Modeling CNS dynamics in HIV infection and cannabinoids with forebrain organoids

项目摘要 艾滋病毒相关的神经认知障碍（HAND）在高达50%的艾滋病毒阳性人群中持续存在，通常在 尽管由于有效的抗逆转录病毒疗法（ART），病毒载量低或检测不到。小胶质细胞是 巨噬细胞在中枢神经系统（CNS），并被认为是一个持久的水库艾滋病毒在艺术 治疗的病人。小胶质细胞分泌一系列影响神经元功能的因子， 在小胶质细胞的HIV感染过程中，这些因子发生了改变。除了HIV病毒-宿主相互作用介导的 小胶质细胞，许多外源性因素可以改变这些相互作用或直接影响中枢神经系统本身，使其 难以剖析导致HAND的神经病理学的根本原因。建立一个生理上的- 相关的平台，以调查艾滋病毒感染的动态背景下，艺术和其他辅助因素，我们 将使用人类诱导多能干细胞来产生人类小胶质细胞，我们可以与我们的 发育中的大脑皮层的良好表征的3D模型。这个集成的3D脑类器官模型将 概括了许多被认为有助于手相关病理学的关键细胞群，包括 神经元、星形胶质细胞和小胶质细胞。我们将评估脑类器官的功效， 通过测量HIV复制动力学和抑制以及HIV蛋白表达来模拟HIV感染 在不同的细胞类型（目标1）。然后，我们将测量HIV对神经元发育和功能的影响， 急性暴露后的几个时间点（目标2）。我们将进行形态学和电生理学检查 在单细胞和群体水平上进行分析和RNA测序，以确定细胞类型特异性 对HIV暴露的转录反应。最后，我们将评估外源性药物对这些 病毒复制和神经元功能的测量（目的3）。作为概念验证，我们将测试首选的 抗逆转录病毒治疗方案，以及大麻素，无论是否有艾滋病毒暴露开始剖析的影响， 这些共同的辅因子对细胞特性的影响可能导致HAND的病理学基础。成功 这些实验的完成将产生一个有效的平台来模拟神经炎症的动力学， 使用与人小胶质细胞整合的脑区域特异性脑类器官的疾病。

PROJECT SUMMARY HIV-Associated Neurocognitive Disorder (HAND) persists in up to 50% of the HIV-positive population, often in spite of low or undetectable viral loads due to effective antiretroviral therapy (ART). Microglia are the resident macrophages in the central nervous system (CNS) and are thought to be a persistent reservoir of HIV in ART treated patients. Microglia secrete a panoply of factors that impact neuronal function and these secreted factors are altered during HIV infection of microglia. In addition to HIV virus-host interactions mediated by microglia, many exogenous factors can modify these interactions or directly impact the CNS itself, making it difficult to dissect the underlying causes of neural pathology leading to HAND. To establish a physiologically- relevant platform to investigate the dynamics of HIV infection in the context of ART and other co-factors, we will use human induced pluripotent stem cells to generate human microglia that we can co-culture with our well-characterized 3D model of the developing cerebral cortex. This integrated 3D cerebral organoid model will recapitulate many of the critical cell populations thought to contribute to HAND-related pathology including glutamatergic neurons, astrocytes, and microglia. We will evaluate the efficacy of the cerebral organoids to model HIV infections by measuring HIV replication kinetics and suppression, as well as HIV protein expression in different cell types (Aim 1). We will then measure the impact of HIV on neuronal development and function at several time points following acute exposure (Aim 2). We will perform morphological and electrophysiological analyses and RNA sequencing at both the single cell and population level to determine the cell type-specific transcriptional responses to HIV exposure. Finally, we will evaluate the impact of exogenous drugs on these measures of viral replication and neuronal function (Aim 3). As a proof-of-concept, we will test a preferred regimen of ART, as well as cannabinoids, both with and without HIV exposure to begin to dissect the impact of these common co-factors on cellular properties that may contribute to pathology underlying HAND. Successful completion of these experiments will result in a validated platform to model the dynamics of neuroinflammatory disorders using brain region-specific cerebral organoids integrated with human microglia.