The Role of Local BDNF Synthesis in Spine Morphogenesis

树突棘是脑内兴奋性谷氨酸能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。脊椎发育包括三个过程：形成、成熟和修剪。修剪似乎是一个依赖于活动的过程，可能在突触连接的精炼中发挥着重要作用。虽然已发现许多蛋白质控制脊柱形成和早期脊柱成熟，但对调节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控机制将对脑发育和突触可塑性的基本过程以及一些神经疾病的病理提供重要的见解。脑源性神经营养因子在突触可塑性中发挥重要作用，尤其是在含有AMPA受体的谷氨酸能突触中。脑源性神经营养因子基因产生两个信使核糖核酸池，有一个短的或长的3‘非翻译区(3’非编码区)。我实验室以前的研究表明，短的3‘UTRBDNF mRNA仅限于胞体，而长的3’UTRBDNF mRNA既存在于胞体中，也存在于树突中，用于局部翻译。本研究的总体目标是了解BDNF调控树突棘形态发生和突触调控的分子机制。具体地说，拟议的实验计划分离躯体和树突合成的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的形态发生，它模拟了啮齿动物海马体内脊柱发育的活体过程。在这项实验中，培养的大鼠海马神经元的肌动蛋白-GFP标记的脊椎在前2周形成，在3周和4周成熟，在4周被修剪。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，即从躯体和树突合成的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，我计划阐明树突合成的BDNF调节脊椎修剪的信号通路(目标2)。我推测，在培养的海马神经元中，该活动诱导树突状细胞翻译和分泌proBDNF，并与p75[NTR]相互作用，通过RhoA介导脊椎修剪。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下拉实验、Western印迹和我最近产生的抗切割的proBDNF构建体。综上所述，这些实验将证明躯体和树突合成的BDNF在脊椎形态发生中的不同作用，并可能识别树突合成的BDNF调节培养的海马神经元中的脊椎修剪的信号级联。

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.