Identifying Cell Type-Specific RNA Expression and Regulatory Changes in Entorhinal Cortex Clinical Samples of Alzheimer's Disease

Spatial and temporal regulation of RNA is key to cellular homeostasis and can become disrupted in disease. The lifecycle of RNA can include splicing, modification, transport, and translation, which all require RNA-binding proteins (RBPs), a class of proteins that bind and regulate RNAs. Specifically in the brain, there are many neuronal and non-neuronal cell types that are each comprised of distinctive sets of transcripts with unique RNA regulatory machineries. While there are genetic tools in mice to parse out cell type-specific RNA regulation, a parallel tool does not currently exist to study this in human postmortem brain tissue, limiting the ability to study human diseases. We have developed crosslinking immunoprecipitation-Fluorescence Activated Nuclei Sorting (CLIP-FANS) which will allow us to overcome this hurdle and for the first time, identify cell-type specific RBP mediated RNA regulation in postmortem samples. CLIP-FANS builds on two previous methodologies: fluorescence-activated nuclei sorting (FANS) to achieve cell type specificity, followed by high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) to analyze gene regulation in sorted populations. Here, we have built an ordered approach and show preliminary data of the similarities and differences in the Hu-binding between excitatory and inhibitory neurons in the human entorhinal cortex in the first iteration of CLIP-FANS. One neurodegenerative disorder with dysregulation of RNAs and RBPs in Alzheimer’s disease (AD), a highly prevalent disease and the leading cause of dementia. For therapeutic interventions, maximum benefit would be achieved by targeting the first brain regions affected in AD, such as the entorhinal cortex, which has few studies on gene expression changes and none on gene regulation changes. Therefore, a combined approach of RNA sequencing of sorted cell types (FANSseq) and CLIP-FANS will generate cell-type specific gene expression and regulatory data of the differences occurring in AD to identify potential targets for therapeutic intervention. Project Number: 1F31AG090056-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: Seungtaek Oh | Institution: ROCKEFELLER UNIVERSITY, NEW YORK, NY | Award Amount: $49,538 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F03C-N (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11164203