Abstract: As of 2014, an estimated 35 million persons worldwide were living with human immunodeficiency virus (HIV). Early after primary infection, HIV enters the CNS and causes long lasting cognitive and motor impairment in 30-60 % of infected individuals, even in the current antiretroviral era. As infected individuals are living longer, the prevalence of neurological complications has been increasing. In the CNS, HIV infects mostly microglia/macrophages, but also a small population of astrocytes. However, despite the key roles of astrocytes in CNS functions, the role of these cells in NeuroAIDS has been relatively ignored. Our studies during the last funding period provide strong evidence for the critical role of astrocytes in the pathogenesis of NeuroAIDS. In particular, we have demonstrated that despite relatively low numbers of infected astrocytes and low to undetectable HIV replication, the HIV infected astrocytes transmit apoptotic and inflammatory signals, including calcium and inositol triphosphate (IP3), to neighboring uninfected cells, promoting neuronal damage and demise. We have also shown that these pro-apoptotic molecules are spread from the few HIV infected astrocytes via connexin-43 (Cx43) containing gap junctions (GJ) and unopposed hemichannels (uHC), whose expression and opening is regulated by HIV. Indeed, blocking GJ or uHC reduced amplification of bystander apoptosis, cellular dysfunction, synaptic compromise, and mitochondrial dysfunction induced by HIV infected astrocytes. Interestingly, HIV infected astrocytes themselves are protected from apoptosis by mechanisms that involve altered apoptosome formation and mitochondrial function. Importantly, we have tested and validated most of the mechanisms operating in HIV infected astrocytes in vivo in human and monkey brain tissue sections. Thus, based on the results obtained during the extremely productive period funded by our first R01 (resulting in over 40 publications in high quality peer reviewed journals) we have formulated our current hypothesis that “HIV infected astrocytes survive HIV infection to become HIV reservoirs, and that these cells send toxic, pro-apoptotic signals to surrounding cells via Cx43 containing channels, leading to the CNS dysfunction and NeuroAIDS”. In this application we propose to characterize the novel pathways of HIV toxicity within the brain and to identify the role of GJ and uHC in CNS dysfunction. The results obtained from this proposal will lead to the identification of potential novel therapeutic targets to limit the devastating consequences of NeuroAIDS.