Examining Responses of RNA Binding Proteins to ALS and Stress in Human Motor Neurons

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterised by rapid progression and relatively selective motor neuron loss. RNA dysregulation and abnormal proteostasis have been shown to play a key role in ALS pathogenesis. In particular, RNA binding protein (RBP) nuclear-to-cytoplasmic mislocalisation and accumulation have been identified as a pathological hallmark of the disease. RBPs are essential contributors to cellular stress responses, highlighting one important intersect between stress and ALS. Here, I have used healthy human induced pluripotent stem cell (hiPSC)-derived motor neurons to model the cellular stress responses. I have observed the assembly and disassembly of stress granules (SGs) in response to various stressors, and identified recurrent heat stress insults does not result in subsequent SG formation. When examining the nucleocytoplasmic localisation of key ALS-linked RBPs in response to stress, I identified stress- and RBP- specific responses. This was also true when examining the kinetics of nuclear relocalisation upon recovery, with TDP-43 and FUS, two of the most recognised RBPs in ALS pathogenesis, as exhibiting delayed nuclear relocalisation following stress. To model ALS, I have used hiPSC-derived motor neurons from patients carrying ALS-causing mutations in valosin containing protein (VCP). I identified TDP-43, FUS and SFPQ nuclear-to-cytoplasmic mislocalisation in VCP mutant motor neurons, with closer analysis finding this mislocalisation extends to within neurites. I showed VCP D2-ATPase inhibition robustly rescued TDP-43 and FUS mislocalisation, suggesting a gain-in-function in the D2 ATPase drives mislocalisation. To gain further insight into possible disease mechanisms, I examined the cellular stress response in terms of SG dynamics and RBP nucleocytoplasmic localisation responses in VCP mutant motor neurons. Across multiple stressors and recovery, there were no aberrant stress responses in VCP mutant motor neurons, suggesting that these are not cellular pathways contributing to early disease pathogenesis in motor neurons. Together, these data provide insights into the pathogenesis of ALS, highlighting possible molecular events for therapeutic intervention.