GSK3, endocytosis, and enhanced HIV infection: abused drugs and novel therapies

描述（由申请人提供）：2008年，全球有3340万人感染艾滋病毒/艾滋病，270万人新感染，200万人死亡。在美国，2008年有140万人感染艾滋病毒/艾滋病，55，000人新感染，25，000人死亡。某些处境不利或脆弱的群体承担着不同的负担，包括全球的妇女和儿童，以及美国的黑人和西班牙裔少数民族。2006年，尽管BAs仅占人口的13%，但却获得了46%的新感染。在女性中，学士学位获得者承担的负担更大，2006年占所有新感染艾滋病毒的女性的61%。在2006年的男性感染者中，76%是男男性行为者。在这些弱势群体中，毒品使用包括安非他明，鸦片制剂，酒精和可卡因，与高风险性行为有关，并加速艾滋病毒的进展和死亡率。药物使用也是血清转换的独立危险因素，并可预测疾病进展、AIDS发展和AIDS死亡率。因此，了解共病药物滥用对艾滋病毒感染的影响越来越重要，特别是因为它影响到弱势群体和治疗发展[1-10]。一些证据表明糖原合成酶激酶3（GSK 3）可能是外周HIV感染的调节因子，GSK 3也可能介导一些滥用药物（如可卡因和甲基苯丙胺）的作用。然而，很少有研究确切地如何或是否GSK 3直接影响HIV感染与或不合并药物滥用，特别是关于关键的HIV嗜性外周CD 4 + T细胞和巨噬细胞。因此，本提案将研究统一的假设，即GSK 3激活增加内吞作用，以增强辅助受体介导的外周来源的人CD 4 + T细胞和巨噬细胞的HIV感染，这是可卡因和安非他明增强这些细胞类型的HIV感染的机制。首先，我们将确定GSK 3转基因过表达或敲低是否直接影响CD 4 + T细胞和巨噬细胞的HIV感染。接下来，使用荧光，免疫细胞化学和蛋白质印迹分析的内吞作用，和siRNA敲低Rab 5，我们将确定是否GSK 3增加HIV感染的CD 4 + T细胞和巨噬细胞通过增强Rab 5介导的内吞作用。在目标2中，使用类似的方法，我们将首先确定甲基或可卡因处理是否增加这些细胞类型中的GSK 3表达和活性以及内吞作用。我们将进一步确定甲基或可卡因是否会增加这些细胞的HIV感染，以及这些影响是否可以通过操纵GSK 3和/或Rab 5表达来消除。在所有这些研究中，有和没有可卡因和甲基，我们将确定GSK 3对CD 4 + T细胞和巨噬细胞的HIV感染的影响是否可以通过GSK 3抑制化合物锂和AR-A014418来改善，以确定GSK 3抑制是否是一种潜在可行的治疗策略，用于减少HIV疾病中的病毒载量。因此，这些研究将提供新的见解，调节HIV感染的高度HIV嗜性外周免疫细胞的分子机制，以确定新的治疗HIV疾病与共病药物滥用。 公共卫生相关性：该项目探索新的机制理论，可能有助于解释艾滋病毒如何进入人体免疫细胞，导致艾滋病毒疾病。更好地了解这些疾病机制，了解艾滋病毒如何引起与艾滋病毒相关的主要细胞类型的感染，将有助于确定用于预防性干预治疗艾滋病毒和艾滋病的其他治疗靶点。

DESCRIPTION (provided by applicant): Worldwide 33.4 million people lived with HIV/AIDS in 2008, with 2.7 million new infections, and 2 million related deaths. In the US, 1.4 million people had HIV/AIDS in 2008, with 55,000 new infections, and 25,000 related deaths. Certain disadvantaged or vulnerable populations bear a disparate share of this burden, including women and children globally, and Black American (BA) and Hispanic minorities in USA. BAs acquired 46% of new infections in 2006, despite comprising only 13% of the population. Among females, BAs share an even more disproportionate burden, acquiring 61% of all new female HIV infections in 2006. For 2006 male infections, 76% were MSM. In these vulnerable groups, drug use including amphetamines, opiates, alcohol, and cocaine, is associated with high-risk sexual behavior, and accelerates progression of and mortality from HIV. Drug use is also an independent risk factor for seroconversion, and predictive of disease progression, development of AIDS, and AIDS mortality. Thus it is increasingly important to understand impacts of comorbid drug abuse on HIV infection, particularly as it affects vulnerable populations and therapy development [1-10]. Some evidence implicates glycogen synthase kinase 3 (GSK3) as a possible regulator of peripheral HIV infection, and GSK3 also may mediate effects of some abused drugs, such as cocaine and methamphetamine. However there has been little investigation of exactly how or whether GSK3 directly affects HIV infection with and without comorbid drug abuse, particularly as regards key HIV tropic peripheral CD4+ T cells and macrophages. Hence, this proposal will investigate the unifying hypothesis that GSK3 activation increases endocytosis to enhance coreceptor mediated HIV infection of peripherally derived human CD4+ T cells and macrophages, and that this is the mechanism by which cocaine and amphetamines enhance HIV infection of these cell types. First we will determine if GSK3 transgene overexpression or knockdown directly affects HIV infection of CD4+ T cells and macrophages. Next, using fluorescent, immunocytochemical, and western blot assays for endocytosis, and siRNA knockdown of Rab5, we will determine whether GSK3 increases in HIV infection in CD4+ T cells and macrophages by enhancing Rab5-mediated endocytosis. In Aim 2, using similar approaches, we will first determine whether Meth or cocaine treatment increases GSK3 expression and activity, and endocytosis, in these cell types. We will further determine whether Meth or cocaine increase HIV infection of these cells, and whether these effects can be abrogated by manipulating GSK3 and/or Rab5 expression. In all of these studies, with and without cocaine and Meth, we will determine whether GSK3's effects on HIV infection of CD4+ T cells and macrophages can be ameliorated by the GSK3-inhibiting compounds lithium and AR-A014418, to determine whether GSK3 inhibition is a potentially viable therapeutic strategy for reducing viral load in HIV disease. Thus these studies will provide new insights into the molecular mechanisms regulating HIV infection of highly HIV-tropic peripheral immune cells, to identify new therapies for treating HIV disease with comorbid drug abuse. PUBLIC HEALTH RELEVANCE: This project explores new mechanistic theories that may help explain how HIV enters human immune cells to cause HIV disease. Better understanding of these disease mechanisms concerning how HIV causes infection of principal cell types involved in HIV, will help identify additional therapeutic targets for preventative intervention to treat HIV and AIDS.