Cell-type specific microglial protein dynamics in models of HIV and opioid use

About This Grant

PROJECT SUMMARY Opioid use disorder (OUD) is common among people living with HIV, and the combination produces more severe neurobehavioral impairments than either condition alone. Both HIV and opioids remodel neural circuits through inflammatory and neuromodulatory pathways, with brain region specific effects. Microglia, the resident immune cells of the brain, are central to these processes, yet it is unknown how HIV and opioid signals converge at the protein level to influence microglial function over time and across circuits. While single-cell RNA sequencing has revealed microglial heterogeneity, transcript levels often correlate poorly with protein abundance. Because proteins are the functional effectors of cellular processes, direct proteomic measurement provides a more accurate view of activity. However, bulk proteomics masks cell- type-specific responses, and targeted protein assays limit discovery of novel effectors. This project employs a state-of-the-art, cell-type-specific proteomic strategy using a Cre-dependent mutant methionyl-tRNA synthetase (MetRS*) system. This approach labels nascent proteins in microglia in vivo with the noncanonical amino acid azidonorleucine (ANL), enabling selective enrichment and mass spectrometry based profiling. It provides unprecedented temporal and spatial resolution of microglial proteomic changes across brain regions and disease stages. Using models of EcoHIV infection and fentanyl exposure, we will define distinct and overlapping microglial protein networks that emerge in each condition and during comorbidity. We hypothesize that HIV and opioids drive both unique and convergent proteomic programs in a brain region and time-specific manner, contributing to behavioral adaptations relevant to addiction and neuroimmune dysfunction. The phased R61/R33 design first validates and optimizes microglial proteomics across brain regions and labeling windows (Aim 1), then identifies EcoHIV-induced proteomic signatures in behaviorally relevant regions (Aim 2). In the R33 phase, we will map microglial proteomic dynamics across the addiction cycle in fentanyl, EcoHIV, and combined models (Aim 3). Finally, we will integrate mouse proteomic data with human single-cell RNA-seq datasets and validate high-priority targets using multiplexed imaging in both mouse and human brain tissue (Aim 4). Impact: This work will produce the first spatiotemporal atlas of microglial proteomic adaptations to HIV, opioids, and their combination. By identifying conserved molecular signatures that link microglial plasticity to behavioral outcomes, the project will yield mechanistic insight into neuroimmune dysregulation in comorbid HIV & OUD and lay the groundwork for targeted therapeutic strategies.