Project summary: Despite combined antiretroviral therapy, HIV proteins like Transactivator of Transcription (Tat) persist at low levels in the brains of HIV-infected individuals, resulting in cognitive decline collectively referred to as HIV-1- associated neurocognitive disorders (HAND). Alcohol use disorder is a common comorbidity in people living with HIV (PLWH) and is also known to potentiate the pathogenesis of HAND. Although the mechanisms underlying HAND are complex and remain poorly understood, low-level, persistent neuroinflammation has been shown to be a correlate of HAND. In the brain, glial cells such as the astrocytes, are major contributors of inflammation, involving the inflammasome NLRP6 (a member of the NLR [nucleotide-oligomerization domain-like receptor) family of proteins that acts as a sensor protein and plays multiple roles in regulating inflammation and host defenses. Based on the premise that alcohol use exacerbates HIV-associated neuroinflammation, we hypothesize that exposure of HIV Tat stimulated astrocytes to alcohol will lead to exacerbated expression of a pro-inflammatory milieu compared to cells exposed to either agent alone or to control cells (HIV protein/drug naïve) and, further that mechanistically this involves increased activation of NLRP6 and its downstream signaling pathways. In fact, our exciting preliminary studies have demonstrated that human A172 astrocyte cells exposed to both HIV Tat (surrogate of HIV infection) and alcohol significantly increased the activation of NLRP6 involving endoplasmic reticulum (ER) stress. The proposed hypothesis will be tested in two aims: SA1 To investigate the molecular mechanism(s) underlying HIV Tat and ethanol-induced astrocytic activation in mouse primary astrocytes (MPAs) and SA2. To validate the findings from SA1 in an in vivo doxycycline-inducible transgenic Tat (iTat) mice model exposed to ethanol These findings could have important implications for the future development of therapeutic interventions aimed at mitigating neuroinflammation in PLWH with HAND and alcohol use disorder.