Cerebral organoid and IPSC derived microglia: Modeling of HIV and methamphetamine co-morbidity

摘要： 估计约有130万成人和青少年感染人类免疫缺陷病毒（HIV） 其中15%的人未被确诊。联合抗逆转录病毒治疗（cART）的启动， 将艾滋病从一种致命的疾病转变为一种慢性和可控制的疾病。艾滋病毒感染者的存在 中枢神经系统（CNS）中的小胶质细胞和巨噬细胞储库以及抗- 逆转录病毒药物在cART后穿过血脑屏障可能是HIV相关疾病持续存在的因素。 神经认知障碍（HAND）。滥用药物，如强效精神兴奋剂甲基苯丙胺（Meth） 据估计，全世界有3000万人滥用这种药物，这进一步降低了cART的疗效， 与HIV结合加重CNS病理。因此，该领域知识的重大差距是为了更好地 了解神经发病机制和病因的临床结果观察艾滋病毒+患者的依赖 关于滥用冰毒目前的拨款提案集中在开发一种基于人类诱导多能干细胞的 脑类器官模型，以研究HIV/Meth相互作用期间潜在的脑功能障碍。脑 类器官是三维的“迷你大脑”，可以自我组织和重演许多里程碑式的事件， in in vivo体内brain脑development发展.我们最近的研究是建立一个新的类器官模型， 控制小胶质细胞比例和类器官形成的微环境以再现脑中的变化 在各种条件下发挥功能（例如病毒和病毒感染诱导的炎症和突触修剪） 使得这个模型非常适合我们的研究。这一提议的中心假设是， 与HIV结合会导致严重的神经元损伤。含有小胶质细胞的脑 类器官模型，细胞外囊泡生物学，单细胞RNA测序，CRISPR/Cas9介导的基因编辑 将破译新的细胞间和分子机制和途径，神经元异常的基础， 功能和连接造成的艾滋病毒感染。我们将在三个具体目标下研究这一假设： 具体目标1将检查神经元功能，突触和线粒体扰动在艾滋病毒/甲基 在3D含小胶质细胞的脑类器官模型中的相互作用;具体目标2将研究 在HIV/甲氨蝶呤条件下处理的小胶质细胞类器官中的细胞外囊泡;而特异性目标3将 描述HIV感染诱导的神经元损伤的分子机制，并进一步评估 我们的小胶质细胞类器官模型作为一个可靠的工具，以确定分子签名的手。我们提出的 实验将破译分子机制，新的信号事件和分子合作伙伴的基础， 手神经元损伤。通过一系列的实验，我们的目标是提出一个易于扩展和 研究HAND发病机制的可重复模型。从这些研究中获得的数据可用于设计 控制艾滋病毒感染的新疗法。

Abstract: An estimated ~1.3 million adults and adolescents are living with human immunodeficiency virus (HIV) infection in the USA of which 15% of them undiagnosed. The initiation of combination antiretroviral therapy (cART) has transformed AIDS from a fatal illness into a chronic and manageable disease. The presence of HIV infected microglia and macrophage reservoirs in the central nervous system (CNS) and variable penetration of anti- retroviral drugs across the blood brain barrier after cART are likely factors for the persistence of HIV associated neurocognitive disorder (HAND). Drugs of abuse such as the potent psychostimulant methamphetamine (Meth) that is abused by an estimated 30 million people in the world further minimize the efficacy of cART and in conjunction with HIV exacerbate CNS pathology. Thus, significant gap in knowledge in the field is to better understand the neuropathogenesis and the etiology of clinical outcomes observed in HIV+ patient’s dependent on Meth abuse. The current grant proposal focuses on developing a human induced pluripotent stem cell based cerebral organoid model to investigate underlying brain dysfunction during HIV/Meth interaction. Cerebral organoids are 3-dimensional “mini brains” that can self-organize and recapitulate many milestone events seen in in vivo brain development. Our recent study on generating a novel organoid model with the feasibility in controlling the microglia ratio and microenvironment of organoid formation to recapitulate changes in brain functions under various conditions (e.g. virus and viral infection-induced inflammation and synaptic pruning) makes this model ideal for our proposed studies. The central hypothesis of this proposal is that Meth in conjunction with HIV causes significant neuronal damage. The combination of microglia-containing cerebral organoid model, extracellular vesicle biology, single-cell RNA-sequencing, CRISPR/Cas9-mediated gene editing will decipher novel intercellular and molecular mechanisms and pathways that underlie abnormalities in neuronal functions and connectivity caused by HIV infection. We will investigate this hypothesis under three specific aims: Specific Aim 1 will examine neuronal function, synaptic and mitochondrial perturbations during HIV/meth interactions in 3D microglia-containing cerebral organoid model; Specific Aim 2 will investigate the role of extracellular vesicles in microglial organoids treated under HIV/meth conditions; and Specific Aim 3 will characterize the molecular mechanisms underlying HIV infection-induced neuronal injury and further evaluate our microglial organoid model as a reliable tool to identify molecular signatures of HAND. Our proposed experiments will decipher molecular mechanisms, novel signaling events and molecular partners underlying the neuronal injury in HAND. Through the series of the experiments we aim to present an easily scalable and reproducible model to study HAND pathogenesis. The data obtained from these studies can be used to design novel therapeutics to control HIV infection.