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（由申请方提供）：本提案的目的是表征人类免疫缺陷病毒（HIV）相关痴呆（HAD）期间主要人星形胶质细胞金属蛋白酶组织抑制剂（TIMP）表达的调节。HIV相关的神经认知障碍是其中HAD最严重的一组障碍。HAD影响美国10-27%的HIV感染者。该疾病的特征在于活化和感染的巨噬细胞/小胶质细胞在中枢神经系统中的积累，其分泌细胞因子、感染性病毒体和病毒蛋白，其活化周围的星形胶质细胞，导致星形胶质细胞增生和基因表达改变。神经功能障碍被认为是导致痴呆症的关键因素。星形胶质细胞在损伤和修复期间维持CNS中的神经稳态中是必不可少的，部分地通过产生神经保护性TIMP。基质金属蛋白酶和TIMP之间的不平衡被认为是导致几种神经退行性疾病，包括HAD。TIMP-1是4种TIMP家族的可诱导形式，是一种多功能蛋白，其通过稳态维持和可能通过抑制凋亡显示神经保护特性。与HIV-1血清阴性对照相比，HAD患者中TIMP-1水平降低，并且用HAD相关刺激物急性刺激的原代人星形胶质细胞增加TIMP-1表达，但暴露3天后水平下降。此外，在TIMP-1启动子中缺失-310处的CAAT位点增加转录。因此，我们提出，在HAD过程中，星形胶质细胞TIMP-1的下调是通过TIMP-1启动子中的CAAT位点的差异转录调节介导的，从而导致星形胶质细胞TIMP-1表达减少并最终加剧神经退行性变。已经设计了两个具体目标来解决HAD期间星形胶质细胞TIMP-1表达的调节：目标1确定活化的原代人星形胶质细胞中CAAT增强子结合蛋白2的表达谱及其对TIMP-1转录的影响，目标2鉴定导致原代人星形胶质细胞中TIMP-1下调的信号转导途径。为了完成目标1，将使用原代人星形胶质细胞的基础细胞培养物和HAD相关刺激物（如细胞因子和病毒蛋白）沿着分析工具，如蛋白质印迹、实时聚合酶链反应、免疫细胞化学和酶联免疫吸附测定。目标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具有和不具有HAD相关刺激的途径指示质粒组合来完成。这些研究的完成将阐明HAD中人类星形胶质细胞TIMP-1表达的调控。 公共卫生关系：HIV-1相关性痴呆（HAD）是HIV-1相关性神经认知障碍的最严重表现，是HIV-1感染的重要神经系统并发症，其特征在于认知、行为和运动功能障碍。据估计，在发达国家如美国，10-27%的HIV血清阳性患者进展为HAD，尽管可获得高效抗逆转录病毒治疗。我们的研究将提供一个更好的理解的具体机制的贡献活化星形胶质细胞的HIV-1-神经发病和神经炎症。