The Role of Local BDNF Synthesis in Spine Morphogenesis

树突棘是脑内兴奋性谷氨酸突触的主要突触后靶点。它们是高度动态的结构，在发育过程中会经历大小、形状和数量的变化，也会对学习等生理刺激做出反应。脊柱发育包括三个过程：形成、成熟和修剪。修剪似乎是一个活动依赖的过程，可能在突触连接的细化中起着重要作用。虽然已经发现许多蛋白质控制脊柱形成和脊柱早期成熟，但对介导脊柱成熟和修剪后期的分子机制知之甚少。因此，了解脊柱形态发生的调节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受体的谷氨酸能突触中。BDNF基因产生两个mRNA库，具有短的或长的3‘非翻译区（3’ utr）。我实验室之前的研究表明，短的3'UTR Bdnf mRNA局限于体细胞，而长3'UTR Bdnf mRNA既存在于体细胞中，也存在于树突中进行局部翻译。本研究计划的总体目标是了解BDNF调控树突棘形态发生和突触调控的分子机制。具体来说，该实验计划分离体细胞和树突合成的BDNF在调节谷氨酸能突触（Aim1）中的作用。我建议使用我最近开发的一种体外实验来研究脊柱形态发生，它模拟了啮齿类动物海马体内脊柱发育的过程。在本实验中，肌动蛋白gfp标记的培养大鼠海马神经元棘在前2周形成，在第3周和第4周成熟，并在第4周修剪。利用免疫细胞化学、NBQX和全细胞膜片钳记录，我将验证我的假设，即体细胞和树突合成的BDNF对ampa型谷氨酸受体的组成、功能和信号传导产生相反的影响。除了这组实验外，我计划阐明树突合成BDNF调节脊柱修剪的信号通路（Aim 2）。我假设活动诱导树突翻译和proBDNF的分泌，proBDNF与p75[NTR]相互作用，通过RhoA介导培养海马神经元的脊柱修剪。为了验证我的假设，我将使用腺相关病毒、shRNA构建物、p75[NTR] KO小鼠、全细胞膜片钳记录、gst下拉试验、Western blot和我最近生成的抗裂解proBDNF构建物。综上所述，这些实验将证明体合成和树突合成BDNF在脊柱形态发生中的不同作用，并可能确定树突合成BDNF调节培养海马神经元脊柱修剪的信号级联。

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.