Abstract: An estimated ~1.3 million adults and adolescents are living with human immunodeficiency virus (HIV) infection in the USA of which 15% of them undiagnosed. The initiation of combination antiretroviral therapy (cART) has transformed AIDS from a fatal illness into a chronic and manageable disease. The presence of HIV infected microglia and macrophage reservoirs in the central nervous system (CNS) and variable penetration of anti- retroviral drugs across the blood brain barrier after cART are likely factors for the persistence of HIV associated neurocognitive disorder (HAND). Drugs of abuse such as the potent psychostimulant methamphetamine (Meth) that is abused by an estimated 30 million people in the world further minimize the efficacy of cART and in conjunction with HIV exacerbate CNS pathology. Thus, significant gap in knowledge in the field is to better understand the neuropathogenesis and the etiology of clinical outcomes observed in HIV+ patient’s dependent on Meth abuse. The current grant proposal focuses on developing a human induced pluripotent stem cell based cerebral organoid model to investigate underlying brain dysfunction during HIV/Meth interaction. Cerebral organoids are 3-dimensional “mini brains” that can self-organize and recapitulate many milestone events seen in in vivo brain development. Our recent study on generating a novel organoid model with the feasibility in controlling the microglia ratio and microenvironment of organoid formation to recapitulate changes in brain functions under various conditions (e.g. virus and viral infection-induced inflammation and synaptic pruning) makes this model ideal for our proposed studies. The central hypothesis of this proposal is that Meth in conjunction with HIV causes significant neuronal damage. The combination of microglia-containing cerebral organoid model, extracellular vesicle biology, single-cell RNA-sequencing, CRISPR/Cas9-mediated gene editing will decipher novel intercellular and molecular mechanisms and pathways that underlie abnormalities in neuronal functions and connectivity caused by HIV infection. We will investigate this hypothesis under three specific aims: Specific Aim 1 will examine neuronal function, synaptic and mitochondrial perturbations during HIV/meth interactions in 3D microglia-containing cerebral organoid model; Specific Aim 2 will investigate the role of extracellular vesicles in microglial organoids treated under HIV/meth conditions; and Specific Aim 3 will characterize the molecular mechanisms underlying HIV infection-induced neuronal injury and further evaluate our microglial organoid model as a reliable tool to identify molecular signatures of HAND. Our proposed experiments will decipher molecular mechanisms, novel signaling events and molecular partners underlying the neuronal injury in HAND. Through the series of the experiments we aim to present an easily scalable and reproducible model to study HAND pathogenesis. The data obtained from these...