Investigating the efficacy of microtubule-targeting agents and uORF ASOs to target NEK1 loss-of-function in ALS

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by the loss of motor neurons (MNs) in the brain and spinal cord. Breakthrough genetic studies of familial ALS patients have enabled the identification of causative mutations in genes that may play a role in disease pathogenesis. Recently, loss-of-function variants in a novel identified gene NEK1, which encodes the NIMA-related kinase 1 (NEK1) protein, have been demonstrated to confer susceptibility for up to 3% of all ALS patients and can thus be considered the third most common genetic cause of ALS. While little is known about the role of NEK1 in normal MN physiology, preliminary evidence from our lab has implicated a dysregulation of microtubule (MT) homeostasis in NEK1 loss-of-function iPSC-derived MN models. We have additionally identified alpha-tubulin (TUBA1B), a major structural component of MTs, as a NEK1-interacting protein that undergoes phosphorylation by NEK1 in vitro. In this application, I propose to utilize multiple human iPSC-derived MN (hiPSC-MN) models to investigate the precise mechanisms by which NEK1 regulates MT homeostasis in human MNs and determine whether MT disruption drives MN degeneration in NEK1-ALS. I will additionally explore the effects of NEK1 loss-of-function on tubulin phosphorylation, via targeted phosphoproteomics, and determine the downstream consequences of altered tubulin phosphorylation on different aspects of MT homeostasis, such as MT stability/polymerization, orientation and cargo transport. Lastly, I propose to employ a diverse array of microtubule-targeting agents (MTAs) to modulate MTs to determine whether NEK1-related impairments and MN degeneration can be rescued in NEK1 loss-of-function models and may be a viable therapeutic avenue in NEK1-ALS.