M-SCORCH: Methamphetamine use disorder data generation center for Single Cell Opioid Responses in the Context of HIV

项目摘要 艾滋病毒和甲基苯丙胺（MA）的使用是全球性的健康问题， 后果艾滋病毒和甲基苯丙胺的使用也产生独立的和累加的损害， 神经认知，以及目前的临床和基础科学研究表明，复杂的，目前不充分 了解HIV和MA病理生理学之间的相互作用。因此，我们建议进行 在单细胞/核水平上对人脑组织进行全面表征， 来源于人诱导多能干细胞的类器官。对于这些单核（sn）RNA-seq和 snATAC-seq分析，我们将对3个大脑区域（前额叶皮层、腹侧纹状体和基底外侧皮层）进行采样。 杏仁核）对MA使用的神经生物学反应至关重要， HIV+MA+和HIV-MA+。这些数据生成工作将补充正在进行的努力，在这些相同的大脑 从HIV-MA-和HIV+MA-供体的区域，使我们能够阐明基因表达的差异和关键 生物学途径，发生在响应MA使用，艾滋病毒，或两者的组合。此外，我们将 分析脑细胞类型的艾滋病毒转录本，使我们能够确定细胞库的艾滋病毒在捐赠者的大脑。 这些努力将有助于使用人类皮质类器官和内侧神经节隆起类器官 文化，它提供了复杂的，区域匹配的模型系统重演体内样细胞多样性， 无潜在混杂因素的微环境，包括患者病史，不同的合并症， 死亡时间延长或者组织退化然后，我们将应用尖端和新颖的数据分析 整合snRNA-seq和snATAC-seq数据并鉴定细胞群和基因表达的管道 细胞簇之间的差异（即，假定的细胞类型）在不同条件下。这些数据也将 与来自SCORCH联盟的外部数据集和其他多组学数据（包括基因型）相结合， 来自健康受试者和HIV感染受试者的RNA-seq、HiC、ChIP-seq和ATAC-seq数据， 神经系统疾病或药物滥用史最后，我们将使用多重免疫组织化学， 单分子荧光原位杂交验证细胞类型特异性表达和共表达 与HIV或MA病因学有关的候选基因、生物学过程和基因调控网络 病理生理学所有数据生成协议和数据分析工具将免费提供给 研究社区，生成的所有数据将作为原始资源和处理后的资源提供。采取 总之，拟议的实验将产生宝贵的资源，并提供新的生物学见解， 人类大脑及其紊乱

PROJECT SUMMARY HIV and methamphetamine (MA) use are global health problems with devastating human and societal consequences. HIV and methamphetamine use also produce independent and additive impairments in neurocognition, and current clinical and basic science research suggest complex and currently inadequately understood interactions between HIV and MA pathophysiologies. We therefore propose to conduct comprehensive characterization, at the single cell/nuclear level, of human brain tissue and regionally specified organoids derived from human induced pluripotent stem cells. For these single nuclear (sn)RNA-seq and snATAC-seq analyses, we will sample 3 brain regions (prefrontal cortex, ventral striatum, and basolateral amygdala) critical for the neurobiological response to MA use in 20 brains from each of two donor groups, HIV+MA+ and HIV-MA+. These data generation efforts will complement ongoing efforts in these same brain regions from HIV-MA- and HIV+MA- donors and allow us to elucidate differences in gene expression and key biological pathways that occur in response to MA use, HIV, or the combination of the two. In addition, we will assay brain cell types for HIV transcripts, allowing us to identify cellular reservoirs of HIV in donor brains. These efforts will be aided by the use of human cortical organoid and medial ganglionic eminence organoid cultures, which offer complex, region-matched model systems recapitulating in vivo-like cellular diversity and microenvironments without potentially confounding factors including patient history, varying co-morbidities, prolonged postmortem intervals, or tissue degradation. We will then apply cutting edge and novel data analysis pipelines to integrate snRNA-seq and snATAC-seq data and identify cell population and gene expression differences between cell clusters (i.e., putative cell types) in different conditions. These data will also be integrated with external datasets from the SCORCH Consortium and other multi-omic data including genotype, RNA-seq, HiC, ChIP-seq, and ATAC-seq data from both healthy subjects and subjects with HIV infection, neurological disease, or a history of drug abuse. Finally, we will use multiplexed immunohistochemistry and single molecular fluorescent in situ hybridization to validate the cell type specific expression and co-expression of candidate genes, biological processes, and gene regulatory networks implicated in the etiology of HIV or MA pathophysiology. All data generation protocols and data analysis tools will be made freely available to the research community, and all data generated will be provided as both raw and processed resources. Taken together, the proposed experiments will generate invaluable resources and offer new biological insights into the human brain and its disorders.