Single cell brain transcriptome changes during chronic HIV infection and opiate use in conventional mice

项目总结 本申请是针对RFA-D-21-019：单细胞阿片类药物反应而提交的 艾滋病毒(SCOCH)计划扩展的背景：慢性阿片类药物的中枢神经系统数据生成， 甲基苯丙胺和/或可卡因暴露。人类免疫缺陷病毒(HIV)可感染 大脑中的非神经细胞，特别是小胶质细胞，这些细胞充当 潜伏感染。HIV感染对非神经元和神经元功能的缺失影响 细胞，包括那些位于与认知、情绪和动机相关的大脑部位的细胞。 已知受艾滋病毒影响的相同大脑部位调节阿片类药物和其他 成瘾药物种类和阿片类药物使用障碍(OUD)在艾滋病毒感染者中更为普遍 个体比一般人群更多。此外，艾滋病毒感染和OUD相互作用， 每一种情况都有可能加剧另一种情况的严重性。感染了生态艾滋病毒的小鼠， 一种针对CD4+T细胞、巨噬细胞和小胶质细胞的改良HIV毒株，重述了 抗逆转录病毒治疗患者慢性HIV感染的主要病理生物学特征 (艺术)。在这里，我们将利用最先进的单细胞测序(ScSeq)、分子、细胞 和行为学方法来定义HIV和阿片类药物之间的细胞类型特异性相互作用 感染EcoHIV的小鼠的大脑。在特定的目标I中，我们优化了静脉注射海洛因 自我给药程序，已经在我们的实验室建立起来，用于生态艾滋病毒- 受感染的小鼠。然后我们将研究EcoHIV感染对阿片类药物表达的影响 与上瘾有关的行为。相反，我们将研究海洛因消费对 与HIV相关的EcoHIV感染小鼠认知异常的表达 人类神经认知障碍(HIV-NCI)。最后，我们将考察艺术对 成瘾和HIV-NCI相关行为异常在EcoHIV感染者中的表达 老鼠。在特定的目标II中，我们将对与阿片成瘾相关的大脑区域进行SCSeq和 HIV-NCI，来自无静脉注射阿片类药物自身史的EcoHIV小鼠 行政行为。我们将研究ART对这些小鼠的scSeq模式的影响。 ScSeq数据将被挖掘以识别大脑中感染EcoHIV的细胞，并确定 在阿片类药物中，哪些细胞对EcoHIV表现出最强的转录反应 阿片类药物经验丰富的小鼠。将使用原位杂交和免疫组织化学来验证 关键发现并确定候选基因的优先顺序，以便进行进一步研究。在具体目标三中，我们将 使用体内CRISPR-Cas9在一种细胞类型和脑中靶向AIM II中确定的优先基因 特定于地区的方式。CRISPR对优先基因的切割对表达的影响 EcoHIV感染小鼠中与成瘾和HIV-NCI相关的行为异常将是 检查过了。这一创新的研究计划可能会产生对 艾滋病毒感染和阿片类药物之间与疾病相关的相互作用。

PROJECT SUMMARY This application is submitted in response to RFA-D-21-019: Single Cell Opioid Responses in the Context of HIV (SCORCH) Program Expansion: CNS Data Generation for Chronic Opioid, Methamphetamine, and/or Cocaine Exposures. Human immunodeficiency virus (HIV) can infect nonneuronal cells in the brain, particularly microglia, with these cells acting as a reservoir of latent infection. HIV infection has deletions effects on the function of nonneuronal and neuronal cells, including those cells located in brain sites relevant to cognition, emotion and motivation. The same brain sites impacted by HIV are known to regulate the actions of opioids and other classes of addictive drugs, and opioid use disorder (OUD) is more prevalent in HIV-infected individuals than the general population. Moreover, HIV infection and OUD reciprocally interact, with each condition potentially exacerbating the severity of the other. Mice infected with EcoHIV, a modified HIV strain that targets CD4+ T cells, macrophages and microglia, recapitulates the major pathobiological features of chronic HIV infection in individuals on antiretroviral therapy (ART). Here, we will leverage state-of-the-art single cell sequencing (scSeq), molecular, cellular and behavioral approaches to define cell type-specific interactions between HIV and opioids in the brains of EcoHIV-infected mice. In Specific Aim I, we optimize the intravenous (IV) heroin self-administration procedure, already well-established in our laboratories, for use in EcoHIV- infected mice. We will then examine the effects of EcoHIV infection on the expression of opioid addiction-relevant behaviors. Conversely, we will examine the effects of heroin consumption on the expression of cognitive abnormalities in EcoHIV-infected mice relevant to HIV-associated neurocognitive impairment (HIV-NCI) in humans. Finally, will examine the effects of ART on the expression of addiction- and HIV-NCI-relevant behavioral abnormalities in EcoHIV-infected mice. In Specific Aim II, we will perform scSeq on brain regions relevant to opioid addiction and HIV-NCI, collected from EcoHIV mice with our without a history of intravenous opioid self- administration behavior. We will investigate the effects of ART on scSeq patterns in these mice. The scSeq data will be mined to identify cells in the brain infected by EcoHIV, and determine which cells show the most robust transcriptional responses to EcoHIV in opioid-naïve and opioid-experienced mice. In situ hybridization and immunohistochemistry will be used to validate key findings and prioritize candidate genes for further investigation. In Specific Aim III, we will use in vivo CRISPR-Cas9 to target prioritized genes identified in Aim II in a cell type- and brain region-specific manner. The impact of CRISPR cleavage of prioritized genes on the expression of addiction- and HIV-NCI-relevant behavioral abnormalities in EcoHIV-infected mice will be examined. This innovative program of research may yield fundamental new insights into disease-relevant interactions between HIV infection and opioid drugs.