Focal Adhesion Kinase in disruption of the blood-brain barrier in encephalitis

描述(由申请人提供)：这项提案的广泛、长期目标是了解艾滋病神经发病机制和血脑屏障(BBS)破坏过程中的分子信号事件。脑炎的特征是感染的单核细胞来源的巨噬细胞(MDM)在血管周围剥离，与血脑屏障破坏有关。目前尚不清楚MDM渗透是导致BBB破坏，还是由破坏促进。我们建议用SIVmac251感染猕猴，并通过耗尽CD8细胞来增加脑炎的发生率，这是恒河猴常规使用的三阶段耗竭策略。然后，我们将确定在艾滋病神经发病过程中触发的内皮间连接中断的机制。粘着斑激酶(FAK)是血管内皮细胞功能的关键调节因子。有待探索的中心假设是：1)粘着斑激酶的激活是神经艾滋病发病过程中血脑屏障破坏的关键机制，2)这种激活需要高效感染的单核细胞的存在。使用恒河猴提出了以下具体目标：SA-1：确定跨越血脑屏障的高效感染的单核/巨噬细胞是否诱导FAK激活和紧密连接的破坏。我们将使用血脑屏障的体外模型来模拟神经侵袭，以确定是否需要生产性感染的细胞在物理上穿过血脑屏障才能进行这种分解。SA2：确定FAK的磷酸化是否是血脑屏障破坏和脑炎发生的必要条件。SA-2a：活体实验。FAK和紧密连接蛋白的表达将使用共聚焦显微镜进行评估。我们还将检查感染SIVmac251的猕猴大脑的厚切片共聚焦显微镜，以确定生产性感染的巨噬细胞与血脑屏障破坏区域的关联。血脑屏障完整性将通过检测少突胶质细胞蛋白5100(3)的酶联免疫吸附试验进行监测，以确定病变形成的时间。SA-2b：体外实验。SA-2b将通过免疫细胞化学和蛋白质表达/激活来评估血脑屏障破坏的时机和机制。FAK是通过关键酪氨酸残基的磷酸化而激活的。我们将使用磷酸酪氨酸抗体和免疫印迹进行FAK的免疫沉淀，然后进行条带密度测定。我们期望通过来自体外和体外研究的实时信息，从体内数据中确定艾滋病发展过程中血脑屏障破裂的时间和机制。这项研究很重要，因为HAART时代正在导致艾滋病相关CMS并发症的累积流行率增加。如果我们能确定血脑屏障在SIV感染中是如何分解的，那么我们就可以开发新的策略来预防蜂房和相关的周围神经疾病。

DESCRIPTION (provided by applicant): The broad, long-term objective of this proposal is to understand the molecular signaling events during AIDS neuropathogenesis and blood-brain barrier (BBS) disruption. Encephalitis, characterized by perivascular cuffing of infected monocyte-derived macrophages (MDMs), is associated with BBB disruption. It is not known if the MDM infiltration results in BBB disruption, or is facilitated by disruption. We propose to infect macaques with SIVmac251, and increase the incidence of encephalitis by depleting CD8 cells using a three-stage depletion strategy routinely used in rhesus macaques. We will then determine the mechanisms of interendothelial junction disruption triggered during AIDS neuropathogenesis. A key regulator of endothelial function is focal adhesion kinase (FAK). The central hypothesis to be explored is that 1) focal adhesion kinase activation is a key mechanism by which BBB breakdown occurs during neuroAIDS pathogenesis, and 2) that this activation requires the presence of productively infected monocytes. The following specific aims are proposed, using the rhesus macaque: SA-1: Determine if a productively- infected monocyte/ macrophage crossing the BBB induces activation of FAK and disruption of tight junctions. We will model neuroinvasion using an in vitro model of the BBB to determine if productively-infected cells are required to have physically crossed the BBB for this breakdown. SA2: Determine if phosphorylation of FAK is a requirement for BBB disruption and development of encephalitis. SA-2a: Experiments in vivo. Expression of FAK and tight junction proteins will be assessed using confocal microscopy. We will also examine thick section confocal microscopy of brains of macaques infected with SIVmac251 to determine association of productively infected macrophages with areas of BBB disruption. BBB integrity will be monitored by ELISA for the oligodendrocyte protein 5100(3 to determine timing of lesion formation. SA-2b: Experiments ex vivo. SA-2b will assess the timing and mechanisms of BBB disruption by immunocytochemistry and protein expression/ activation. FAK is activated by phosphorylation of key tyrosine residues. We will immunoprecipitate using phosphotyrosine antibodies and immunoblot for FAK, followed by band densitometry. We expect to determine the timing and mechanisms of BBB breakdown during the development of AIDS from the in vivo data, with real time information coming from ex vivo and in vitro studies. This research is important because the era of HAART is resulting in an increased cumulative prevalence of AIDS-related CMS complications. If we can determine how BBB is breaking down in SIV infection then we can develop new strategies for prevention of HIVE and the associated peripheral neuropathies.