Background:  Mononuclear phagocyte (macrophages and microglia) dysfunction plays a significant role in the pathogenesis of HIV-1-associated dementia (HAD).  Elevated concentrations of the excitatory neurotransmitter glutamate found in the conditioned media of HIV-1-infected monocyte-derived macrophage (MDM) may contribute to neuronal toxicity. Zidovudine (AZT), an inhibitor of HIV-1 replication, inhibits glutamate generation, demonstrating a dependence on productive HIV-1 infection. The observed glutamate production is dependent upon the presence of glutamine, implicating the involvement of a glutamate-generating enzyme, glutaminase, in macrophage-mediated glutamate production. Glutamine is a widely available substrate and its conversion to glutamate by glutaminase is energetically favorable. We investigated whether glutaminase is responsible for this glutamate increase and potential mechanisms.

Methods:  Various HIV-1 strains including ADA, BAL, JR-FL, and 89.6 were used to infect primary human MDM. Conditioned media was analyzed by reversed phase high performance liquid chromatography (RP-HPLC), RNA was quantified with real time polymerase chain reaction (RT-PCR), and protein was evaluated by Western blotting. A panel of novel small molecule glutaminase inhibitors and siRNA for glutaminase were used to block glutaminase function.

Results:  After observing glutamine-dependent generation of glutamate by various HIV-1 strains, microarray analysis revealed up-regulation of the GAC isoform of glutaminase, an observation confirmed with RT-PCR. Using novel small-molecule inhibitors of glutaminase, glutamate production by infected MDM was reduced to control levels. Conditioned media from infected MDM mediates calcium influx and neurotoxicity in rat neuronal culture; this effect was partially blocked by the removal of glutamine as well as treatment with glutaminase inhibitors prior to MCM collection. Furthermore, glutaminase-specific siRNA blocked glutamate production by MDM cultures.

Conclusions:  HIV-1 infection results in pathogenically significant levels of glutamate from mononuclear phagocytes. This glutamate production is mediated by glutaminase. Excitotoxic damage is a phenomenon found in various neurodegenerative disorders including HAD, and glutaminase-mediated excitotoxicity is a potentially significant disease mechanism. These studies identify a potential new therapeutic avenue to neuroinflammation, the blocking of excess glutamate production by mononuclear phagocytes.