Abnormal modifications to mutant superoxide dismutase 1 (SOD1) are linked to familial amyotrophic lateral sclerosis (fALS). Misfolding of wild-type SOD1 (SOD1 WT ) is also observed in postmortem tissue of a subset of sporadic ALS (sALS) cases, but cellular and molecular mechanisms generating abnormal SOD1 WT species are unknown. We analyzed aberrant human SOD1 WT species over the lifetime of transgenic mice and found the accumulation of disulfidecross-linked high-molecular-weight SOD1 WT

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... tes during aging. Subcellular fractionation of spinal cord tissue and protein overexpression in NSC-34 motoneuron-like cells revealed that endoplasmic reticulum (ER) localization favors oxidation and disulfide-dependent aggregation of SOD1 WT . We established a pharmacological paradigm of chronic ER stress in vivo, which recapitulated SOD1 WT aggregation in young transgenic mice. These species were soluble in nondenaturing detergents and did not react with a SOD1 conformation-specific antibody. Interestingly, SOD1 WT aggregation under ER stress correlated with astrocyte activation in the spinal cord of transgenic mice. Finally, the disulfide-cross-linked SOD1 WT species were also found augmented in spinal cord tissue of sALS patients, correlating with the presence of ER stress markers. Overall, this study suggests that ER stress increases the susceptibility of SOD1 WT to aggregate during aging, operating as a possible risk factor for developing ALS. amyotrophic lateral sclerosis | wild-type SOD1 | ER stress | aging | protein aggregation A myotrophic lateral sclerosis (ALS) is characterized by selective degeneration of motoneurons from cerebral cortex, brainstem, and spinal cord leading to muscle weakness, atrophy, paralysis, and premature death (1, 2). Most ALS cases are considered sporadic (sALS), while 10% are familial (fALS), involving mutations in superoxide dismutase 1 (SOD1), transactive response DNA binding protein 43 (TARDBP or TDP-43), fused in sarcoma/ translocated in sarcoma (FUS/TLS), and the hexanucleotide repeat expansions in C9ORF72 as the most common alterations (1-3). More than 150 mutations in SOD1 have been linked to ALS, with varying degrees of aggressiveness and aggregation propensity (4). SOD1 folding depends on complex posttranslational modifications, including the insertion of zinc and copper ions and intramolecular disulfide-bond formation, followed by homodimerization (4). Biochemical studies have shown that fALS-linked mutations destabilize SOD1 polypeptide, preventing its posttranslational processing with resultant accumulation of aggregation-prone species (5). Mutant SOD1 toxicity may be the result of disturbed cellular homeostasis at many levels, such as axonal transport, the cytoskeleton, mitochondrial function, and the secretory pathway, in addition to cell-nonautonomous mechanisms marked by the abnormal activation of microglia and astrocytes (6, 7). Several studies have reported the presence of abnormal SOD1 WT species in postmortem sALS tissue using conformation-specific antibodies that recognize misfolded forms of the protein, possibly corresponding to small and diffusible oligomeric species that do not accumulate into high-molecular-weight (HMW) aggregates (8-10). Misfolded SOD1 WT can exhibit toxic properties by impairing axonal transport, inhibiting endoplasmic reticulum (ER)associated degradation, and self-propagating its misfolding in a prion-like fashion (9-12). At the molecular level, altered posttranslational maturation of SOD1 WT can trigger its misfolding and aggregation (9, 13, 14) . Interestingly, coexpression of human SOD1 WT together with fALS-linked SOD1 mutants aggravates disease severity in ALS mouse models, and homozygous SOD1 WT transgenic mice develop late-onset motor disease (15, 16). Despite representing an attractive hypothesis of convergent disease mechanisms of sALS and fALS, the overall relevance of SOD1 WT to sALS has been questioned because of low frequency or lack of staining with conformation-specific antibodies in some studies (17, 18) . Altered proteostasis is a common pathogenic hallmark of both sALS and many different forms of fALS (19). One of the main nodes of the proteostasis network affected in ALS is the ER (20), the major subcellular compartment involved in protein folding and quality control mechanisms. ER stress triggers an adaptive reaction known as the unfolded protein response (UPR) to restore Significance The identification of aberrant SOD1 WT species accumulating in the spinal cord during aging could reveal pathogenic species involved in sporadic (s)ALS. Using a combination of biochemical approaches, we discovered that disulfide-cross-linked SOD1 WT aggregates rise before other abnormal protein species during aging and are significantly increased in sALS spinal cord tissue. We also found that endoplasmic reticulum stress stimulates accumulation of these species, with involvement of tryptophan-32 oxidation. These results establish a connection between SOD1 WT aggregation and a major proteostasis network affected in ALS.