Disentangling the HIV/opioid intertwined mechanisms on mitochondrial mediated inflammasome activation, neurotoxicity and targeting strategies: In vivo and in vitro analyses of fresh human brain

Summary. Despite effective antiretrovirals (ARV), Human Immunodeficiency Virus (HIV) spreads within the central nervous system (CNS) and establishes persistent infection precipitating cognitive impairment (CI). Recent findings suggest that HIV protein (namely VPR) expression in the brain activates the inflammasome pathway in neurons leading to gasdermin E pore formation as a key mechanism of neurodegeneration. Mitochondrial damage is a critical mediator of inflammasome activation and may be a primary catalyst for the heightened inflammation and neurotoxic effects induced by HIV. This already complex scenario is further compounded by opioids. Yet, no studies have investigated the individual and combined impact of HIV and opioids on inflammasome-mediated neuro-inflammation and neurotoxicity at the cellular level using fresh, high-quality human CNS specimens. To address this knowledge gap, we have assembled a team with complementary expertise in virology, inflammasome, pharmacology, neuropsychology, and biostatistics. Through a comprehensive investigation employing neuropathology and complementary ex vivo and in vitro methodologies, our overarching goal is to uncover the mechanistic intersection through which HIV and opioids heighten inflammation, offering insights for targeted interventions to improve CI. Using a unique set of archived and fresh brain specimen from persons with HIV (PWH) and persons without HIV (PWoH) with thorough evaluation of opioid use and neurological conditions enrolled in the Last Gift cohort, we will analyze CNS specimens across 5 brain regions (frontal, occipital and parietal cortex, basal ganglia, and hippocampus) collected during rapid research autopsy. • Using flash frozen brain specimens with varying tissue concentrations of opioids from PWH (n=30) and PWoH (n=10) and with a defined history of opioid use, we will determine the relationship between HIV transcriptional activity, opioid exposure and inflammasome activity in neurons and BrMCs (Aim 1). • Next, we will use BrMC cultures generated from freshly autopsied brain tissues (n=10 PWH/5 PWoH) and in vitro models for neurons to probe underlying mechanisms of inflammasome activation by using small molecules to promote mitochondrial function and siRNA to knockdown inflammasome pathway proteins (Aim 2). Through this comprehensive examination of the separate and combined effects of HIV and opioids, our study endeavors to elucidate how the convergence of these factors leads to more severe neurological consequences. By understanding the nuanced interactions and synergies between HIV infection and opioid use, we aim to contribute valuable insights into the mechanisms underlying neurotoxicity in the context of HIV-associated CI ultimately paving the way for targeted interventions and therapeutic strategies.