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缺陷有关。我们的目标是为理解表达Rett综合征相关MECP2突变的神经元中树突棘的形成和维持以及突触后Ca2+稳态做出贡献。Rett综合征（RTT）是一种X连锁的发育障碍，是女性智力迟钝的主要原因。在90%的RTT病例中发现了转录抑制因子MECP2的突变。MeCP2的靶基因之一是脑源性神经营养因子bdnf。考虑到BDNF最近被认为是出生后大脑活动依赖性突触发育和可塑性的有效调节剂，包括基本的神经元特性，如树突棘密度和形状以及神经元Ca2+信号，我们假设BDNF信号的失调可能是RTT中观察到的树突病理的基础。需要验证的具体假设是双重的：1)rtt相关的MECP2突变导致树突棘丢失，通过减少BDNF信号传导导致海马锥体神经元树突Ca2+信号受损；2) BDNF处理可以恢复MECP2突变神经元的树突结构受损。MECP2突变表达的后果将在保持在器官型切片培养和通过颗粒介导的基因转移转染的神经元中进行评估。与产生转基因或敲除小鼠相比，生物学基因转移方法提供了一种更灵活的方法来引入不同形式的MECP2突变体，此外还允许共同转染其他cdna或敲低感兴趣的siRNA构建体。因此，它代表了一种新的RTT细胞模型。转染的神经元将通过激光扫描共聚焦和延时多光子显微镜，以及同时Ca2+成像和全细胞胞内记录进行研究。这种最先进的方法组合从未用于研究MECP2功能，或应用于RTT的动物模型。我们希望提出的研究能够为RTT细胞模型中海马神经元MECP2突变表达的后果提供新的见解。

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.