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: 8542439
• 负责人: Jerel Adam Fields
• 金额: $ 4.92万
• 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/8542439
• 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; Interleukins; 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：该提案的目的是表征与HIV相关的神经认知障碍（手）期间神经元线粒体功能以及HIV蛋白破坏稳态神经元线粒体裂变/融合过程的机制。尽管进行了30年的研究以及高度活跃的抗逆转录病毒疗法（HAART）的出现，但仍在手中。此外，HAART增加了预期寿命，越来越多的HIV患者正在管理因衰老过程而混淆的病毒感染。因此，非常需要能够改善艾滋病毒渗透中枢神经系统（CNS）的毁灭性影响的疗法。 HIV进入单核细胞中的中枢神经系统，随后再现，感染CNS细胞并引发一系列炎症级联反应，最终导致突触降解，神经变性和手。已经取得了进展，但是有效的手处理仍然难以捉摸。最近的研究表明，HIV蛋白可能会干扰线粒体裂变/融合蛋白DRP1和MFN2的正常功能，这可能会影响裂变/融合和线粒体。我们的初步数据表明，DRP1增加，MFN1和MFN 2在艾滋病毒感染患者的死后脑组织中降低，这表明是促膜的环境。我们发现重组HIV阴性调节因子（NEF）和糖蛋白（GP）120类似地增加神经元DRP1并降低神经元MFN1和MFN2。这些和其他研究表明，在HIV感染期间，中枢神经系统可能会损害线粒体裂变/融合。潜在的贡献者。因此，我们假设HIV蛋白NEF和/或GP120结合DRP1，MFN1和/或MFN2，并破坏神经元中的稳态线粒体裂变/融合过程。为了探讨这种可能性，我们提出以下目的：目标1：表征DRP1，MFN1和MFN2表达模式及其与HIV感染供体的后脑组织中的HIV蛋白相互作用。目标2：研究HIV蛋白在线粒体裂变/融合功能障碍和产生的神经毒性的细胞机制中的作用。将分析来自加利福尼亚神经辅助联盟的AIM 1组织，将通过共免疫沉淀，免疫印迹，实时聚合酶链反应（RT2PCR），酶 - 链接免疫体的抗衡剂（Elisotiant andiserment）（Elissoicer）（Elissoy）（Elissoy）（Elissoy）（Elissoy）（ELISAY）（ELISARINIC）（ELISARINIC）（ELISAIN）（ELISAREM）（ELISARINIC）（ELISARINIC）（elisa）和Quantionsay（Elissa）（Elissa）（Elissa）（elisa）和量化。 AIM 2将使用表达NEF，GP120的慢病毒（LV）向量完成，在神经元和小胶质细胞系中的主导负（DN）DRP1，MFN1和MFN2。 DRP1，MFN1和MFN 2表达水平和功能将通过共免疫沉淀，免疫印迹，RT2PCR和定量免疫细胞化学评估。这些研究的完成将揭示HIV感染期间的线粒体功能。方案A [是]感染了HIV的小胶质细胞分泌后代病毒，细胞因子和有毒病毒蛋白，导致神经退行性变性和手。 [方案] B [是]适当的线粒体裂变/融合过程对于维持和构建新的神经途径是必要的。干扰可能导致通过有毒病毒蛋白的神经退行性变化：1。结合神经元受体，并影响DRP1表达和/或2。直接（DRP1，MFN1，MFN2）和改变功能或更改功能或半寿命。这些研究将为手和其他中枢神经系统疾病期间神经元线粒体动力学提供宝贵的见解，并可能提供新颖的治疗靶标。