Investigating Metabolic Dysfunction and Microbiome Imbalance as a Driver in the neurodegenerative Disease Amyotrophic Lateral Sclerosis

The neurodegenerative disorder Amyotrophic lateral Sclerosis (ALS) leads to a progressive loss of motor neurons controlling voluntary muscles ultimately leading to paralysis and death usually through respiratory paralysis. Recent findings suggest that metabolic defects in combination with lower abundance and diversity of the microbial profile in ALS patients play an important role in the onset and progression of ALS. Dietary compounds are metabolised by the gut microbiota yielding metabolites altering the metabolic status of the host influencing host health. In addition, research also suggests that a cell's response to metabolic changes can contribute to the toxicity associated with ALS when placed in the context of mutations in the protein Superoxide dismutase 1 (Sod1) and the regulation of calcium homeostasis, mediated by Calcineurin. Understanding these interactions may help in our understanding of familial and sporadic forms of ALS. In this thesis we used a multi-model approach to investigate how SOD1 mutations interrupt calcium and energy homeostasis, particularly in the absence of specific metabolites and vacuolar membrane transporters using S. cerevisiae as a simple ALS model. We further examined the therapeutic potential of butyrate, a short-chain fatty acid reduced in ALS patients, which has been linked to gut health, microbial diversity, and histone deacetylase (HDAC) inhibition. Using C. elegans and human neuroblastoma cell models, we assessed the effects of butyrate on Sod1mut aggregation, motor function, and neuromuscular junction integrity. In addition, we investigated the antifungal properties of butyrate against C. albicans, considering fungal dysbiosis as an emerging factor in ALS pathology. Our findings show that butyrate supplementation could reduce Sod1mut aggregation, rescue motor and neuromuscular defects in C. elegans, and decrease aggregation in human cell, thereby validating these systems as platforms for testing HDAC inhibitors. We could show that the mode of action of butyrate is pH-dependent: under acid conditions it interrupts calcium homeostasis, impairs mitochondrial function, and induces ROS accumulation, whilst simultaneously exerting anti-fungal activity against C. albicans. Finally, we could show that the Sod1A4V mutation alters calcium sensitivity and impairs calcineurin-Crz1 signalling, exacerbating neurodegenerative processes. In summary, my work demonstrates how a single mutated protein (Sod1A4V) can interrupt multiple, highly conserved cellular systems including vacuolar (lysosomal) function, autophagy, metabolic signalling, and calcium homeostasis, each of which are implicated in ALS. Our findings reinforce the concept of ALS as both proteopathy and an endosomal- lysosomal disorder. Importantly, by linking reduced butyrate levels and gut mycobiome dysbiosis in ALS patients to cellular dysfunction, this thesis underscores the potential role of gut-derived factors in ALS pathology.