Defining molecular mechanisms by which stimulant evoked dopamine drives inflammation and neuronal dysfunction in neuroHIV

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The HIV pandemic is increasingly driven by the spread of infection in vulnerable sub-populations with a relatively high prevalence of substance abuse disorders (SUD), including the use of stimulants such as Methamphetamine (Meth) and Cocaine (Coc). Stimulant use accelerates systemic disease, and can drive changes in neuropathogenesis, increase risk neuropsychiatric comorbidities and accelerate cognitive decline, despite effective ART. Despite the high prevalence of stimulant use among PLWH, the mechanisms by which stimulants impact disease progression are poorly defined. This is particularly true in the CNS, as there are substantial technical challenges involved in modeling microglial infection and interaction with neurons, as well as the subsequent changes that this has on neuronal function. All stimulants increase CNS dopamine release, exposing myeloid populations to highly elevated dopamine levels. Data indicate that it is the exposure to released dopamine, rather than the stimulants themselves, which drives changes in microglial infection and function. Our data support this, showing stimulant induced dopamine levels increase HIV entry and enhance myeloid inflammation, increasing cytokine release, and NF-κB and NRLP3 inflammasome activity in vitro and in the NHP CNS. Neuroinflammation driven by infected CNS myeloid populations is central to HIV neuropathogenesis in the ART era, underlying the neuronal dysfunction and disruptions in neuroimmune communication that lead to cognitive impairment and behavioral changes. We hypothesize that stimulant use exacerbates HIV- associated microglial inflammation through dopamine receptor activation, leading to neuronal dysfunction in neuroHIV. To address this, we propose to develop tractable, syngeneic co-cultures of human iPSC-derived microglia (iMG) and iPSC-derived dopamine neurons (iDAN). Critically, these iDAN will release dopamine in vitro in response to stimulant exposure. Co-cultures will be based on our existing protocols for iMG and iDAN differentiation and will be developed and optimized for high-throughput analysis during the R61 phase. During the R61, we will infect with HIV and treat with stimulants +/- ART, then use high content imaging, single cell RNA-seq / ATAC-seq and Alphalisas to evaluate changes in viral dynamics (Aim 1b), gene expression and chromatin accessibility (Aim 1c) and inflammation pathways (NF-κB, AP-1, STAT and NLRP3 activity, Aim 1d). Using the R61 results as readouts, the R33 phase will use pharmacologic inhibition and CRISPR to identify the specific dopamine receptors involved in each readout (Aim 2), and to examine neuronal function and neuroimmune interaction (Aim 3) by evaluating; resting membrane potential and neuron firing rate with patch clamp electrophysiology, dendritic spine density and morphology and microglial-neuronal contacts using confocal imaging and Neurolucida360 analysis, and neuronal network activity using multielectrode arrays. This will define specific dopamine receptors and microglial or neuronal functions that can be targeted to ameliorate the impact of SUD on inflammation in PLWH by manipulating dopaminergic activity.