Aggregation of Cu, Zn superoxide dismutase in amyotrophic lateral sclerosis : kinetic, mechanistic, and therapeutic approaches.

Abstract

Investigating in vitro kinetics of protein aggregation using high-throughput microplate-based assays provides open venues for obtaining valuable information regarding mechanism(s) of pathogenesis of protein aggregates in neurodegenerative diseases, and facilitates development of effective therapies. In this dissertation, I use high-throughput microplate-based assays for studying the real-time kinetics of wild type and ALS-variant Cu, Zn superoxide dismutase (SOD1) aggregation in the context of amyotrophic lateral sclerosis (ALS). ALS is a neurodegenerative disease that is hallmarked with selective death of motor neurons, which leads to muscle paralysis, and eventually death. Mutations in SOD1 gene are believed to underlie ~ 3 % of cases of ALS via triggering the misfolding and aggregation of SOD1 protein. These SOD1 aggregates render toxicity in motor neurons via interfering with and disrupting normal functions of cells such as cytoplasmic and axonal transport or membrane integrity. In this dissertation, I first show that aspirin (the quintessential acylating pharmacon) can inhibit the amyloidogenesis of wild-type (WT) and ALS-variant apo-SOD1 by increasing the intrinsic net negative charge of the polypeptide, via acetylation of multiple lysines. In the third chapter, I measure rates of fibrillar and amorphous SOD1 aggregation at high iteration and show that rates of oligomerization were intrinsically irreproducible and populated continuous probability distributions. In the fourth chapter, I used Kaplan-Meier estimators to quantify the probability of apo-SOD1 fibrillization (in vitro) from ~ 103 replicate amyloid assays of WT SOD1 and nine ALS variants, and showed that the probability of apo-SOD1 fibrillization is non-uniformly altered by different mutations. I found a linear correlation between the Hazard ratios of SOD1 fibrillization and those of patient survival in SOD1-linked ALS. The fifth chapter answers a very fundamental question: “how do gyrating beads accelerate amyloid fibrillization?” I found that increasing the mass in beads from non-polymeric materials (e.g., steel) increases the nucleation rate of SOD1 fibrillization, whereas hydrophobicity and surface adhesion affected rate of SOD1 fibrillization in the case of polymeric beads. In chapter six, I study the mechanism behind Hofmeister series in proteins. Chapter seven includes a project dedicated to early detection of leukocoria in children with retinoblastoma, during recreational photography.

Description
Keywords
Cu,Zn superoxide. Amyotrophic lateral sclerosis. Amyloid. Fibril. Stochasticity. Acylation. Aspirin. Bead. Aggregation.
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