Date of Award

Summer 8-21-2015

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry and Molecular Biology

Advisor

Roger Sher

Second Committee Member

Julie Gosse

Third Committee Member

Clarissa Henry

Abstract

Amyotrophic lateral sclerosis (ALS) is a devastating and deadly neurodegenerative disease. It affects upper and lower motoneurons leading to muscle atrophy, weakness, progressive paralysis, and death with a mean survival after diagnosis of only 4 years.1 Approximately 10% of ALS cases are familial or heritable (FALS), and the genetic mutations contributing to the disease vary immensely. There are hundreds of mutations on 27 known Mendelian genes that cause FALS.2 The other 90% of cases are sporadic (SALS). ALS patients, even those with familial ALS, exhibit vast heterogeneity of disease phenotypes including location of disease onset, rate and pattern of progression, relative levels of upper and lower motor neuron degeneration,3 age of onset, and disease duration.4 This heterogeneity indicates that the combination of genetic and environmental exposures play a major role in the development and course of ALS. Increased ALS incidence has been linked with exposure to various environmental neurotoxins, including Beta-methylamino-L-alanine (BMAA),5 heavy metals,6 pesticides, and solvents.7 This thesis focuses on BMAA, which is a non-proteinogenic amino acid produced by cyanobacteria, dinoflaggelates,8 and diatoms9 worldwide. So far, exposure to BMAA has been linked to clustered ALS cases in Guam,10 southern France,11 and Annapolis, Maryland.12 To explore the interactions between genetics and environmental exposures in ALS, we utilized zebrafish models transiently and transgenically overexpressing a FALS causing SOD1 mutation. We also used healthy transient/transgenic models overexpressing wild type SOD1 and uninjected AB zebrafish controls. These fish were exposed to a series of environmentally relevant doses of BMAA during early embryonic development and surveyed for early indicators of motor neuron disruption and late stage ALS-like phenotypes. Previously established early indicators of zebrafish motoneuron disruption include developing nerve length and neuromuscular junction architecture changes. Late stage ALS-like phenotypes include reduced endurance and increased fatigability in the 5-month old zebrafish. We established earlier developmental time points for each of these phenotypes than has previously been reported. Establishing earlier time points during which ALS-like symptoms and early indicators of motor neuron disruption in the zebrafish can be found increases the power of the model for toxicological and drug screens. We found that in each of these early and late stage phenotypes, there was a distinct BMAA dose-dependent and genotype dependent significance pattern. Our control AB zebrafish had distinct dose-dependent phenotypic patterns in early nerve length and later neuromuscular junction colocalization. In the transient mutant model, distinct dose-dependent early nerve length changes were mirrored in later nerve colocalization changes, while in the transgenic model distinct dose-dependent early nerve length phenotypes were mirrored in 5-month fatigability. Strains overexpressing healthy SOD1 were largely resistant to any BMAA dose-wise changes in any phenotype, indicating that overexpression of healthy SOD1 serves a neuroprotective function. Like the phenotypic heterogeneity found among ALS patients, zebrafish with differing genetic influences and environmental exposures exhibit a vast heterogeneity of early and late stage ALS indicators. It appears that etiology of ALS, even among individuals with a genetic form of the disease, is highly influenced by environmental exposures.

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