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.

PROJECT NARRATIVE Methamphetamine use and HIV infection share a common route of transmission (i.e., injection) and some common long-term adverse outcomes including impaired cognitive and immune functions. However, neither the specific types of cells nor the precise molecular causes of these outcomes are well understood, and little is known about how the effects of HIV and methamphetamine might exacerbate these symptoms. We will therefore use cutting edge genomic techniques to develop critical new insights into the human brain and its dysfunction in response to HIV and methamphetamine use.