Innovating next generation technologies to define mechanisms of neurodegenerative disease and devise therapeutic strategies - Project Summary/Abstract: This is a proposal to consolidate our NINDS research projects into one larger R35 award, enabling us the time and flexibility to explore important biological questions relevant to human neurodegenerative diseases. My laboratory has used a combination of yeast and human genetics to define novel mechanisms of ALS, FTD, and Parkinson’s disease. These experiments have led to the discovery of ataxin 2 intermediate-length polyglutamine expansions as a major genetic risk factor for ALS. We found that reduction of ataxin 2 in mouse profoundly extends survival of TDP-43 transgenic mice (either by genetic knockout or using ASOs). These preclinical studies have led to the initiation of a clinical trial in human to test ataxin 2 ASOs in human ALS. We have pursued ataxin 2 as a therapeutic target in ALS and have identified additional regulators of ataxin 2 levels including a small molecule that can lower ataxin 2 levels in vitro and in vivo. Given the success of these genetic screens for finding regulators of ataxin 2 levels, I propose performing genomewide modifier screens in human cells using CRISPR/Cas9 for regulators of additional neurodegenerative disease proteins. We recently identified dozens of cryptic splicing events that occur in neurons harboring TDP-43 pathology, including one in the UNC13A gene, one of the strongest GWAS hits for FTD/ALS. Importantly, we found that the genetic variations in UNC13A that increase risk for disease increase cryptic exon inclusion in the face of TDP-43 pathology. We propose functional studies to elucidate the role of additional cryptic splicing targets in ALS pathogenesis. Finally, we have expanded efforts to include single-cell sequencing and have performed RNA- sequencing on adult mouse spinal cord, revealing remarkable heterogeneity and new markers of skeletal motor neuron subtypes. We propose studies to apply this technology to mouse models of ALS and to human spinal cord. Together, we present an ambitious research program aimed at defining novel mechanisms of human neurodegenerative diseases and then intensely working to translate those mechanisms to novel therapies to help treat these devastating conditions.
