IMPORTANT ROLES OF EXOSOMES IN HIV-1 TAT-MEDIATED NEUROTOXICITY

Purpose: Our purpose is to determine the significance of astrocyte-released exosomes in regulation of HIV-1 Tat neurotoxicity. We hypothesize that the exosomes produced by astrocytes contribute to Tat neurotoxicity. Methods: Primary astrocyte cultures were prepared from the embryos of brain-specific and doxycycline-inducible Tat-transgenic and wild type mice and treated with 5 ug/ml doxycycline for 3 days to induce Tat expression. The Tat transgenic mice express HIV Tat gene under the dual control of a doxycycline-inducible promoter and the glial fibrillary acidic protein (GFAP) gene promoter. Addition of doxycycline induces the expression of HIV-1 Tat protein in mouse astrocytes. Supernatants from doxycycline-treated cultures were collected and incubated with SHSY5Y neuroblastoma cells for 3 days. MTT assay was performed to determine the survival of the neurons. Briefly, tetrazolium reagent was added to cultures and incubated for 5 hours in 37oC. The reduction of this reagent by NAPDH-Oxidase in the mitochondria of healthy cells will form a blue color which can be measured spectrophotometrically after dissolution with acid-isopropanol. The intensity of the blue color indicates the number of healthy cells. To determine the roles of exosomes in Tat-induced neurotoxicity, astrocyte cultures were first induced with doxycycline, the culture media were replaced with fresh complete media, the cell continued to culture for 48 hours in the presence of 5 uM or 10 uM GW4869, an inhibitor of neutral sphingomyelinase which is critical for the production of exosomes in astrocytes. The supernatants from GW4869-treated cultures were collected and determined for its neurotoxicity using the MTT assay described above. Results: Supernatants of doxycycline-induced Tat-transgenic astrocyte cultures decreased the survival of neuroblastoma cells compared to those of wild type astrocytes. Inhibition of exosome production in doxycycline-treated astrocytes with GW4869 improved the survival of neuroblastoma cells compared to those that were not treated. Conclusions: These findings confirmed that Tat expression in astrocytes in the absence of HIV infection caused neurotoxicity and showed for the first time that exosomes produced from Tat-expressing astrocytes were involved in induction of Tat neurotoxicity.