Abstract Body: INTRODUCTION: Ischemic injury to the brain results in both acute and long-lasting effects that may trigger progressive neurodegenerative cascades mediated by glial cells such as astrocytes, the most common glial cell in the brain. However, a comprehensive understanding of proteomic changes occurring in astrocytes following ischemic injury, while retaining their native state in vivo, is lacking.

METHODS: We applied cell type-specific in vivo bio-orthogonal non-canonical amino acid tagging (ciBONCAT) to label newly-synthesized (nascent) proteins, specifically in astrocytes. BONCAT uses Cre-mediated recombination to express mutant Methionyl-tRNA synthetase (MetRS) that tags nascent proteins with the Methionine analog Azidonorleucine (Anl) for purification using Click-chemistry, along with GFP reporter expression. Experimental diffuse cortical ischemia was induced in Aldh1l1-Cre-ert2/MetRS-floxed mice via a 20-minute bilateral common carotid artery occlusion (BCCAO), followed by 3-week Anl water supplementation. Azide-tagged proteins from cortices were Click-labeled with biotin-alkyne, then enriched and analyzed by label-free Mass Spectrometry (LFQ-MS).

RESULTS: Immunofluorescence (IF) microscopy confirmed astrocyte-specific GFP expression in the brains of astrocyte-BONCAT mice (A). Western blot confirmed proteomic labeling in astrocyte-BONCAT mice as compared to controls (B). After enrichment of azide-tagged proteins, LFQ-MS identified an astrocyte-specific nascent proteome of over 1,200 proteins, including canonical astrocyte markers (eg. GFAP, Aqp4, Aldh1l1). BCCAO resulted in reactive astrocytosis in the cortex and hippocampus as verified by IF. LFQ-MS identified distinct proteomic changes occurring specifically in astrocytes, including 84 upregulated (eg. Fus, Adrbk1) and 100 downregulated (eg. Ndufs8, Necap1) proteins (C). Gene set enrichment analyses revealed increased translation and mitochondrial metabolic changes in astrocytes in response to BCCAO.

CONCLUSIONS: We used ciBONCAT to obtain native-state astrocyte-specific nascent proteomes from the adult mouse brain. This novel approach identified unique astrocyte-specific proteomic alterations related to mitochondria, metabolism, and translation following BCCAO. These astrocytic proteomic changes are likely indicative of long-lasting progressive changes several weeks following transient global ischemic injury, representing potential targets for drug therapy to facilitate and improve stroke recovery.