A DUAL CHIMERIC HUMAN ASTROGLIAL-MICROGLIAL MODEL OF HIV AND HAND

摘要突触衰竭是HIV脑感染的一个重要特征，也可能是HIV的一个关键因素。 相关神经认知障碍(手)。然而，目前的动物模型已被证明在定义 这一过程的机制，部分是因为艾滋病毒感染的物种特异性，但也是因为 人类星形胶质细胞比小鼠的复杂程度更高。为了解决这个问题，我们将利用MICE 人小胶质细胞和人星形胶质细胞的嵌合体，以评估HIV感染对中枢神经系统的影响 神经元。为此，我们将把人胶质前体细胞(HGPC)和小胶质细胞分别移植到小鼠体内 来源于胚胎干细胞(HESCs)。我们已经建立了产生这些人类神经胶质细胞的方法 嵌合体，通过新生儿植入hGPC，它胜过并最终取代宿主小鼠 GPC，产生成体嵌合体，与人类星形1-5广泛定居。这一过程在以下方面尤其强大 新纹状体，允许与纹状体奖赏和成瘾回路密切相关的区域的胶质人性化。 我们最近扩展了这一方法，包括与hESC来源的小胶质细胞的嵌合化，与 利用缺乏宿主小胶质细胞的CSF1R缺失小鼠，与NSG SGM3小鼠杂交，以允许稳定的异种移植 HGPC。因此，在T和B细胞缺陷的情况下，小鼠是hGPC来源的星形胶质细胞和小胶质细胞的嵌合体 背景，使胶质细胞HIV感染对神经元的影响被分离出来，在脑内 接种感染艾滋病毒的小胶质细胞。这些嵌合的人类星形胶质-小胶质(CHAM)小鼠 很有吸引力，因为它们融合了人类星形胶质细胞特有的特征，而这些特征本身就是关键 中枢突触的组成部分。使用这个模型，我们将测试星形胶质细胞既成为 小胶质细胞HIV感染导致结构和功能受损，导致突触参与丢失 受影响的星形胶质细胞，随之而来的树突内陷和网络破坏。通过感染Cham小鼠 并使用狂犬病病毒-EGFP追踪纹状体树突，我们将评估星形细胞HIV感染的影响 关于常驻中棘神经元的树突构筑和突触结构。同时，我们会研究 HIV感染合并使用甲基苯丙胺对Cham小鼠的影响--一种常见的致残作用 抑制纹状体多巴胺能输入的共病--侧重于结构和转录 人脑胶质细胞对感染和成瘾的反应以及对行为的影响 这种组合。为此，我们将使用单细胞RNA-Seq通过以下方式评估基因表达的变化 人类星形胶质细胞及其配对的小鼠神经元由艾滋病毒感染引起的单独和共同的 长期使用甲基苯丙胺，以确定那些导致纹状体突触中断的变化，并 这些小鼠的行为病理学。我们的目标是测试这一假设，即感染艾滋病毒的纹状体，通过 尤其是星形细胞纤维从多巴胺能突触中分离出来，尤其容易受到这种影响。 安非他明滥用，同时定义了这种脆弱性背后的神经胶质病理的转录基础。

Abstract Synaptic failure is an important feature of HIV infection of the brain, and a likely key contributor to HIV- associated neurocognitive disorders (HAND). Yet current animal models have proven of limited utility in defining the mechanisms of this process, in part because of the species-specific nature of HIV infection, but also because of the greater complexity of human astrocytes relative to those of mice. To address this issue, we will utilize mice chimeric for both human microglia and human astrocytes, to assess the effects of HIV infection on central neurons. To that end, we will engraft mice with both human glial progenitor cells (hGPCs) and microglia, each derived from embryonic stem cells (hESCs). We have established the methods of generating these human glial chimeras, by the neonatal implantation of hGPCs, which outcompete and ultimately replace the host mouse GPCs, yielding adult chimeras broadly colonized with human astroglia1-5. This process is especially robust in the neostriatum, allowing the glial humanization of regions critically involved in striatal reward and addiction circuits. We have recently extended this approach to include chimerization with hESC-derived microglia, paired with the use of CSF1R null mice lacking host microglia, crossed to NSG SGM3 mice to allow the stable xenograft of hGPCs. The mice are thus chimeric for hGPC-derived astrocytes as well as microglia, in a T- and B-cell deficient background that allows the effects of glial HIV infection on neurons to be isolated, following intracerebral inoculation with HIV-infected microglia. These chimeric human astroglial-microglial (CHAM) mice are especially attractive, since they incorporate the hominid-specific features of human astroglia, which are themselves key components of central synapses. Using this model, we will test the postulate that astrocytes become both structurally and functionally impaired by microglial HIV infection, resulting in the loss of synaptic engagement by affected astrocytes, with consequent dendritic involution and network disruption. By infecting CHAM mice with HIV, and using rabies viral-EGFP to trace striatal dendrites, we will assess the effects of astrocytic HIV infection on the dendritic architecture and synaptic structure of resident medium spiny neurons. In parallel, we will study the effects in CHAM mice of HIV infection complicated by methamphetamine use – a common and disabling comorbidity that suppresses dopaminergic input to the striatum – focusing on the structural and transcriptional responses of human glia to the combination of infection and addiction, as well as on the behavioral effects of that combination. To that end, we will use single cell RNA-Seq to assess the changes in gene expression by human astrocytes and their partnered mouse neurons caused by HIV infection, both alone and together with chronic methamphetamine use, to identify those changes that contribute to the striatal synaptic disruption and behavioral pathology of these mice. Our goal is to test the hypothesis that the HIV-infected striatum, by virtue of astrocytic fiber disengagement from dopaminergic synapses in particular, is especially vulnerable to the effects of amphetamine abuse, while defining the transcriptional basis for the glial pathology underlying that vulnerability.