Regulating BDNF Action in Postnatal Development.

描述(申请人提供)：神经营养因子诱导神经元的结构和功能变化，以调节突触的疗效；我们的长期目标是确定调节BDNF靶向和在突触释放的分子机制，以调节神经元结构和神经传递。BDNF最初被合成为前体形式(ProBDNF)，它被分类到受调节的分泌途径，并以依赖于活性的方式释放。当proBDNF在突触处释放时，它可以与p75受体结合来诱导LTD，并有可能降低脊椎密度和树突的复杂性。然而，如果在分泌囊泡或突触裂隙中将proBDNF转化为成熟的BDNF，TrkB就会被选择性地激活，以增强突触传递，促进轴突分支和树突生长。TrkB受体存在于Schaffer侧支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的TrkB受体，促进神经传递。因此，调节ProBDNF向成熟BDNF转化的分子机制，以及调节细胞内转运到树突或轴突的分子机制，对于调节结构和功能神经元的可塑性至关重要。我们已经开发了新的遗传工具来促进内源性BDNF的检测，并发现了指导BDNF细胞内转运的新的分选受体。具体地说，我们已经产生了表达HA标记的BDNF的敲入小鼠，以显著增强对内源性BDNF的检测。我们还鉴定了包括山梨素在内的细胞内伴侣，以及其他与proBDNF结合的山梨素家族成员。利用这些工具，提出了三个目标来剖析BDNF的运输、切割和去极化依赖的释放：(1)使用BDNF-HA标记的小鼠的神经元，确定ProBDNF向成熟BDNF的转化是否发生在合成和分选到分泌囊泡的过程中，或者转化是否发生在囊泡释放之后。我们预测，不同神经元亚型和出生后早期时间点的BDNF转换位置可能不同，此时突触连接正在完善，突触发生强劲。(2)我们将研究Sortilin家族成员在神经元培养中如何改变proBDNF的胞内切割，以及如何调节前体和成熟BDNF的释放。(3)我们将确定山梨素家族成员的伴侣原BDNF的构成或调节的分泌途径，以及树突或轴突。我们推测，不同的sortilin家族成员将细胞内转运引导到不同的亚细胞室，并调节成熟BDNF的切割和释放。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度表达或shRNA敲除。这些研究将确定调控BDNF加工和运输的分子机制，以诱导发育中的后天中枢神经系统的结构和功能变化。 公共卫生相关性：神经营养因子BDNF在调节神经元存活、形态和活性依赖形式的突触可塑性方面发挥关键作用。有趣的是，BDNF最初是作为前体proBDNF合成的，它表现出截然不同的生物学作用，甚至与其成熟形式的生物学作用相反。在这里，我们将确定以下分子机制：(1)调节BDNF向突触的运输，(2)调节ProBDNF向成熟BDNF的转化，(3)确定这些过程在出生后早期发育中如何调节，这是突触发生的关键期。众所周知，即使是神经元分泌的BDNF水平的轻微变化也会对学习、记忆、焦虑状态和抑郁产生重大影响，这些研究将检验BDNF的运输和释放受细胞内伴侣的不同使用调控的假说。

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.