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

项目总结 持续免疫激活是体内HIV-1感染的决定性特征和进展的驱动力 到艾滋病末期。艾滋病毒携带者的系统免疫激活被假设为 慢性炎症性疾病的发病率较高，包括与艾滋病毒相关的神经认知 精神障碍(手)。虽然与HIV-1感染相关的重大神经系统并发症发生 在血清转换后数年，通常与进行性免疫抑制和高 病毒载量，在中枢神经系统建立病毒库时发生原发感染。此外，急性 中枢神经系统的感染被认为启动了一连串的促炎事件，导致炎症- 诱导的神经元损伤和相关的神经认知障碍，即使在现在也是明显的 联合抗逆转录病毒疗法(CART)时代。全球约有1200万人注射毒品，13% 其中包括艾滋病毒携带者。阿片类药物滥用是艾滋病毒感染的途径和有效的障碍 抗逆转录病毒疗法(ART)。然而，目前尚不清楚阿片类药物滥用是否会改变艾滋病毒的进程 发病机制，特别是与艾滋病毒相关的神经认知障碍。我们的中心假设是 阿片类药物的滥用通过失调大脑中的神经胶质细胞数量而加剧了中枢神经系统中艾滋病毒的发病。 我们的总体目标是在单核水平上利用细胞类型特定的转录信息 患者大脑样本，以表征阿片类药物使用障碍对中枢神经系统神经元和神经胶质细胞的影响， HIV感染和手。我们将确定单核基因表达的特征，并识别异常调节 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 艾滋病毒感染者和/或手部感染。我们还将进行计算分析，以确定 神经元和神经胶质细胞调节网络因阿片类药物滥用而改变。在验证组件中，我们将 从单细胞转录图谱中选择前20个目标，首先验证 用固定的脑组织进行免疫组织化学，然后选择几个顶级靶点，并使用2D和3D 培养iPS来源的神经元、小胶质细胞和星形胶质细胞以鉴定CRISPR的功能 淘汰赛。这些目标的成功完成将在1)年前产生重大的研究和临床影响 阐明阿片类药物滥用如何改变中枢神经系统中HIV/Hand的发病机制，以及2)发现候选 在阿片类药物滥用的背景下，调节艾滋病毒感染或在中枢神经系统持续存在的分子。

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.