Dissecting the Role of Oxidative Stress in Spinal Muscular Atrophy (SMA)

Spinal muscular atrophy (SMA) is a fatal inherited neurodegenerative disorder of childhood characterised by progressive motor neuron degeneration, extreme muscle weakness and atrophy. SMA results from biallelic mutations or deletions in the survival motor neuron 1 (SMN1) gene, which lead to diminished levels of the survival motor neuron (SMN) protein. SMN is a ubiquitously expressed protein localised in the nucleus and cytoplasm of cells and plays an essential role in several cellular processes. Its canonical function lies in the biogenesis of small nuclear ribonucleoproteins (snRNPs) required for pre-mRNA splicing. In addition, SMN regulates neuron-specific processes such as mRNA trafficking, local translation at synaptic terminals, and synaptic vesicle dynamics. These specialised functions contribute to the heightened vulnerability of spinal cord motor neurons to SMN deficiency. The approval of three therapeutic interventions for SMA patients in recent years has markedly improved patient outcome by significantly extending their lifespan. However, variability in treatment response among patients and the limited evidence of long-term effects has indicated that current therapies, despite being beneficial, do not represent a definitive cure for SMA. In recent years, mitochondrial dysfunction and oxidative stress have emerged as potential contributors to SMA pathology. Various in vitro and in vivo models of SMA have highlighted that SMN deficiency leads to a range of mitochondrial abnormalities, such as increased mitochondrial fragmentation and altered mitochondrial membrane potential, impairing energy production and elevating reactive oxygen species (ROS) levels. Consequently, markers of oxidative damage to proteins, lipids and DNA have been detected in both SMA patients and preclinical SMA models. Additionally, oxidative stress can destabilise SMN and interfere with its primary function in pre-mRNA splicing, suggesting a bidirectional relationship between SMN depletion and oxidative stress that may accelerate SMA progression. Despite this evidence, the involvement of oxidative stress in SMA pathology remains poorly understood. This thesis investigates the role of oxidative stress in SMA by utilising a combination of targeted pharmacological interventions, genetic manipulation and behavioural assays in the Caenorhabditis elegans (C. elegans) smn-1(ok355) SMA model, which is known to be characterised by neuromuscular defects and reduced lifespan. Reminiscent to mammalian studies, mutant smn-1(ok355) animals displayed transcriptional downregulation of mitochondrial energy metabolism pathways and elevated levels of cytoplasmic ROS. Interestingly, these mutants displayed increased sensitivity to oxidative stress, despite maintaining resistance to other types of stressors, suggesting a selective impairment of oxidative stress response mechanisms in SMA. To evaluate whether ROS reduction could improve neuromuscular function in SMA, five known antioxidant compounds were administered to smn-1(ok355) mutant animals. N-acetylcysteine was found to reduce ROS levels and improved motor phenotypes, as previously documented in the mammalian system. Interestingly, other chemical compounds unexpectedly increased ROS yet enhanced neuromuscular function (i.e. epigallocatechin gallate and melatonin). Additionally, SMN-1 protein levels were increased under both ROS-reducing and ROS-elevating conditions, indicating that redox state does not strictly govern SMN-1 abundance in vivo. Finally, the role of specific oxidative stress-response genes in SMA pathology was examined. A set of antioxidant genes from the superoxide dismutase (SOD) family were identified to be dysregulated in smn-1(ok355) animals. Pharmacological and behavioural analysis revealed that deletion of sod-1 and sod-2 increased smn-1 oxidative stress sensitivity, although only sod-1 deletion exacerbated neuromuscular defects. In contrast, loss of sod-4 or sod-5 improved both smn-1 oxidative stress resilience and motor function. These findings demonstrated that modulating specific ROS-detoxifying pathways can influence SMA phenotypes. Overall, our study highlights the intricate regulation of ROS in SMA, emphasising the necessity for precise modulation of oxidative stress responses. Our findings may inform the development of more effective, targeted antioxidant therapies for SMA in the future.