Regulating BDNF Action in Postnatal Development.

描述（由申请人提供）：神经营养素诱导神经元的结构和功能改变以调节突触效能；我们的长期目标是确定调节BDNF在突触的靶向和释放以调节神经元结构和神经传递的分子机制。BDNF最初作为前体形式（proBDNF）合成，被分类到一个受调节的分泌途径，并以活性依赖的方式释放。当proBDNF在突触释放时，它可以结合p75受体诱导LTD，并可能降低脊柱密度和树突复杂性。然而，如果proBDNF在分泌囊泡或突触间隙中转化为成熟的BDNF， TrkB被选择性激活，增强突触传递，促进轴突分支和树突生长。TrkB受体同时存在于Schaffer侧通路的突触前和突触后，成熟的BDNF可以激活突触前和突触后TrkB受体以促进神经传递。因此，调节proBDNF向成熟BDNF转化的分子机制，以及调节细胞内向树突或轴突运输的分子机制，对于调节神经元的结构和功能可塑性至关重要。我们已经开发了新的遗传工具来促进内源性BDNF的检测，并鉴定了指导BDNF细胞内运输的新的分选受体。具体来说，我们已经产生了表达HA标记BDNF的敲入小鼠，以显着提高内源性BDNF的检测。我们还发现了细胞内的伴侣，包括sortilin和其他与proBDNF结合的sortilin家族成员。利用这些工具，提出了三个目标来解剖BDNF运输、切割和去极化依赖释放：(1)使用BDNF- ha标记小鼠的神经元，确定proBDNF是否在合成和分选到分泌囊泡时转化为成熟的BDNF，或者是否在囊泡释放后转化。我们预测BDNF转换的位置可能在不同的神经元亚型和出生后早期时间点有所不同，此时突触连接正在完善，突触发生稳健。(2)我们将研究sortilin家族成员如何在神经元培养中改变proBDNF的细胞内切割和调节BDNF的成熟和成熟释放。(3)我们将确定sortilin家族成员，这些成员将陪伴proBDNF进入构成或调节的分泌途径，以及树突或轴突。我们假设不同的sortilin家族成员指导细胞内转运到不同的亚细胞区室，并调节BDNF成熟及其释放的裂解。这些研究将依赖于BDNF- HA标记的小鼠，以及不同伴侣蛋白的过表达或shRNA敲低。这些研究将确定调节BDNF加工和运输的分子机制，以诱导发育中的产后中枢神经系统的结构和功能变化。

DESCRIPTION (provided by applicant): Neurotrophins induce structural and functional changes in neurons to modulate synaptic efficacy; our long term goal is to identify molecular mechanisms that regulate BDNF targeting and release at synapses to modulate neuronal structure and neurotransmission. BDNF is initially synthesized as a precursor form (proBDNF) that is sorted to a regulated secretory pathway, and released in an activity-dependent manner. When proBDNF is released at the synapse, it can bind to p75 receptors to induce LTD, and potentially reduce spine density and dendritic complexity. However, if proBDNF is converted to mature BDNF in the secretory vesicle or synaptic cleft, TrkB is selectively activated to enhance synaptic transmission and promote axonal branching and dendritic growth. TrkB receptors are present both pre- and post-synaptically in the Schaffer collateral pathway, and mature BDNF can activate both pre- and post-synaptic TrkB receptors to facilitate neurotransmission. Thus, the molecular mechanisms that regulate conversion of proBDNF to mature BDNF, and that regulate intracellular trafficking to dendrites or axons are critical to modulate structural and functional neuronal plasticity. We have developed new genetic tools to facilitate detection of endogenous BDNF, and identified new sorting receptors that direct BDNF intracellular trafficking. Specifically, we have generated knock-in mice that express HA tagged BDNF to markedly enhance detection of endogenous BDNF. We have also identified intracellular chaperones, including sortilin, and other sortilin family members that bind to proBDNF. With these tools, three aims are proposed to dissect BDNF trafficking, cleavage, and depolarization dependent release: (1) Using neurons from the BDNF-HA tagged mouse, identify if conversion of proBDNF to mature BDNF occurs during synthesis and sorting to secretory vesicles, or whether conversion occurs following vesicle release. We predict that the location of BDNF conversion may differ among neuronal subtypes and across early postnatal time points when synaptic connections are being refined and synaptogenesis is robust. (2) We will investigate how sortilin family members alter intracellular cleavage of proBDNF and modulate pro- vs. mature BDNF release in neuronal cultures. (3) We will identify the sortilin family members that chaperone proBDNF to the constitutive or regulated secretory pathway, and to dendrites or axons. We posit that different sortilin family members direct intracellular trafficking to different subcellular compartments and regulate cleavage to mature BDNF and its release. These studies will rely on the BDNF- HA tagged mouse, and overexpression or shRNA knockdown of different chaperones. These studies will identify molecular mechanisms that regulate BDNF processing and trafficking, to induce structural and functional changes in the developing postnatal central nervous system. PUBLIC HEALTH RELEVANCE: The neurotrophin BDNF plays critical roles in regulating neuronal survival, morphology, and activity-dependent forms of synaptic plasticity. Interestingly, BDNF is initially synthesized as a precursor, proBDNF, which exhibits biological actions that are distinct, and even opposing those of its mature form. Here we will identify molecular mechanisms that (1) regulate the trafficking of BDNF to synapses, (2) regulate the conversion of proBDNF to mature BDNF and (3) determine how these processes are regulated in early postnatal development, a critical period of robust synaptogenesis. It is well established that even modest changes in the level of BDNF secreted by neurons has significant effects on learning, memory, anxiety states and depression, and these studies will test the hypothesis that BDNF trafficking and release is regulated by differential use of intracellular chaperones.