The Role of Local BDNF Synthesis in Spine Morphogenesis

局部 BDNF 合成在脊柱形态发生中的作用

基本信息

批准号：
8254913
负责人：
Lauren Lynn Orefice
金额：
$ 4.18万
依托单位：
GEORGETOWN UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-09-30 至 2014-09-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8254913
关键词：
3&apos Untranslated Regions AMPA Receptors Actins Affect Affinity Alzheimer&apos s Disease Antibodies Biological Assay Brain Brain-Derived Neurotrophic Factor Cell Maintenance Cell Survival Cells Data Dendrites Dendritic Spines Dependovirus Development Epilepsy Frequencies Genes Glutamate Receptor Glutamates Goals Guanidines Guanine Nucleotide Exchange Factors Hippocampus (Brain)Label Laboratories Learning Length Measures Mediating Mental Retardation Messenger RNA Molecular Morphogenesis Morphology Mus NGFR gene Neurodegenerative Disorders Neurons Nucleotides Pathology Phase Phenotype Physiological Play Population Process Proteins Rattus Receptor Signaling Regulation Research Research Project Grants Research Proposals Resistance Rodent Role Shapes Signal Pathway Signal Transduction Stimulus Structure Synapses Synaptic plasticity System Testing Transcript Translations Untranslated Regions Vertebral column Western Blotting density immunocytochemistry in vitro Assay in vivo insight knock-down nervous system disorder neuronal cell body overexpression patch clamp postsynaptic receptor research study response small hairpin RNA synaptic function synaptogenesis

项目摘要

Dendritic spines are the primary postsynaptic targets for excitatory glutamatergic synapses in the brain. They are highly dynamic structures that undergo changes in size, shape, and number during development, as well as in response to physiological stimuli such as learning. Spine development involves three processes: formation, maturation, and pruning. Pruning appears to be an activity-dependent process and likely plays a significant role in the refinement of synaptic connections. While many proteins have been found to control spine formation and early spine maturation, very little is known about the molecular mechanisms that mediate the late phase of spine maturation and pruning. Therefore, understanding the mechanisms regulating spine morphogenesis will provide significant insight into processes fundamental for brain development and synaptic plasticity, as well as the pathology of some neurological diseases. Brain-derived neurotrophic factor (BDNF) plays a critical role in synaptic plasticity, particularly at glutamatergic synapses containing AMPA receptors. The gene for BDNF produces two pools of mRNA, with either a short or long 3' untranslated region (3'UTR). Previous studies from my lab show that short 3'UTR Bdnf mRNA is restricted to the soma, while long 3'UTR Bdnf mRNA is present in both the soma as well as dendrites for local translation. The overall objective of this research proposal is to understand the molecular mechanisms through which BDNF regulates dendritic spine morphogenesis and synapse regulation. Specifically, the proposed experiments plan to dissociate the roles of somatically and dendritically synthesized BDNF in the regulation of glutamatergic synapses (Aim1). I propose to use an in vitro assay I have recently developed for the study of spine morphogenesis, which mimics the in vivo course of spine development in the rodent hippocampus. In this assay, actin-GFP-labeled spines of cultured rat hippocampal neurons form during the first 2 weeks, mature during weeks 3 and 4, and are pruned during week 4. Using immunocytochemistry, NBQX and whole-cell patch-clamp recordings, I will test my hypothesis that somatically and dendritically synthesized BDNF exert opposing effects on AMPA-type glutamate receptor composition, function and signaling. In addition to this set of experiments, I plan to elucidate the signaling pathway by which dendritically synthesized BDNF regulates spine pruning (Aim 2). I hypothesize that activity induces dendritic translation and secretion of proBDNF, which interacts with p75[NTR] to mediate spine pruning through RhoA in cultured hippocampal neurons. To test my hypothesis, I will employ adeno-associated viruses, shRNA constructs, p75[NTR] KO mice, whole-cell patch-clamp recordings, GST-pull down assay, Western blot and a cleavage-resistant proBDNF construct I have recently generated. Taken together, these experiments will demonstrate distinct roles of somatically and dendritically synthesized BDNF in spine morphogenesis and may identify a signaling cascade through which dendritically synthesized BDNF regulates spine pruning in cultured hippocampal neurons.

树突棘是大脑中兴奋性谷氨酸突触的主要突触后目标。它们是高度动态的结构，在发育过程中以及对学习等生理刺激的反应中，它们的大小、形状和数量都会发生变化。脊柱发育涉及三个过程：形成、成熟和修剪。修剪似乎是一个依赖于活动的过程，并且可能在突触连接的细化中发挥着重要作用。虽然已发现许多蛋白质可以控制脊柱形成和早期脊柱成熟，但人们对介导脊柱成熟和修剪后期的分子机制知之甚少。因此，了解调节脊柱形态发生的机制将为了解大脑发育和突触可塑性的基本过程以及一些神经系统疾病的病理学提供重要的见解。脑源性神经营养因子 (BDNF) 在突触可塑性中发挥着关键作用，特别是在含有 AMPA 受体的谷氨酸能突触中。 BDNF 基因产生两个 mRNA 库，分别具有短或长的 3' 非翻译区 (3'UTR)。我实验室之前的研究表明，短 3'UTR Bdnf mRNA 仅限于体细胞，而长 3'UTR Bdnf mRNA 则存在于体细胞和树突中，用于局部翻译。本研究计划的总体目标是了解 BDNF 调节树突棘形态发生和突触调节的分子机制。具体来说，所提出的实验计划分离体细胞和树突状合成的 BDNF 在谷氨酸能突触调节中的作用 (Aim1)。我建议使用我最近开发的一种用于研究脊柱形态发生的体外测定法，该测定法模拟啮齿动物海马体中脊柱发育的体内过程。在此测定中，培养的大鼠海马神经元的肌动蛋白-GFP 标记的棘在前 2 周内形成，在第 3 周和第 4 周成熟，并在第 4 周修剪。使用免疫细胞化学、NBQX 和全细胞膜片钳记录，我将检验我的假设，即体细胞和树突状合成的 BDNF 对 AMPA 型谷氨酸受体的组成、功能和信号传导发挥相反的作用。除了这组实验之外，我还计划阐明树突状合成的 BDNF 调节脊柱修剪的信号通路（目标 2）。我假设该活性诱导树突翻译和 proBDNF 分泌，proBDNF 与 p75[NTR] 相互作用，通过培养的海马神经元中的 RhoA 介导脊柱修剪。为了检验我的假设，我将使用腺相关病毒、shRNA 构建体、p75[NTR] KO 小鼠、全细胞膜片钳记录、GST 下拉测定、蛋白质印迹和我最近生成的抗裂解 proBDNF 构建体。总而言之，这些实验将证明体细胞和树突状合成的 BDNF 在脊柱形态发生中的不同作用，并可能确定树突状合成的 BDNF 通过该信号级联调节培养的海马神经元中的脊柱修剪。