Investigating metabolic dysfunction in a Saccharomyces cerevisiae model of SOD1-associated amyotrophic lateral sclerosis

Superoxide dismutase 1 (Sod1) is an enzyme that converts superoxides into hydrogen peroxide and water. Over 200 mutations in the gene SOD1 which encodes Sod1, causes familial amyotrophic lateral sclerosis (ALS), a neurodegenerative disease (ND) that involves the progressive death of motor neurons (MNs). The precise mechanism as to how mutations in SOD1 give rise to ALS is not yet known. Many cellular and animal models of SOD1-associated ALS (SOD1-ALS) have been developed in organisms ranging from baker's yeast (Saccharomyces cerevisiae) to mice (Mus musculus) which have led to many discoveries into the function of Sod1. In recent years, there has been a focus on developing stable single-copy models of ALS, so as to study mutant isoforms of Sod1 when it is expressed at a physiologically relevant level. The first aim of this project was to develop a stable single-copy SOD1-ALS model in S. cerevisiae. Gateway cloning and CRISPR/Cas9 gene editing techniques were used to generate an integrated plasmid-based model, and a model with the mutations A4V, G37R and H48Q introduced into the endogenous yeast SOD1 gene. The stable integrated plasmid model was used in this study to carry out phenotypic and genetic screens. In this thesis, I investigated previous findings that were made using a S. cerevisiae model of SOD1-ALS. It was found that overexpression of ALS-linked mutant isoforms of Sod1 into S. cerevisiae causes cytotoxicity, metabolic dysfunction, and a vacuole acidification defect. Vacuole acidification is mediated by the V-ATPase, a highly conserved proton pump. Using an in vivo protein complementation assay (PCA) and in vitro V-ATPase assays, I found that Sod1 may interact with the Vma2, Vma4 and Vma8 subunits of the V1 domain of the V-ATPase. I also found that altering the assembly state of the V-ATPase either by modulating glucose levels, or by deleting Vma2 or Vma4, affected the interaction between Sod1 and the V-ATPase. The interaction between Sod1 and calcineurin was also explored in S. cerevisiae using the in vivo PCA assay and a flow cytometry based calcineurin activity assay. In summary, the stable SOD1-ALS model that was developed allowed for the study of mutant Sod1 when expressed in the cell at a physiologically low level. A novel interaction between Sod1 and the V-ATPase in S. cerevisiae was observed which could potentially be relevant towards ALS pathogenesis. It was found that in S. cerevisiae, Sod1 plays a protective role for calcineurin and that cells expressing mutant Sod1 alleles have reduced calcineurin signalling