Astrocyte connexin43 containing channels amplify CNS dysfunction in NeuroAIDS

描述（由申请人提供）：截至2010年，全球估计有3400万人感染人类免疫缺陷病毒（HIV）（世界卫生组织和联合国估计）。在初次感染后早期，HIV进入CNS并在30-60%的感染个体中引起认知和运动障碍，即使在抗逆转录病毒时代也是如此。随着感染者的寿命延长，HIV CNS感染引起的神经系统并发症的患病率也增加了。HIV-1引起神经发病机制或NeuroAIDS的细胞基础和机制仍然没有很好地理解。星形胶质细胞是中枢神经系统中调节血脑屏障完整性、中枢神经系统炎症、免疫反应和神经元存活的关键细胞。HIV仅感染这些细胞中的一小部分，并且检测到最小到不可检测的病毒产生。然而，我们的数据首次表明，HIV感染的星形胶质细胞，通过间隙连接通道，也许半通道，可以放大炎症和中枢神经系统损伤。我们假设含有间隙连接（GJ）和半通道（uHC）的Cx43将在少数HIV感染的星形胶质细胞中产生的细胞间信号放大到周围未感染的细胞，导致细胞毒性、BBB破坏和DKK 1分泌，导致即使在病毒复制最小的当前抗逆转录病毒时代，在HIV感染人群中也经常观察到CNS功能障碍。为了解决这一假设，我们将扩大我们广泛的初步研究，证明这些通道参与星形胶质细胞，神经元和血脑屏障（BBB）功能障碍，以及放大细胞活化和炎症 在HIV感染的星形胶质细胞和未感染的细胞中。这些数据将表征脑内HIV毒性的新途径，并将确定这些通道在CNS功能障碍中的作用。从这个提议中获得的结果应该表明潜在的新的治疗靶点，以限制NeuroAIDS的破坏性后果。PHS 398/2590（Rev.06/09） 公共卫生相关性：由于抗逆转录病毒疗法的成功，艾滋病毒感染者的寿命延长，因此这些感染人群中认知和运动缺陷的患病率也在增加。星形胶质细胞在维持中枢神经系统功能中起关键作用。然而，它们在NeuroAIDS发病机制中的作用尚未得到很好的表征，主要是由于缺乏特异性分子工具来检查星形胶质细胞的HIV感染。研究体内和体外星形胶质细胞的HIV感染及其在脑中的后果的新技术的发展使我们能够证明星形胶质细胞中的间隙连接（GJ）和半通道（uHC）在NeuroAIDS的发病机制中是重要的。我们的研究已经表明，GJ和连接蛋白43（Cx43）的uHC是至关重要的，尽管HIV感染的细胞数量很少，病毒复制最小，但它会将损伤扩散到邻近细胞。我们建议扩展这些研究，以表征未感染的星形胶质细胞，神经元和脑内皮细胞的旁观者失调。此外，我们还将通过检测GJ和uHC的激活和调节来研究它们向邻近细胞传递和放大毒性信号的分子机制。最后，我们将扩大我们在体内获得的初步数据，通过使用一种新的旁观者毒性的动物模型介导的微量注射少数人HIV感染的人星形胶质细胞到动物的大脑中与星形胶质细胞基因缺失的Cx43。我们将使用这些动物，在显微注射的HIV感染的星形胶质细胞周围的相邻细胞中的凋亡和BBB破坏进行评估。这些研究结果可能为开发治疗HIV感染者神经功能障碍的疗法提供信息。

DESCRIPTION (provided by applicant): As of 2010, an estimated 34 million persons worldwide were living with human immunodeficiency virus (HIV) infection (Word Health Organization and UN estimations). Early after primary infection, HIV enters the CNS and causes cognitive and motor impairment in 30-60% of infected individuals, even in the antiretroviral era. As infected individuals are living longer, the prevalence of neurological complications due to HIV CNS infection has increased. The cellular basis and mechanisms by which HIV-1 causes neuropathogenesis, or NeuroAIDS, are still not well understood. Astrocytes are key cells in the CNS that regulate BBB integrity, CNS inflammation, immune responses and neuronal survival. HIV only infects a small percentage of these cells and minimal to undetectable viral production is detected. Nevertheless, our data demonstrate for first time that HIV infected astrocytes, through gap junction channels and perhaps hemichannels, can amplify inflammation and CNS damage. We hypothesize that Cx43 containing gap junctions (GJ) and hemichannels (uHC) amplify intercellular signals generated in few HIV infected astrocytes to surrounding uninfected cells resulting in cellular toxicity, BBB disruption and secretion of DKK1 leading to the CNS dysfunction often observed in the HIV infected population even in the current antiretroviral era, where viral replication is minimal. To address this hypothesis we will expand upon our extensive Preliminary Studies demonstrating the participation of these channels in astrocyte, neuronal and blood brain barrier (BBB) dysfunction, as well in amplification of cell activation and inflammation in HIV infected astrocytes and in uninfected cells. These data will characterize novel pathways of HIV toxicity within the brain and will identify the role of these channels in CNS dysfunction. The results obtained from this proposal should indicate potential novel therapeutic targets to limit the devastating consequences of NeuroAIDS. PHS 398/2590 (Rev. 06/09) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: As individuals with HIV are living longer due to the success of antiretroviral therapies, the prevalence of cognitive and motor deficits in this infected populatio is increasing. Astrocytes play a key role in maintaining CNS functions. However, their role in the pathogenesis of NeuroAIDS has not been well characterized, mainly due to the lack of specific molecular tools to examine HIV infection of astrocytes. The development of novel techniques to examine HIV infection of astrocytes in vivo and in vitro and their consequences in brain enable us to demonstrate that gap junction (GJ) and hemichannels (uHC) in astrocytes are important in the pathogenesis of NeuroAIDS. Our studies already showed that GJ and uHC of Connexin43 (Cx43) are critical to spread damage to neighboring cells despite the few numbers of HIV infected cells and minimal viral replication. We propose to expand these studies to characterize bystander dysregulation of uninfected astrocytes, neurons and brain endothelial cells. In addition, we will study the molecular mechanism by which GJ and uHC transmit and amplify toxic signals to neighboring cells by examining their activation and regulation. Lastly, we will expand upon our preliminary data obtained in vivo by using a novel animal model of bystander toxicity mediated by microinjection of few human HIV infected human astrocytes into the brain of animals with astrocytes genetically deleted for Cx43. We will evaluate using these animals, apoptosis and BBB disruption in neighboring cells around the microinjected HIV infected astrocytes. The results of these studies may provide information for the development of therapies to treat the neurologic dysfunctions in HIV infected individuals.