HIV-1 TAT EXPRESSION ALTERS HUMAN ASTROCYTE INFLAMMATORY BIOMARKER PROFILES AND GLUTAMATE METABOLISM

Purpose (a): More than 50% of the human immunodeficiency virus type 1 (HIV-1) infected individuals exhibit some form of HIV-associated neurocognitive disorders (HAND). Several studies reported that HIV-1 transactivator of transcription (Tat) protein was associated with HAND pathophysiology. HIV-1 Tat induces apoptosis and dysregulates cytokine/chemokine profiles leading to neurotoxicity. Previous studies have studied the in vivo HIV-1 Tat regulation using transgenic animal models. Although animal models have helped determine the in vivo disease pathology, application of in vitro neural cell models will be critical to decipher cellular and molecular mechanisms associated with HAND. Here, we report an in vitro model system developed by transfecting human astrocytes with a full-length (101 AA) HIV-1 Tat protein expressing plasmid (pTat). HIV-1 Tat expressing in vitro system was used to evaluate HIV-1 Tat regulation of astrocyte inflammatory responses and altered neuroprotective function i.e. glutamate uptake from synapse. Methods (b): Primary human astrocytes were transfected with pTat by nucleofection and HIV-1 Tat expression was evaluated by immunocytochemistry. Effects of HIV-1 Tat on cell viability and replication were determined with metabolic activity and cell proliferation assays. Proinflammatory cytokines and chemokines were assayed using ELISAs. HIV-1 Tat regulation of glutamate clearing ability of astrocytes was determined using a modified amplex red glutamic acid/glutamate oxidase assay. Additionally, mRNA and protein expression of excitatory amino acid transporter-2 (EAAT-2), the major glutamate transporter in astrocytes, was measured by RT-PCR and western blot analysis respectively. Results (c): The immunostaining confirmed HIV-1 Tat expression in transfected astrocytes, while glial fibrillary acidic protein (GFAP) staining indicated morphological alterations. The pTat transfection did not significantly change cell metabolism as compared to controls. However, HIV-1 Tat expression altered chemokine and cytokine levels; specifically HIV-1 Tat increased CCL2 levels significantly (P. Conclusions (d): HIV-1 Tat expression upregulated inflammatory biomarkers and altered glutamate clearing ability of astrocytes, implicating a direct role for astrocyte-expressed HIV-1 Tat in HAND neuropathogenesis.