Modeling HIV and methamphetamine-induced neuroinflammation in cerebral organoids

联合抗逆转录病毒疗法（cART）已导致艾滋病毒感染者的寿命显着增加， 个体然而，尽管cART有效，但HIV相关的发病率仍造成重大损失。艾滋病毒- 相关的神经认知障碍（HAND）发生在高达50%的慢性感染个体中， 推车HAND的发病机制仍在研究中。神经炎症是HAND的标志， 如临床研究、尸检研究和动物模型所确定的。持续或间歇性复制 中枢神经系统中的HIV可能通过直接作用于受感染的细胞而导致神经炎症， 通过释放病毒蛋白和炎症介质。使用神经兴奋剂，包括 甲基苯丙胺可以加剧HAND中观察到的神经认知下降，但其机制 这一现象的根本原因还不清楚。小胶质细胞是大脑中主要的骨髓细胞， 在急性感染HIV或SIV后的早期感染，可作为CNS病毒储库， 被认为在HAND的发展中起着核心作用。负责小胶质细胞的通路 HIV感染后的激活和功能障碍仍然没有完全确定。小胶质细胞来自 诱导多能干细胞（iPSC）提供了一个独特的机会，研究分子机制， 潜在的小胶质细胞激活iPSC衍生的小胶质细胞将被引入脑类器官， 确定小胶质细胞活化对周围星形胶质细胞，神经元， 和其他细胞。Tetherin是一种宿主限制因子，在组装过程中捕获HIV， 感染的细胞并在感染的细胞内产生促炎信号级联。实验目的 该项目的1将评估艾滋病毒引起的神经炎症都在一个公正的方式，并通过 Tetherin-mediated signaling作为小胶质细胞炎症触发的作用的直接评估。 将采用RNAseq、细胞因子产生和免疫荧光显微镜来定义小胶质细胞。 艾滋病毒感染后激活。在目标2中，我们将把HIV感染的小胶质细胞引入大脑类器官中， 确定HIV诱导的神经炎症和神经元功能障碍的分子基础。单细胞 RNAseq和神经元健康和电生理学的评价将在代表性的模型中进行。 急性感染和ART抑制的慢性脑感染。甲基苯丙胺的潜力， 在HIV感染的小胶质细胞/类器官模型中， 然后定义，并确定相关路径。总之，这些研究将提供深入了解 HAND的发病机制和甲基苯丙胺对神经认知功能下降的潜在贡献。

Combination antiretroviral therapy (cART) has led to dramatic increases in lifespan among HIV-infected individuals. Despite effective cART, however, HIV-associated morbidities exert a significant toll. HIV- associated neurocognitive disorders (HAND) occur in up to 50% of chronically infected individuals despite cART. The pathogenesis of HAND remains under investigation. Neuroinflammation is a hallmark of HAND, as established by clinical studies, autopsy studies, and animal models. Ongoing or intermittent replication of HIV in the CNS is likely to contribute to neuroinflammation through direct effects on the infected cells or through release of viral proteins and inflammatory mediators. Use of neural stimulants including methamphetamine can exacerbate the neurocognitive decline seen in HAND, but the mechanisms underlying this comorbidity are not understood. Microglia are the primary resident myeloid cells of the brain, are infected at early times following acute infection with HIV or SIV, can act as a CNS viral reservoir, and are thought to play a central role in the development of HAND. The pathways responsible for microglial activation and dysfunction following HIV infection remain incompletely defined. Microglia derived from induced pluripotent stem cells (iPSCs) provide a unique opportunity to examine the molecular mechanisms underlying microglial activation. iPSC-derived microglia will be introduced into cerebral organoids, providing the additional opportunity to define the effects of microglial activation on surrounding astrocytes, neurons, and other cells. Tetherin is a host restriction factor that captures HIV during the assembly process in infected cells and generates a proinflammatory signaling cascade within infected cells. Experiments in Aim 1 of this project will evaluate HIV-induced neuroinflammation both in an unbiased way and through a directed evaluation of the role of tetherin-mediated signaling as a trigger of microglial inflammation. RNAseq, cytokine production, and immunofluorescence microscopy will be employed to define microglial activation following HIV infection. In Aim 2, we will introduce HIV-infected microglia into cerebral organoids to define the molecular basis of HIV-induced neuroinflammation and neuronal dysfunction. Single-cell RNAseq and evaluation of neuronal health and electrophysiology will be performed in models representing acute infection and in ART-suppressed, chronic infection of the brain. The potential of methamphetamine to contribute to neuroinflammation and neuronal damage in the HIV-infected microglia/organoid model will then be defined, and the relevant pathways identified. Together, these studies will provide insights into the pathogenesis of HAND and the potential contribution of methamphetamine to neurocognitive decline.