Role of BDNF in dendritic pathologies caused by Rett-associated MeCP2 mutations

描述（由申请人提供）：精神发育迟滞的神经病理学被认为与突触结构和功能的缺陷有关。我们的目标是作出贡献，了解形成和维护树突棘和突触后Ca 2+稳态的神经元表达Rett综合征相关突变的MECP 2。Rett综合征（RTT）是一种X染色体连锁的发育障碍，是女性智力低下的主要原因.在>90%的RTT病例中已经鉴定出转录抑制因子MECP 2的突变。MeCP 2的靶基因之一是bdnf，脑源性神经营养因子。考虑到BDNF最近已成为一个有效的调制器的活性依赖性突触发育和可塑性在出生后的大脑，包括基本的神经元特性，如树突棘密度和形式和神经元钙离子信号，我们假设，BDNF信号的失调可能是在RTT中观察到的树突病理。待检验的具体假设有两个方面：1）RTT相关的MECP 2突变导致树突棘缺失，通过减少BDNF信号传导导致海马锥体神经元中树突Ca 2+信号传导受损; 2）MECP 2突变神经元中受损的树突结构可通过BDNF处理恢复。将在保持在器官型切片培养物中并通过颗粒介导的基因转移转染的神经元中评价突变型MECP 2表达的后果。与转基因或敲除小鼠的产生相比，生物射弹基因转移方法提供了更灵活的方式来引入不同突变形式的MECP 2，此外还允许共转染其他感兴趣的cDNA或敲低siRNA构建体。因此，它代表了RTT的一种新的细胞模型。将通过激光扫描共聚焦和延时多光子显微镜，以及通过同时Ca 2+成像和全细胞细胞内记录来研究转染的神经元。这种最先进方法的组合从未用于研究MECP 2功能，或应用于RTT的动物模型。我们期望所提出的研究提供新的见解突变MECP 2表达在RTT的细胞模型中的海马神经元的后果。

DESCRIPTION (provided by applicant): The neuropathology of mental retardation is thought to be associated with deficits in synaptic structure and function. Our goal is to make contributions into understanding the formation and maintenance of dendritic spines and of postsynaptic Ca2+ homeostasis in neurons expressing Rett Syndrome-associated mutations in MECP2. Rett syndrome (RTT) is an X- linked developmental disorder and the leading cause of mental retardation in females. Mutations in the transcriptional represser MECP2 have been identified in >90% of RTT cases. One of the target genes of MeCP2 is bdnf, brain-derived neurotrophic factor. Considering that BDNF has recently emerged as a potent modulator of activity-dependent synaptic development and plasticity in the postnatal brain, including fundamental neuronal properties such as dendritic spine density and form and neuronal Ca2+ signaling, we hypothesize that a deregulation of BDNF signaling may underlie the dendritic pathologies observed in RTT. The specific hypothesis to be tested is twofold: 1) RTT-associated MECP2 mutations cause dendritic spine loss leading to Impaired dendritic Ca2+ signaling in hippocampal pyramidal neurons through reduced BDNF signaling; 2) impaired dendritic structure in MECP2 mutant neurons can be reverted by BDNF treatment. The consequences of mutant MECP2 expression will be evaluated in neurons maintained in organotypic slice cultures and transfected by particle-mediated gene-transfer. The biolistic gene-transfer approach provides a more flexible way to introduce different mutant forms of MECP2 compared to the generation of transgenic or knockout mice, in addition to allow the co-transfection of other cDNAs or knockdown siRNA constructs of interest. Thus, it represents a novel cellular model of RTT. Transfected neurons will be studied by laser-scanning confocal and time-lapse multiphoton microscopy, as well as by simultaneous Ca2+ imaging and whole-cell intracellular recordings. This combination of state-of-the-art approaches has never been used to investigate MECP2 function, or applied to animal models of RTT. We expect the proposed studies to provide novel insights into the consequences of mutant MECP2 expression in hippocampal neurons in a cellular model of RTT.