Amyotrophic lateral sclerosis (ALS) is a devastating neurological disease that is characterized by a progressive inability to stimulate and control muscle movement. The clinical manifestation of the disease is mediated by the selective dysfunction and degeneration of upper and lower motor neurons that connect the central nervous system (CNS) to the musculature. The overwhelming majority of ALS is sporadic in nature, while 10-12% of patients suffer from familial forms of disease, which have enabled the identification of causative genetic variants. Such genetic studies have demonstrated that ALS can be caused by mutations in genes that encode proteins involved in diverse cellular functions ranging from RNA processing, vesicle transport, cytoskeletal homeostasis, mitochondrial function and the processing of unfolded proteins. A series of recent genetic studies have highlighted a novel gene, NIMA-related kinase 1 (NEK1), as a major genetic contributor to ALS. In particular, loss-of-function genetic variants in NEK1 confer susceptibility to ALS in as many as 3% of all cases. While NEK1 is a well-characterized kinase implicated in multiple cellular functions including the DNA damage response, its specific role and function in the CNS remains unresolved. What also remains elusive is the cellular mechanism(s) that lead to NEK1-related ALS pathophysiology, and the causal relationship of NEK1 variants to ALS. In the present study, we will use mutant NEK1 cellular models, patient iPSC-derived neurons, CRISPR/Cas9 gene-editing approaches and ALS patient CNS tissue to elucidate how NEK1 genetic variants associated with ALS, contribute towards neuronal dysfunction and degeneration. To assess both the necessity and sufficiency of NEK1 genetic variants, we will use CRISPR/Cas9 to correct mutations in patient iPSC lines, as well as to introduce mutations into a healthy genetic background.
In Aim 1 we will determine whether ALS- related NEK1 variants exhibit signatures of ALS pathophysiology, infer susceptibility to DNA damage and alter cytoskeletal homeostasis.
In Aim 2 we will perform a global phosphoproteomic analysis in mutant NEK1 and isogenic control motor neurons to identify protein targets that are differentially phosphorylated. We will validate our findings in postmortem patient CNS tissue, as well as brain and spinal cord from mutant NEK1 mouse models. Our studies will shed light into the cellular mechanisms that are compromised by NEK1 haploinsufficiency in patients and will likely uncover potential therapeutic targets for a significant percentage of ALS patients.
ALS is a devastating and untreatable neurological disease characterized by motor neuron loss. The main goal of this proposal is to determine how mutations in a newly identified ALS causal gene termed NEK1 compromise motor neuron function and lead to their degeneration. These efforts will highlight new neurotoxic mechanisms and identify potential points of therapeutic intervention that may be broadly relevant for treating ALS.
