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

Authors


Dr H. H. Kerschbaum, as above.
E-mail: hubert.kerschbaum@sbg.ac.at

Abstract

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-H2DCFDA. Apoptosis, induced by l-glutamine, was inhibited either by the radical scavenger α-tocopherol or by the nitric oxide synthase blocker NG-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.

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