Probing the Effects of Pathogenic Mutations on Prion-like Conversion of Superoxide dismutase-1

Abstract

Multiple neurodegenerative diseases feature the accumulation of misfolded proteins in and around neurons. In some cases, misfolding propagates through a prion-like mechanism whereby interactions between misfolded conformers and natively folded conformers induce the latter to convert into the misfolded conformer. We developed a novel assay to monitor the prion-like conversion of superoxide dismutase-1 (SOD1), whose misfolding is linked to amyotrophic lateral sclerosis (ALS). In this assay, a misfolded SOD1 mutant monomer that is associated with familial ALS is tethered to a wild-type (wt) SOD1 monomer, and the enzymatic activity of the wt SOD1 domain is monitored over time to detect its conversion into inactive misfolded conformers. Tethering the mutant vastly increases the effective local concentration of misfolded protein in the assay while keeping the global concentration low, decoupling conversion from aggregation. Our study tested this assay on a range of SOD1 heterodimers, including wt-G85R, wt-G127X, wt-D76V, wt-G93A, wt-G41D, wt-I104F, wt-G41S, and wt-A4V. The results varied significantly among different mutants. We observed distinct patterns in the enzymatic activity of these heterodimers, with some exhibiting a sigmoidal decline, suggestive of a direct conversion process. This decline was characterized by a stable phase of activity, followed by a decrease representing the conversion phase, and finally reaching stability again, suggesting the completion of the conversion. In contrast, other heterodimers demonstrated a pattern of stability with no obvious change in activity, suggesting either a slower conversion rate or retention of some activity in the mutant domain. One specific heterodimer, wt-I104F, showed a more complex or biphasic decline in its activity, suggesting the heterodimer may undergo two separate processes. Our findings challenge our initial assumptions about the relationship between the rapidity of ALS progression and the conversion rate of wt SOD1. The lack of a direct correlation between these factors suggests a more intricate mechanism underpinning ALS progression than previously understood. This research lays the groundwork for future studies to develop a more nuanced understanding of the prion-like conversion process in ALS. Future directions include refining our assay technique to explore a broader range of SOD1 mutants and investigating the potential therapeutic applications of targeting specific stages or mechanisms of the SOD1 conversion process. By deepening our understanding of SOD1 misfolding and its prion-like propagation in ALS, we aim to open new avenues for developing effective treatments for this disabling disease.