Role of enteric glia in the death of neurons during gut inflammation

 描述(申请人提供)：肠道的反射行为，包括蠕动，是由肠道神经系统(ENS)协调的，这是一个嵌入肠壁的复杂神经网络。炎症通过促进肠道运动而深刻改变ENS控制运动的回路 神经节炎；一种以肠道神经元死亡为特征的炎症性神经病。神经病被越来越多地认为是胃肠道(GI)运动和功能性肠病中持续性肠道功能障碍的触发因素，但调节神经病的机制尚不清楚。这项建议研究了肠神经胶质细胞在调节肠神经病变中的作用。肠神经胶质细胞是一种星形细胞样细胞，环绕在神经细胞周围。拟议的研究将使用胃肠道炎症的活体模型、转基因小鼠、免疫组织化学、带有荧光探针的活细胞成像、生物传感分析和功能测试来研究神经元-神经胶质细胞的相互作用。中心假设是，肠神经胶质细胞的嘌呤能激活依赖于ADP或腺苷激活的神经胶质细胞而不同地调节神经元的存活。这项提案有两个具体目标，每个目标都有三个子目标。每个目标都将把人类和小鼠组织的体外机制研究与转基因小鼠的体内功能研究联系起来。目的1验证ADP驱动的神经胶质细胞钙反应导致反应性胶质细胞增厚、神经元死亡和肠道功能障碍的假说。特定目标1A将测试GFAP：hM3Dq小鼠或人类组织中胶质钙反应的激活如何影响反应性胶质增生和神经元死亡的诱导。目的1B将测试神经胶质细胞是否通过释放神经毒性物质直接导致神经元死亡 神经元死亡需要免疫细胞的募集。在胶质细胞中可诱导地去除连接蛋白-43或MHC-II的小鼠将分别被用于特异性干扰免疫细胞募集的胶质递质释放。目的1C将测试使用上述转基因小鼠操作胶质细胞增生症对体内和体外肠道功能的影响。目的2验证腺苷抑制反应性胶质细胞增生和刺激神经胶质细胞保护机制以保护ENS功能的假说。Aim 2A将使用药物和CD73基因缺失的小鼠来测试胶质腺苷受体的激活是否必要和/或足以逆转反应性胶质增生。目的2B研究CD73基因缺失小鼠体内炎症后，神经胶质细胞A2受体活化的神经保护作用是通过改变胶质细胞介质的释放还是通过减少炎症细胞的浸润来实现的。目的2C将利用体内炎症、药物和CD73缺失小鼠来确定操纵胶质腺苷信号如何影响急性炎症后的体内和体外肠道功能。意义：肠道炎症可导致肠神经病变，在胃肠动力障碍中导致持续性肠道功能障碍。了解胶质机制如何促进和限制肠道神经病变是重要的，因为它可能导致发现新的治疗靶点，以及在胃肠动力障碍、功能性肠病和炎症性肠病中神经元死亡的常见致病机制。

 DESCRIPTION (provided by applicant): Reflex behaviors of the intestine including peristalsis are orchestrated by the enteric nervous system (ENS); a complex neural network embedded in the gut wall. Inflammation profoundly alters ENS circuits controlling motility by promoting enteric ganglionitis; an inflammatory neuropathy characterized by the death of enteric neurons. Neuropathy is increasingly recognized as a trigger for persistent gut dysfunction in gastrointestinal (GI) motility and functional bowel disorders but the mechanisms that regulate neuropathy are not understood. This proposal investigates the role of enteric glial cells, astrocyte-like cells that surround neurons in the ENS, in the regulation of enteric neuropathy. The proposed studies will use in vivo models of GI inflammation, transgenic mice, immunohistochemistry, live-cell imaging with fluorescent probes, biosensing assays and functional tests to study neuron-glia interactions. The central hypothesis is that purinergic activation of enteric glial cells differentially regulates neuron survival depending on glial activation by ADP or adenosine. There are 2 specific aims in this proposal, each with three sub-aims. Each aim will link in vitro mechanistic studies in tissue from humans and mice with in vivo functional studies in transgenic mice. Aim 1 will test the hypothesis that glial Ca2+ responses driven by ADP cause reactive gliosis, neuron death and gut dysfunction. Specific aim 1A will test how activation of glial Ca2+ responses in GFAP:hM3Dq mice or human tissue transduced with glial- specific vectors affects the induction of reactive gliosis and neuron death. Aim 1B wil test whether glial cells directly drive neuron death by releasing neurotoxic substances or if glial driven neuron death requires immune cell recruitment. Mice with an inducible ablation of connexin-43 or MHC-II in glia will be used to specifically interfere with gliotransmitter release o immune cell recruitment, respectively. Aim 1C will test how manipulation of gliosis using the transgenic mice listed above affects in vivo and ex vivo intestinal function. Aim 2 will test the hypothesis that adenosine inhibits reactive gliosis and stimulates protective mechanisms in glia to preserve ENS function. Aim 2A will use drugs and CD73 null mice to test if activation of glial adenosine receptors is necessary and/or sufficient to reverse reactive gliosis. Aim 2B will test whether the neuroprotective actions of glial A2BR activation are mediated by altering the release of glial mediators or by decreasing the inflammatory infiltrate following in vivo inflammation in CD73 null mice. Aim 2C will use in vivo inflammation, drugs and CD73 null mice to determine how manipulation of glial adenosine signaling impacts in vivo and ex vivo assays of gut function following acute inflammation. Significance: Intestinal inflammation can drive enteric neuropathy, leading to persistent gut dysfunction in GI motility disorders. Understanding how glial mechanisms both promote, and limit enteric neuropathies is important because it could lead to the discovery of novel therapeutic targets and a common causative mechanism of neuron death in GI motility disorders, functional bowel disorders and inflammatory bowel disease.