Modeling drugs of abuse-HIV interactions using iPSC-derived human cerebral organoids

摘要 感染艾滋病毒的小胶质细胞释放细胞因子、趋化因子和病毒基因产物，如gp120和tat， 对神经元有毒性。在这项提议中，我们将使用人类诱导的多能干细胞(IPSCs)来产生 皮质和多巴胺能神经元以及小胶质细胞，它们将被培养成有机体来模拟HIV药物 滥用相互作用结合深入的基因组方法和电生理学。我们展示了 电生理结果表明，我们的器官中神经元的分化水平 可与体内和体外制剂相媲美，是科学上可以找到的最好的制剂之一 文学。确定HIV表达的候选宿主调控因子和HIV诱导的组织损伤的介导者 和疾病的进展，我们将使含有小胶质细胞的脑器官暴露于甲基苯丙胺 兴奋剂(冰毒)和吗啡(鸦片类药物)，这是两类滥用药物的成员 在艾滋病毒/艾滋病患者(PLWHA)中更为普遍。脑有机化合物会暴露在有毒物质中 艾滋病毒产品，如TAT、gp120、滥用药物和联合抗逆转录病毒疗法(CART)。在一些 我们还将在实验中将IPSC来源的星形胶质细胞加入含有小胶质细胞的脑器官中，以 测试星形胶质细胞在神经保护和神经退行性变中的作用。我们将进行单细胞基因 药物滥用、HIV产物和CART和WE对iPSC衍生有机化合物的表达谱 将使用先进的系统生物学策略来生成可测试的机械假说 神经HIV神经退行性变和组织损伤的发病机制(目标1)和确定候选调节因子 这可能有助于阐明HIV前病毒潜伏期和重新激活的调节(AIM 2)。在初步研究中，这种计算实验方法使我们能够确定候选司机 与神经性艾滋病毒和包括阿尔茨海默氏症在内的神经退行性疾病相关的基因表达变化 疾病、吸毒和酗酒。这些发现支持了一个重要的假设，即解剖 中枢神经系统的基因调控网络将为新基因的鉴定铺平道路 改善PLWHA和PLWHA神经心理功能的机制假说和可用药靶点 药物滥用共病。

Summary HIV-infected microglia release cytokines, chemokines and viral gene products, such as gp120 and tat, that are toxic to neurons. In this proposal, we will use human induced pluripotent stem cells (iPSCs) to generate cortical and dopaminergic neurons as well as microglia, which will be cultured as organoids to model HIV-drug abuse interactions in conjunction with in-depth genomic approaches and electrophysiology. We show electrophysiological results that demonstrate that the levels of differentiation of the neurons in our organoids are comparable to in vivo and ex vivo preparations and are among the best that can be found in the scientific literature. To identify candidate host regulators of HIV expression and mediators of HIV-induced tissue damage and disease progression, we will expose microglia-containing cerebral organoids to methamphetamine (METH), a stimulant, and morphine, an opiate, which are members of two of the classes of drugs of abuse that are more prevalent among People Living with HIV/AIDS (PLWHA). Cerebral organoids will be exposed to toxic HIV products such as Tat, gp120, drugs of abuse and combination antiretroviral therapy (cART). In some experiments we will also incorporate iPSC-derived astrocytes into microglia-containing cerebral organoids to test the role of astrocytes in neuroprotection and neurodegeneration. We will carry out single-cell gene expression profiling of iPSC-derived organoids exposed to drugs of abuse, HIV products, and cART and we will employ an advanced systems biology strategy to generate testable mechanistic hypotheses on the pathogenesis of neurodegeneration and tissue damage in neuroHIV (Aim 1) and identify candidate regulators of the LTR promoter that may shed light on the regulation of latency and reactivation of the HIV provirus (Aim 2). In preliminary studies, this computational experimental approach allowed us to identify candidate drivers of gene expression changes associated with neuroHIV and neurodegenerative diseases, including Alzheimer’s disease and drug and alcohol abuse. These findings support the overarching hypothesis that dissection of the gene regulatory network of the central nervous system will pave the way for the identification of novel mechanistic hypotheses and druggable targets to improve neuropsychological functioning of PLWHA and substance abuse comorbidity.