Microglial P2Y6 receptor calcium signaling as a core regulator of epileptogenesis

项目摘要/摘要 小胶质细胞是中枢神经系统的常驻免疫细胞，利用G蛋白信号完成不同的 任务。GI信令用于执行过程扩展，而Gs信令在 神经元活动不足(如麻醉)。然而，对小胶质细胞GQ钙信号转导知之甚少。 部分原因是小胶质细胞很少在静息状态下显示钙瞬变。另一方面，小胶质细胞 在癫痫持续状态后的一段时间内，大大提高了它们的钙信号 预示着未来癫痫的发展。小胶质细胞钙信号在癫痫发病中的作用 发育尚不清楚，但推测是小胶质细胞吞噬作用的一个组成部分。最小胶质细胞 癫痫发生中的钙信号转导归因于P2Y6受体信号转导--一种GQ-钙受体 由关键的嘌呤激活。对P2Y6最好的描述是它在吞噬作用中的作用，但这一点还没有被充分研究 在癫痫的发展中。(创新)目前的提案将利用一种新的鼠标系列，允许 活体动物小胶质细胞钙活性和突起运动的同时检测(使用两种方法) 光子显微镜)。(目的1)该品系将被用来测试P2Y6在小胶质细胞吞噬中的作用， 利用光遗传技术通过兴奋毒性选择性地杀死神经元。与此同时，组织研究将 在癫痫发生的关键时间点上进行，以确定是否丢失了P2Y6通路 阻止癫痫持续状态后死亡/垂死神经元的适当清除。死亡的长期存在 神经元是促炎的，并假设从长期来看会对神经元网络动力学产生负面影响。 (目标2)出于这些原因，我们将测试癫痫期间长期的P2Y6信号丢失的多个方面 发展。我们将确定P2Y6信号缺失的长期促炎效应。此外，我们 将使用迷你示波器技术和全天候视频脑电来确定P2Y6钙信号和 其假定的吞噬作用改变了网络调节失调或癫痫的风险。 这项拟议的研究将增强候选人使用先进技术探测神经元的经验 电路功能。这样的训练直接关系到考生自主学习关键神经元语的目标 在癫痫的发展过程中，通路及其如何影响复杂的神经元回路。这些研究将是 在梅奥诊所进行，由神经胶质(吴龙俊博士)、癫痫(格雷格博士)专家监督 (Worrell和Peyman Golshani博士)和微型望远镜技术(Luis Lujan博士和Peyman Golshani博士)。

Project Summary/Abstract Microglia, resident immune cells of the central nervous system, utilize G-protein signaling to accomplish different tasks. Gi-signaling is used to perform process outgrowth, while Gs signaling is engaged during periods of neuronal hypoactivity (such as anesthesia). However, very little is known about microglial Gq calcium signaling. Partially, this is due to the fact that microglia rarely display calcium transients at rest. On the other hand, microglia greatly elevate their calcium signaling in the period that follows status epilepticus—a prolonged seizure state predictive of later epilepsy development. The purpose of microglial calcium signaling during epilepsy development is not known, but is hypothesized to be a component of microglial phagocytosis. Most microglial calcium signaling in epilepsy development is attributable to P2Y6 receptor signaling—a Gq-calcium receptor activated by a key purine. P2Y6 is best described for its role in phagocytosis, but that has not been fully explored in epilepsy development. (Innovation) The current proposal will utilize a novel mouse line that allows for the simultaneous examination of microglial calcium activity and process movement in the living animal (using two- photon microscopy). (Aim 1) This line will be used to test the role of P2Y6 in microglial phagocytosis, after neurons are selectively killed through excitotoxicity using optogenetic techniques. In parallel, tissue studies will be conducted across key time points in epilepsy development to determine if the loss of the P2Y6 pathway prevents proper clearance of dead/dying neurons after status epilepticus. The prolonged presence of dying neurons is pro-inflammatory and hypothesized to negatively affect neuronal network dynamics in the long term. (Aim 2) For these reasons, we will test multiple aspects of long-term P2Y6 signaling loss during epilepsy development. We will determine the long-term pro-inflammatory effects of P2Y6 signaling loss. Additionally, we will use miniscope technology and 24/7 video EEG to determine whether the loss of P2Y6 calcium signaling and its putative phagocytosis alters network dysregulation or epilepsy risk. The proposed research will enhance the candidate’s experience in using advanced techniques to probe neuronal circuit function. Such training is directly related to the candidate’s goal of independently studying key glial pathways and how they influence complex neuronal circuits during epilepsy development. These studies will be performed at the Mayo Clinic under the supervision of experts in glia (Dr. Long-Jun Wu), epilepsy (Dr. Greg Worrell and Dr. Peyman Golshani), and miniscope technology (Dr. Luis Lujan and Dr. Peyman Golshani).