In many infected individuals, human immunodeficiency virus type 1 (HIV-1) enters the brain and can cause a broad spectrum of HIV-1-associated neurocognitive disorders (HAND) ranging from mild impairments to severe HIV-associated dementia (HAD). While widespread use of combination antiretroviral therapy (cART) has effectively increased the life span of people living with HIV-1/AIDS (acquired immunodeficiency syndrome), the prevalence of milder forms of HAND has also increased in the cART era. Indeed, recent epidemiological studies indicate that greater than 50% of HIV-1 infected people in the USA develop HAND. As HIV-1 does not infect neurons, HIV-1 infected macrophages and microglia (and possibly astrocytes) are thought to contribute to neuronal dysfunction and death via a direct mechanism (production of viral proteins) or an indirect “bystander” effect (production of cytokines and chemokines). Pathology studies have also shown that a chronic state of HIV replication in the brain increases intra and possibly extracellular β-amyloid (Aβ), a classic hallmark of Alzheimer's disease (AD) and dementia. However, how and why Aβ production is elevated by HIV-1 infection, and whether this contributes to neurodegeneration, remains unclear. Our preliminary data identifies amyloid precursor protein (APP) as an innate restriction factor for both early and late HIV-1 infection in human cells, including microglia. Moreover, we establish that HIV-1 overcomes this restriction by reducing APP levels, but in doing so generates Aβ40 and Aβ42 products that are toxic to primary cortical neurons. In identifying the underlying mechanism for further study, we find that APP is a novel HIV-1 matrix (MA) associated protein that blocks early infection. By transfection of an infectious HIV-1 clone or Gag-expressing plasmids, we find that increasing APP expression also binds HIV-1 Gag through its MA domain and potently suppresses late stage HIV-1 budding, trapping Gag in specific membrane compartments. To escape this restriction, HIV-1 infection or Gag alone promotes γ-secretase-dependent processing of APP. This results in elevated secretion of Aβ40 and Aβ42 as determined by western blotting and ELISA, and can be blocked by γ-secretase inhibitors. Fractionated supernatants from Gag-expressing cells cause toxicity in cultured primary cortical neurons, and is blocked by treating Gag-expressing cells with γ-secretase inhibitors. Finally, regression analysis shows that neurotoxicity in supernatants correlates precisely with Aβ levels under a variety of conditions in this system. In this proposal we aim to determine the underlying mechanisms by which APP restricts HIV-1 infection, and how viral evasion of this restriction through degradation of APP results in altered Aβ metabolism and neuronal damage. These aims will make use of a wide range of innovative approaches including high-resolution live cell imaging and co- culturing of microglia with neuronal cells in microfluidic chambe...