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

描述(申请人提供)：2008年，全球有3340万艾滋病毒/艾滋病患者，新增感染者270万人，相关死亡200万人。在美国，2008年有140万人感染艾滋病毒/艾滋病，新增感染者5.5万人，相关死亡2.5万人。某些弱势群体承担着不同的负担，包括全球的妇女和儿童，以及美国的美国黑人(BA)和西班牙裔少数族裔。2006年，尽管仅占总人口的13%，BAS却获得了46%的新感染病例。在女性中，bas分担的负担甚至更不成比例，2006年感染了所有新的女性艾滋病毒感染者的61%。在2006年男性感染者中，76%是男男性接触者。在这些弱势群体中，使用包括安非他明、鸦片类药物、酒精和可卡因在内的药物与高危性行为有关，并加速艾滋病毒的进展和死亡。药物使用也是血清转换的独立危险因素，并预测疾病进展、艾滋病的发展和艾滋病死亡率。因此，了解药物滥用对艾滋病毒感染的影响日益重要，特别是因为它影响到弱势人群和治疗发展[1-10]。一些证据表明糖原合成酶激酶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在有或没有药物滥用的情况下究竟如何或是否直接影响艾滋病毒感染的研究很少，特别是关于关键的嗜艾滋病毒的外周CD4+T细胞和巨噬细胞。因此，这项建议将调查一个统一的假设，即GSK3激活增加内吞作用，以增强辅受体介导的人类外周血CD4+T细胞和巨噬细胞的HIV感染，这就是可卡因和苯丙胺增强这些细胞类型的HIV感染的机制。首先，我们将确定GSK3转基因过表达或敲除是否直接影响HIV对CD4+T细胞和巨噬细胞的感染。接下来，我们将利用荧光、免疫细胞化学和免疫印迹方法检测内吞作用，并下调Rab5的siRNA，以确定GSK3是否通过增强Rab5介导的内吞作用而增加了HIV在CD4+T细胞和巨噬细胞中的感染。在目标2中，使用类似的方法，我们将首先确定Meth或可卡因处理是否增加了这些细胞类型中GSK3的表达和活性，以及内吞作用。我们将进一步确定Meth或可卡因是否会增加这些细胞的HIV感染，以及是否可以通过操纵GSK3和/或Rab5的表达来消除这些影响。在所有这些研究中，无论有没有可卡因和蛋氨酸，我们都将确定Gsk3抑制化合物锂和AR-A014418能否改善Gsk3对HIV感染CD+T细胞和巨噬细胞的影响，以确定Gsk3抑制是否是降低HIV疾病病毒载量的潜在可行的治疗策略。因此，这些研究将为调控高度亲HIV的外周免疫细胞感染HIV的分子机制提供新的见解，为治疗HIV疾病合并药物滥用寻找新的治疗方法。 与公共卫生相关：该项目探索新的机制理论，可能有助于解释艾滋病毒是如何进入人类免疫细胞导致艾滋病毒疾病的。更好地了解这些关于艾滋病毒如何导致艾滋病毒涉及的主要细胞类型感染的疾病机制，将有助于确定预防干预治疗艾滋病毒和艾滋病的额外治疗目标。

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.