Microglial P2Y6 receptor calcium signaling as a core regulator of epileptogenesis

小胶质细胞 P2Y6 受体钙信号传导作为癫痫发生的核心调节因子

• 批准号: 10630958
• 负责人: ANTHONY DAVID UMPIERRE
• 金额: $ 11.71万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10630958
• 关键词: Address; Anesthesia procedures; Animals; Area; Automobile Driving; Benchmarking; Brain; Calcium; Calcium Signaling; Calcium-Sensing Receptors; Cells; Cellular Morphology; Central Nervous System; Clinic; Cognition; Complex; Cytoskeleton; Development; Disease; Electroencephalography; Epilepsy; Epileptogenesis; GTP-Binding Proteins; Genetic Transcription; Goals; Hippocampus; Histology; Image; Immune; Inflammation; Inflammatory; Internal Ribosome Entry Site; Knock-out; Knockout Mice; Knowledge; Link; Lysosomes; Metabolic Clearance Rate; Microglia; Microscope; Modeling; Morphology; Movement; Mus; National Institute of Neurological Disorders and Stroke; Neuroglia; Neurons; Pathway interactions; Phagocytes; Phagocytosis; Process; Purines; Purinoceptor; Receptor Signaling; Research; Rest; Risk; Risk Reduction; Role; Seizures; Signal Pathway; Signal Transduction; Signaling Protein; Source; Status Epilepticus; Supervision; Techniques; Technology; Testing; Therapeutic; Time; Tissues; Training; Visualization; awake; cell motility; confocal imaging; cytokine; excitotoxicity; experience; in vivo; innovation; insight; kainate; lens; miniaturize; negative affect; network dysfunction; neuronal circuitry; next generation sequencing; novel; optogenetics; prevent; protective effect; purinoceptor P2Y6; tissue fixing; two photon microscopy; two-photon

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).

项目总结/摘要 小胶质细胞，中枢神经系统的常驻免疫细胞，利用G蛋白信号传导来完成不同的免疫功能。 任务Gi-信号传导用于执行过程生长，而Gs信号传导在过程生长期间参与。 神经元功能减退（如麻醉）。然而，很少有人知道小胶质细胞Gq钙信号。 部分原因是小胶质细胞在静息时很少显示钙瞬变。另一方面， 在癫痫持续状态（一种长时间的癫痫发作状态）后， 预测癫痫的后期发展。小胶质细胞钙信号在癫痫发作中的作用 发育尚不清楚，但假设是小胶质细胞吞噬作用的一个组成部分。大多数小胶质细胞 癫痫发生中的钙信号传导可归因于P2 Y 6受体信号传导--一种Gq-钙受体 由关键嘌呤激活P2 Y 6最好的描述是其在吞噬作用中的作用，但尚未得到充分探讨 癫痫的发展。（创新）目前的提案将利用一种新颖的鼠标线， 在活体动物中同时检查小胶质细胞钙活性和突起运动（使用两个- 光子显微术）。(Aim 1）该细胞系将用于测试P2 Y 6在小胶质细胞吞噬作用中的作用， 使用光遗传学技术通过兴奋性毒性选择性地杀死神经元。同时，组织研究将 在癫痫发展的关键时间点进行，以确定P2 Y 6通路的丢失是否 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 神经元是促炎性的，并且被假设为长期负面影响神经元网络动力学。 (Aim 2）出于这些原因，我们将测试癫痫期间长期P2 Y 6信号丢失的多个方面。 发展我们将确定P2 Y 6信号丢失的长期促炎作用。另外我们 将使用微型内窥镜技术和24/7视频EEG来确定P2 Y 6钙信号的丢失和 其假定的吞噬作用改变网络失调或癫痫风险。 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候选人独立研究关键神经胶质细胞的目标 途径以及它们如何影响癫痫发展过程中复杂的神经元回路。这些研究报告将 在马约诊所的神经胶质专家（吴龙军博士）、癫痫专家（格雷格博士）的监督下进行 Worrell和Peyman Golshani博士）和微型显微镜技术（Luis Lujan博士和Peyman Golshani博士）。