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

反应性星形胶质细胞在局灶性缺血性中风后神经再生和大脑恢复中的作用

• 批准号: 10220140
• 负责人: Shinghua Ding
• 金额: $ 35.11万
• 依托单位: UNIVERSITY OF MISSOURI-COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-05-15 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10220140
• 关键词: Action Potentials; Acute; Adult; Affect; Affinity Chromatography; Apoptotic; Astrocytes; Attenuated; Behavioral; Biological Assay; Brain; Brain Infarction; Brain Injuries; Cell Line; Cell Proliferation; Cessation of life; DLG4 gene; Dendrites; Dependovirus; Electrophysiology (science); Exhibits; Gene Expression; Glutamates; Goals; Hindlimb; Histologic; Human; Image; Infarction; Injections; Ischemia; Ischemic Stroke; Knockout Mice; Lead; Measurement; Mediating; Methods; Microscopy; Molecular; Molecular Genetics; Morphology; Motor Cortex; Mus; Natural regeneration; Nerve Regeneration; Neuroglia; Neurons; Play; Process; Proteins; Quantitative Reverse Transcriptase PCR; Recombinants; Recovery; Recovery of Function; Ribosomes; Role; Signal Transduction; Structure; Synapses; Synaptic plasticity; Synaptophysin; Technology; Testing; Therapeutic; Time; Tissues; Transcript; Translating; Vertebral column; Viral; Virus; Western Blotting; adeno-associated viral vector; adult neurogenesis; base; behavior test; behavioral outcome; brain repair; conditional knockout; disability; functional outcomes; gain of function; genetic approach; glial cell-line derived neurotrophic factor; immunocytochemistry; improved; in vivo; insight; loss of function; motor behavior; mouse model; neuron loss; neurotrophic factor; neurotropic; novel; overexpression; patch clamp; promoter; stroke outcome; stroke therapy; synaptic function; synaptogenesis; tool; treatment strategy; two-photon

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.

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 中枢神经系统中的神经胶质细胞数量众多，种类繁多，并与神经元密切相互作用，以支持和调节其功能。 功能协调发展的FIS后，PIR中的星形胶质细胞在形态、增殖和基因表达方面发生动态变化， 表达，尤其是在梗死周围区（PIR）。这些星形胶质细胞被称为反应性星形胶质细胞（RA）。 然而，在以下情况下，反应性星形胶质细胞（RA）是否以及如何影响FIS后的脑恢复， 星形胶质细胞-神经元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，一种有效的 在光血栓形成后，缺血半球和RA中神经营养因子显著上调 （PT）诱导的FIS。此外，我们发现星形胶质细胞GDNF的缺失减少了成年人的神经发生， 在PT后的PIR中增加脑梗塞并减弱细胞增殖。基于 这些强有力的初步结果，我们假设，RAS衍生的GDNF在神经细胞中起着重要作用。 FIS后的再生和功能性脑恢复。项目目标是确定RA是否以及如何- 衍生的GDNF刺激PIR中存活神经元的突触再生和重塑， FIS后的长期卒中结局。为了实现这一目标，我们开发了跨学科的技术 包括自身互补腺相关病毒（scAAV）载体和Glast-CreERT 2：GDNF f/f小鼠， 在FIS后时间，在体内双光子（2-P）长期， 显微镜、电生理学、免疫细胞化学、Western印迹（WB）分析、脑损伤和神经元损伤 死亡分析和行为测试我们提出三个具体目标。在目标1中，我们将检验以下假设： RAS源性GDNF可促进FIS后PIR的突触发生，刺激神经再生。我们将 确定RAS衍生的GDNF对涉及突触功能的神经元蛋白表达的影响 和可塑性的PIR;使用TRAP（翻译核糖体亲和纯化）方法，我们将进一步确定 PIR中神经元转录物在翻译状态下的变化。在目标2中，我们将检验RA的假设- 衍生的GDNF可以促进PIR中存活神经元的结构和功能突触重塑， FIS。使用体内长期2-P成像，我们将确定RAS衍生的GDNF对脊柱转换的影响。 (i.e.,棘的形成和消除），谷氨酸释放和钙信号在同一树突的存活 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衍生GDNF对功能性突触可塑性的影响。在目标3中，我们将检验以下假设： 星形胶质细胞GDNF可以改善卒中的长期预后。我们将评估RAS衍生的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-神经元相互作用的背景下，对FIS后大脑恢复过程的功能性见解揭示了 潜在的中风治疗策略，因此具有科学和翻译意义。