A Novel Genome-Wide Screen to Identify and Characterize Regulators of ALS Disease Modifier Gene Ataxin-2

项目摘要/摘要

Project Summary / Abstract Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with an estimated lifetime risk of 1 in 400 individuals. ALS is clinically characterized by motor deficits, and pathologically by the selective loss of motor neurons in the brain and spinal cord, as well as deposition of ubiquitinated proteinaceous aggregates of TDP- 43. Despite the presence of TDP-43 pathology in nearly all (~97%) brains of ALS patients, the genetic underpinnings of the disease is highly heterogeneous, with ~90% being considered to be ‘sporadic,’ or having no known genetic cause. The variable nature of the underlying causes has made treatment of the disease historically difficult due to a lack of clear therapeutic targets. In the past decade, Ataxin-2 (ATXN2) has emerged as a promising therapeutic target for ALS, as a potent genetic modifier of TDP-43 aggregation and toxicity across multiple models of TDP-43 proteinopathy. Most excitingly, decreasing ATXN2 levels using anti-sense oligonucleotides (ASOs) in a mouse model of TDP-43 overexpression led to a marked rescue of motor impairments and dramatic extension of lifespan. Despite the promise of ASOs, having an orthogonal method to reduce ATXN2 levels—such as a small molecule drug that can target one of its regulators—could have immense practical benefit in the clinical context. Moreover, little remains known on how ATXN2 is normally regulated, as well as its true role in disease. To gain mechanistic insight as well as to identify additional therapeutic targets, I developed a novel FACS (fluorescence activated cell sorting)-based CRISPR/Cas9 genome-wide knockout screening strategy. The idea was to identify suppressors and enhancers of ATXN2 protein levels in a reliable and efficient way; genes that decrease ATXN2 levels upon knockout could serve as novel therapeutic targets for ALS, while those that increase ATXN2 levels upon knockout could potentially contribute to heightened risk for the disease. The screen yielded a multitude of promising hits, with many acting in same biological pathways, or sometimes encoding subunits of one protein complex. One example of this is the lysosomal vacuolar ATPase (v-ATPase), for which genes encoding nearly every subunit were found to be significant suppressors of ATXN2 protein levels in my screens. In addition to validating hits from the initial screens across multiple disease relevant systems—such as in mouse primary neurons and human iPSC-derived neurons—I will expand the analysis to delve deeper into the mechanism of how the v-ATPase is regulating ATXN2 protein levels. Moreover, given that several FDA-approved small molecule drugs are available that inhibit v-ATPase subunits, I will test their safety and efficacy in reducing ATXN2 levels and rescuing disease phenotypes in a mouse model of ALS in vivo. If this approach is successful, there are a multitude of exciting possibilities for this screening platform and overall target discovery approach that I believe could help to uncover regulators of many other neurodegenerative diseases genes (e.g., tau and Ab in FTD and Alzheimer’s Disease, a-synuclein in Parkinson’s Disease) to empower the discovery of novel therapeutic targets in contexts not limited to ALS.