# A multiplexable in vivo perturbation toolkit to identify genes affecting neurodegeneration in a model of synucleinopathy > **NIH NIH R21** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2024 · $101,927 ## Abstract Project Summary/Abstract Neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease with dementia (PDD) and dementia with Lewy bodies (DLB) have no cure, but all three share signs of α-synuclein (α-syn) pathology and a pattern of neurodegeneration in which there is variation in neuronal loss. Our long-term goal is to examine this differential neuronal susceptibility to identify features of neurodegeneration in synucleinopathy, and to use this knowledge for subsequent development of neuroprotective strategies and biomarkers. Our central hypothesis is that CRISPR-Cas9 library screening can be used as a cell-type specific, high-throughput in vivo method to elucidate determinants of neurodegeneration in synucleinopathy. The rationale for the proposed research is that, once susceptibility modifiers are identified, therapeutic strategies can be devised for the treatment of PDD, DLB and even perhaps AD. Thus, in AIM 1, we propose to begin by identifying candidate modifiers of neuronal susceptibility in the transgenic mThy1-α-syn (L61Tg) mouse model of synucleinopathy using cell-type-enriched single-nucleus profiling of dopaminergic (DA) neurons. Given the known differential susceptibility among ventral midbrain DA neurons in synucleinopathies, we posit that transcriptomic profiling of these neurons will not only identify signatures of neuronal resilience to stress, but also identify candidate modifiers for inclusion in our subsequent CRISPR library screen. For this work, our cell-specific nuclei tagging system will allow to selectively profile, by single-nucleus RNA-sequencing, DA neurons in L61Tg and their non- transgenic (NTg) littermates. The resulting sequencing data will then be clustered and investigated using differential composition and gene expression analysis to generate a final set of candidate genes that are expressed in subsets of DA neurons, differentially regulated in L61Tg mice, and relevant to human DA subtypes. These candidate genes, which will be included in the list of perturbation candidates in AIM 2, will also serve as a set of genes for future investigations in neurodegeneration research. Next, in AIM 2, we will perform a targeted in vivo CRISPR library screening of DA-neurons in the same mouse model of synucleinopathy. Here, single guide RNA (sgRNA) libraries will be delivered by systemic AAV injection, before brains are harvested six months later and processed for sgRNA sequencing, to identify which genes from our list, upon being silenced, are functional modifiers of neuronal loss. The candidate gene list will be made of our reported hits (Brichta et al., Nat Neurosci, 2015) and those identified in AIM 1. Successful completion of this project will reveal determinants of neuronal survival in vivo and in response to α-syn, a stressor relevant to human neurological disorders. Thus, our anticipated results will have an important positive impact as they will provide opportunities for preventive and therapeutic... ## Key facts - **NIH application ID:** 11082857 - **Project number:** 3R21AG085144-01S1 - **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES - **Principal Investigator:** SERGE E PRZEDBORSKI - **Activity code:** R21 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2024 - **Award amount:** $101,927 - **Award type:** 3 - **Project period:** 2023-09-30 → 2025-09-29 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/11082857 ## Citation > US National Institutes of Health, RePORTER application 11082857, A multiplexable in vivo perturbation toolkit to identify genes affecting neurodegeneration in a model of synucleinopathy (3R21AG085144-01S1). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/11082857. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*