Endothelial Regulation of Astrocyte Trans-differentiation in Stroke

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Neurogenesis plays an important role in stroke recovery. In models of cerebral ischemia, neurogenesis is amplified in the sub-ventricular zone (SVZ) and the sub-granular zone (SGZ). In addition to these responses in the standard neurogenic niches, some reports suggest that parenchymal astrocytes may convert into neurons after stroke. Within neurogenic niches in SVZ and SGZ, brain endothelial cells play a vital role by secreting trophic factors to support neural stem cells. Is it possible that brain endothelium may also help regulate astrocyte trans-differentiation? In this proposal, we will investigate this idea, i.e. brain endothelial cells act as an unexpected source of signals to convert astrocytes into neurons, thus improving outcomes after stroke. Our pilot data suggest that (i) oxygen-glucose deprivation (OGD)-stimulated brain endothelial cells convert astrocytes into neural progenitor cells (NPC) in vitro, (ii) transfer of microvesicles derived from OGD-stimulated brain endothelial cultures reprogram astrocytes into NPC in vitro, (iii) microvesicles derived from OGD- stimulated brain endothelial cells also convert astrocytes into NPC in vivo, and promote recovery in mouse focal cerebral ischemia, (iv) pro-neural transcription factor Ascl1 in brain endothelial cells may be involved in astrocyte trans-differentiation, (v) methods to dissect these mechanisms (mouse genetics, brain endothelial cell-specific viral infection, lineage tracing, single-nuclei RNA-seq, imaging etc) are feasible in our labs. Based on our pilot data, we hypothesize that up-regulation of Ascl1 in brain endothelial cell-derived microvesicles induces astrocyte trans-differentiation, thus contributing to neurogenesis, tissue repair, and recovery after stroke. We have 3 specific aims. In Aim 1, we will compare effects of major neurovascular unit cells on astrocyte trans- differentiation. In Aim 2, we will dissect mechanisms of brain endothelial cell-mediated astrocyte trans- differentiation, and use gain- and loss-of-function experiments to manipulate Ascl1 expression in brain endothelial cells, and track astrocyte trans-differentiation after focal ischemia. In Aim 3, we will investigate the effect of microvesicles derived from Ascl1-overexpressed brain endothelial cells on astrocyte trans- differentiation, and assess neurogenesis, tissue repair, and sensorimotor/cognitive outcomes in young, older, male or female mice after focal ischemia. Our experiments will utilize mouse genetic and molecular tools including a combination of AAV-induced brain endothelial cell-specific gene expression (Tie2-Cre or Cdh5-Cre mice), lineage tracing of astrocytes by using tamoxifen-inducible Aldh1l1-CreERT2;R26R-YFP mice, scRNA- seq or snRNA-seq to fully map transcriptomes of astrocytes in mice treated with microvesicles post stroke. Translational relevance will be assessed with in vivo optical imaging and long-term outcomes post-stroke. This project should define a new form of endothelial signaling in stroke, identify a unique vascular-regulated mechanism of neuroplasticity, and hopefully may lead to novel therapeutic opportunities for brain repair.