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Mutations in superoxide dismutase 1 (SOD1) are linked to amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder predominantly affecting upper and lower motor neurons leading to progressive paralysis and atrophy of skeletal muscles ultimately resulting in quadriplegia and fatal respiratory failure. The SOD1 protein is primarily responsible for defence against damaging superoxide free radicals. SOD1 loss of function was initially thought to play a role in ALS due to the discovery of disease- causing mutations in the SOD1 gene and the well-established link between oxidative stress and neurodegeneration. However, a reduction in SOD1 activity is not causative for ALS, although it is likely to play a modifying role. The major effect of SOD1 mutations in ALS is linked to the protein aggregation of misfolded SOD1 protein species, a process that has been termed toxic gain of function. While numerous genetic and molecular discoveries have deepened the understanding of ALS pathophysiology the precise mechanism of SOD1-ALS pathogenesis remains unclear.

Established animal and cellular models of SOD1-associated ALS (SOD1- ALS) have relied upon overexpression systems, including in vivo transgenic mouse models and in vitro plasmid-based transfection cell models. In mouse models, the presence of numerous transgene copies resulting in overexpression of the mutant SOD1 protein has been advantageous to accelerate the neurodegenerative phenotype and examine late-stage ALS pathology. Furthermore, the utility of overexpression in vitro models of SOD1-ALS has enabled identification of key pathological features associated with SOD1 mutations and recapitulated the hallmark proteinopathies seen in ALS patients. However, the presence of non-physiological levels of SOD1 expression in these models makes it difficult to study the early disease process and hinders the discovery of early biomarkers for disease.

The principal goal of this thesis was to develop and utilise new in vivo and in vitro models of SOD1-ALS. These models were used to elucidate early pathogenic mechanisms of ALS linked to loss or gain of function of the SOD1 protein, under conditions of endogenous levels of expression from single loci. Further, given the multistep nature of ALS pathogenesis, the goal of this work was to highlight temporal signatures of disease in SOD1-ALS mouse models and human induced pluripotent stem cell-derived motor neurons (iPSC-MNs).

The SOD1-ALS genetically humanised mouse models developed in this thesis work led to the discovery of pre-symptomatic transcriptomic and metabolomic signatures of disease whilst dysregulated proteome degradation dynamics and alteration of axonal mitochondrial integrity was highlighted in SOD1-ALS patient derived iPSC-MNs. This thesis also describes the establishment of a custom-built semi-automated tool (Axon-OI) for analysing organelle interactions in iPSC-MN axons leveraging machine learning and live cell imaging to provide a unique resource to understand the impact of axonal transport disruptions in ALS pathogenesis.

Collectively, the work described in this thesis establishes a novel foundation for future investigations into the early stages of ALS pathogenesis and identification of therapeutic targets for pre- symptomatic treatment of SOD1-ALS.