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

项目摘要 本申请是为了响应RFA-D-21-019：单细胞阿片样物质反应， HIV（SCORCH）计划扩展的背景：慢性阿片类药物的CNS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 脑中的非神经元细胞，特别是小胶质细胞，这些细胞充当神经元的储存库。 潜伏感染HIV感染对非神经元和神经元的功能有缺失效应 细胞，包括那些位于与认知、情感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药物的作用。 类成瘾药物，阿片类药物使用障碍（OUD）在艾滋病毒感染者中更为普遍， 个人比一般人。此外，艾滋病毒感染和OUD感染相互作用， 每种情况都可能加重另一种情况的严重程度。感染EcoHIV的小鼠， 一种针对CD 4 + T细胞、巨噬细胞和小胶质细胞的改良HIV毒株， 接受抗逆转录病毒治疗的慢性HIV感染者的主要病理生物学特征 （ART）。在这里，我们将利用最先进的单细胞测序（scSeq），分子，细胞 和行为方法来定义艾滋病毒和阿片类药物之间的细胞类型特异性相互作用， HIV感染小鼠的大脑。在特定目标I中，我们优化了静脉注射（IV）海洛因 自我管理程序，已经在我们的实验室建立，用于EcoHIV- 感染的老鼠然后，我们将研究EcoHIV感染对阿片样物质表达的影响。 成瘾相关行为。相反，我们将研究海洛因消费对 EcoHIV感染小鼠中与HIV相关的认知异常的表达 神经认知障碍（HIV-NCI）。最后，将研究ART对 EcoHIV感染者中成瘾和HIV-NCI相关行为异常的表达 小鼠在Specific Aim II中，我们将对与阿片类药物成瘾相关的大脑区域进行scSeq， HIV-NCI，收集自EcoHIV小鼠，无静脉注射阿片类药物自我治疗史。 行政行为我们将研究ART对这些小鼠中scSeq模式的影响。 scSeq数据将被挖掘以识别被EcoHIV感染的大脑细胞，并确定 哪些细胞在阿片类药物初治和初治中对EcoHIV表现出最强的转录应答， 阿片类药物经验丰富的小鼠原位杂交和免疫组织化学将用于验证 关键发现，并优先考虑候选基因进行进一步研究。在具体目标三中，我们将 使用体内CRISPR-Cas9靶向Aim II中鉴定的优先基因， 特定区域的方式。CRISPR切割优先基因对表达的影响 在EcoHIV感染的小鼠中，成瘾和HIV-NCI相关的行为异常将被 考察这项创新的研究计划可能会产生根本的新见解， 艾滋病毒感染与阿片类药物之间的疾病相关相互作用。

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.