Molecular Analysis of BDNF-TrkB Regulation of Synapse Formation and Maintenance

描述（由申请人提供）：我们将表征TrkB调节小脑中抑制性突触形成和维持的机制，期望所获得的见解将证明对于理解TrkB在大脑许多区域中的作用是重要的。扩展我们先前的证明，TrkB控制抑制性突触形成整个小脑，并具有重要的前和突触后细胞自主的作用，我们将使用高分辨率随机光学重建显微镜（STORM）成像，以表征抑制性突触相关的细胞表面和突触支架蛋白的定位，并确定TrkB激活和抑制对它们在突触上的存在的影响。扩展我们最近的观察，成人TrkB活性需要维持抑制性突触和早期抑制后的TrkB信号转导的重新激活导致在许多突触蛋白的突触处重新出现，我们将研究成人TrkB抑制和重新激活对这些突触的分子组成的影响。使用细胞培养，我们将更详细地研究与TrkB激活和失活后突触相关的蛋白质的出现和消失。我们将确定TrkB的功能是否部分通过控制蛋白质合成或周转。我们还将研究TrkB活性对桥蛋白稳定性、插入和去除细胞培养物中突触位点的动力学的影响。最后，我们更严格地检查我们的模型，TrkB通过控制形成突触支架的蛋白质的分子组装而起作用。我们将确定TrkB的激活和失活在体内和体外的分布f gephyrin和其他突触后支架蛋白在洗涤剂可溶性和耐药性的馏分，这些蛋白质与结合伙伴的相互作用和磷酸化和其他翻译后修饰的影响。 公共卫生相关性：本申请将描述一种神经营养因子通过一种受体酪氨酸激酶（TrkB）调节小脑中突触形成和维持的机制，该神经营养因子参与神经元存活，称为脑源性神经营养因子（BDNF）信号传导，小脑是一个控制运动表现和协调（如行走和跑步）的大脑区域。虽然拟议中的研究集中在小脑，但机制的见解可能有助于理解这种蛋白质及其受体在大脑许多区域的功能，包括那些涉及记忆，情绪和成瘾行为的区域。人类遗传学研究表明，BDNF与精神分裂症和其他精神疾病有关。TrkB中罕见的人类突变导致严重的精神发育迟滞、肥胖和心脏疾病。该提案的主要目的是了解这些基因控制大脑内突触形成和神经元回路功能的机制，期望这些发现将确定药物的新靶点，以缓解这些神经元障碍，并增加我们对大脑如何在健康和疾病中工作的知识。

DESCRIPTION (provided by applicant): We will characterize mechanisms through which TrkB regulates inhibitory synapse formation and maintenance in the cerebellum with the expectation that the insights obtained will prove important for understanding the role of TrkB in many regions of the brain. Extending our prior demonstrations that TrkB controls inhibitory synapse formation throughout the cerebellum and has important pre- and postsynaptic cell- autonomous roles, we will use high resolution stochastic optical reconstruction microscopy (STORM) imaging to characterize the localizations of inhibitory synapse-associated cell surface and synaptic scaffold proteins and determine the effects of TrkB activation and inhibition on their presence at the synapse. Extending our recent observation that adult TrkB activity is required to maintain inhibitory synapses and that reactivation of TrkB signaling after earlier inhibition results in reappearance at the synapse of many synaptic proteins, we will examine the effects of adult TrkB inhibition and reactivation on the molecular composition of these synapses. Using cell culture we will examine in more detail the appearance and disappearance of proteins associated with the synapse following TrkB activation and inactivation. We shall determine whether TrkB functions in part through control of protein synthesis or turnover. We shall also examine the effects of TrkB activity on the kinetics of gephyrin stability, insertion and removal a synaptic sites in cell culture. Finally, we more critically examine our model that TrkB acts in par through control molecular assembly of the proteins that form the synaptic scaffold. We will determine the effects of TrkB activation and inactivation in vivo and in vitro on the distribution f gephyrin and other postsynaptic scaffold proteins in detergent soluble and resistant fractions, the interactions of these proteins with binding partners and on phosphorylation and other post-translational modifications. PUBLIC HEALTH RELEVANCE: This application will characterize mechanisms through which a neurotrophic factor implicated in neuronal survival named brain-derived neurotrophic factor (BDNF) signaling through a receptor tyrosine kinase named (TrkB) regulate synapse formation and maintenance in the cerebellum, a brain region that controls motor performance and coordination, such as walking and running. While the proposed studies focus on the cerebellum, the mechanistic insights are likely to be useful in understanding the function of this protein and its receptor in many regions of the brain, including those involved in memory, mood, and addictive behaviors. Human genetic studies have implicated BDNF in schizophrenia and other mental disorders. Rare human mutations in TrkB result in severe mental retardation, obesity, and cardiac disorders. The major aim of this proposal is to understand the mechanisms through which these genes control synapse formation and neuronal circuit function within the brain with the expectation that discoveries will identify novel targets for drugs to alleviate these devastatig disorders as well as increasing our knowledge of how the brain works in health and disease.