Semaphorin-Dependent GABAergic Synapse Formation: A Novel Approach to Increasing Inhibition in the Intact Brain

我们建议利用Sema 4D信号的突触发生潜力来增加 GABA能突触数量，从而增强神经回路中的抑制作用并抑制 癫痫发作这种方法可能有利于防止癫痫的建立，停止癫痫发作。 其进展，或抑制癫痫发作期间的过度兴奋。 本实验室先前发现跨膜蛋白的胞外区 Semaphorin 4D（Sema 4D）在非常快的时间尺度上驱动抑制性突触形成 (i.e.在海马神经元和培养的脑片中， 海马体。我们还证明了海马内输注纯化的Sema 4D， 细胞外结构域进入成年海马迅速促进新的 GABA能突触。此外，我们报告说，Sema 4D治疗可以防止癫痫发作， 通过电刺激齿状回或静脉输注 戊四氮 鉴于这些研究的成功，我们采取了一个新的实验方向， 确定Sema 4D治疗是否对人类癫痫有治疗潜力。的首要目标 该提案旨在研究Sema 4D治疗作为啮齿动物急性抗癫痫治疗的作用 癫痫持续状态（SE）模型。SE是需要立即干预的医疗紧急情况 在人类中;大约30%的SE患者对目前的治疗难治 包括苯二氮卓类药物。本提案提供的初步数据显示， Sema 4D治疗恢复了地西泮在难治性SE啮齿动物模型中的疗效。这 结果与我们的假设一致，即Sema 4D治疗急性增加了 海马中的GABA能突触，其维持或重建苯二氮卓类 大脑的敏感性。 我们建议的第二个目的是确定长期接触 Sema 4D（通过病毒递送至CNS）抑制突触形成并最终抑制癫痫发作 在啮齿动物慢性癫痫模型中的频率和严重程度。作为实现这一目标的第一步，我们 将探索Sema 4D给药的替代方法（例如， 脑内注射编码Sema 4D的病毒）。在第三个目标中，我们将开始 通过询问Sema 4D是否可以解释我们在啮齿类动物中使用Sema 4D的研究结果， 促进人类神经元培养物中抑制性突触形成和网络活动。

We propose to harness the synaptogenic potential of Sema4D signaling to increase GABAergic synapse number, thus enhancing inhibition in neural circuits and suppressing seizures. This approach could be beneficial to preventing the establishment of epilepsy, halting its progression, or suppressing hyperexcitability during a seizure event. Previously our lab discovered that the extracellular domain of transmembrane protein Semaphorin 4D (Sema4D) drives inhibitory synapse formation on a remarkably fast time scale (i.e. minutes) in hippocampal neurons and slice cultured from the pre-natal and neonatal hippocampus. We also demonstrated that intra-hippocampal infusion of purified, Sema4D extracellular domain into the adult hippocampus rapidly promotes the formation of new GABAergic synapses. Further, we reported that Sema4D treatment protects against seizures induced by electrical stimulation of the dentate gyrus or by intravenous infusion of the proconvulsant drug pentylenetetrazol. Given the success of these studies, we undertook a new experimental direction to determine if Sema4D treatment has therapeutic potential for human epilepsies. The first aim of this proposal is to investigate Sema4D treatment as an acute, anti-seizure therapeutic in rodent models of status epilepticus (SE). SE is a medical emergency requiring immediate intervention in humans; approximately 30% of patients with SE are refractory to treatment with current medications including benzodiazepines. Preliminary data presented in this proposal shows that Sema4D treatment restored the efficacy of diazepam in a rodent model of refractory SE. This result is consistent with our hypothesis that Sema4D treatment acutely increases the number of GABAergic synapses in hippocampus, which maintains or re-establishes benzodiazepine sensitivity in the brain. The second aim of our proposal is to determine the effect of chronic exposure of Sema4D (via viral delivery to the CNS) on inhibitory synapse formation and ultimately, seizure frequency and severity in rodent models of chronic epilepsy. As a first step towards this goal we will explore the efficacy and time-course of alternative methods of administering Sema4D (e.g. intracerebral injection of virus encoding Sema4D) to mice. In the third aim, we will begin to address the translatability of our findings with Sema4D in rodents by asking if Sema4D promotes inhibitory synapse formation and network activity in cultures of human neurons.