The Role of Local BDNF Synthesis in Spine Morphogenesis

树突棘是大脑中兴奋性突触的主要突触后靶点。它们是高度动态的结构，在发育过程中以及对生理刺激（如学习）的反应中经历大小，形状和数量的变化。脊椎的发育包括三个过程：形成、成熟和修剪。修剪似乎是一个活动依赖性的过程，并可能在突触连接的细化中发挥重要作用。虽然已经发现许多蛋白质控制刺的形成和早期刺成熟，但很少有人知道介导刺成熟和修剪的后期阶段的分子机制。因此，了解调节脊柱形态发生的机制将为大脑发育和突触可塑性的基础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）在突触可塑性中起关键作用，特别是在含有AMPA受体的突触能突触中。BDNF基因产生两个mRNA库，具有短或长的3'非翻译区（3' UTR）。本实验室以前的研究表明，短的3 'UTR Bdnf mRNA仅限于索马，而长的3' UTR Bdnf mRNA既存在于索马中，也存在于树突中，用于局部翻译。这项研究的总体目标是了解BDNF调节树突棘形态发生和突触调节的分子机制。具体来说，拟议的实验计划解离的作用，体细胞和树突合成的BDNF在调节的神经元突触（Aim 1）。我建议使用一种体外试验，我最近开发的研究脊柱形态发生，它模仿在啮齿动物海马体的脊柱发育的体内过程。在该试验中，肌动蛋白-GFP标记的培养大鼠海马神经元的棘在前2周形成，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，我将测试我的假设，体细胞和树突合成的BDNF对AMPA型谷氨酸受体的组成，功能和信号产生相反的影响。除了这一组实验，我计划阐明的信号通路，其中树突状合成的BDNF调节脊柱修剪（目标2）。我推测，活动诱导树突状翻译和分泌的proBDNF，这与p75[NTR]介导的脊修剪通过RhoA在培养的海马神经元相互作用。为了验证我的假设，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-下拉分析，Western印迹和我最近产生的抗切割proBDNF构建体。两者合计，这些实验将证明不同的作用，体细胞和树突合成的BDNF在脊柱形态发生，并可能确定一个信号级联通过树突合成的BDNF调节培养的海马神经元脊柱修剪。

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.