Astrocyte-mediated HIV-1 Tat neurotoxicity and its molecular mechanisms: astrocyte activation and impaired neurogenesis

Human immunodeficiency virus type 1 (HIV-1) invasion of the central nervous system (CNS) often causes motor and cognitive dysfunction, which is termed HIV-associated neurocognitive disorders (HAND). Although the introduction of combination antiretroviral therapy (cART) has effectively suppressed viral replication, improved immune function and increased life expectancy among HIV-infected individuals, it has failed to provide complete protection from HAND or to reverse the disease. HIV-1 Tat protein is a major pathogenic factor in HAND. Studies including ours have demonstrated that Tat is taken up by HIV-1 uninfected brain cells and alters the function of these cells, especially astrocytes, neurons and neural progenitor cells. However, the underlying mechanisms are still unclear. There are two parts to my dissertation research. In the first part, we determined the roles of signal transducer and activator of transcription 3 (STAT3) in Tat-induced glial fibrillary acidic protein (GFAP) transactivation. Astrocytes, the most abundant cells in the brain, not only provide the scaffold support in the brain, but also are essential for maintaining the homeostasis of the brain. GFAP is the specific molecular marker for astrocytes, but it also modulates astrocyte function both physiologically and pathologically. In this study, we first showed that STAT3 expression and phosphorylation led to significant increases in GFAP transcription and protein expression. Then we determined that Tat expression was associated with increased STAT3 expression and phosphorylation in Tat-expressing astrocytes and HIV-infected astrocytes. In addition, we showed that GFAP, Egr-1 and p300 transcription all showed positive response to STAT3 and its phosphorylation. Moreover, knockdown of STAT3 resulted in significant decreases in Tat-induced GFAP and Egr-1 transcription and protein expression. Taken together, these findings show that STAT3 is involved in and acts upstream of Egr-1 and p300 in the Tat-induced GFAP transactivation cascade and suggest important roles of STAT3 in controlling astrocyte proliferation and activation in the HIV-infected CNS. In the second part of the dissertation research, we took advantage of the doxycyclineinducible and astrocyte-specific HIV-1 Tat transgenic mice (iTat) and determined the relationship between Tat expression and neurogenesis. Tat expression in astrocytes was associated with detection of fewer neuron progenitor cells (NPC), fewer immature neurons and fewer mature neurons in the dentate gyrus of the hippocampus of the mouse brain. In vitro NPC-derived neurosphere assays showed that Tat-containing conditioned media from astrocytes and recombinant Tat protein inhibited NPC proliferation and migration and altered NPC differentiation, while immunodepletion of Tat from Tat-containing conditioned media or heat inactivation of recombinant Tat abrogated those effects. Notch signaling downstream gene Hes1 promoter-driven luciferase reporter gene assay and Western blotting showed that recombinant Tat and Tat-containing conditioned media activated Hes1 transcription and protein expression, which were abrogated by Tat heat inactivation, immunodepletion, and cysteine mutation at position 30. Moreover, Notch signaling inhibitor DAPT significantly rescued Tat-impaired NPC differentiation in vitro and neurogenesis in vivo. Taken together, these results show that Tat adversely affects NPC and neurogenesis through Notch signaling and point to the potential of developing Notch signaling inhibitors as HAND therapeutics.