Integrative Single Cell isoform and chromatin accessibility Mapping of Chronic Opioid Exposure in Cognitive Brain Areas in HIV

阿片类药物导致的神经病理学事件恶化和HIV转录的改变导致HIV 相关的CNS功能障碍有很多报道。尽管多年来持续的抑制性抗逆转录病毒治疗 (ART)潜伏的艾滋病毒持续存在，并在许多与阿片类药物使用障碍有关的大脑区域中找到庇护所 (OUD)表明艾滋病病毒和阿片类药物在脑细胞中相互作用。然而，在我们的国家， 了解OUD如何影响人类ART中HIV感染大脑中的细胞反应和病毒持续存在 或相关的模型器官。这项建议旨在生成地形数据集和证据， 海马体和前额叶皮层（PFC）的细胞分辨率，这两个大脑区域以偏爱 HIV在非人灵长类动物（NHP）和死后人脑中的持久性和OUD。这些数据将提供 前所未有的多种模式的细胞景观，可以利用这些模式来制定限制 病毒持久性和恢复和保持最佳的大脑健康的人与艾滋病毒。在我们的出版和 初步工作，我们已经开发出创新的单细胞方法：（A）单细胞亚型RNA测序 （ScISOr-Seq），其能够对数千个多聚腺苷酸化RNA进行单细胞长读段RNA测序 单细胞的;（B）载玻片同种型测序（Sl-ISO-Seq）以在脑切片中空间定位同种型，以及（C）a 在单细胞水平鉴定HIV序列的单细胞平台（ScHIV-Seq）。在演唱会这些小说 测序和计算方法，沿着用于染色质可及性的scATAC-Seq，将允许 绘制细胞基因表达、开放染色质区域、同种型和跨单个细胞的HIV检测图， 最近的文献支持艾滋病毒在大脑中的存在，更具体地说， 在海马和PFC内存在的小胶质细胞和星形胶质细胞中。重要的是，这些大脑区域也 参与OUD的联想学习过程。此外，我们先前在啮齿动物海马体中的研究 通过建立阿片样物质的区域和细胞特异性分布，为拟议的研究奠定了基础 肽和受体以及相关的信号分子，以及这些分布如何受到性别的影响， 压力和阿片类药物相关的学习。在进一步的初步研究中，我们进行阿片受体绘图，脑 空间转录组学、NHP认知行为评估和当前ART的药理学特征分析 组织中的方案。这些方法将提供一个全面的区域景观，以支持我们的单一 细胞特异性表型我们提出了一个总体假设，即：（i）我们新的集成单细胞 方法将绘制单细胞和细胞类型特异性人类和NHP转录组和表观基因组签名， NHP和死后人脑中S/HIV的海马和PFC;（ii）慢性阿片类药物暴露增加了 可区分的签名S/HIV感染与长期ART，并定义细胞亚型，其中这些 签名是植根于;和（iii）这些签名是从未感染的大脑慢性阿片类药物暴露不同。 这些研究进一步了解了HIV和脑内OUD的分子机制。

Opioid driven exacerbations of neuropathological events and alterations in HIV transcription contributing to HIV associated CNS dysfunction are well-reported. Despite years of continuous suppressive antiretroviral therapy (ART), latent HIV persists and finds sanctuary in many of the same brain regions involved in opioid use disorder (OUD) suggesting interactions between HIV and opioids in brain cells. However, there is a sizeable gap in our knowledge on how OUD impacts cellular responses and viral persistence in HIV-infected brain on ART in humans or relevant model organsims. This proposal seeks to generate topographical data sets and evidence at single cell resolution across the hippocampus and prefrontal cortex (PFC), two brain regions known for predilection for HIV persistence and OUD in non-human primate (NHP) and in post-mortem human brain. These data will provide an unprecedented cellular landscape of multiple modalities that can be harnessed to develop strategies to limit viral persistence and restore and retain optimal brain health in people living with HIV. In our published and preliminary work we have developed innovative single-cell approaches: (A) Single-cell isoform RNA sequencing (ScISOr-Seq), which enables single-cell long-read RNA sequencing of polyadenylated RNAs across thousands of single cells; (B) Slide-isoform sequencing (Sl-ISO-Seq) to spatially locate isoforms in brain slices and (C) a single-cell platform that identifies HIV sequences at single cell level (ScHIV-Seq). In concert these novel sequencing and computational methods, along with scATAC-Seq for chromatin accessibility, will permit the mapping of cellular gene expression, open chromatin regions, isoforms and the detection of HIV across single- cells of hippocampus and PFC. Recent literature supports the presence of HIV in the brain and more specifically in microglia and astrocytes present within the hippocampus and PFC. Importantly, these brain regions are also involved in associative learning processes for OUD. Moreover, our prior studies in rodent hippocampus have laid the groundwork for the proposed studies by establishing the regional and cell-specific distributions of opioid peptides and receptors as well as related signaling molecules, and how these distributions are impacted by sex, stress and opioid-associated learning. In further preliminary studies, we conduct opioid receptor mapping, brain spatial transcriptomics, NHP cognitive behavioral assessment and pharmacological profiling of current ART regimens in tissues. These approaches will provide a comprehensive regional landscape to support our single cell specific phenotypes. We propose an overarching hypothesis that: (i) our new integrated single-cell methods will map single-cell and cell-type specific human and NHP transcriptome and epigenome signatures in the hippocampus and PFC of S/HIV in NHPs and post-mortem human brain; (ii) chronic opioid exposure adds a distinguishable signature to S/HIV infection with long-term ART and defines cell subtypes in which these signatures are rooted; and (iii) these signatures are different from chronic opioid exposure on uninfected brain. These studies further an understanding of molecular mechanisms in HIV and OUD in brain.