FOCAL ADHESION KINASE IN DISRUPTION OF THE BLOOD-BRAIN BARRIER IN ENCEPHALITIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 尽管在接受高效抗逆转录病毒治疗(HAART)的患者中，艾滋病毒痴呆症的发病率有所下降，但存活率的提高导致了艾滋病神经系统并发症的累积患病率增加。许多根本性的空白仍然存在于与机制和途径有关的知识中 与艾滋病毒相关的神经系统疾病的发展。这种疾病的发病机制尚不清楚，但被认为与血脑屏障的改变有关。这项提案的重点是血脑屏障(BBB)如何以及何时发生破坏。了解这种情况是如何以及何时发生的，对于阐明神经艾滋病的发病机制具有深远的影响。 在这个项目中，我们正在研究紧密连接蛋白的作用，从ZO-1开始，以及导致紧密连接中断的细胞内机制。我们最初的重点是粘着斑激酶(FAK)，它在暴露于HIV蛋白TAT后在脑内皮细胞中被激活。当FAK定位于整合素聚集的位点时，通过磷酸化的酪氨酸残基启动下游信号转导。我们正试图确定这些负责破坏血脑屏障的细胞内蛋白在艾滋病的神经发病机制中是如何调节的。我们认为，FAK的激活和由此产生的破坏将促进细胞相关的和无细胞的病毒通过血脑屏障进入中枢神经系统 导致神经系统并发症。我们的主要假设是，粘着斑激酶(FAK)的激活是SIV神经发病过程中紧密连接蛋白破坏的关键调节事件。 为了验证这一假设，我们提出了以下具体目标： 具体目标1：确定FAK的磷酸化是否是脑炎血脑屏障破坏和发展的必要条件。这一目标将通过体内、体外和体外方法进行测试。为了增加脑炎的发病率，并加速发展到晚期疾病，我们将在感染前耗尽猕猴的CD8细胞。 具体目标2：确定通过血脑屏障的高效感染的单核/巨噬细胞是否诱导FAK的激活和紧密连接的破坏。这些研究将利用我们的血脑屏障体外3D模型。我们将使用体内使用安全的特定FAK抑制剂，目标是将研究扩大到 检查FAK抑制剂是否能抑制脑炎的发展。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite a reduced incidence of HIV dementia in patients on highly active antiretroviral therapy (HAART), the improved survival has resulted in increased cumulative prevalence of nervous system complications of AIDS. Many fundamental gaps remain in knowledge relating to mechanisms and pathways involved in the development of HIV-associated neurologic disease. The pathogenesis of the disease is unknown, but it is thought to be associated with alterations in the BBB. The focus of this proposal is on how and when disruption of the blood-brain barrier (BBB) occurs. Understanding how and when this occurs could have profound impact on elucidating the pathogenesis of neuroAIDS. In this project we are examining the roles of tight junction proteins, starting with zo-1, and the intracellular mechanisms that lead to the disruption of tight junctions. We have initially focused on focal adhesion kinase (FAK), which is activated in cerebral endothelial cells following exposure to the HIV protein Tat. When localized to sites of integrin clustering, FAK initiates downstream signaling via a phosphorylated tyrosine residue. We are attempting to determine how these intracellular proteins responsible for the disruption of the BBB are regulated in the neuropathogenesis of AIDS. We propose that activation of FAK and the resultant disruption will facilitate entry of cell-associated and cell-free virus into the CNS compartment through the BBB leading to neurological complications. Our primary hypothesis is that activation of focal adhesion kinase (FAK) is a key regulatory event in disruption of tight junction proteins during SIV neuropathogenesis. To test this hypothesis we proposed the following specific aims: Specific Aim 1: Determine if phosphorylation of FAK is a requirement for BBB breakdown and development of encephalitis. This Aim will be tested using in vivo, ex vivo and in vitro approaches. To increase the incidence of encephalitis, and to accelerate progression to terminal disease, we will deplete macaques of CD8 cells prior to infection. Specific Aim 2: Determine if a productively-infected monocyte/macrophage crossing the BBB induces activation of FAK and disruption of tight junctions. These studies will utilize our in vitro 3D model of the BBB. We will utilize specific inhibitors of FAK that are safe for in vivo use, with a goal of extending the study to examine if FAK inhibitors can inhibit the development of encephalitis.