HIV-1 TAT INDUCES ER STRESS IN ASTROCYTES AND CAUSES NEUROTOXICITY THROUGH GFAP ACTIVATION AND AGGREGATION

Purpose: HIV-1 Tat is a major pathogenic factor for HIV-associated neurodegenerative diseases. One of the consistent hallmarks of HIV-1 infection of the central nervous system (CNS) is astrocytosis, which is characterized by increased cytoplasmic accumulation of intermediate filament glial fibrillary acidic protein (GFAP). Our previous studies show that Tat induces GFAP expression in astrocytes and GFAP is a critical regulator of Tat neurotoxicity. However, the molecular mechanisms responsible for GFAP activation-mediated Tat neurotoxicity is not known. Thus, we attempted in this study to determine the underlying unknown molecular mechanisms. Methods: Brain-targeted inducible Tat transgenic and GFAP knockout mice were used in the study. Primary astrocytes were either transfected with pTat.cMyc or treated with doxycycline to induce Tat expression. Cells were harvested to detect GFAP, eIF2a, ATF6, Oasis and Bip expression by Western blotting to , or to detect Tat, GFAP and XBP-1 by RT-PCR. Culture supernatants were collected and analyzed for neurotoxicity toward primary mouse or human neurons using MTT assay. GFAP, MAP-2 and Bip expression in astrocytes, Tat-expressing mouse brain and the brains of HIV-infected individuals were also determined by IF and IHC staining. Flow cytometry was performed to determine proteasomal activity by monitor expression intensity of proteasomal activity reporter pZsProSensor-1. Results: We showed that HIV-1 Tat-induced GFAP up-regulation and aggregation in astrocytes activated endoplasmic reticulum (ER) stress and unfolded protein response (UPR) pathways, such as PERK, IRE1, ATF6 and OASIS, We further showed that supernatants from Tat-expressing astrocytes were neurotoxic. Importantly, we showed that the neurotoxicity in these culture supernatants were significantly diminished when GFAP was null or when the cultures were treated with 4-sodium phenyl butyrate (4-PBA), a chemical chaperon capable of blocking the ER stress, suggesting that GFAP activation and aggregation-induced ER stress in the presence of Tat expression is at least in part responsible for Tat neurotoxicity. Lastly, we showed that GFAP activation and aggregation resulted in lower proteasome activity and induction of autophagy in astrocytes. Conclusions: Taking together, this study 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.