Abstract: 3D human neurocircuits to determine the role of microglia in AUD and Alzheimer’s neuronal pathology. Altered neuronal metabolism, neuronal activity, and neurodegeneration are key features in both alcohol use disorder (AUD) and Alzheimer’s disease (AD). AUD and AD are linked, with alcohol related brain damage (ARBD) being one of the strongest risk factors for AD. However, the mechanisms underlying altered neuronal metabolism in these diseases, and its impacts on neuronal activity and neurodegeneration are unknown. We reported that proinflammatory microglia promote ARBD and alcohol-enhancement of AD pathology. We now find that microglia promote lipid accumulation in neurons with alcohol. Therefore, we hypothesize that alcohol alters neuronal activity and survival to promote AUD and AD through a novel microglia- neuronal metabolic link. In AUD and AD, brain glucose metabolism is reduced. ARBD correlates with this reduction, suggesting metabolic changes promote pathology. Recent studies suggest microglia may also contribute. Proinflammatory microglia undergo a glycolytic burst that can produce high levels of lactate and express the lactate exporter MCT4. We hypothesize that the proinflammatory microglia induced by alcohol deliver excess lactate to neurons, causing a metabolic imbalance that alters neuronal activity and promotes neurodegeneration. Alcohol (i.e. ethanol) is metabolized by oxidative and non-oxidative pathways (OME and NOME) to produce acetate and fatty-acid ethyl esters (FAEEs) respectively. Acetate is converted to lipids through acetyl-coA. Microglia, but not neurons, have OME machinery. Thus, we hypothesize microglial OME releases acetate to disrupt neuronal lipid metabolism. We recently found that accumulation of neuronal lipids caused by alcohol promotes AD pathology. Amyloid-β (Aβ) is normally degraded within neurons by lysosomes. Ethanol increased neuronal lysosomal lipid to cause lysosomal damage and prevent neuronal degradation of intraneuronal Aβ. Inhibition of proinflammatory microglia prevented neuronal lipidosis, identifying microglia as regulators of AD-promoting changes in neuronal metabolism. However, the mechanism underlying this microglia- neuronal link in AD are unknown. Given the impact of ethanol on microglial activation, and consequences of ethanol metabolism, we hypothesize energetic metabolites produced by microglial glycolysis as well as microglial ethanol metabolism combine to produce neuronal lipidosis, lysosomal dysfunction, altered neuronal activity, and neurodegeneration associated with AUD and AD. We propose to employ novel human 3D reciprocal brain circuits (h3D-rC) to test the role of microglia in neuronal metabolism and activity (Aim 1-2), alcohol-induced neurotoxicity (Aim 1) and Aβ accumulation (Aim 2). These reciprocal circuits are formed using human IPSC- derived neurons grown in proprietary microfluidic culture platforms (XonaTM) that enable the bidirectional growth of axons between two di...