Functional analysis of variant BDNF (Val66Met)

描述(申请人提供)：脑源性神经营养因子(BDNF)在脊椎动物神经系统的发育和功能中起着关键作用。最近，BDNF基因的一个单核苷酸多态(Val66Met)导致了66位的原结构域从Valine(Val)替换为Met(Met)，这导致了人类的海马区依赖性记忆障碍和神经精神障碍的易感性。这种BDNF基因多态性代表了与临床病理相关的神经营养因子的第一个变化。对BDNFMet功能变异的分子机制知之甚少。当BDNFMet在海马神经元过表达时，BDNFMet减少了活性依赖的分泌，这表明BDNF前域中存在一个特定的信号，这是有效地将BDNF转运到受调节的分泌途径所必需的。初步研究表明，BDNFMet异常地参与了高度专业化的生化机制，该机制调节BDNF向分泌途径的运输，而这些途径是BDNF生物学反应的关键决定因素。拟议的研究旨在确定调节BDNFMet异常运输的特定蛋白质，并检查体内对海马体结构和功能的影响。在这项提议中的实验将主要利用一种新型的转基因敲入小鼠来表达BDNFNF9e变异体(BDNF9e)的表位标记版本，以便能够在内源性表达条件下评估BDNFMet转运事件，并分析体内对海马体结构和功能的影响。这些研究的具体目的是1)确定内源性表达BDNFMet的神经元的转运缺陷和功能后果，2)识别参与BDNFMet异常转运的蛋白质，以及3)确定BDNFMet对海马区功能的体内后果。这些研究将有助于从分子水平上了解BDNFMet在神经元中的异常转运机制，并直接探讨这种变异的BDNF与海马区功能的生理相关性。

DESCRIPTION (provided by applicant): Brain derived neurotrophic factor (BDNF) plays critical roles in vertebrate nervous system development and function. Recently, a single nucleotide polymorphism (Val66Met) in the BDNF gene leading to a prodomain substitution at position 66 from a valine (Val) to methionme (Met) has been shown to lead in humans to hippocampal dependent memory impairments and susceptibility to neuropsychiatric disorders. This BDNF polymorphism represents the first alteration in a neurotrophin that has been linked to clinical pathology. Less is known about the molecular mechanisms underlying altered variant BDNF (BDNFMet) functioning. When overexpressed in hippocampal neurons, BDNFMet has reduced activity dependent secretion, suggesting the presence of a specific signal in the BDNF prodomain that is required for efficient BDNF trafficking to the regulated secretory pathway. Preliminary studies suggest the hypothesis that BDNFMet aberrantly engages the highly specialized biochemical mechanisms that regulate BDNF trafficking to secretory pathways, which are critical determinants of BDNF's biological responses. The proposed studies are designed to identify specific proteins that regulate aberrant BDNFMet trafficking, and to examine the in vivo consequences on hippocampal structure and function. Experiments in this proposal will primarily utilize a novel transgenic knock-in mouse expressing an epitope tagged version of variant BDNF (BDNF 9 e) to enable assessment of BDNFMet trafficking events under endogenously expressed conditions and analysis of in vivo consequences on hippocampal structure and function. The Specific Aims of the proposed studies are to 1) define the trafficking defect and functional consequences in neurons endogenously expressing BDNFMet, 2) identify proteins that are involved in aberrant BDNFMet trafficking, and 3) determine the in vivo consequences of BDNFMet on hippocampal function. These studies will contribute to a fundamental molecular understanding of the mechanisms that underlie aberrant BDNFMet trafficking in neurons, and directly address the physiological relevance of this variant BDNF on hippocampal function.