Endothelial Regulation of Astrocyte Trans-differentiation in Stroke

中风中星形胶质细胞转分化的内皮调节

基本信息

批准号：
10712315
负责人：
Wenlu Li
金额：
$ 47.23万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-17 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10712315
关键词：
Adult Astrocytes Blood Vessels Brain Brain Injuries Brain region Calcium Cell Culture Techniques Cells Cerebral Ischemia Cerebrovascular system Coculture Techniques Cognitive DNA Data Down-Regulation Embryo Endothelial Cells Endothelium Etiology Event Excision Female Gene Expression Generations Genes Glial Fibrillary Acidic Protein Glucose Human Image In Vitro Infarction Ischemia Knock-out Liposomes Maps Measures Mediating Methods Microglia Modeling Molecular Mus Neuronal Plasticity Neurons Outcome Oxygen Pericytes Play Population Potassium Process Proteins RNA Rattus Recovery Regulation Reporting Reverse Transcriptase Polymerase Chain Reaction Role Signal Transduction Small Interfering RNA Sodium Source Stains Stroke Synapses Tamoxifen Testing Therapeutic Therapeutic Effect Time Up-Regulation Virus Diseases adeno-associated viral vector brain cell brain endothelial cell brain repair cell type deprivation experimental study foot functional improvement gain of function improved improved outcome in vivo in vivo calcium imaging in vivo optical imaging ischemic injury loss of function male microvesicles mouse genetics nerve stem cell neural neurogenesis neurovascular neurovascular unit newborn neuron novel novel therapeutics overexpression polysialyl neural cell adhesion molecule post stroke programs promoter response single nucleus RNA-sequencing single-cell RNA sequencing stroke patient stroke recovery subventricular zone tissue repair tool transcription factor transcriptome transcriptome sequencing transdifferentiation

项目摘要

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.

神经发生在中风恢复中起着重要作用。在脑缺血模型中，神经发生是在室下区（SVZ）和颗粒下区（SGZ）中放大。除了这些回应之外标准的神经源性生态位，一些报告表明实质星形胶质细胞可能转化为神经元中风后。在 SVZ 和 SGZ 的神经源性微环境中，脑内皮细胞通过分泌支持神经干细胞的营养因子。脑内皮是否也可能有助于调节星形胶质细胞转分化？在这个提案中，我们将研究这个想法，即脑内皮细胞充当将星形胶质细胞转化为神经元的意想不到的信号来源，从而改善中风后的预后。我们的试验数据表明，(i) 氧糖剥夺 (OGD) 刺激的脑内皮细胞转化为在体外将星形胶质细胞转化为神经祖细胞 (NPC)，(ii) 转移源自 OGD 刺激的微泡脑内皮培养物在体外将星形胶质细胞重编程为 NPC，(iii) 来自 OGD-的微泡受刺激的脑内皮细胞还在体内将星形胶质细胞转化为NPC，并促进小鼠的康复局灶性脑缺血，(iv)脑内皮细胞中的前神经转录因子Ascl1可能参与星形胶质细胞转分化，(v) 剖析这些机制的方法（小鼠遗传学、脑内皮细胞细胞特异性病毒感染、谱系追踪、单核 RNA 测序、成像等）在我们的实验室是可行的。根据我们的试验数据，我们假设脑内皮细胞来源的 Ascl1 上调微泡诱导星形胶质细胞转分化，从而有助于神经发生、组织修复和中风后的恢复。我们有 3 个具体目标。在目标 1 中，我们将比较主要神经血管单位细胞对星形胶质细胞转运的影响差异化。在目标 2 中，我们将剖析脑内皮细胞介导的星形胶质细胞转导机制分化，并使用功能获得和丧失实验来操纵大脑中 Ascl1 的表达内皮细胞，并追踪局灶性缺血后星形胶质细胞的转分化。在目标 3 中，我们将调查 Ascl1过表达脑内皮细胞来源的微泡对星形胶质细胞转运的影响分化，并评估年轻人、老年人、老年人的神经发生、组织修复和感觉运动/认知结果局灶性缺血后的雄性或雌性小鼠。我们的实验将利用小鼠遗传和分子工具包括 AAV 诱导的脑内皮细胞特异性基因表达的组合（Tie2-Cre 或 Cdh5-Cre 小鼠），使用他莫昔芬诱导的 Aldh1l1-CreERT2;R26R-YFP 小鼠进行星形胶质细胞谱系追踪，scRNA- seq 或 snRNA-seq 来完整绘制中风后接受微泡治疗的小鼠星形胶质细胞的转录组图谱。将通过体内光学成像和中风后的长期结果来评估转化相关性。这该项目应定义中风中内皮信号传导的新形式，确定独特的血管调节神经可塑性机制，有望为大脑修复带来新的治疗机会。