NEUROTOXICITY OF HIV COAT PROTEIN-GP120, NMDA RECEPTORS, AND PROTEIN-KINASE-C - A STUDY WITH RAT CEREBELLAR GRANULE CELL-CULTURES

The neuronal loss observed in AIDS patients may be partly due to the neurotoxicity of HIV coat protein gp120, whose mechanism of action has been suggested to involve an interaction with voltage-dependent Ca2+ channels and NMDA receptors (Lipton, Trends Neurosci 15:75-79, 1992). In the present investigation we analyzed the acute neurotoxicity of gp120 on a purified neuronal population (rat cerebellar granule cell cultures) amply used for studies on glutamate toxicity. Cultures of 7-8 days were exposed for 15 min to a buffered Locke's solution containing the substances under study, washed, and cultured for another 24 hr in their original medium. The cells were stained with the nuclear dyes propidium iodide (for dead cells) and Hoechst 33258 (for total cells) and counted. Average cell death in controls was 8%. gp120 (1 pM-10 nM) caused an increase of cell death of about 80%. The effect was totally antagonized by NMDA antagonists (1 mM APV and 10 muM MK-801), by 1 muM nifedipine, and by anti-gp120 antibodies. At a concentration of 100 muM glutamate caused an average 130% increase of cell death, which was totally antagonized by APV. The effect of gp120 or glutamate did not appear to be mediated by the secretion of neurotoxins by nonneuronal cells present in a low proportion in the cultures nor to be due to the inactivation of (or competition with) neurotrophic factors present in the medium. The simultaneous administration of gp120 and glutamate (in various combinations of concentrations) had an effect that was less than additive. The combined effect of the two substances was partially antagonized by anti-gp120 antibodies and totally prevented by the combined action of anti-gp120 antibodies and NMDA antagonists. Surprisingly, however, NMDA antagonists prevented only partially the neuronal death elicited by gp120 + glutamate. In order to determine whether gp120 affected one of the events potentially involved in NMDA-mediated neurotoxicity [protein kinase C (PKC) translocation to the plasma membrane], we studied the effect of gp120 and glutamate on the binding of 4beta-[H-3]phorbol-12,13-dibutyrate, a specific ligand for membrane PKC. While glutamate caused a dose-dependent, APV-sensitive increase in binding, gp120 determined a decrease in basal binding, suggesting that its mechanism of action does not involve PKC translocation.