furthermore, treatment of mutant G93A-SOD1 transgenic mice with DMPO significantly delayed paralysis and increased survival. These findings suggest a causal relationship between enhanced oxidative stress and mutant SOD1-mediated motor neuron degeneration, considering that enhanced oxygen free radical production results from the SOD1 structural alterations. Molecular approaches aimed at increasing mitochondrial antioxidative activity or effectively blocking oxidative stress propagation can be potentially useful in the clinical management of human ALS disease.     

Transgenic mouse models of amyotrophic lateral sclerosis

The discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases was rapidly followed by the generation of transgenic mice expressing various forms of SOD1 mutants. The mice overexpressing high levels of mutant SOD1 mRNAs do develop motor neuron disease but unraveling the mechanisms of pathogenesis has been very challenging. Studies with mouse lines suggest that the toxicity of mutant SOD1 is unrelated to copper-mediated catalysis but rather to propensity of a subfraction of mutant SOD1 proteins to form misfolded protein species and aggregates. However, the mechanism of toxicity of SOD1 mutants remains to be elucidated. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Here, we describe how transgenic mouse models have been used for understanding pathogenic pathways of motor neuron disease and for pre-clinical drug testing.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Astroglial inhibition of NF-κB does not ameliorate disease onset and progression in a mouse model for amyotrophic lateral sclerosis (ALS)

Motor neuron death in amyotrophic lateral sclerosis (ALS) is considered a "non-cell autonomous" process, with astrocytes playing a critical role in disease progression. Glial cells are activated early in transgenic mice expressing mutant SOD1, suggesting that neuroinflammation has a relevant role in the cascade of events that trigger the death of motor neurons. An inflammatory cascade including COX2 expression, secretion of cytokines and release of NO from astrocytes may descend from activation of a NF-κB-mediated pathway observed in astrocytes from ALS patients and in experimental models. We have attempted rescue of transgenic mutant SOD1 mice through the inhibition of the NF-κB pathway selectively in astrocytes. Here we show that despite efficient inhibition of this major pathway, double transgenic mice expressing the mutant SOD1(G93A) ubiquitously and the dominant negative form of IκBα (IκBαAA) in astrocytes under control of the GFAP promoter show no benefit in terms of onset and progression of disease. Our data indicate that motor neuron death in ALS cannot be prevented by inhibition of a single inflammatory pathway because alternative pathways are activated in the presence of a persistent toxic stimulus.     

Familial amyotrophic lateral sclerosis-linked mutant SOD1 aberrantly interacts with tubulin

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause 20-25% of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes motor neuron degeneration through toxic gain-of-function(s). However, the direct molecular targets of mutant SOD1, underlying its toxicity, are not fully understood. In this study, we found that α/β-tubulin is one of the major mutant SOD1-interacting proteins, but that wild-type SOD1 does not interact with it. The interaction between tubulin and mutant SOD1 was detected in the spinal cords of mutant G93A SOD1 transgenic mice before the onset of symptoms. Tubulin interacted with amino acid residues 1-23 and 116-153 of SOD1. Overexpression of mutant SOD1 resulted in the accumulation of tubulin in detergent-insoluble fractions. In a cell-free system, mutant SOD1 modulated tubulin polymerization, while wild-type SOD1 did not. Since tightly regulated microtubule dynamics is essential for neurons to remain viable, α/β-tubulin could be an important direct target of mutant SOD1.     

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.     

Dorfin ubiquitylates mutant SOD1 and prevents mutant SOD1-mediated neurotoxicity

Amyotrophic lateral sclerosis (ALS) is a progressive paralytic disorder resulting from the degeneration of motor neurons in the cerebral cortex, brainstem, and spinal cord. The cytopathological hallmark in the remaining motor neurons of ALS is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. In this paper we report that Dorfin, a RING finger-type E3 ubiquitin ligase, is predominantly localized in the inclusion bodies of familial ALS with a copper/zinc superoxide dismutase (SOD1) mutation as well as sporadic ALS. Dorfin physically bound and ubiquitylated various SOD1 mutants derived from familial ALS patients and enhanced their degradation, but it had no effect on the stability of the wild-type SOD1. The overexpression of Dorfin protected against the toxic effects of mutant SOD1 on neural cells and reduced SOD1 inclusions. Our results indicate that Dorfin protects neurons by recognizing and then ubiquitylating mutant SOD1 proteins followed by targeting them for proteasomal degradation.     

Alsin, the product of ALS2 gene, suppresses SOD1 mutant neurotoxicity through RhoGEF domain by interacting with SOD1 mutants

Mutation of the ALS2 gene encoding alsin is linked to the onset of autosomal recessive motor neuron diseases, including juvenile-onset amyotrophic lateral sclerosis (ALS). Alsin long form (LF) belongs to the family of the guanine nucleotide exchanging factor (GEF) for small GTPases. Expression of alsin LF, but not alsin short form, protected motor neuronal cells from toxicity induced by mutants of the Cu/Zn-superoxide dismutase (SOD1) gene, which cause autosomal dominant ALS. In contrast, expression of alsin did not suppress neurotoxicity by other neurodegenerative insults such as Alzheimer's disease-related genes. Deletion analysis of alsin LF demonstrated that the RhoGEF domain is essential for alsin-mediated neuroprotection. Furthermore, we found that alsin LF bound to SOD1 mutants, but not to wtSOD1, via the RhoGEF domain. Such functional and physical interaction between two ALS-related genes will become a promising clue to clarify the pathogenesis of ALS and other motor neuron diseases.     

Cu,Zn-Superoxide Dismutase Increases Toxicity of Mutant and Zinc-deficient Superoxide Dismutase by Enhancing Protein Stability

When replete with zinc and copper, amyotrophic lateral sclerosis (ALS)-associated mutant SOD proteins can protect motor neurons in culture from trophic factor deprivation as efficiently as wild-type SOD. However, the removal of zinc from either mutant or wild-type SOD results in apoptosis of motor neurons through a copper- and peroxynitrite-dependent mechanism. It has also been shown that motor neurons isolated from transgenic mice expressing mutant SODs survive well in culture but undergo apoptosis when exposed to nitric oxide via a Fas-dependent mechanism. We combined these two parallel approaches for understanding SOD toxicity in ALS and found that zinc-deficient SOD-induced motor neuron death required Fas activation, whereas the nitric oxide-dependent death of G93A SOD-expressing motor neurons required copper and involved peroxynitrite formation. Surprisingly, motor neuron death doubled when Cu,Zn-SOD protein was either delivered intracellularly to G93A SOD-expressing motor neurons or co-delivered with zinc-deficient SOD to nontransgenic motor neurons. These results could be rationalized by biophysical data showing that heterodimer formation of Cu,Zn-SOD with zinc-deficient SOD prevented the monomerization and subsequent aggregation of zinc-deficient SOD under thiol-reducing conditions. ALS mutant SOD was also stabilized by mutating cysteine 111 to serine, which greatly increased the toxicity of zinc-deficient SOD. Thus, stabilization of ALS mutant SOD by two different approaches augmented its toxicity to motor neurons. Taken together, these results are consistent with copper-containing zinc-deficient SOD being the elusive “partially unfolded intermediate” responsible for the toxic gain of function conferred by ALS mutant SOD.     

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.     

Hydrogen-deuterium exchange in vivo to measure turnover of an ALS-associated mutant SOD1 protein in spinal cord of mice

Mutations of cytosolic Cu/Zn superoxide dismutase 1 (SOD1) in humans and overexpression of mutant human SOD1 genes in transgenic mice are associated with the motor neuron degenerative condition known as amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease). Gain-of-function toxicity from the mutant protein expressed in motor neurons, associated with its misfolding and aggregation, leads to dysfunction and cell death, associated with paralyzing disease. Here, using hydrogen-deuterium exchange in intact mice in vivo, we have addressed whether an ALS-associated mutant protein, G85R SOD1-YFP, is subject to the same rate of turnover in spinal cord both early in the course of the disease and later. We find that the mutant protein turns over about 10-fold faster than a similarly expressed wild-type fusion and that there is no significant change in the rate of turnover as animals age and disease progresses.     

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.     

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.     

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.     

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.     

Guanabenz Treatment Accelerates Disease in a Mutant SOD1 Mouse Model of ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons. The mechanisms leading to motor neuron degeneration in ALS are unclear. However, there is evidence for involvement of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in ALS, notably in mutant SOD1 mediated models of ALS. Stress induced phosphorylation of the eIF2 alpha subunit by eukaryotic translation initiation factor 2-alpha kinase 3 Perk activates the UPR. Guanabenz is a centrally acting alpha2 adrenergic receptor agonist shown to interact with a regulatory subunit of the protein phosphatase, Pp1/Gadd34, and selectively disrupt the dephosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eif2alpha). Here we demonstrate that guanabenz is protective in fibroblasts expressing G93A mutant SOD1 when they are exposed to tunicamycin mediated ER stress. However, in contrast to other reports, guanabenz treatment accelerated ALS-like disease progression in a strain of mutant SOD1 transgenic ALS mice. This study highlights challenges of pharmacological interventions of cellular stress responses in whole animal models of ALS.