Regulation of Astrocyte TIMP-1 in HIV-Associated Dementia

星形胶质细胞 TIMP-1 在 HIV 相关痴呆中的调节

• 批准号: 8141024
• 负责人: Jerel Adam Fields
• 金额: $ 2.78万
• 依托单位: UNIVERSITY OF NORTH TEXAS HLTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8141024
• 关键词: AIDS Dementia Complex; Acute; Address; Affect; Apoptosis; Astrocytes; Base Pairing; Behavioral; Binding; Boxing; CCAAT-Enhancer-Binding Proteins; Categories; Chronic; Cognitive; Complication; Data; Dementia; Disease; Disease Progression; Down-Regulation; Elements; Enzyme-Linked Immunosorbent Assay; Family; Functional disorder; Gene Expression; Genetic Transcription; Glycoproteins; Goals; HIV; HIV-1; Highly Active Antiretroviral Therapy; Homeostasis; Human; In Vitro; Indium; Individual; Infection; Inflammatory; Interleukins; Investigation; Light; Luciferases; Maintenance; Mediating; Messenger RNA; Metalloproteases; Microglia; Mitogen-Activated Protein Kinase Inhibitor; Mitogen-Activated Protein Kinases; Molecular Profiling; Motor; Nerve Degeneration; Neuraxis; Neurocognitive; Neurodegenerative Disorders; Neuroglia; Neurologic; Neurophysiology - biologic function; Pathology; Pathway interactions; Patients; Plasmids; Polymerase Chain Reaction; Primary Cell Cultures; Production; Promoter Regions; Property; Protein Isoforms; Proteins; RNA; Regulation; Reporter; Role; Signal Transduction Pathway; Site; Stimulus; Technology; Time; Tissues; Trans-Activators; Transcriptional Regulation; United States; Viral Proteins; Virion; Virus Diseases; Western Blotting; astrogliosis; cytokine; design; falls; immunocytochemistry; inhibitor/antagonist; injury and repair; insight; knock-down; macrophage; mutant; neuroinflammation; new therapeutic target; novel; overexpression; prevent; promoter; relating to nervous system; response; tool

DESCRIPTION (provided by applicant): The goal of this proposal is to characterize regulation of primary human astrocyte tissue inhibitors of metalloproteases (TIMP) expression during Human Immunodeficiency Virus (HIV)-associated dementia (HAD). HIV-associated neurocognitive disorders are a group of disorders of which HAD is the most severe. HAD affects 10-27% of HIV-infected individuals in the United States. The disease is characterized by an accumulation of activated and infected macrophages/microglia in the central nervous system, which secrete cytokines, infectious virions and viral proteins that activate surrounding astrocytes leading to astrogliosis and altered gene expression. Neural dysfunction is believed to be the key component causing dementia. Astrocytes are essential in maintaining neural homeostasis in the CNS during injury and repair, in part through the production of neuroprotective TIMPs. An imbalance between matrix metalloproteases and TIMPs is thought to contribute to several neurodegenerative disorders, including HAD. TIMP-1, the inducible form of a family of 4 TIMPs, is a multifunctional protein that displays neuroprotective properties via homeostatic maintenance and possibly by inhibiting apoptosis. TIMP-1 levels are decreased in HAD patients compared to HIV-1 seronegative controls and primary human astrocytes acutely stimulated with HAD-relevant stimuli increase TIMP-1 expression, but levels fall after 3 days of exposure. Additionally, deleting the CAAT site at -310 in the TIMP-1 promoter increases transcription. Thus, we propose that down regulation of astrocyte TIMP-1 during HAD is mediated through differential transcriptional regulation via a CAAT site in the TIMP-1 promoter leading to reduced astrocyte TIMP-1 expression and ultimately exacerbating neurodegeneration. Two specific aims have been designed to address regulation of astrocyte TIMP-1 expression during HAD: Aim 1 determine the expression profile of CAAT enhancer binding protein 2 in activated primary human astrocytes and their influence on TIMP-1 transcription and Aim 2 identify signal transduction pathways leading to TIMP-1 down regulation in primary human astrocytes. To complete Aim 1 basic cell culture of primary human astrocytes and HAD-relevant stimuli like cytokines and viral proteins will be used along with analysis tools such as western blot, real-time polymerase chain reaction, immunocytochemistry and enzyme linked immunosorbent assay. Aim 2 will be completed by combining pathway-specific inhibitors with a panel of luciferase expression plasmids, CAAT-binding factor overexpression plasmids and pathway-indicating plasmids with and without HAD-relevant stimuli. Completion of these studies will shed light on the regulation of human astrocyte TIMP-1 expression in HAD. PUBLIC HEALTH RELEVANCE: HIV-1-associated dementia (HAD), the most severe manifestation of HIV-1-associated neurocognitive disorders, is an important neurological complication of HIV-1 infection and is characterized by cognitive, behavioral and motor dysfunction. An estimated 10-27% of HIV-seropositive patients progress to develop HAD in developed worlds such as the United States, despite the availability of highly active antiretroviral therapy. Our studies will provide a better understanding of the specific mechanistic contributions of activated astrocytes to HIV-1-neuropathogensis and neuroinflammation.

描述（由申请人提供）：该提案的目的是表征人类免疫缺陷病毒（HIV）相关痴呆（HAS）期间金属蛋白酶（TIMP）表达的原代人星形胶质细胞组织抑制剂（TIMP）的调节。艾滋病毒相关的神经认知疾病是一组疾病，其中最严重。在美国影响了10-27％的艾滋病毒感染者。该疾病的特征是中枢神经系统中激活和感染的巨噬细胞/小胶质细胞的积累，这些巨噬细胞/小胶质细胞分泌细胞因子，传染性病毒元和病毒蛋白，这些蛋白激活周围的星形胶质细胞，导致星形胶质细胞和基因表达改变。据信神经功能障碍是引起痴呆的关键成分。星形胶质细胞对于在受伤和修复过程中维持中枢神经系统中的神经稳态至关重要，部分是通过产生神经保护性的TIMP。基质金属蛋白酶和TIMP之间的不平衡被认为会导致包括HAT在内的几种神经退行性疾病。 TIMP-1是4个TIMP家族的诱导形式，是一种多功能蛋白，通过稳态维持以及可能通过抑制凋亡来显示神经保护性能。与HIV-1血清阴性对照相比，HAD患者的TIMP-1水平降低，而与抗硫磺相关刺激急性刺激的原发性星形胶质细胞会增加TIMP-1的表达，但暴露3天后的水平下降。另外，在TIMP -1启动子中删除-310的CAAT位点会增加转录。因此，我们提出，通过在TIMP-1启动子中的CAAT位点进行差异转录调节，在HAD中的星形胶质细胞TIMP-1的下调是介导的，导致星形胶质细胞TIMP-1表达降低，并最终加剧神经变性。已经设计了两个具体目的，以解决AIM 1在AIM 1中的调节TIMP-1表达的调节：确定激活原代人星形胶质细胞中CAAT增强子结合蛋白2的表达曲线及其对TIMP-1转录的影响，AIM 2识别信号转导途径，导致原代人类星形胶质细胞中TIMP-1下调的信号转导途径。为了完成目标1原代人星形胶质细胞和相关刺激（例如细胞因子和病毒蛋白）的基本细胞培养，将与分析工具一起使用，例如蛋白质印迹，实时聚合酶链反应，免疫细胞化学化学和酶连接的免疫吸收分析。 AIM 2将通过将途径特异性抑制剂与一组荧光素酶表达质粒，CAAT结合因子过表达质粒和途径指示质粒相结合，从而完成AIM 2。这些研究的完成将阐明HAD中人类星形胶质细胞TIMP-1表达的调节。 公共卫生相关性：HIV-1相关痴呆（HAD），HAD（HAD）是HIV-1相关神经认知疾病的最严重表现，是HIV-1感染的重要神经系统并发症，并具有认知，行为和运动功能障碍的特征。尽管有高度活跃的抗逆转录病毒疗法，但估计有10-27％的HIV - 呼吸阳性患者在美国等发达国家中发展。我们的研究将更好地理解活化的星形胶质细胞对HIV-1神经性疾病和神经炎症的特定机理贡献。