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

HIV认知脑区慢性阿片类药物暴露的综合单细胞亚型和染色质可及性图谱

基本信息

批准号：
10879756
负责人：
Teresa A Milner
金额：
$ 16.76万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-30 至 2026-06-30
项目状态：
未结题

项目摘要

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.

阿片类药物导致的神经病理事件的加重和艾滋病毒转录的改变导致艾滋病毒相关的中枢神经系统功能障碍的报道也很多。尽管持续多年的抑制性抗逆转录病毒治疗 (ART)，潜伏的艾滋病毒持续存在，并在与阿片类药物使用障碍有关的许多相同的大脑区域找到避难所 (OUD)表明艾滋病毒与脑细胞中的阿片类药物之间的相互作用。然而，我们在这方面存在着相当大的差距。人类抗逆转录病毒治疗对HIV感染脑内细胞反应和病毒持久性的影响或相关的模型器官。这项提议寻求生成地形数据集和证据海马区和前额叶皮质(PFC)的细胞分辨率，这两个脑区是已知的偏爱 HIV在非人灵长类动物(NHP)和死后人脑中的持久性和OUD。这些数据将提供史无前例的多种模式的蜂窝格局，可以利用这些模式来制定战略，以限制病毒的持久性，恢复和保持艾滋病毒携带者的最佳大脑健康。在我们出版的和初步工作我们开发了创新的单细胞方法：(A)单细胞异构体RNA测序 (ScISOr-Seq)，它使数千个多腺化RNA的单细胞长读RNA测序成为可能单细胞；(B)幻灯片异构体测序(Sl-ISO-Seq)以在脑片中空间定位异构体；以及(C)a 在单细胞水平识别艾滋病毒序列的单细胞平台(ScHIV-Seq)。这些小说不约而同测序和计算方法，以及染色质可及性的SCATAC-SEQ，将允许细胞基因表达、染色质开放区域、异构体的作图和HIV的检测海马区和前额叶皮质的细胞。最近的文献支持艾滋病毒在大脑中的存在，更具体地说在小胶质细胞和星形胶质细胞中存在于海马区和前额叶皮质内。重要的是，这些大脑区域也是参与OUD的联想学习过程。此外，我们之前对啮齿动物海马体的研究通过建立阿片类药物的区域和细胞特异性分布，为拟议的研究奠定了基础多肽和受体以及相关的信号分子，以及这些分布是如何受性别影响的，压力和阿片类药物相关的学习。在进一步的初步研究中，我们进行阿片受体映射，大脑现代抗逆转录病毒治疗的空间转录、NHP认知行为评估和药理学研究组织中的养生法。这些方法将提供一个全面的区域格局，以支持我们的细胞特有的表型。我们提出了一个重要的假设：(I)我们新的集成单细胞方法将绘制单细胞和细胞类型特定的人类和NHP转录组和表观基因组特征图 S/艾滋病毒在NHPS和死后人脑中的海马区和PFC；(Ii)慢性阿片类药物暴露增加了长期抗逆转录病毒治疗可区分S/艾滋病毒感染的特征，并定义了这些细胞亚型签名是根深蒂固的；以及(Iii)这些签名不同于在未感染的大脑中长期接触阿片类药物。这些研究进一步了解了HIV和OUD在大脑中的分子机制。