NeuroAIDS is an HIV infection-associated neurological comorbidity that significantly affects life quality of infected subjects. Introduction of combination anti-retroviral therapy (cART) has markedly reduced the incidence of HIV-associated dementia (HAD), a severe form of neuroAIDS, but cognitive impairment remains prevalent even in subjects with undetectable viral load. The mild forms of neuroAIDS are not associated with neurodegeneration or severe inflammation, but are characterized by neuronal demyelination and functional impairment. Therefore, pathogenic mechanisms responsible for the neurologic comorbidities in the cART era are different from those previously implicated in HAD. In this application, we propose a novel mechanism for pathogenesis of cognitive dysfunction in HIV-infected subjects: we hypothesize that Nef induces CNS demyelination by impairing cholesterol metabolism in glial cells and promoting pro-inflammatory status of brain macrophages. Studies from our group demonstrated Nef-induced inhibition of cholesterol efflux and characterized the mechanism of this effect, which involves downregulation and inactivation of the main cellular cholesterol transporter ABCA1. Importantly, ABCA1 downregulation occurs not only in HIV-infected cells, but also in bystander cells affected by Nef released from infected cells. Given that microglial cells and astrocytes in the brain are a recognized HIV reservoir that may produce Nef even under successful cART therapy, it is likely that ABCA1 in astrocytes and oligodendrocytes is also affected by Nef. This may be the reason for persistence of cognitive impairment despite cART, even in subjects with undetectable viral load. Our preliminary results show that treatment of brain slices with recombinant Nef induces demyelination. The mechanisms behind the Nef effects on astrocytes and oligodendrocytes will be investigated in Aim 1 of the proposal. The bioinformatics and protein interaction studies in this aim will be performed in the laboratory of our Russian partners, who are experts in neurological disorders and with whom we have established a long-standing productive collaboration in studies of Nef-induced impairment of ABCA1. Aim 2 will be devoted to studies of Nef's contribution to persistent neuroinflammation characteristic to HIV infection. Our preliminary results suggest that Nef induces pro-inflammatory memory in differentiating monocytes, leading to hyperresponsiveness of these cells to inflammatory stimuli. Together with our Russian collaborators, we will extend these findings to brain macrophages and will characterize gene expression and epigenetic changes induced by Nef. In Aim 3, we will perform screening for compounds targeting the pathogenic effect of Nef on cholesterol metabolism and inflammation. Our Russian collaborators will carry out this screening, and testing of identified compounds in vitro and in vivo will be performed at both sites. These studies will provide a comprehensive phenotypical and mechanistic characterization of the important pathogenic activity of HIV Nef, and will identify new anti-HIV agents that may be developed to treat HIV-associated neurocognitive disease.