Fig. 5: Astrocyte protein biotinylation reveals region-specific proteomic signatures and differences between Camk2a neurons.

a Study design for neuron-specific and astrocyte-specific proteomic biotinylation. Rosa26^TurboID/wt, Rosa26^TurboID/wt/Camk2a-Cre^Ert2, Rosa26^TurboID/wt/Aldh1l1-Cre^Ert2 mice received tamoxifen intraperitoneally for 5 days. After 4 weeks, mice received biotin water for 2 weeks. b Representative Western blots from brain region lysates (n = 2 mice/group), probed for biotin (streptavidin Alexa488), V5 and Gapdh are shown. c Representative images (n = 2 mice/group) showing biotinylation in the hippocampus. d Representative immunofluorescence images (n = 2 mice/group) showing overlap between astrocytic biotinylation (streptavidin Alexa488), Gfap, and Ndrg2 in the hippocampus region from Rosa26^TurboID/wt/Aldh1l1 mice. e Representative immunofluorescence images (n = 2 mice/group) showing no overlap between biotinylation, Iba1, and βIII-tubulin in astrocytes and blood vessels in the hippocampus region from Rosa26^TurboID/wt/Aldh1l1 mice. f Clustering analysis of protein abundance data of region-enriched proteins with at least 4-fold enrichment over other regions in Rosa26^TurboID/wt/Aldh1l1 mice. STRING analysis identified networks of direct (protein-protein) and indirect (functional) interactions within core regional protein signatures in cortex/hippocampus and cerebellum. g Heatmap representation, based on enrichment Z-scores, of gene ontologies enriched in core regional proteins. h Volcano plot showing differentially enriched proteins comparing Rosa26^TurboID/wt/Aldh1l1 and Rosa26^TurboID/wt/Camk2a mice. For this analysis, all six brain regions were combined for both groups. Orange symbols (two-tailed T test unadjusted p ≤ 0.05 and ≥ 2-fold change) represent biotinylated proteins enriched in Camk2a neurons with neuron-specific proteins highlighted. Blue symbols (two-tailed T test unadjusted p ≤ 0.05 and ≥ 2-fold change) represent biotinylated proteins enriched in Aldh1l1 astrocytes and examples of astrocyte-specific proteins are highlighted in dark blue. i HCA of GSEA showing over-represented ontologies within neuronal-enriched proteins and astrocyte-enriched proteins. Representative gene ontology terms are highlighted. j Volcano plot showing enrichment (two-tailed T test unadjusted p ≤ 0.05) of MAPK and Akt/mTOR phospho-proteins in Rosa26^TurboID/wt/Camk2a compared to Rosa26^TurboID/wt/Aldh1l1 brains. For related MS data and additional analyses, see Supplementary Data 19–24. The figure was partly generated using Servier Medical Art, provided by Servier, licensed under a Creative Commons Attribution 3.0 unported license. Source data are provided as a Source Data file. Source data are provided as a Source Data file.