Illuminating Reactive Astrocyte Contributions to Alzheimer’s Pathology via PIP-sort Technology
PROJECT SUMMARY Astrocytes—supportive glial cells in the brain—undergo significant changes in Alzheimer’s disease (AD), becoming reactive and contributing to disease progression. These reactive astrocytes, characterized by markers such as SERPINA3, GFAP δ, and PLXNB1, are scarce in postmortem brain tissues, making them difficult to study with conventional methods. Traditional single-nucleus RNA sequencing requires analyzing vast numbers of cells to capture these rare populations, and standard fluorescence-activated cell sorting (FACS) techniques are ineffective on nuclei due to the absence of surface markers. To overcome these challenges, we introduce PIP-sort, an innovative, microfluidic-free method that enables the isolation and detailed analysis of rare astrocyte nuclei from frozen brain samples. PIP-sort utilizes droplet digital PCR (ddPCR) to amplify specific mRNA transcripts within droplets, causing them to fluoresce. This fluorescence allows for the rapid sorting of targeted nuclei using standard FACS equipment. Each nucleus is paired with a barcoded bead, facilitating the capture of its full-length cDNA and, with additional processing, its chromatin accessibility profile. This approach yields high- resolution, single-nucleus RNA and ATAC sequencing data, providing insights into both gene expression and epigenetic regulation. Our project comprises three specific aims: · Validation of PIP-sort: We will optimize and validate the PIP-sort technique using synthetic mixtures of human and mouse cells to demonstrate its capability in enriching rare cell types based on mRNA markers. · Multi-omic Profiling: We will extend PIP-sort to simultaneously profile transcriptomic and epigenomic landscapes of isolated nuclei, enabling a comprehensive understanding of the regulatory mechanisms in reactive astrocytes. · Functional Analysis: Applying PIP-sort to human AD brain samples, we will isolate reactive astrocyte populations and identify key regulatory genes. Subsequent CRISPR/Cas9-mediated perturbations in mouse models will assess the causal roles of these genes in amyloid and tau pathologies and cognitive decline. By facilitating the study of elusive astrocyte subpopulations directly from archival human tissues, PIP-sort aims to uncover novel therapeutic targets and advance our understanding of glial contributions to Alzheimer’s disease.
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Imported from NIH RePORTER | Grant 1R01AG100367-01.
Open sourceOverview
Grant agreement 1R01AG100367-01
Open sourcePROJECT SUMMARY Astrocytes—supportive glial cells in the brain—undergo significant changes in Alzheimer’s disease (AD), becoming reactive and contributing to disease progression. These reactive astrocytes, characterized by markers such as SERPINA3, GFAP δ, and PLXNB1, are scarce in postmortem brain tissues, making them difficult to study with conventional methods. Traditional single-nucleus RNA sequencing requires analyzing vast numbers of cells to capture these rare populations, and standard fluorescence-activated cell sorting (FACS) techniques are ineffective on nuclei due to the absence of surface markers. To overcome these challenges, we introduce PIP-sort, an innovative, microfluidic-free method that enables the isolation and detailed analysis of rare astrocyte nuclei from frozen brain samples. PIP-sort utilizes droplet digital PCR (ddPCR) to amplify specific mRNA transcripts within droplets, causing them to fluoresce. This fluorescence allows for the rapid sorting of targeted nuclei using standard FACS equipment. Each nucleus is paired with a barcoded bead, facilitating the capture of its full-length cDNA and, with additional processing, its chromatin accessibility profile. This approach yields high- resolution, single-nucleus RNA and ATAC sequencing data, providing insights into both gene expression and epigenetic regulation. Our project comprises three specific aims: · Validation of PIP-sort: We will optimize and validate the PIP-sort technique using synthetic mixtures of human and mouse cells to demonstrate its capability in enriching rare cell types based on mRNA markers. · Multi-omic Profiling: We will extend PIP-sort to simultaneously profile transcriptomic and epigenomic landscapes of isolated nuclei, enabling a comprehensive understanding of the regulatory mechanisms in reactive astrocytes. · Functional Analysis: Applying PIP-sort to human AD brain samples, we will isolate reactive astrocyte populations and identify key regulatory genes. Subsequent CRISPR/Cas9-mediated perturbations in mouse models will assess the causal roles of these genes in amyloid and tau pathologies and cognitive decline. By facilitating the study of elusive astrocyte subpopulations directly from archival human tissues, PIP-sort aims to uncover novel therapeutic targets and advance our understanding of glial contributions to Alzheimer’s disease.