Effects of Neuroinflammation on Gap Junction Communication in Glia

描述(由申请人提供)：缝隙连接是接触细胞之间的直接细胞间通道，允许小分子(&gt；1 kDa)通过，包括离子、代谢前体和第二信使。在中枢神经系统(CNS)中观察到广泛的细胞间连接和大量的缝隙连接，表明神经胶质细胞间的组织是合胞体样的。一种中枢神经系统感染性疾病，其对神经胶质细胞缝隙连接通讯(GJC)的影响尚不清楚，它是由化脓性细菌引起的脑实质感染导致的脑脓肿。最近的研究表明，金黄色葡萄球菌激活的胶质细胞产生的几种促炎介质，包括白介素1(IL-1)、肿瘤坏死因子-α(TNF-α)和一氧化氮(NO)，能够调节星形胶质细胞和小胶质细胞的GJC。具体地说，这些分子减弱了星形胶质细胞中的GJC，而激活的小胶质细胞则变得功能偶联。我们将这一现象称为“合胞开关”，并提出在脑脓肿过程中形成的炎症环境可能对重塑常驻神经胶质细胞之间的相互作用类型具有重要意义，而偏离生理耦合可能会影响远离主要感染灶的脑区的完整性。这些变化可能是由脓肿内Cx表达的区域差异所决定的。本研究的目的是探讨IL-1、TNF-α和NO在调节神经胶质细胞开关中的功能重要性，以及Cx43在脑脓肿发病机制中的作用。为实现这一目标，将针对以下具体目标：(1)评估金黄色葡萄球菌及其细胞壁产物PGN对原代星形胶质细胞和小胶质细胞高细胞GJC的影响以及参与合胞开关的信号通路；(2)利用基因敲除(KO)小鼠的原始胶质细胞，确定促炎介质IL-1、TNF-α和NO在调节神经胶质细胞对金黄色葡萄球菌刺激反应中的功能重要性；(3)在金黄色葡萄球菌诱导的小鼠实验性脑脓肿模型中，利用遗传KO模型研究促炎症介质对连接蛋白表达的影响以及Cx43在疾病发病机制中的作用。由于正常中枢神经系统中神经胶质细胞群广泛的缝隙连接偶联，正常神经胶质合胞网络的神经炎性破坏可能在一定程度上导致脑脓肿消退后观察到的一些长期效应，包括癫痫发作和认知障碍。这些实验将对促炎介质如何影响脑脓肿中胶质细胞GJC的程度提供有意义的见解。

DESCRIPTION (provided by applicant): Gap junctions represent direct intercellular conduits between contacting cells that permit the passage of small molecules (> 1 kDa) including ions, metabolic precursors, and second messengers. The observation of extensive intercellular coupling and large numbers of gap junctions in the central nervous system (CNS) suggests a syncytium-like organization of glial compartments. One CNS infectious disease in which nothing is known regarding its impact on glial gap junction communication (GJC) is parenchymal infection with pyogenic bacteria leading to the establishment of brain abscess. Recent studies have revealed that several proinflammatory mediators detected in developing brain abscesses and produced by S. aureus activated glia, including interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-a), and nitric oxide (NO) are capable of modulating GJC in astrocytes and microglia. Specifically, these molecules attenuate GJC in astrocytes whereas activated microglia become functionally coupled. We have coined this phenomenon a "syncytial switch" and propose that the inflammatory milieu that develops during the course of brain abscess may be important for remodeling the types of interactions between resident glia and that deviation from physiological coupling may impact the integrity of brain regions distant from the primary focus of infection. These changes may be dictated by regional variations in Cx expression within the abscess. The objective of the proposed work is to investigate the functional importance of IL-1, TNF-a, and NO in regulating the glial syncytial switch and the role of Cx43 in brain abscess pathogenesis. To address this objective the following Specific Aims will be addressed: (1), to evaluate the consequences of S. aureus and its cell wall product PGN on homocellular GJC in primary astrocytes and microglia and the signaling pathways responsible for the syncytial switch; (2), to establish the functional importance of the proinflammatory mediators IL-1, TNF-a, and NO on modulating glial GJC in response to S. aureus stimulation using primary glia from knockout (KO) mice; and (3), to investigate the role of proinflammatory mediators on connexin expression and the functional importance of Cx43 in disease pathogenesis in a mouse model of S. aureus-induced experimental brain abscess using genetic KO models. Due to the extensive gap junctional coupling of glial cell populations in the normal CNS, neuroinflammatory disruption of normal glial syncytial networks could contribute, in part, to some of the long-term effects observed in patients following brain abscess resolution including seizures and cognitive deficits. These experiments will provide meaningful insights into how proinflammatory mediators influence the extent of glial GJC in brain abscess.