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

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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 administrative supplement proposal seeks to generate new topographical data sets from the parent award and evidence at single cell resolution across the hippocampus, a brain region known for predilection for HIV persistence and OUD in non-human primate (NHP). 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 including Single-cell isoform RNA sequencing (ScISOr-Seq), which enables single-cell long-read RNA sequencing of polyadenylated RNAs across thousands of single cells. 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 SIV across single-cells of hippocampus. Recent literature supports the presence of HIV in the brain and more specifically in microglia and astrocytes present within the hippocampus. Importantly, this brain region is 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. Using new cutting-edge single-cell mulitomic integrated genomics, we propose in this supplemental application to generate multi-dimensional single-cell data sets and define the relevance of such observations in a controlled SIV infected NHP model with and without a different opioid of documented chronic morphine exposure for comparison with the parent study evaluating oxycodone. We also propose to validate any changes observed in single-cell expression in select genes using in situ hybridization. We propose to achieve these goals under the following specific aims: Aim 1. Generation of genome-wide single-cell transcriptomic profiles in the hippocampus in a controlled setting of SIV infection of NHPs monitored on ART and exposed to chronic morphine. Aim 2. Establish genome-wide single-cell epigenomic profiles of the hippocampus in the groups examined in aim 1.