Superoxide dismutase 1 mutants related to amyotrophic lateral sclerosis induce endoplasmic stress in neuro2a cells.

One of the common features of damaged neurons in many neurodegenerative diseases is the presence of abnormal aggregates of the disease-related proteins. In amyotrophic lateral sclerosis (ALS) of both sporadic and familial forms, protein aggregates are found in the affected spinal cords. In familial ALS with mutations in copper-zinc superoxide dismutase 1 (SOD1), the propensity of SOD1 for aggregation is known to increase with the mutation. In the present study, we examined whether the aggregate-prone SOD1 mutants induce endoplasmic reticulum (ER) stress and the inhibition of the ER stress protects the cells. The ALS-related mutant G85R SOD1 and G93A SOD1 formed visible aggregates and caused cell death possibly by apoptosis when over-expressed in neuro2a cells. Interestingly, the rate of the mutant SOD1-induced cell death was greater than that of the visible aggregate formation. Expression of the mutant SOD1 caused signs of both early and late ER stress responses, namely, RNA-dependent protein kinase-like ER kinase and eukaryotic initiation factor alpha phosphorylation, Jun amino-terminal kinase activation, activating transcription factor 6-translocation, X-box binding protein 1 mRNA splicing, and caspase 12 activation. The X-box binding protein 1 mRNA splicing activation was also detected in the mutant SOD1-expressing cells even without the visible aggregates. The cell death induced by the mutant SOD1 over-expression looked like apoptosis as evidenced by nuclear morphology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling. Importantly, an ER stress inhibitor, salubrinal delayed the formation of insoluble aggregates of the mutant SOD1 and suppressed the mutant-induced cell death. In addition, over-expression of the ER-targeted Bcl-xL protected the cells from the mutant SOD1-induced cytotoxicity. These results suggest that the misfolding of ALS-related mutant SOD1 induces ER stress possibly prior to the formation of visible aggregates, which may contribute to the motor neuron degeneration in ALS pathogenesis.