Modeling HIV-associated neurocognitive disorders and encephalopathy in human iPSC brain organoids containing microglia

项目总结/摘要 即使随着成功的联合抗逆转录病毒疗法（c-ART）的出现， 免疫缺陷病毒（HIV-1）仍然是一个全球性的公共卫生危机。艾滋病毒感染大脑导致 重要的临床症状，包括HIV相关的神经认知障碍或HAND， 影响认知、行为和运动功能的症状，以及小儿HIV脑病（PHE） 导致神经发育缺陷。这些疾病甚至可能发生在接受c- 抗逆转录病毒治疗，虽然严重程度较低。HIV病理生理学的分子和细胞机制- HAND和PHE仍然知之甚少，这在很大程度上是因为现有模型系统的局限性， 研究他们。动物模型一般不感染艾滋病毒，昂贵和不切实际的或不好概括 人类大脑感染培养的细胞模型提供了一些见解，但不能概括细胞的许多方面。 在人脑中发现的细胞和细胞-基质相互作用。干细胞技术的进展，包括使用 人类诱导多能干细胞（iPSC或iPS细胞）现在能够产生患者特异性神经干细胞。 谱系细胞和小胶质细胞（脑免疫细胞），以及这些细胞组装成三维（3D） 脑类器官这些脑类器官模仿免疫、神经胶质和神经元细胞的重要特征 相互作用，独特地使我们能够研究这些相互作用如何受到艾滋病毒感染的影响，并建立模型， HAND和PHE发病机制的关键方面。这个项目的首要目标是利用我们的优势 在神经科学，病毒学，干细胞生物学和基因表达，以了解手的病理生理学 和PHE使用人类神经小胶质细胞系统。我们已经建立了人类3D大脑类器官， iPS细胞衍生的小胶质细胞来研究神经发育和神经免疫相互作用， 成功地用HIV感染了这些混合的类器官。我们假设小胶质细胞感染艾滋病毒导致 改变的神经元功能，可以在这些含有小胶质细胞的脑类器官中建模， HAND和PHE发病机制的机制理解系统。在本提案中，我们将 表征HIV感染对小胶质细胞活化和细胞因子产生，神经元细胞群， 组织和基因表达，以及候选细胞因子介质的特异性作用。我们会进行切割- 从干细胞技术、脑类器官模型和单细胞RNA测序中提取的边缘方法， 开发这个模型系统，这将允许长期的，详细的分子和神经生理学分析。 HIV相关神经系统疾病发病机制的研究。

Project Summary/Abstract Even with the advent of successful combination antiretroviral therapy (c-ART), infection by Human Immunodeficiency Virus (HIV-1) remains a global public health crisis. HIV infection of the brain results in important clinical symptoms including HIV-associated neurocognitive disorder or HAND, a constellation of symptoms affecting cognitive, behavioral, and motor functions, as well as pediatric HIV encephalopathy (PHE) in children, leading to neurodevelopmental deficits. These disorders may still occur even in patients receiving c- ART, although with less severity. The molecular and cellular mechanisms underlying the pathophysiology of HIV- HAND and PHE remain poorly understood, in large part because of limitations of the existing model systems to study them. Animal models are generally not infected by HIV, expensive and impractical or poorly recapitulate human brain infection. Cultured cell models provide some insights, but do not recapitulate many aspects of cell- cell and cell-matrix interactions found in human brain. Advances in stem cell technologies, including the use of human induced pluripotent stem cells (iPSC or iPS cells), now enable generation of patient-specific neural lineage cells and microglia (brain immune cells), as well as the assembly of these cells into 3-dimensional (3D) brain organoids. These cerebral organoids mimic important features of immune, glial and neuronal cell interactions, uniquely enabling us to examine how these interactions are affected by HIV infection and to model key aspects of HAND and PHE pathogenesis. The overarching goal of this project is to leverage our strengths in neurosciences, virology, stem cell biology and gene expression to understand the pathophysiology of HAND and PHE using a human neural-microglial system. We have established human 3D brain organoids prepared with iPS cell-derived microglial cells to study neurodevelopment and neuroimmune interactions and have successfully infected these mixed organoids with HIV. We hypothesize that microglial infection by HIV causes altered neuronal function which can be modeled in these microglial-containing brain organoids, providing a new system for mechanistic understanding of the pathogenesis of HAND and PHE. In this proposal, we will characterize the effects of HIV infection on microglia activation and cytokine production, neuronal cell populations, organization and gene expression, and the specific effects of candidate cytokine mediators. We will apply cutting- edge approaches drawn from stem cell technologies, brain organoid models, and single cell RNA sequencing to develop this model system, which will allow long-term, detailed analyses of the molecular and neurophysiologic mechanisms responsible for the pathogenesis of HIV-associated neurological disorders.