Single Cell Transcriptomics of the Opioid Use Disorder and HIV Syndemic in the Human Brain

项目摘要 持续的免疫激活是体内HIV-1感染的定义特征，也是进展的驱动力 到晚期艾滋病HIV感染者的全身免疫激活被假设为 慢性炎症性疾病的发病率较高，包括艾滋病毒相关的神经认知功能障碍。 疾病（手）。虽然与HIV-1感染相关的严重神经系统并发症发生 血清转换后10年，通常与进行性免疫抑制和高 病毒载量，CNS中病毒储库的建立与原发性感染一起发生。此外，急性 CNS中的感染被认为启动了导致炎症的促炎事件级联- 诱导的神经元损伤和相关的神经认知障碍，即使在目前也很明显， 联合抗逆转录病毒治疗（cART）时代。全球约有1200万人注射毒品，13% 其中有艾滋病毒感染者。阿片类药物滥用是艾滋病毒感染的一种途径，也是有效预防艾滋病毒感染的一个障碍。 抗逆转录病毒疗法（ART）。然而，目前还不清楚阿片类药物滥用是否会改变艾滋病毒的病程。 发病机制，特别是对艾滋病毒相关的神经认知障碍。我们的核心假设是， 阿片类药物滥用通过调节脑中的神经胶质细胞群而加剧CNS中的HIV发病机制。 我们的总体目标是在单个核水平上利用细胞类型特异性转录组信息， 患者脑样品以表征阿片样物质使用障碍对CNS神经元和神经胶质细胞的影响， 艾滋病毒感染和手。我们将描述单个核基因表达的特征， 与阿片类药物滥用相关的神经元和神经胶质细胞群体中的基因调控网络， HIV感染者和/或HANDs患者。我们还将进行计算分析，以确定 阿片类药物滥用改变了神经元和神经胶质细胞的调节网络。在验证组件中，我们将 从单细胞转录组学谱中选择前20个靶标，首先用 使用固定的脑组织进行免疫组织化学，然后选择几个顶级靶标，并使用2D和3D iPS衍生的神经元、小胶质细胞和星形胶质细胞的培养物，以表征CRISPR的功能 漂亮这些目标的成功完成将对研究和临床产生重大影响， 阐明阿片类药物滥用如何改变CNS中的HIV/HAND发病机制，以及2）发现候选药物 在阿片类药物滥用的情况下，调节HIV感染或在CNS中持续存在的分子。

PROJECT SUMMARY Persistent immune activation is the defining feature of HIV-1 infection in vivo and a driver of progression to end-stage AIDS. Systemic immune activation in people living with HIV has been hypothesized to account for higher incidence of chronic inflammatory diseases, including HIV-associated neurocognitive disorders (HAND). While significant neurological complications associated with HIV-1 infection occur years after seroconversion and is commonly coincident with progressive immunosuppression and high viral loads, establishment of a virus reservoir in the CNS occurs with primary infection. Furthermore, acute infection in the CNS is thought to initiate a cascade of pro-inflammatory events that result in inflammation- induced neuronal injury and associated neurocognitive disorders that are evident even in the present combination antiretroviral therapy (cART) era. Approximately 12 million people inject drugs globally, 13% of these are people living with HIV. Opioid misuse is a route of HIV acquisition and a barrier to effective antiretroviral therapy (ART). However, it is unclear whether opioid misuse changes the course of HIV pathogenesis, especially on HIV-associated neurocognitive disorders. Our central hypothesis is that opioid misuse exacerbates HIV pathogenesis in the CNS by dysregulating the glial population in the brain. Our overall objective is to exploit cell type specific transcriptomic information at the single nuclei level from patient brain samples to characterize the effects of opioid use disorder on CNS neuronal and glial cells, HIV infection and HANDs. We will characterize single nuclei gene expression and identify dysregulated gene regulatory networks in each of the neuronal and glial populations associated with opioid misuse in HIV infected individuals and/or with HANDs. We will also perform computational analysis to identify neuronal and glial cell regulatory networks altered by opioid misuse. In the validation component, we will select the top 20 targets from single cell transcriptomics profiles, first validating with immunohistochemistry with fixed brain tissue, then selecting a few top targets and using 2D and 3D cultures of iPS derived neurons, microglia and astrocytes to characterize functionality with CRISPR knockout. Successful completion of these aims will have significant research and clinical impact by 1) elucidating how opioid misuse alters HIV/HAND pathogenesis in the CNS, and 2) discovering candidate molecules to regulate HIV infection or persistence in the CNS in the context of opioid misuse.