Semaphorin4D and PlexinB1 mediate GABAergic synapse development in mammalian CNS

描述(申请人提供)：Semaphorin4D和PlexinB1在哺乳动物中枢神经系统中介导GABA能突触的发育。正确的神经元交流依赖于神经元之间突触连接的精确组装和发展。许多神经疾病是由突触连接的紊乱引起的，例如智力低下、自闭症和雷特综合征(Fernandez和Garner，2007；Rubenstein和Marzenich，2003；Tabuchi等，2007；Zoghbi，2003)。此外，许多突触蛋白，包括Semaphorin家族成员，都与神经发育障碍有关(Weiss等人，2009年)，缺乏信号素会导致啮齿动物中不正确的突触连接(Morita等人，2006年；O‘Connor等人，2009年；Paradis等人，2007年；Sahay等人，2005年)。这个 跨膜蛋白Sema4D是正常的GABA能突触形成所必需的，因为RNAi下调突触后神经元的表达导致培养神经元中GABA能突触密度的下降(Paradis等人，2007年)。我们的初步结果表明，在培养的海马神经元中加入可溶性的、胞外区的Sema4D足以驱动GABA能突触的形成。重要的是，这种增加依赖于Sema4D的受体PlexinB1的表达。此外，我们的电压钳实验表明，PlexinB1是正常的GABA能突触传递所必需的。因此，我们的工作将PlexinB1定义为一种新的受体，介导哺乳动物中枢神经系统中Sema4D反应的GABA能突触的形成。这项建议的目的是阐明Sema4D及其受体PlexinB1在GABA能突触发育中的作用。在这里提出的实验中，我们假设Sema4D通过其受体PlexinB1启动GABA能突触蛋白的组装，如GABAA受体和GePhyrin。这将在培养的海马神经元中使用各种成像技术进行测试，包括共聚焦和时间推移成像，以测量在可溶性Sema4D处理后神经元中GABA能突触蛋白的迁移和积累。此外，初步数据表明，PlexinB1对GABA能突触传递很重要。电生理学将被用来确定PlexinB1功能的作用部位，以调节GABA能突触传递。这里提出的实验不仅将极大地扩展我们对GABA能突触发育中的新型受体-配体对的理解，还将使我们了解GABA能突触发生的一些基本机制。好了！

DESCRIPTION (provided by applicant): Semaphorin4D and PlexinB1 mediate GABAergic synapse development in mammalian CNS. Proper neuronal communication depends on the precise assembly and development of synaptic connections between neurons. Many neurological disorders arise from perturbations in synaptic connectivity, such as mental retardation, autism, and Rett Syndrome (Fernandez and Garner, 2007; Rubenstein and Marzenich, 2003; Tabuchi et al., 2007; Zoghbi, 2003). In addition, many synaptic proteins, including Semaphorin family members, have been linked to neurodevelopmental disorders (Weiss et al, 2009) and the lack of Semaphorins results in improper synaptic connectivity in rodents (Morita et al., 2006; O'Connor et al., 2009; Paradis et al., 2007; Sahay et al., 2005). The transmembrane protein Sema4D is necessary for proper GABAergic synapse formation, as knockdown of expression in the postsynaptic neuron by RNAi leads to a decrease in GABAergic synaptic density in cultured neurons (Paradis et al 2007). Our preliminary results demonstrate that addition of the soluble, extracellular domain of Sema4D to cultured hippocampal neurons is sufficient to drive GABAergic synapse formation. Importantly, this increase is dependent on the expression of Sema4D's receptor, PlexinB1. In addition, our voltage-clamp experiments indicate that PlexinB1 is necessary for proper GABAergic synaptic transmission in the hippocampus. Thus, our work defines PlexinB1 as a novel receptor mediating GABAergic synapse formation in response to Sema4D in the mammalian CNS. The goal of this proposal is to elucidate the role of Sema4D and it's receptor, PlexinB1 in GABAergic synapse development. In experiments proposed here, we hypothesize that Sema4D acts to initiate assembly of GABAergic synaptic proteins such as GABAA receptors and gephyrin through its receptor PlexinB1. This will be tested using a variety of imaging techniques in cultured hippocampal neurons, including confocal and time- lapse imaging, to measure the mobility and accumulation of GABAergic synaptic proteins in neurons after treatment with soluble Sema4D. In addition, preliminary data suggests that PlexinB1 is important for GABAergic synaptic transmission. Electrophysiology will be used to determine the site of action of PlexinB1 function to regulate GABAergic synaptic transmission. The experiments proposed here will not only greatly expand our understanding of a novel receptor-ligand pair in GABAergic synapse development; it will inform us as to some of the basic mechanisms underlying GABAergic synaptogenesis. !