In vivo TurboID proximity labeling to capture astrocyte proteomic and transcriptomic signatures under homeostatic and neuroinflammatory conditions

Abstract

AbstractBackgroundDespite decades of research into the cellular and molecular contributions of brain cell types to Alzheimer’s disease (AD) pathogenesis, there are no glial‐specific disease‐modifying therapies. To address this, our group has developed the innovative cell‐specific in vivo biotinylation of proteins (CIBOP) approach to quantify astrocyte‐specific in vivo proteomic and transcriptomic contributions to AD under homeostatic and neuroinflammatory conditions.MethodIn the CIBOP approach, TurboID, a biotin ligase, is selectively expressed in the cell type of interest using a conditional Cre/lox genetic strategy to label the cellular proteome. Using mass spectrometry (MS)‐based proteomics, we have found that TurboID biotinylates many RNA‐binding and ribosomal proteins. Leveraging this, we extended the CIBOP approach to obtain representative cell type‐specific transcriptomes, along with the proteome. We crossed astrocyte specific Cre (Aldh1l1‐Cre‐ert2) and Rosa26TurboID/wt floxed mice for astrocyte‐specific proteomic labeling (astrocyte‐CIBOP). Astrocyte‐CIBOP and control (Cre‐only) mice received tamoxifen, followed by biotin‐containing water and systemic lipopolysaccharide (via intraperitoneal injections) to induce a neuroinflammatory state. Biofluids (CSF and plasma) and cortical tissue were collected. While maintaining RNA‐protein interactions, cortical tissue was lysed, biotinylated proteins were enriched via streptavidin beads, and then RNA was eluted. Immunofluorescent microscopy (IF) and biochemical assays were performed to confirm astrocyte‐specific proteomic biotinylation.ResultWestern blot analysis of the cortex and CSF confirmed biotinylation of the cellular proteome of astrocyte‐CIBOP mice under homeostatic and neuroinflammatory conditions when compared to controls. IF images show that the biotinylation signal is astrocyte specific, proteomic labeling does not impact astrocyte morphology, and high levels of signal was detected at astrocytic end‐feet. RNA gel electrophoresis shows low RNA yield from control mouse brain streptavidin pulldowns, and high levels of RNA from astrocyte‐CIBOP brain pulldowns. MS‐based proteomics of astrocyte‐derived biotinylated proteins and RNA‐sequencing from biotinylated protein‐RNA interactions are underway.ConclusionWe validate the CIBOP approach to capture the cortical Aldh1l1‐positive astrocyte proteome and transcriptome in vivo under homeostatic and neuroinflammatory conditions and demonstrate labeling of astrocyte‐derived CSF proteins. Our novel in vivo cell type‐specific and native‐state dual ‐omics approach provides complementary transcriptomic and proteomic information to investigate disease mechanisms, discover new biomarkers, and identify novel therapeutic targets.