Expanding therapeutic targets for ALS : a novel CRISPR screen for regulators of ALS protein Ataxin-2
Abstract/Contents
- Abstract
- Neurodegenerative diseases are devastating for all involved, with no cures and few treatments. One feature of neurodegeneration is the accumulation of misfolded proteins in the brain, and strategies to eliminate these proteins are emerging. For amyotrophic lateral sclerosis (ALS), TDP-43 is the major protein aggregate, presenting in ~97% of patient brains. In this context, ataxin-2 (encoded by the gene ATXN2) has become a compelling therapeutic target for ALS due to 1) its ability to modify TDP-43 aggregation and toxicity in vitro and in vivo, and 2) the discovery of mutations in ATXN2 that cause heightened risk for ALS. Genetic reduction of ataxin-2 ameliorates motor impairments and profoundly extends lifespan in a mouse model of TDP-43 proteinopathy, and anti-sense oligonucleotides (ASOs) targeting ATXN2 are now being tested in humans in an ongoing phase 1 clinical trial. My doctoral dissertation involves taking a genes-to-small molecule therapy approach in a project aiming to identify additional ways to therapeutically reduce ataxin-2 levels. I begin by establishing a genome-wide screening strategy in human cells using CRISPR-Cas9 and applying this approach to uncover modifiers of ataxin-2 protein levels. Using antibody staining in fixed cells and fluorescence as a read-out via fluorescence-activated cell sorting (FACS), I identified numerous previously unknown modifiers of endogenous ataxin-2 levels, with many acting in the same biological pathways or encoding subunits of a single protein complex. After individually validating numerous hit genes, I honed in on one class of hits—genes encoding components of the lysosomal v-ATPase—which can be targeted by multiple FDA-approved small molecule inhibitors. Using both genetic and pharmacologic inhibition, I found that perturbing the v-ATPase results in decreased ataxin-2 protein levels in human and mouse neurons, as well as in vivo in the brains of mice upon oral administration of one of the drugs—Etidronate. These results demonstrate not only the tractability and efficacy of a FACS-based genetic screening approach in discovering regulators of proteins of interest, but also the potential to identify and repurpose existing drugs for diseases impacted by protein levels. This screening platform and overall target discovery approach may also be useful in uncovering regulators of many other disease-associated genes, to empower the discovery of novel therapeutic targets and normal cell biological mechanisms in contexts not limited to ALS.
Description
| Type of resource | text |
|---|---|
| Form | electronic resource; remote; computer; online resource |
| Extent | 1 online resource. |
| Place | California |
| Place | [Stanford, California] |
| Publisher | [Stanford University] |
| Copyright date | 2022; ©2022 |
| Publication date | 2022; 2022 |
| Issuance | monographic |
| Language | English |
Creators/Contributors
| Author | Kim, Garam |
|---|---|
| Degree supervisor | Gitler, Aaron D |
| Thesis advisor | Gitler, Aaron D |
| Thesis advisor | Bassik, Michael |
| Thesis advisor | Reimer, Richard J |
| Thesis advisor | Wyss-Coray, Anton |
| Degree committee member | Bassik, Michael |
| Degree committee member | Reimer, Richard J |
| Degree committee member | Wyss-Coray, Anton |
| Associated with | Stanford University, Neurosciences Program |
Subjects
| Genre | Theses |
|---|---|
| Genre | Text |
Bibliographic information
| Statement of responsibility | Garam Kim. |
|---|---|
| Note | Submitted to the Neurosciences Program. |
| Thesis | Thesis Ph.D. Stanford University 2022. |
| Location | https://purl.stanford.edu/vn395pq5712 |
Access conditions
- Copyright
- © 2022 by Garam Kim
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