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

描述（由申请人提供）：2008年，全世界有3340万人感染艾滋病毒/艾滋病，有270万新感染者，200万人因此死亡。2008年，美国有140万人感染艾滋病毒/艾滋病，其中5.5万人新感染，2.5万人死亡。某些处境不利或弱势群体承担着这一负担的不同份额，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，尽管仅占人口的13%，但新感染人数却占46%。在女性中，艾滋病患者的负担更大，占2006年所有女性艾滋病毒新感染病例的61%。2006年男性感染者中，76%是男男性行为者。在这些弱势群体中，包括安非他明、阿片类药物、酒精和可卡因在内的药物使用与高危性行为有关，并加速艾滋病毒的进展和死亡。药物使用也是血清转化的独立危险因素，并可预测疾病进展、艾滋病发展和艾滋病死亡率。因此，了解合并症药物滥用对艾滋病毒感染的影响变得越来越重要，特别是因为它影响弱势群体和治疗发展[1-10]。一些证据暗示糖原合成酶激酶3 （GSK3）可能是外周HIV感染的调节因子，GSK3也可能介导一些滥用药物的作用，如可卡因和甲基苯丙胺。然而，关于GSK3究竟如何或是否直接影响HIV感染（伴或不伴药物滥用）的研究很少，特别是对关键的HIV外周CD4+ T细胞和巨噬细胞的影响。因此，本研究将探讨GSK3激活增加内吞作用以增强外周来源的人CD4+ T细胞和巨噬细胞的共受体介导的HIV感染的统一假设，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GSK3转基因过表达或敲低是否直接影响CD4+ T细胞和巨噬细胞的HIV感染。接下来，利用荧光、免疫细胞化学和western blot检测Rab5的内吞作用和siRNA敲低，我们将确定GSK3是否通过增强Rab5介导的内吞作用而增加CD4+ T细胞和巨噬细胞的HIV感染。在Aim 2中，使用类似的方法，我们将首先确定甲基苯丙胺或可卡因治疗是否会增加这些细胞类型中的GSK3表达和活性以及内吞作用。我们将进一步确定冰毒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GSK3和/或Rab5的表达来消除。在所有这些研究中，无论是否使用可卡因和冰毒，我们将确定GSK3对CD4+ T细胞和巨噬细胞感染的影响是否可以通过GSK3抑制化合物锂和AR-A014418来改善，以确定GSK3抑制是否是一种潜在可行的降低HIV疾病病毒载量的治疗策略。因此，这些研究将为研究高嗜HIV性外周免疫细胞调控HIV感染的分子机制提供新的见解，并为治疗伴药物滥用的HIV疾病提供新的治疗方法。

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.