Effects of HIV-1 Tat protein and methamphetamine on VMAT2-mediated dopamine transmission in the context of neuroHIV and drug abuse

项目总结 全世界有超过3800万人感染了人类免疫缺陷病毒(HIV) 这仍然是一个全球公共卫生问题。尽管广泛使用有效的组合 抗逆转录病毒疗法(CART)，高达70%的艾滋病毒阳性患者患有认知和行为缺陷 统称为HIV相关性神经认知障碍(HAND)。尽管急性病毒复制已经 在HIV感染早期用CART得到很好的控制，长期接触HIV-1病毒蛋白在 尽管CART的出现，中枢神经系统仍会导致多巴胺能缺陷和神经认知障碍。物质 滥用甲基苯丙胺(冰毒)已被证明会增加手和 加剧了它的严重性。在病毒蛋白中，HIV-1转录反式激活因子(TAT)蛋白起着重要的作用 HIV感染早期病毒复制的重要作用及其病理生理效应 手的发育。多巴胺(DA)转运体(DAT)将细胞外多巴胺(DA)转运到 突触终末的胞浆间隙，而囊泡单胺转运体2(VMAT2)转运 胞浆中的DA进入突触小泡，因此DA转运体和VMAT2对正常的DA至关重要 动态平衡。我们已发表的研究表明，TAT蛋白增加了细胞外多巴胺 然而，通过直接抑制DAT浓度，TAT诱导抑制 VMAT2介导的DA释放/摄取仍未被探索。我们假设HIV-1TAT通过 独特的结合位点，干扰VMAT2调节网络，该网络通常维持集中的DA运输 并增强冰毒对VMAT2功能的影响，导致DA相关的神经精神障碍 在手中的显著特征。研究单个TAT蛋白使我们能够确定TAT结合的靶标和 开发治疗方法以防止TAT介导的神经损伤。建议的目的是 研究是为了了解VMAT2介导的DA调节失调的分子机制 HIV-1 TAT和冰毒诱导的传播及新化合物的潜在预防作用 手中的。拟议调查的具体目标是：(1)通过计算 建模和实验验证，识别人VMAT2上TAT的识别结合口袋， 甲氨蝶呤或新型VMAT2抑制剂，并探索这些抑制剂与TAT和冰毒的潜在相互作用， (2)促进VMAT2在TAT和甲基失调的DA系统中的病理生理作用；(3) 使用药理学和遗传学方法作为生物探针执行概念验证研究 确定其在同时吸食冰毒患者中的治疗应用潜力。

PROJECT SUMMARY More than 38 million people are living with Human Immunodeficiency Virus (HIV) infection worldwide which continues to be a global public health problem. Despite the widespread use of efficacious combination of antiretroviral therapy (cART), up to 70% of HIV-positive individuals suffer from cognitive and behavioral deficits collectively known as HIV associated neurocognitive disorders (HAND). Although acute viral replication has been well controlled with cART in the early stage of HIV infection, long-term HIV-1 viral protein exposure within the CNS causes dopaminergic deficits and neurocognitive impairments despite the advent of cART. Substance abuse such as methamphetamine (METH) have been shown to increase the incidence of HAND and exacerbate its severity. Among the viral proteins, HIV-1 transactivator of transcription (Tat) protein plays a significant role in viral replication in the early stage of HIV infection and the pathophysiological effects on development of HAND. Dopamine (DA) transporter (DAT) transports the extracellular dopamine (DA) into cytosolic space of the synaptic terminals, whereas the vesicular monoamine transporter2 (VMAT2) transports the cytosolic DA into synaptic vesicles, whereby both DA transporter and VMAT2 are critical for normal DA homeostasis. Our published studies have demonstrated that Tat protein increases extracellular DA concentration by directly inhibiting DAT, however, the cellular mechanisms underlying Tat-induced inhibition of VMAT2-mediated DA release/uptake remains unexplored. We hypothesize that HIV-1 Tat, acting via the unique binding sites, perturbs the VMAT2 regulatory network that normally sustains concentrative DA transport and potentiates METH’s effect on VMAT2 function, resulting in DA-linked neuropsychiatric dysfunction prominently featured in HAND. Studying single Tat protein allow us to identify targets for Tat binding and develop therapeutic approaches to prevent Tat-mediated neurological damages. The purpose of the proposed investigation is to understand the molecular mechanisms of dysregulation of VMAT2-mediated DA transmission induced by HIV-1 Tat and METH and explore the potential of novel compounds for the prevention of HAND. The specific aims to be pursued in the proposed investigation are: (1) through computational modeling and experimental validation, identify the recognition binding pockets on human VMAT2 for Tat, METH, or novel VMAT2 inhibitors and explore the potential interactions of the inhibitors with Tat and METH, (2) accelerate the pathophysiological roles of VMAT2 in Tat- and METH-dysregulated DA system, and (3) perform proof-of-concept studies using pharmacological and genetic approaches as biological probes to establish their potential for therapeutic application in HAND in concurrent METH users.