Mechanisms of mitochondrial fission/fusion dysregulation during HIV-1-associated

HIV-1相关期间线粒体裂变/融合失调的机制

• 批准号: 8652197
• 负责人: Jerel Adam Fields
• 金额: $ 4.45万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-04-01 至 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8652197
• 关键词: AIDS neuropathy; Affect; Age; Aging-Related Process; Alzheimer' s Disease; American; Amino Acids; Apoptotic; Autopsy; Binding; Biochemical; Biological Assay; Brain; Buffers; Calcium; California; Cell Culture Techniques; Cell Line; Cells; Centers for Disease Control and Prevention (U.S.); Central Nervous System Diseases; Chimeric Proteins; Chronic; Co-Immunoprecipitations; Conditioned Culture Media; Confocal Microscopy; Data; Disease; Dominant-Negative Mutation; Dynamin; Electron Microscopy; Environment; Enzyme-Linked Immunosorbent Assay; Event; Functional disorder; Fusion Protein Expression; Genetic Transcription; Glycoproteins; Goals; HIV; HIV Envelope Protein gp120; HIV-1; Half-Life; Highly Active Antiretroviral Therapy; Homeostasis; Immune; Immunoblotting; Immunohistochemistry; Inflammation; Inflammatory; Interleukin-1; Lead; Lentivirus Vector; Life; Life Expectancy; Light; Methods; Microglia; Mitochondria; Nerve Degeneration; Nervous System Physiology; Neural Pathways; Neuraxis; Neurocognitive; Neuroglia; Neuronal Plasticity; Neurons; Oxidative Stress; Parkinson' s Dementia; Pathway interactions; Patients; Pattern; Persons; Polymerase Chain Reaction; Prevalence; Process; Protein Binding; Proteins; Recombinant Proteins; Recombinants; Recovery; Regimen; Research; Role; Sampling; Scheme; Synapses; Time; Tissues; Trans-Activators; United States; Viral Proteins; Virion; Virus; Virus Diseases; age related cognitive disorder; aged; aging population; brain tissue; cytokine; effective therapy; immunocytochemistry; insight; macrophage; monocyte; mutant; neurotoxicity; new therapeutic target; overexpression; public health relevance; receptor; virus pathogenesis

DESCRIPTION: The goal of this proposal is to characterize neuronal mitochondria function during HIV-associated neurocognitive disorders (HAND) and the mechanisms by which HIV proteins disrupt homeostatic neuronal mitochondria fission/fusion processes. Despite 30 years of research and the advent of highly active antiretroviral therapy (HAART), HAND persist. Moreover, HAART increases life expectancy and an increasingly large pool of HIV patients are managing viral infection confounded by processes of aging. Accordingly, there is great need for therapies capable ameliorating the devastating effects of HIV infiltrating the central nervous system (CNS). HIV enter the CNS in monocytes and subsequently reproduce, infect CNS cells and initiate a battery of inflammatory cascades that ultimately result in synaptic degradation, neurodegeneration and HAND. Progress has been made, but effective treatment for HAND remains elusive. Recent studies show that HIV proteins may interfere with normal function of mitochondrial fission/fusion proteins DRP1 and Mfn2, which may affect fission/fusion and mitophagy. Our preliminary data show DRP1 is increased and Mfn1 and Mfn 2 are decreased in postmortem brain tissue of HIV infected patients, suggesting a pro-fission environment. We found that recombinant HIV negative regulatory factor (Nef) and glycoprotein (gp)120 similarly increase neuronal DRP1 and decrease neuronal Mfn1 and Mfn2. These and other studies suggest mitochondria fission/fusion may be impaired in the CNS during HIV infection; a potential contributor to HAND. Therefore, we hypothesize that HIV proteins, Nef and/or gp120 bind DRP1, Mfn1 and/or Mfn2 and disrupt homeostatic mitochondria fission/fusion processes in neurons. To explore this possibility we propose the following aims: Aim 1: To characterize DRP1, Mfn1 and Mfn2 expression patterns and their interactions with HIV proteins in postmortem brain tissues from HIV infected donors. Aim 2: To investigate the role of HIV proteins in the cellular mechanisms of mitochondrial fission/fusion dysfunction and resulting neurotoxicity. To complete Aim 1 tissue from the California NeuroAIDS consortium will be analyzed for DRP1, Mfn1 and Mfn2 expression and their interactions with HIV proteins by co-immunoprecipitation, immunoblot, real-time polymerase chain reaction (RT2PCR), enzyme-linked immunosorbent assay (ELISA) and quantitative immunohistochemistry. Aim 2 will be completed using lentiviral (LV) vectors expressing Nef, gp120, dominant negative (DN) DRP1, Mfn1 and Mfn2 in neuronal and microglial cell lines. DRP1, Mfn1 and Mfn 2 expression levels and function will be assessed via coimmunoprecipitation, immunoblot, RT2PCR, and quantitative immunocytochemistry. Completion of these studies will shed light on mitochondrial function during HIV infection. Scheme A [is that] HIV-infected microglia secrete progeny virus, cytokines and toxic viral proteins that lead to neurodegeneration and HAND. [Scheme] B [is that] proper functioning mitochondria fission/fusion processes are necessary for maintaining and constructing new neural pathways. Interference may lead to neurodegeneration via toxic viral proteins: 1. Binding neuron receptor and affecting DRP1 expression and/or 2. Binding fission/fusion machinery directly (DRP1, Mfn1, Mfn2) and altering function or half-life. These studies will provide valuable insight into neuronal mitochondria dynamics during HAND and other CNS disease, and possibly provide novel therapeutic targets.

描述：本提案的目的是表征 HIV 相关神经认知障碍 (HAND) 期间神经元线粒体的功能以及 HIV 蛋白破坏稳态神经元线粒体裂变/融合过程的机制。尽管经过 30 年的研究和高效抗逆转录病毒疗法 (HAART) 的出现，HAND 仍然存在。此外，HAART 可延长预期寿命，并且越来越多的艾滋病毒患者正在控制与衰老过程相混淆的病毒感染。因此，非常需要能够减轻HIV浸润中枢神经系统(CNS)的破坏性影响的疗法。 HIV 进入单核细胞中的 CNS，随后繁殖、感染 CNS 细胞并引发一系列炎症级联反应，最终导致突触退化、神经变性和 HAND。已经取得了进展，但 HAND 的有效治疗方法仍然难以实现。最近的研究表明，HIV蛋白可能干扰线粒体裂变/融合蛋白DRP1和Mfn2的正常功能，从而可能影响裂变/融合和线粒体自噬。我们的初步数据显示，HIV 感染患者死后脑组织中 DRP1 增加，Mfn1 和 Mfn 2 减少，表明存在促裂变环境。我们发现重组 HIV 负调节因子 (Nef) 和糖蛋白 (gp)120 同样会增加神经元 DRP1 并减少神经元 Mfn1 和 Mfn2。这些和其他研究表明，在 HIV 感染期间，中枢神经系统中的线粒体裂变/融合可能会受到损害。 HAND 的潜在贡献者。因此，我们假设 HIV 蛋白、Nef 和/或 gp120 结合 DRP1、Mfn1 和/或 Mfn2，并破坏神经元中线粒体的稳态裂变/融合过程。为了探索这种可能性，我们提出以下目标： 目标 1：表征 HIV 感染捐献者死后脑组织中的 DRP1、Mfn1 和 Mfn2 表达模式及其与 HIV 蛋白的相互作用。目标 2：研究 HIV 蛋白在线粒体裂变/融合功能障碍及由此产生的神经毒性的细胞机制中的作用。为了完成目标 1，将通过免疫共沉淀、免疫印迹、实时聚合酶链式反应 (RT2PCR)、酶联免疫吸附测定 (ELISA) 分析加州 NeuroAIDS 联盟组织的 DRP1、Mfn1 和 Mfn2 表达及其与 HIV 蛋白的相互作用。 ）和定量免疫组织化学。目标 2 将使用在神经元和小胶质细胞系中表达 Nef、gp120、显性失活 (DN) DRP1、Mfn1 和 Mfn2 的慢病毒 (LV) 载体来完成。 DRP1、Mfn1 和 Mfn 2 表达水平和功能将通过免疫共沉淀、免疫印迹、RT2PCR 和定量免疫细胞化学进行评估。这些研究的完成将揭示艾滋病毒感染期间线粒体的功能。方案 A 认为，感染 HIV 的小胶质细胞会分泌子代病毒、细胞因子和有毒病毒蛋白，从而导致神经变性和 HAND。 [方案] B [认为] 正常运作的线粒体裂变/融合过程对于维持和构建新的神经通路是必要的。干扰可能通过有毒病毒蛋白导致神经变性： 1. 结合神经元受体并影响 DRP1 表达和/或 2. 直接结合裂变/融合机制（DRP1、Mfn1、Mfn2）并改变功能或半衰期。这些研究将为手部疾病和其他中枢神经系统疾病期间的神经元线粒体动力学提供有价值的见解，并可能提供新的治疗靶点。