Kaplan–Meier Meets Chemical Kinetics: Intrinsic Rate of SOD1 Amyloidogenesis Decreased by Subset of ALS Mutations and Cannot Fully Explain Age of Disease Onset

Abstract

Over 150 mutations in SOD1 (superoxide dismutase-1) cause amyotrophic lateral sclerosis (ALS), presumably by accelerating SOD1 amyloidogenesis. Like many nucleation processes, SOD1 fibrillization is stochastic (in vitro), which inhibits the determination of aggregation rates (and obscures whether rates correlate with patient phenotypes). Here, we diverged from classical chemical kinetics and used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ∼10 replicate amyloid assays of wild-type (WT) SOD1 and nine ALS variants. The probability of apo-SOD1 fibrillization (expressed as a Hazard ratio) is increased by certain ALS-linked SOD1 mutations but is decreased or remains unchanged by other mutations. Despite this diversity, Hazard ratios of fibrillization correlated linearly with (and for three mutants, approximately equaled) Hazard ratios of patient survival (R = 0.67; Pearson's r = 0.82). No correlation exists between Hazard ratios of fibrillization and age of initial onset of ALS (R = 0.09). Thus, Hazard ratios of fibrillization might explain rates of disease progression but not onset. Classical kinetic metrics of fibrillization, i.e., mean lag time and propagation rate, did not correlate as strongly with phenotype (and ALS mutations did not uniformly accelerate mean rate of nucleation or propagation). A strong correlation was found, however, between mean ThT fluorescence at lag time and patient survival (R = 0.93); oligomers of SOD1 with weaker fluorescence correlated with shorter survival. This study suggests that SOD1 mutations trigger ALS by altering a property of SOD1 or its oligomers other than the intrinsic rate of amyloid nucleation (e.g., oligomer stability; rates of intercellular propagation; affinity for membrane surfaces; and maturation rate).

Lysine acylation in superoxide dismutase-1 electrostatically inhibits formation of fibrils with prion-like seeding

“…The absence of (or decrease in) ThT fluorescence is not definitive proof that a modification inhibited the fibrillization of SOD1. Amyloid fibrils of apo-SOD1 with minimal ThT fluorescence have been reported (34).…”

“…In the present study, Kaplan-Meier estimators express the probability of fibrillization of acylated SOD1 into ThT-positive species relative to unacylated SOD1. The principal readout in this analysis is a hazard ratio (HR) of fibrillization (34). Hazard ratios Ͼ 1 indicate that acylation promoted fibrillization into ThT-positive species; ratios Ͻ 1 demonstrate that modifications inhibited fibrillization into ThT-positive species; and ratios ϭ 1 demonstrate no effect.…”

“…Analysis of SOD1 aggregation data, including fitting kinetic traces, extraction of mean kinetic parameters (i.e. lag time, propagation rate, ThT/ANS fluorescence intensity), construction of Kaplan-Meier plots, and calculation of Hazard ratios, were performed as previously described (1,34).…”

“…To determine the morphology of SOD1 aggregates after the termination of each aggregation assay, we performed TEM microscopy using a JEOL JEM-1010 transmission electron microscope (JEOL Ltd.). TEM samples for SOD1 aggregates were prepared, as previously described (34).…”

Large SOD1 aggregates, unlike trimeric SOD1, do not impact cell viability in a model of amyotrophic lateral sclerosis

“…In contrast, Proctor et al (9) identified trimer-stabilizing SOD1 mutants and demonstrated that the elevated population of SOD1 trimers promoted cell death. For the fibrillar form of SOD1 aggregates, studies revealed that the intrinsic rate of SOD1 amyloidogenesis was increased by certain ALS-linked mutations and that the fibrillization rate might explain the speed of disease progression (10). The perturbations introduced through various mutations, however, have an impact on the disparate nonnative states of SOD1 (monomers, oligomers, and fibrils).…”