HIV-1 TAT INDUCED LYSOSOMAL EXOCYTOSIS IN ASTROCYTES AND ITS CONTRIBUTIONS TO TAT NEUROTOXICITY

Purpose (a): HIV-1 Tat protein is considered to be the critical reason in the processing of HIV-associated neuropathogenesis. Our previous studies demonstrates that HIV-1 Tat expression leads to ER stress in astrocytes through GFAP aggregation and suggest that disruption of ER homeostasis, i.e., ER stress may be involved in HIV-associated neuropathogenesis. But what the neurotoxic factor is in this indirect astrocyte-mediated Tat neurotoxicity system is still unknown. In this study, we take advantage of our Tat-inducible transgenic mice, and proteomic analysis was performed to explore the neurotoxic factor in the astrocyte-mediated Tat neurotoxicity system. Methods (b): Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes and neurons cultures were prepared from mouse embryos. U373 cells and primary astrocytes and were either transfected with pTat.Myc or treated with doxycycline to induce Tat expression. Cells were prepared and analyzed for β–hexosaminidase activity by NAG assay after ionomycin mediated Ca2+ influx. Culture supernatants were collected for immunodepletion and analyzed for their neurotoxicity toward primary mouse or human neurons using MTT assay. TIRF microscopy is utilized to visualize lysosome exocytosis events. Results (c): Base on the protein sequencing and pathway analysis, we proposed that HIV-1 Tat induces lysosomal exocytosis in astrocytes. Then, NAG assay and TIRF microscopy provide consistent evidences that verified our hypothesis. Moreover, we attested that inhibition of Tat induced lysosomal exocytosis in astrocytes by vaculin-1 can abolish Tat induced neuron death. More interestingly, we observed that Tat induced lysosomal exocytosis and neuron death only appears in astrocytes but not in other cell types, such as 293T and Huh 7.5.1 cells. Further more, we proved that GFAP plays a critical role in Tat induced lysosomal exocytosis and neuron death. Conclusions (d): Taken together, these results demonstrate that HIV-1 Tat induces lysosomal exocytosis and hydrolytic enzymes contained within lysosome are suggested to contribute to neuronal death, which is a novel insight into astrocytes-mediated Tat neurotoxicity.