Exploring the mechanisms of Semaphorin/Plexin-mediated synapse formation in the intact hippocampus

项目摘要： 在哺乳动物的中枢神经系统中，两种主要类型的突触--谷氨酸能和GABA能--发挥作用。 兴奋或抑制突触后细胞的相反作用。虽然关键的是，在细胞与细胞接触时， 突触形成正确的突触后特化（兴奋性或抑制性），分子通路 确定这种身份仍然是发育神经生物学中的一个基本谜团。这个过程是 推测由属于蛋白质家族的跨突触配体/受体配偶体（例如， 神经连接蛋白/神经毒素、肝配蛋白/Ephs、脑信号蛋白/丛状蛋白），其已被证明调节这两者 兴奋性和抑制性突触形成。 脑信号蛋白（Semas）和丛蛋白是广泛表达的和功能多样的跨膜或跨膜转运蛋白家族。 分泌的配体和它们的跨膜受体，首先由于它们在轴突导向中的作用而受到关注 在神经系统发育过程中。4类Sema和丛蛋白-B受体在哺乳动物中表达 海马（在兴奋性和抑制性神经元和神经胶质细胞中），并促进突触形成， 发展和成年。 我们以前的研究表明Sema4D是少数几种具有突触发生功能的分子之一， 抑制性突触; Sema4D的细胞外结构域诱导快速突触形成抑制性突触。 时间尺度（约30分钟）通过丛蛋白-B1受体。此外，Sema4A促进了 分别通过丛蛋白-B1或丛蛋白-B2受体在海马中的抑制性和兴奋性突触。的 通过应用Sema4A或Sema4D快速驱动突触形成的能力，以及观察到 这些Semas以独特的方式调节抑制性和兴奋性突触的形成，需要不同的丛蛋白-B 受体，是我们提出的识别不同信号传导构象的策略的核心， 指导丛蛋白-B受体下游突触特性（兴奋性或抑制性 订婚此外，我们先前表明，Sema4D蛋白的应用增加， 体内成年小鼠对癫痫发作的恢复力，这一发现支持了 这项工作 这项研究使用了转基因小鼠，海马切片培养，基因 敲除和表达丛蛋白-B受体的突变形式，以检查1）独特的 丛蛋白-B信号传导结构域，2）顺式门控丛蛋白-B1信号传导的新机制，以及3） 丛蛋白B辅助受体，在4类信号素促进的兴奋性和抑制性突触形成中的作用。

Project Summary: In the mammalian central nervous system, two main types of synapses – glutamatergic and GABAergic – play opposing roles in exciting or inhibiting the postsynaptic cell. While it is critical that upon cell-cell contact, new synapses form the correct postsynaptic specialization (excitatory or inhibitory), the molecular pathways specifying this identity remain a fundamental mystery in developmental neurobiology. This process is presumably regulated by trans-synaptic ligand/receptor partners that belong to protein families (e.g. Neuroligins/Neurexins, Ephrins/Ephs, Semaphorins/Plexins) which have been demonstrated to regulate both excitatory and inhibitory synapse formation. Semaphorins (Semas) and Plexins are families of widely expressed and functionally versatile transmembrane or secreted ligands and their transmembrane receptors that first gained attention for their roles in axon guidance during nervous system development. Class 4 Semas and Plexin-B receptors are expressed in mammalian hippocampus (in excitatory and inhibitory neurons and in glia) and promote synapse formation both during development and in adulthood. Our previous studies revealed Sema4D to be one of few molecules having synaptogenic function restricted to inhibitory synapses; the extracellular domain of Sema4D induces inhibitory synapse formation on a rapid timescale (~30 mins) through the Plexin-B1 receptor. Additionally, Sema4A promotes formation of both inhibitory and excitatory synapses in hippocampus via the Plexin-B1 or Plexin-B2 receptors, respectively. The ability to rapidly drive synapse formation by application of Sema4A or Sema4D, as well as the observations that these Semas regulate inhibitory and excitatory synapse formation in unique ways requiring different Plexin-B receptors, are central to our proposed strategy to identify the distinct signaling conformations that instruct synapse identity (excitatory or inhibitory) downstream of Plexin-B receptor engagement. Further, we previously showed that Sema4D protein application increases resilience to seizure in adult mice in vivo, a finding that supports the potential clinical impact of this work. The proposed research uses a combination of transgenic mice, organotypic hippocampal slice culture, gene knockdown, and expression of mutant forms of Plexin-B receptors to examine the divergent roles of 1) unique Plexin-B signaling domains, 2) a novel mechanism gating Plexin-B1 signaling in cis, and 3) contributions from Plexin-B coreceptors, in excitatory and inhibitory synapse formation promoted by class 4 semaphorins.