Protein aggregation is a hallmark of many diseases, including amyotrophic lateral sclerosis (ALS) where aggregation of copper/zinc superoxide dismutase (SOD1) is implicated in pathogenesis. We report here that fully metallated (holo) SOD1 under physiologically relevant solution conditions can undergo changes in metallation and/or dimerization over time and form aggregates that do not exhibit classical characteristics of amyloid. The relevance of the observed aggregation to disease is demonstrated by structural and tinctorial analyses, including the novel observation of binding of an anti-SOD1 antibody that specifically recognizes aggregates in ALS patients and mice models. ALS-associated SOD1 mutations can promote aggregation but are not essential. The SOD1 aggregation is characterized by a lag phase, which is diminished by self-or cross-seeding and by heterogeneous nucleation. We interpret these findings in terms of an expanded aggregation mechanism consistent with other in vitro and in vivo findings that point to multiple pathways for the formation of toxic aggregates by different forms of SOD1. Amyotrophic lateral sclerosis (ALS)2 is a devastating, rapidly progressive, and invariably fatal neurodegenerative disease, characterized by motor neuron degeneration and paralysis (1). Approximately 10% of ALS cases are familial (fALS), the remaining cases being sporadic (sALS). The familial and sporadic diseases are clinically indistinguishable and so have been proposed to share common disease mechanisms (1). Mutations in copper/zinc superoxide dismutase (SOD1) account for ϳ20% of fALS and represent a major known cause of the disease. It is generally accepted that fALS-linked SOD1 mutations result in a toxic gain of function rather than a loss of function (1). Much attention has focused on toxic protein aggregation as causing ALS, analogous to pathogenic protein aggregation in other neurodegenerative diseases, including Alzheimer, Huntington, Parkinson, and prion diseases (1-5). SOD1 is present in aggregates in motor neurons of SOD1-linked fALS patients (3, 6) and mice models (3, 7-9) and in some sALS patients (4, 5, 10).Mature SOD1 is a homodimeric protein, with each ␤-barrel subunit containing one catalytic copper ion, one structural zinc ion, one intrasubunit disulfide bond, and two free cysteines (11) (see Fig. 1). More than 147 mainly missense mutations throughout the SOD1 structure have been associated with fALS. Aggregation has been reported previously only for immature (metal and/or disulfide deficient) or aberrant forms of SOD1, often under highly destabilizing conditions, which favor aggregation in general (12-19). The relevance to human disease of such aggregation is not known, nor is it known what forms of SOD1 may give rise to or be present in aggregates in patients (20). Mature (holo) SOD1 is a major form of SOD1 in cells for wild type and most mutant SOD1s (11). Although this form has been proposed to be incompatible with aggregation because of its very high stability (15, 21), several studies have rep...