Abstract Body: Background: Astrocyte endfeet, which ensheathe the brain vasculature to regulate the blood-brain barrier (BBB) and cerebral blood flow, are implicated in the cellular response to cerebral ischemia. Acute responses in the endfoot may depend on local protein synthesis from the pool of actively translating mRNA in this subcellular compartment, known as the endfoot translatome. The effect of ischemia on the endfoot translatome is unknown. Here, we characterized the endfoot translatome during early phases of reperfusion following ischemia in mice.

Methods: The study was conducted using a transgenic RiboTag mouse line (Aldh1l1-eGFP/Rpl10a), which expresses a tagged ribosomal protein under an astrocyte-specific promoter. Cerebral ischemia was induced for 2-hours via an intraluminal middle cerebral artery occlusion and reperfusion for 6-hours (MCAo/R) by removing the filament occluder. Extraction of the endfoot translatome was performed in two phases: 1) mechanical isolation of brain microvessels that retain the astrocyte endfeet, and 2) immunoprecipitation of the tagged polysomes. RNA sequencing was performed from this pool of ribosome-bound RNA. Differentially expressed genes (DEGs) were identified by comparing post-ischemic and contralateral control samples.

Results: We verified that astrocyte endfeet were retained in mechanically isolated microvessels in our MCAo/R model. Ribosome-bound RNA from post-ischemic microvessels were enriched with astrocytic genes (Aqp4, Gfap) while depleted of vascular genes (Pecam1, Cldn5, Pdgfrb, Acta2). We identified 205 DEGs in the post-ischemic astrocyte endfeet. Pathways associated with the DEGs included proteostasis, inflammation, cell cycle, and metabolism. Transcription factors whose targets were enriched amongst upregulated translating genes included HSF1, the master regulator of the heat shock response. The most highly upregulated genes in the translatome were HSF1-dependent Hspa1a and Hspa1b, which encode the inducible HSP70. We found that the HSP70 protein is upregulated in post-ischemic astrocyte endfeet, coinciding with an increase in ubiquitination across the proteome.

Conclusion: These findings suggest that proteotoxic stress in the post-ischemic astrocyte endfoot results in the activation of the heat shock response. Modulating proteostasis of the endfeet may be a strategy to preserving endfeet function and BBB integrity during ischemic stroke.