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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 pathophysiologyoffamilialALS.ThepossibilitythatPDImaybeatherapeutic target to prevent SOD1 aggregation is also raised by this study.Mutations in the Cu/Zn-superoxide dismutase (SOD1) 2 gene are associated with 20% of familial amyotrophic lateral sclerosis (FALS) cases (1), and when these mutations are overexpressed in transgenic rodents (2, 3), motor neuron degeneration reminiscent of ALS results. Although SOD1 is thermally very stable (4), abnormal mutant SOD1 (mSOD1) aggregates are present in spinal cords of FALS patients and transgenic mice (5). The mechanism of mSOD1-mediated toxicity is unclear but is non-cell autonomous and involves apoptotic signaling (reviewed in Ref. 6). The selective toxicity for motor neurons also remains unresolved.SOD1 is an intracellular homodimeric metalloprotein that forms an unusually stable intrasubunit disulfide bond between two highly conserved cysteines, Cys 57 and Cys 146 . Recent evidence implicates the disulfide-reduced monomer as the aggregation-prone and common neurotoxic intermediate for over 100 mSOD1 proteins (7-11). Hence, modulation of disulfide bond formation may be important in mSOD1-linked toxicity.The disulfide status of proteins is largely regulated by ER stress-inducible enzymes. ER stress is triggered when misfolded proteins accumulate within the lumen, inducing the unfolded protein response (UPR) (12). The 78-kDa chaperone immunoglobulin-binding protein (BiP) controls activity of the three major UPR sensors: the kinase and endonuclease IRE1, the basic leucine-zipper transcription factor ATF6, and the PERK kinase (13). The combined effect of the activation of these three molecules is the up-regulation of genes encoding ER-resident chaperones and down-regulation of protein synthesis. Proteindisulfide isomerase (PDI) and en...

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