Semaphorin-dependent GABAergic synapse formation: A novel approach to increasing inhibition in the intact brain

我们建议利用Sema4D信号的突触生成潜力来增加 GABA能突触数目，从而增强神经回路的抑制和抑制 癫痫发作。这种方法可能有利于防止癫痫的建立，停止 它的进展，或在癫痫发作期间抑制过度兴奋。 此前，我们的实验室发现跨膜蛋白的胞外区 信号素4D(Sema4D)以非常快的时间尺度驱动抑制性突触的形成 (即分钟)在海马神经元和切片培养的出生前和新生儿 海马体。我们还证明了纯化的Sema4D在海马区内注入 胞外区进入成年海马区迅速促进新的 GABA能突触。此外，我们还报道了Sema4D治疗可以预防癫痫发作。 由电刺激齿状回或静脉输注 抗惊厥药物戊四唑。 鉴于这些研究的成功，我们进行了一个新的试验方向 确定Sema4D治疗是否具有治疗人类癫痫的潜力。的第一个目标 这项建议是为了研究Sema4D作为一种急性、抗癫痫治疗方法在啮齿类动物中的应用。 癫痫持续状态(SE)模型。SE是一种需要立即干预的医疗紧急情况 在人类中；大约30%的SE患者对电流治疗无效 包括苯二氮卓类在内的药物。这份提案中提出的初步数据表明， 在难治性SE的啮齿动物模型中，Sema4D治疗恢复了安定的疗效。这 结果与我们的假设一致，即Sema4D治疗显著增加了 海马区GABA能突触维持或重建苯二氮 大脑的敏感度。 我们建议的第二个目标是确定长期接触铅的影响 Sema4D(通过病毒传递到中枢神经系统)抑制突触形成并最终导致癫痫发作 慢性癫痫啮齿动物模型的频率和严重程度。作为迈向这一目标的第一步，我们 将探索使用Sema4D的替代方法的有效性和时间进程(例如 小鼠脑内注射编码Sema4D的病毒)。在第三个目标中，我们将开始 通过询问Sema4D在啮齿类动物中的可译性来解决我们的研究结果在啮齿类动物中的可译性 促进人类神经元培养中抑制性突触的形成和网络活动。

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.