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

描述：这项建议的目的是描述HIV相关神经认知障碍(HAND)中神经元线粒体的功能，以及HIV蛋白破坏稳态神经线粒体分裂/融合过程的机制。尽管经过了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延长了预期寿命，越来越多的艾滋病毒患者正在管理因老龄化过程而混乱的病毒感染。因此，迫切需要能够改善艾滋病毒侵入中枢神经系统(CNS)的破坏性影响的治疗方法。HIV以单核细胞的形式进入CNS，随后繁殖、感染CNS细胞，引发一连串的炎性级联反应，最终导致突触退化、神经变性和手部病变。已经取得了进展，但手部的有效治疗仍然难以捉摸。最近的研究表明，HIV蛋白可能干扰线粒体分裂/融合蛋白Drp1和Mfn2的正常功能，从而可能影响分裂/融合和有丝分裂。我们的初步数据显示，HIV感染者死后脑组织中Drp1增加，Mfn1和Mfn2减少，提示存在促进分裂的环境。我们发现，重组HIV负性调节因子(Nef)和糖蛋白(GP)120类似地增加了神经元Drp1，降低了神经元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分裂/融合可能受到损害；这是Hand的潜在贡献者。因此，我们假设HIV蛋白、Nef和/或gp120与Drp1、Mfn1和/或Mfn2结合，并破坏神经元内稳态的线粒体分裂/融合过程。为了探索这种可能性，我们提出了以下目标：目的1：研究HIV感染者死后脑组织中Drp1、Mfn1和Mfn2的表达模式及其与HIV蛋白的相互作用。目的：探讨人类免疫缺陷病毒(HIV)蛋白在线粒体分裂/融合功能障碍及其神经毒性机制中的作用。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)、酶联免疫吸附试验(ELISA)和定量免疫组织化学方法分析来自加州神经艾滋病联盟的组织中Drp1、Mfn1和Mfn2的表达及其与HIV蛋白的相互作用。目的2利用慢病毒(LV)载体在神经细胞和小胶质细胞系中表达Nef、gp120、显性阴性(DN)DRp1、Mfn1和Mfn2。将通过免疫共沉淀、免疫印迹、RT2PCR和定量免疫细胞化学来评估Drp1、Mfn1和MFn2的表达水平和功能。这些研究的完成将有助于揭示艾滋病毒感染过程中线粒体的功能。方案A[是]感染艾滋病毒的小胶质细胞分泌后代病毒、细胞因子和有毒病毒蛋白，导致神经退化和手部疾病。[方案]B[是]正常运作的线粒体分裂/融合过程对于维持和构建新的神经通路是必要的。干扰可能通过毒性病毒蛋白导致神经变性：1.结合神经元受体，影响Drp1的表达和/或2.直接结合分裂/融合机制(Drp1、Mfn1、Mfn2)，改变功能或半衰期。这些研究将对手部和其他中枢神经系统疾病过程中神经元线粒体的动力学提供有价值的见解，并可能提供新的治疗靶点。

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.