Single cell transcriptomic and epigenomic changes during chronic HIV infection and cocaine self-administration

项目总结 本申请是根据RFA-D-21-019：艾滋病毒背景下的单细胞阿片反应提交的 (SCOCH)计划扩展：慢性阿片类药物、甲基苯丙胺和/或可卡因的CNS数据生成 曝光。我们组建了一支在艾滋病毒病毒学方面拥有世界级专业知识的杰出调查团队， 成瘾神经生物学和单细胞转录学。我们将利用最先进的单核RNA 测序(SnRNA-seq)连接到单核表观遗传学(SnATAC-seq)以定义转录和 HIV感染小鼠成瘾相关脑细胞中神经细胞和非神经细胞的表观遗传格局 强迫自己吸食可卡因。通过这种方式，我们将确定艾滋病毒的细胞类型和大脑部位 和可卡因相互作用，加剧艾滋病毒感染对大脑的负面影响，并有助于 感染者持续存在可卡因使用障碍。为了促进这一目标，我们开发了一种扩展的 获得小鼠静脉注射可卡因的自我给药程序，该程序可使小鼠对 抵抗负面后果的可卡因(意外产生的有害脚电击)。我们将利用生态艾滋病毒， 这是一种经过改造的艾滋病毒毒株，可以感染具有常规免疫能力的小鼠。铬单电池 多组ATAC和基因表达分析及多重稳健荧光原位杂交 (MerFish)将被用来识别大脑中对可卡因和/或艾滋病毒反应的细胞，具有前所未有的细胞学 和空间分辨率。我们将使用可卡因的死后脑组织来证实我们的主要发现- 有经验的艾滋病毒感染者。我们的目标将通过以下四个具体目标来实现。在……里面 目的1，我们将描述对照组和HIV感染小鼠的强迫性类可卡因摄入的特征，并确定 抗逆转录病毒治疗(ART)对这些小鼠可卡因反应的影响。在目标2中，我们将使用SnRNA-seq 和多倍体分析，以确定那些显示出最强大的转录和表观遗传可塑性的细胞 可卡因消费和艾滋病毒感染。在目标3中，我们将使用MerFish来确认和验证我们的主要SnRNA- SEQ和Multiome分析结果，并为这些细胞生成更精细的空间分辨图谱 受到可卡因和/或艾滋病毒的影响。在目标4中，我们将使用死后人脑来确认我们的主要发现 纸巾。这一极具创新性、协作性和多学科的研究计划有望产生 对艾滋病毒感染和可卡因使用之间与疾病相关的相互作用的基本新见解。

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. We have assembled an outstanding team of investigators with world-class expertise in HIV virology, addiction neurobiology, and single cell transcriptomics. We will leverage state-of-the-art single-nuclei RNA sequencing (snRNA-seq) coupled to single-nuclei epigenetics (snATAC-seq) to define the transcriptional and epigenetic landscape of neuronal and nonneuronal cells in addiction-relevant brain cells of HIV-infected mice that compulsively self-administer cocaine. In this manner we will identify cell types and brain sites in which HIV and cocaine interact to exacerbate the negative impact of HIV infection on the brain and contribute to the persistence of cocaine use disorder in infected individuals. To facilitate this goal, we have developed an extended access intravenous cocaine self-administration procedure for mice that precipitates compulsive-like response for cocaine that is resistant to negative outcome (contingently delivered noxious foot shocks). We will utilize EcoHIV, which is a modified HIV strain that can infect conventional immunocompetent mice. The Chromium Single Cell Multiome ATAC and Gene Expression assay and Multiplexed error-robust fluorescence in situ hybridization (MERFISH) will be used to identify cocaine and/or HIV-responsive cells in the brain with unprecedented cellular and spatial resolution. We will confirm our major findings using postmortem brain tissues from cocaine- experienced HIV-infected individuals. Our goals will be accomplished through the following four specific aims. In Aim 1, we will characterize compulsive-like cocaine intake in control and HIV-infected mice and determine the impact of antiretroviral treatment (ART) on cocaine response in these mice. In Aim 2, we will use snRNA-seq and Mulitome assays to identify those cells that show the most robust transcriptional and epigenetic plasticity to cocaine consumption and HIV infection. In Aim 3, we will use MERFISH to confirm and validate our major snRNA- seq and Multiome assay findings, and generate more refined spatially resolved maps of those cells most impacted by cocaine and/or HIV. In Aim 4, we will confirm our major findings using post-mortem human brain tissues. This highly innovative, collaborative and multidisciplinary program of research promises to yield fundamental new insights into disease-related interactions between HIV infection and cocaine use.