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 成年大脑具有从局灶性缺血性中风（FIS）中恢复的显着能力。星形胶质细胞最多 中枢神经系统中的众多和多样的神经胶质细胞，并与神经元紧密相互作用以支持和调节其 功能。 FIS之后，PIR中的星形胶质细胞暴露了形态，增殖和基因的动态变化 尤其是在侵入周围区域（PIR）中的表达。这些星形胶质细胞称为反应性星形胶质细胞（RAS）。 但是，在FIS的背景下，反应性星形胶质细胞（RAS）是否影响FIS后的大脑恢复 星形胶质细胞 - 神经元相互作用在很大程度上仍然是出乎意料的。在我们的初步研究中，我们发现了GDNF 神经营养因子，在缺血性半球和RAS中大大更新 （PT）诱导的FIS。此外，我们发现星形胶质细胞GDNF的缺失减少了成人神经发生 正常的大脑，并增加脑梗塞并减轻PT后PIR中细胞的增殖。基于 这些强烈的初步结果，我们假设RAS衍生的GDNF在神经中起着重要作用 FIS后的再生和功能性脑恢复。预测的目标是确定是否以及如何ras- 衍生的GDNF刺激PIR中生存神经元的突触再生和重塑，并改善 FIS之后的长期中风结果。为了实现这一目标，我们开发了跨学科技术 包括自我平衡腺相关病毒（SCAAV）向量和Glast-Creert2：gdnff/f小鼠到 在FIS后时间，特定于RAS中的过度表达或删除GDNF，在Vivo Two Photoson（2-P）长期 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 死亡测定和行为测试。我们提出了三个具体目标。在AIM 1中，我们将检验以下假设 Ras衍生的GDNF可以增强突触发生，以刺激FIS后PIR的神经再生。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PIR中的可塑性；使用陷阱（翻译核糖体亲和力纯化）我们将进一步识别 神经元转录本在PIR中的翻译状态下变化。在AIM 2中，我们将检验以下假设。 衍生的GDNF可以促进PIR中生存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对脊柱更新的影响 （即脊柱形成和消除），在同一生存的树突中释放谷氨酸和Ca2+信号 PIR中的神经元。我们将对PIR中的生存神经元进行贴片钳记录，以确定 RAS衍生的GDNF对功能突触可塑性的影响。在AIM 3中，我们将检验以下假设 星形胶质细胞GDNF可以改善长期中风结果。我们将评估RAS衍生的GDNF对 长期组织学和行为结果。我们的项目将提供新颖的分子，细胞和 在胶质神经元相互作用的背景下，FIS后对大脑恢复过程的功能见解揭示 中风治疗的潜在策略，因此具有科学和翻译意义。