L-Glutamine-induced apoptosis in microglia is mediated by mitochondrial dysfunction

Under physiological conditions, astrocytes take up L-glutamate from the synaptic gap, metabolize it to L-glutamine and return it to neurons, where L-glutamine is metabolized to L-glutamate and stored in neurotransmitter vesicles. However, under pathological conditions, such as hepatic failure, L-glutamine and ammonium are elevated globally in the brain. The Trojan horse hypothesis of L-glutamine toxicity assumes that intramitochondrial hydrolysis of L-glutamine enhances ammonium locally and leads to mitochondrial dysfunction. In the present study, we show that exposure of murine primary microglia as well as of the microglial cell-line BV-2 to L-glutamine promotes chromatin condensation and formation of crescent-like structures in the nucleus. Furthermore, L-glutamine induced an increase in annexin-V labelling, cell shrinkage (apoptotic volume decrease), cell fragmentation and formation of apoptotic bodies. Inhibition of the phosphate-activated glutaminase with 6-diazo-5-oxo-L-norleucine suppressed chromatin condensation and annexin-V labelling in L-glutamine-exposed cells. In addition, inhibition of the glutamine synthetase with L-methionine sulfoximine suppressed chromatin condensation and annexin-V labelling in ammonium-exposed cells. L-glutamine and ammonium enhanced production of reactive oxygen species, as detected with CM-H(2)DCFDA. Apoptosis, induced by L-glutamine, was inhibited either by the radical scavenger alpha-tocopherol or by the nitric oxide synthase blocker N (G)-methyl-L-arginine. Cyclosporin A, a ligand of the permeability transition pore complex component cyclophilin D, prevented L-glutamine-triggered apoptosis. Furthermore, blockade of caspase-9 activity with Z-LEHD-FMK prevented L-glutamine-triggered apoptosis. Taken together, our results indicate that hydrolysis of l-glutamine and, accordingly, accumulation of ammonium in mitochondria induce the intrinsic pathway of apoptosis, characterized by mitochondrial dysfunction and activation of caspase-9, which activates caspase-3.