The impact of poly-substance use on the crosstalk between microglia, astrocytes, and neurons that regulates HIV latency

Background. This proposal entitled “The impact of poly-substance use on the crosstalk between microglia, astrocytes, and neurons that regulates HIV latency" is submitted in response to RFA-DA-24-001: Ex Vivo Models for Studies at the Intersection of HIV and Poly-Substance Use”. Although combination antiretroviral therapy (ART) dramatically lowers the levels of HIV RNA in the brain, it does not reduce the prevalence of HIV- associated neurocognitive disorders (HAND), which still develops in up to 50% of people with HIV (PWH). Poly- substance use in PWH increases the risk of developing HAND, creating a significant unmet medical challenge. Our goal. Our proposed ex vivo studies of poly-substance exposure are based on newly developed methods to derive authentic microglia, astrocytes, and neurons from induced pluripotent stem cells (iPSC). We will study the responses of these cells to HIV and poly-substance exposure in mono-, di-, tri-cultures, and organoids. Our previous work has shown that HIV latency is established due to signals from healthy neurons and astrocytes that drive infected microglia into a homeostatic state. The latent virus becomes reactivated in response to inflammatory cytokines and neuronal damage. For example, adding methamphetamine (METH) at a physiologically relevant concentration (100 nM) to co-cultures of microglia and neurons induces HIV reactivation and neuronal damage. Also, it sensitizes HIV-infected microglia to reactivation by inflammatory stimuli such as TNF-α. We will test the hypothesis that poly-substance use (combinations of methamphetamine and opioids) deregulates the cortical/dopaminergic system and disrupts the homeostatic regulation of microglia, leading to HIV reactivation and increased neuronal damage. In addition to measuring the impact of poly-substance exposure on HIV reactivation, we will use single-cell RNA sequencing to study the changes in cellular signaling and activation induced by substance exposure. How will we advance the field? To define how intracellular signaling between neurons, astrocytes, and microglia is dysregulated by poly-substance exposure, we will first investigate how METH and opioids affect the expression of crucial signal/receptor pairs (CD200/CD200R, CX3XL1/CX3CR1, glucocorticoid/GR, DA/DRD1-5) in the presence or absence of HIV. In addition, since we discovered unexpectedly that adenosine is a potent silencer of HIV in iMG cells, we will investigate how extracellular ATP produced by neurotoxicity and astrocyte activation is metabolized to adenosine by surface receptors on microglial cells and promotes HIV silencing. Understanding these mechanisms at the molecular level will set the stage for devising effective anti-inflammatory strategies to ameliorate HAND in the context of poly-substance use. challenging studies because it is only by working within the context of We are undertaking these technically authentic human microglial cells in an environment that better reflects the HIV-infected CNS that the underlying physiology is accurately represented.