Tat-induced Neurotoxicity Research Articles

Human immunodeficiency virus type 1 Tat protein decreases cyclic AMP synthesis in rat microglia cultures.

We have studied the modulation of cyclic AMP (cAMP) accumulation by the human immunodeficiency virus type 1 (HIV 1) protein Tat in microglia and astrocyte cultures obtained from neonatal rat brain. Pretreatment of microglia with recombinant Tat resulted in a dose- and time-dependent decrease of cAMP accumulation induced by subsequent exposure to isoproterenol (1 microM). The inhibitory action of 100 ng/mL Tat approached 50% after 4 h of preincubation and reached a maximum of 70% after 24 h. The Tat-induced time- and dose-dependent decrease of cAMP accumulation was observed also when microglial cultures were stimulated with the adenylyl cyclase activator forskolin (100 microM). In both cases, Tat inhibitory action was 70% reverted by a specific monoclonal anti-Tat antibody, but was not prevented either by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xantine (100 microM) or by a 16-h pretreatment of microglial cultures with the Gi protein inhibitor pertussis toxin (10 ng/mL). All these results suggested that the viral protein acts at a step of the cAMP transduction pathway other than receptors, G proteins and phosphodiesterases. The target of Tat appeared to be adenylyl cyclase, whose activity was markedly reduced (up to 60%) in membranes prepared from Tat-treated microglial cells, both in basal conditions and after stimulation with isoproterenol and forskolin. The inability of the competitive inhibitor of nitric oxide synthase N(G)-monometyl- L-arginine (20 and 200 microM) to revert Tat action on forskolin-induced cAMP accumulation, and of two potent nitric oxide donors, PAPA and DETA (0.1-2 m M), to alter forskolin-induced cAMP accumulation, excluded an involvement of nitric oxide in Tat-induced adenylyl cyclase inhibition. On the contrary, two inhibitors of nuclear factor kappaB activation, N-tosyl-( L)-phenylalanine chloromethyl ketone (10 microM) and SN50 (25 microM), markedly prevented the reduction of forskolin-evoked cAMP accumulation by Tat, suggesting a possible role for this nuclear transcriptional factor in the regulation of adenylyl cyclase by Tat in microglia. This assumption was strengthened by the ability of lipopolysaccharide (100 ng/mL, 4 h) to mimic the inhibitory effect of the viral protein. Conversely, astrocyte cAMP accumulation was unaffected by the viral protein, as tested at various concentrations and time points. Finally, Tat inhibition of microglial adenylyl cyclase was not due to non-specific cytotoxicity. As cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, and microglia is believed to mediate Tat-induced neurotoxicity, these results suggest that the ability of Tat to inhibit cAMP synthesis in microglia may contribute to neuronal degeneration and cell death associated with HIV infection.

Involvement of inositol 1,4,5-trisphosphate-regulated stores of intracellular calcium in calcium dysregulation and neuron cell death caused by HIV-1 protein tat.

HIV-1 infection commonly leads to neuronal cell death and a debilitating syndrome known as AIDS-related dementia complex. The HIV-1 protein Tat is neurotoxic, and because cell survival is affected by the intracellular calcium concentration ([Ca2+]i), we determined mechanisms by which Tat increased [Ca2+]i and the involvement of these mechanisms in Tat-induced neurotoxicity. Tat increased [Ca2+]i dose-dependently in cultured human fetal neurons and astrocytes. In neurons, but not astrocytes, we observed biphasic increases of [Ca2+]i. Initial transient increases were larger in astrocytes than in neurons and in both cell types were significantly attenuated by antagonists of inositol 1,4,5-trisphosphate (IP3)-mediated intracellular calcium release [8-(diethylamino)octyl-3,4,5-trimethoxybenzoate HCI (TMB-8) and xestospongin], an inhibitor of receptor-Gi protein coupling (pertussis toxin), and a phospholipase C inhibitor (neomycin). Tat significantly increased levels of IP3 threefold. Secondary increases of neuronal [Ca2+]i in neurons were delayed and progressive as a result of excessive calcium influx and were inhibited by the glutamate receptor antagonists ketamine, MK-801, (+/-)-2-amino-5-phosphonopentanoic acid, and 6,7-dinitroquinoxaline-2,3-dione. Secondary increases of [Ca2+]i did not occur when initial increases of [Ca2+]i were prevented with TMB-8, xestospongin, pertussis toxin, or neomycin, and these inhibitors as well as thapsigargin inhibited Tat-induced neurotoxicity. These results suggest that Tat, via pertussis toxin-sensitive phospholipase C activity, induces calcium release from IP3-sensitive intracellular stores, which leads to glutamate receptor-mediated calcium influx, dysregulation of [Ca2+]i, and Tat-induced neurotoxicity.