Nonamyloid Aggregates Arising from Mature Copper/Zinc Superoxide Dismutases Resemble Those Observed in Amyotrophic Lateral Sclerosis

Abstract

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...

Decreased stability and increased formation of soluble aggregates by immature superoxide dismutase do not account for disease severity in ALS

“…Other forms of SOD1 have also been shown previously to form amyloid under destabilizing conditions caused by denaturant, sonication, trifluoroethanol, or low pH (8,14,20,40), and formation of intermolecular disulfide bonds (25). In contrast, other studies under less extreme conditions have also reported evidence for distinct aggregation processes from native-like states (41)(42)(43). Protein aggregation is generally strongly dependent on solution conditions, and many destabilizing and often nonphysiological conditions can result in the formation of amyloid.…”

SOD1 aggregation in ALS mice shows simplistic test tube behavior

“…Accordingly, it has been shown that the in vitro fibrillation of apoSOD1 displays the characteristic fingerprint of fragmentation-assisted growth (15) with a square root dependence on [D] (7), consistent with the requirement of sample agitation to expedite the reaction (1)(2)(3)(4)10). Analogous fibrillation behavior is found for β2-microglobulin (2), yeast prions Sup35 (16) and Ure2p (17), insulin (18), WW domain (19), TI 127 (20), and α-synuclein (21).…”

Folding and Aggregation of Cu, Zn-Superoxide Dismutase

“…A number of studies have used different solution conditions (increased temperature, decreased pH, increased ionic strength, sonication or agitation) to promote the formation of well-structured, fibrillar amyloid aggregates (see 1.2.3) by various forms of SOD1 (Stathopulos et al 2004;Chattopadhyay et al 2008;Chattopadhyay and Valentine 2009;Oztug Durer et al 2009). Other studies have demonstrated soluble oligomer and small aggregate formation by various forms of SOD1 in quiescent, physiologically relevant solution conditions (Vassall, 2011, Hwang, 2010, Banci, 2008. Thus, it is evident that multiple factors can greatly influence protein folding and aggregation and these factors must be considered when investigating the molecular mechanisms of protein aggregation.…”

“…The overwhelming majority of fALS-associated mutations destabilize the holo state, but because of its extremely high thermodynamic stability the absolute increase in the amount of unfolded species will still be very small, and thus unlikely to affect aggregation. What seems more likely to impact disease is increased local structural fluctuations that can arise from www.intechopen.com metal loss and/or dimer dissociation, exposing regions of the SOD1 structure that can make favourable contacts with other SOD1 molecules, and thereby give rise to aggregation from native-like states (Elam et al 2003;Hwang et al 2010). …”

“…However, even subtle decreases in global protein stability are often accompanied by local destabilization and recent investigations have provided evidence for aggregate formation from native-like species (N*, Figure 1) that have undergone much more restricted unfolding Chiti and Dobson 2009). Examples of aggregate formation from native-like states include various proteins associated with disease such as mutant lysozyme (Chiti and Dobson 2009), 2-microglobulin (Chiti and Dobson 2009), and SOD1 (Hwang et al 2010). The propensity of a given globular protein to aggregate depends on how energetically feasible it is for the protein to access locally, partially or fully unfolded aggregation-prone state(s).…”