Regulating BDNF Action in Postnatal Development.

调节 BDNF 在产后发育中的作用。

• 批准号: 8001977
• 负责人: BARBARA L HEMPSTEAD
• 金额: $ 36.02万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-01-01 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8001977
• 关键词: Adolescent; Affect; Animals; Anxiety; Axon; Binding; Biological; Brain-Derived Neurotrophic Factor; Cleaved cell; Dendrites; Detection; Development; Electrons; Epitopes; Exhibits; Family member; Genetic; Goals; Growth; In Vitro; Knock-in Mouse; Learning; Light; Location; Mediating; Memory; Mental Depression; Microscopic; Molecular; Molecular Chaperones; Morphology; Mus; Neonatal; Neuraxis; Neuronal Differentiation; Neuronal Plasticity; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 2; Pathway interactions; Play; Process; Protein Isoforms; Protein Tyrosine Kinase; Reagent; Role; Secretory Vesicles; Signal Transduction; Site; Sorting - Cell Movement; Structure; Synapses; Synaptic Cleft; Synaptic Transmission; Synaptic plasticity; Testing; Time; Vertebral column; Vesicle; critical period; density; in vivo; member; nerve stem cell; neuronal survival; neurotransmission; neurotrophic factor; overexpression; postnatal; postsynaptic; presynaptic; promoter; public health relevance; receptor; research study; small hairpin RNA; sortilin; synaptogenesis; tool; trafficking; transcriptional coactivator p75

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.

描述（由申请人提供）：神经营养蛋白诱导神经元的结构和功能变化以调节突触功效；我们的长期目标是确定调节BDNF靶向和释放突触以调节神经元结构和神经传递的分子机制。 BDNF最初合成为前体形式（probDNF），该形式被分类为调节的分泌途径，并以活动依赖性方式释放。当ProbDNF在突触处释放时，它可以与P75受体结合以诱导LTD，并可能降低脊柱密度和树突复杂性。但是，如果在分泌囊泡或突触裂口中将ProbDNF转换为成熟的BDNF，则TRKB被选择性地激活以增强突触传播并促进轴突分支和树突状生长。 TRKB受体在Schaffer侧支途径中均存在前和突触后，并且成熟的BDNF可以激活前和突触后TRKB受体以促进神经传递。因此，调节probdnf转化为成熟BDNF的分子机制，并调节细胞内运输到树突或轴突对于调节结构和功能性神经元可塑性至关重要。我们开发了新的遗传工具来促进内源性BDNF的检测，并确定了直接bdnf细胞内运输的新分选受体。具体而言，我们已经生成了表达HA标记BDNF的敲门型小鼠，以显着增强内源性BDNF的检测。我们还鉴定了细胞内伴侣，包括托氨基蛋白和其他与probdnf结合的托硅蛋白家族成员。使用这些工具，提出了三个目的，以剖析BDNF运输，裂解和依赖于去极化的释放：（1）使用BDNF-HA标记的鼠标的神经元，确定在合成过程中是否会在合成过程中出现ProbDNF转换为成熟BDNF的转换，并在秘密的囊泡中进行分类，还是发生vesicle释放。我们预测，在精炼突触连接并且突触发生时，BDNF转化的位置在神经元亚型之间以及在早期产后时间点之间可能有所不同。 （2）我们将研究托硅蛋白家庭成员如何改变probdnf的细胞内切割，并调节神经元培养物中的BDNF释放。 （3）我们将确定与构型或受调节分泌途径伴侣的替代蛋白家族成员，以及树突或轴突。我们认为，不同的Tortilin家族成员将细胞内贩运引导到不同的亚细胞室，并调节成熟BDNF及其释放的裂解。这些研究将依靠BDNF-HA标记的小鼠，以及不同伴侣的过表达或shRNA敲低。这些研究将确定调节BDNF处理和运输的分子机制，以诱导发育中的产后中枢神经系统的结构和功能变化。 公共卫生相关性：神经营养蛋白BDNF在调节神经元存活，形态和活动依赖性突触可塑性中起着关键作用。有趣的是，BDNF最初被合成为前体ProbDNF，它表现出不同的生物学作用，甚至反对其成熟形式的生物学作用。在这里，我们将确定（1）调节BDNF向突触的运输的分子机制，（2）调节probdnf向成熟的BDNF的转化，（3）确定如何在早期产后发育中调节这些过程，这是一个关键的鲁棒突触发生。众所周知，即使是神经元分泌的BDNF水平的适度变化对学习，记忆，焦虑状态和抑郁症也有重大影响，这些研究将检验以下假设：BDNF运输和释放受细胞内伴侣的不同使用受到调节。