Reactive astrocytes in neural regeneration and brain recovery after focal ischemic stroke

项目摘要 成人大脑具有从局灶性缺血性中风(FIS)中恢复的非凡能力。星形胶质细胞是最 中枢神经系统内大量多样的神经胶质细胞与神经元密切相互作用，以支持和调节其 功能。FIS后，PIR中的星形胶质细胞在形态、增殖和基因方面都发生了动态变化 尤其是在梗塞周围区(PIR)的表达。这些星形胶质细胞被称为反应性星形胶质细胞(RAS)。 然而，反应性星形胶质细胞(RAS)是否以及如何影响FIS后的脑恢复 星形胶质细胞与神经元之间的相互作用在很大程度上仍未被探索。在我们的初步研究中，我们发现GDNF是一种有效的 光血栓形成后缺血半球和RAS中神经营养因子的显著上调 (PT)诱导的FIS。此外，我们发现星形细胞GDNF的缺失减少了成年大鼠的神经发生。 正常脑组织，PT后PIR区脑梗塞增加，细胞增殖减弱。基于 这些强有力的初步结果，我们假设RAS来源的GDNF在神经中起重要作用 FIS后的再生和脑功能恢复。计划的目标是确定RAS是否以及如何- 衍生的GDNF刺激PIR存活神经元的突触再生和重塑并改善 FIS后的长期卒中结果。为了实现这一目标，我们开发了跨学科的技术 包括自补充腺相关病毒(ScAAV)载体和Glast-CreERT2：GDNFf/f小鼠 FIS后长期体内双光子(2-P)在RAS中特异地过表达或缺失GDNF 显微镜、电生理学、免疫细胞化学、蛋白质印迹(WB)分析、脑损伤和神经元 死亡分析和行为测试。我们提出了三个具体目标。在目标1中，我们将检验假设 RAS来源的GDNF可促进FIS后PIR的突触发生，刺激神经再生。我们会 确定RAS来源的GDNF对突触功能相关神经元蛋白表达的影响 和可塑性；利用TRAP(翻译核糖体亲和纯化)方法进一步鉴定 在PIR中，神经元转录在翻译状态下发生变化。在目标2中，我们将检验RAS- 衍生GDNF可促进PIR后存活神经元的结构和功能突触重构 菲斯。使用活体长期2-P成像，我们将确定RAS来源的GDNF对脊柱翻转的影响 (即棘突的形成和消除)、谷氨酸释放和钙信号在存活的同一树突中 PIR中的神经元。我们将对PIR中存活的神经元进行膜片钳记录，以确定 RAS源性胶质细胞源性神经营养因子对功能性突触可塑性的影响在目标3中，我们将检验假设 星形细胞胶质细胞源性神经营养因子可以改善中风的长期预后。我们将评估RAS来源的GDNF对 长期的组织学和行为学结果。我们的项目将提供新的分子、细胞和 在神经胶质细胞-神经元相互作用的背景下，对FIS后大脑恢复过程的功能洞察揭示 中风治疗的潜在策略，因此具有科学和翻译意义。

Project Summary The adult brain has a remarkable capacity to recover from focal ischemic stroke (FIS). Astrocytes are the most numerous and diverse glial cells in CNS and intimately interact with neurons to support and regulate their functions. After FIS, astrocytes in the PIR exhibit dynamic changes in morphology, proliferation and gene expression especially in the peri-infarct region (PIR). These astrocytes are called reactive astrocytes (RAs). However, whether and how reactive astrocytes (RAs) affect brain recovery after FIS in the context of astrocyte—neuron interactions largely remain unexplored. In our preliminary study, we found GDNF, a potent neurotrophic factor, is dramatically upregulated in the ischemic hemisphere and RAs after photothrombosis (PT)-induced FIS. Furthermore, we found that deletion of astrocytic GDNF reduces adult neurogenesis in normal brain, and increases brain infarction and attenuates cell proliferation in the PIR after PT. Based on these strong preliminary results, we hypothesize that RAs-derived GDNF plays an important role in neural regeneration and functional brain recovery after FIS. The prohect goal is to determine whether and how RAs- derived GDNF stimulates synaptic regeneration and remodeling of surviving neurons in the PIR and improves long-term stroke outcomes after FIS. To achieve this goal, we have developed interdisciplinary technologies including self-complementary adeno-associated virus (scAAV) vectors and Glast-CreERT2:GDNFf/f mice to specifically overexpress or delete GDNF in RAs during post FIS time, in vivo two photon (2-P) long-term microscopy, electrophysiology, immunocytochemistry, Western blot (WB) analysis, brain damage and neuronal death assays and behavioral tests. We propose three specific aims. In Aim 1, we will test the hypothesis that RAs-derived GDNF can enhance synaptogenesis to stimulate neural regeneration in the PIR after FIS. We will determine the effects of RAs-derived GDNF on the expression of neuronal proteins involving synaptic function and plasticity in the PIR; using TRAP (translating ribosome affinity purification) method we will further identify neuronal transcript changes at translational status in the PIR. In Aim 2, we will test the hypothesis that RAs- derived GDNF can promote structural and functional synaptic remodeling of surviving neurons in the PIR after FIS. Using in vivo long-term 2-P imaging we will determine the effect of RAs-derived GDNF on spine turnover (i.e., spine formation and elimination), glutamate release and Ca2+ signaling in the same dendrites of surviving neurons in the PIR. We will conduct patch-clamp recording on surviving neurons in the PIR to determine the effect of RAs-derived GDNF on functional synaptic plasticity. In Aim 3, we will test the hypothesis that astrocytic GDNF can improve long-term stroke outcomes. We will evaluate the effect of RAs-derived GDNF on long-term histological and behavioral outcomes. Our project will provide novel molecular, cellular and functional insights into the brain recovery processes after FIS in the context of glia-neuron interactions, reveal potential strategies for stroke therapy, and thus has both scientific and translational significances.