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）：神经营养素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 在突触靶向和释放以调节神经元结构和神经传递的分子机制。 BDNF 最初被合成为前体形式 (proBDNF)，被分类到受调节的分泌途径，并以活性依赖性方式释放。当 proBDNF 在突触处释放时，它可以与 p75 受体结合以诱导 LTD，并可能降低树突棘密度和树突复杂性。然而，如果proBDNF在分泌小泡或突触间隙转化为成熟的BDNF，TrkB就会被选择性激活，以增强突触传递并促进轴突分支和树突生长。 TrkB 受体同时存在于 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 受体以促进神经传递。因此，调节 proBDNF 转化为成熟 BDNF 以及调节细胞内向树突或轴突运输的分子机制对于调节结构和功能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，并确定了指导 BDNF 细胞内运输的新分选受体。具体来说，我们已经培育出表达 HA 标记的 BDNF 的敲入小鼠，以显着增强内源性 BDNF 的检测。我们还鉴定了细胞内伴侣，包括分拣蛋白和其他与 proBDNF 结合的分拣蛋白家族成员。利用这些工具，提出了三个目标来剖析 BDNF 运输、裂解和去极化依赖性释放：（1）使用来自 BDNF-HA 标记小鼠的神经元，确定在合成和分选到分泌囊泡的过程中是否发生 proBDNF 向成熟 BDNF 的转化，或者囊泡释放后是否发生转化。我们预测，BDNF 转换的位置可能因神经元亚型和出生后早期时间点而异，此时突触连接正在细化且突触发生稳健。 (2) 我们将研究分拣蛋白家族成员如何改变 proBDNF 的细胞内裂解并调节神经元培养物中 pro-vs. 成熟 BDNF 的释放。 (3) 我们将鉴定分拣蛋白家族成员，其将 proBDNF 陪伴至组成型或调节性分泌途径以及树突或轴突。我们假设不同的分拣蛋白家族成员将细胞内运输引导至不同的亚细胞区室并调节成熟 BDNF 的裂解及其释放。这些研究将依赖于 BDNF-HA 标记的小鼠，以及不同伴侣的过度表达或 shRNA 敲低。这些研究将确定调节 BDNF 加工和运输的分子机制，以诱导出生后中枢神经系统发育中的结构和功能变化。 公共健康相关性：神经营养蛋白 BDNF 在调节神经元存活、形态和突触可塑性的活动依赖性形式中发挥着关键作用。有趣的是，BDNF 最初是作为前体 proBDNF 合成的，它表现出独特的生物作用，甚至与其成熟形式相反。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，以及 (3) 确定如何在出生后早期发育（突触发生的关键时期）中调节这些过程。众所周知，即使神经元分泌的 BDNF 水平发生微小变化，也会对学习、记忆、焦虑状态和抑郁产生显着影响，这些研究将检验 BDNF 运输和释放受细胞内伴侣的差异使用调节的假设。