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

粘着斑激酶破坏脑炎血脑屏障

• 批准号: 8032674
• 负责人: ANDREW G MACLEAN
• 金额: $ 11.11万
• 依托单位: TULANE UNIVERSITY OF LOUISIANA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8032674
• 关键词: AIDS Dementia Complex; AIDS neuropathy; Acquired Immunodeficiency Syndrome; Animals; Antibodies; Area; Belief; Binding; Blood - brain barrier anatomy; Brain; C-terminal; CD8-Positive T-Lymphocytes; Cells; Cerebrum; Confocal Microscopy; Data; Densitometry; Development; Disease; Dissociation; Encephalitis; Endothelial Cells; Endothelium; Enzyme-Linked Immunosorbent Assay; Event; Exposure to; Focal Adhesion Kinase 1; Giant Cells; Goals; HIV tat Protein; HIV-1; Highly Active Antiretroviral Therapy; Immunoblotting; In Vitro; Incidence; Infection; Infiltration; Inflammatory; Integrins; Knowledge; Lead; Lesion; Macaca; Macaca mulatta; Modeling; Molecular; Monitor; Nervous system structure; Neurologic; Neuropathogenesis; Oligodendroglia; Pathogenesis; Pathway interactions; Patients; Peripheral Nervous System Diseases; Phosphorylation; Phosphotyrosine; Prevalence; Prevention strategy; Proteins; Research; Role; SIV; SIV encephalitis; Signal Transduction; Site; Staging; Talin; Testing; Thick; Tight Junctions; Time; Tyrosine; Urticaria; Viral Load result; Virus; immunocytochemistry; improved; in vitro Model; in vivo; inhibitor/antagonist; kinase inhibitor; macrophage; monocyte; paxillin; prospective; protein expression; research study; three-dimensional modeling

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.

描述（由申请人提供）：本提案的广泛、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。以感染的单核细胞衍生的巨噬细胞（MDM）的血管周围套囊为特征的脑炎与BBB破坏相关。尚不清楚MDM浸润是否导致BBB破坏，或者破坏是否促进了MDM浸润。我们建议用SIVmac 251感染猕猴，并通过使用恒河猴中常规使用的三阶段耗竭策略耗竭CD 8细胞来增加脑炎的发病率。然后我们将确定艾滋病神经发病过程中触发的内皮细胞间连接中断的机制。内皮功能的关键调节因子是粘着斑激酶（FAK）。待探索的中心假设是：1）粘着斑激酶激活是神经AIDS发病过程中BBB破坏的关键机制，2）这种激活需要存在有效感染的单核细胞。使用恒河猴提出了以下具体目标：SA-1：确定穿过BBB的有效感染的单核细胞/巨噬细胞是否诱导FAK的活化和紧密连接的破坏。我们将使用BBB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物理地穿过BBB以进行这种分解。SA 2：确定FAK的磷酸化是否是BBB破坏和脑炎发展的必要条件。SA-2a：体内实验。将使用共聚焦显微镜评估FAK和紧密连接蛋白的表达。我们还将检查SIVmac 251感染的猕猴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BBB破坏区域的关联。BBB完整性将通过ELISA监测少突胶质细胞蛋白5100 β以确定损伤形成的时间。SA-2b：离体实验。SA-2b将通过免疫细胞化学和蛋白质表达/活化评估BBB破坏的时机和机制。FAK通过关键酪氨酸残基的磷酸化而活化。我们将使用磷酸酪氨酸抗体进行免疫沉淀，并对FAK进行免疫印迹，然后进行条带密度测定。我们希望通过体内数据，以及来自体外和离体研究的真实的时间信息，确定AIDS发展过程中血脑屏障破坏的时间和机制。这项研究很重要，因为HAART时代导致艾滋病相关CMS并发症的累积患病率增加。如果我们能够确定SIV感染时血脑屏障是如何被破坏的，那么我们就可以开发新的策略来预防HIVE和相关的周围神经病变。