N-methyl-D-aspartate receptor-mediated mitochondrial Ca(2+) overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca(2+) influx.
Mitochondrial uptake of Ca(2+) has recently been found to play an important role in glutamate-induced neurotoxicity (GNT) as well as in the activation of Ca(2+)-dependent molecules, such as calmodulin and neuronal nitric oxide synthase (nNOS), in the cytoplasm. Prolonged exposure to glutamate injures motor neurons predominantly through the activation of Ca(2+)/calmodulin-nNOS, as previously reported, and is, in part, associated with the pathogenesis of amyotrophic lateral sclerosis (ALS). In the present study, we investigated how mitochondrial uptake of Ca(2+) is involved in GNT in spinal motor neurons. Acute excitotoxicity induced by exposure to 0.5 mM glutamate for 5 min was found in both motor and nonmotor neurons in cultured spinal cords from rat embryos and was dependent on extracellular Ca(2+) and on N-methyl-D-aspartate (NMDA) receptor activation. Mitochondrial uncouplers markedly blocked acute excitotoxicity, and membrane-permeable superoxide dismutase mimics attenuated acute excitotoxicity induced by glutamate and NMDA but not by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) or kainate. Fluorimetric analysis showed that mitochondrial Ca(2+) was elevated promptly with subsequent accumulation of reactive oxygen species (ROS) in the mitochondria. An NMDA receptor antagonist and a mitochondrial uncoupler eliminated the increase in fluorescence of mitochondrial Ca(2+) and ROS indicators. These data indicate that acute excitotoxicity in spinal neurons is mediated by mitochondrial Ca(2+) overload and ROS generation through the activation of NMDA receptors. This mechanism is different from that of chronic GNT.
Urushitani
M
M
Department of Neurology, Graduate School of Medicine, Kyoto University, Sakyoku, Kyoto, Japan.
Nakamizo
T
T
Inoue
R
R
Sawada
H
H
Kihara
T
T
Honda
K
K
Akaike
A
A
Shimohama
S
S
D019297:2,4-Dinitrophenol / Q000494:pharmacology; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone / Q000494:pharmacology; D002478:Cells, Cultured / Q000187:drug effects; D016572:Cyclosporine / Q000494:pharmacology; D003992:Dibucaine / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018698:Glutamic Acid / Q000494:pharmacology / Q000633:toxicity*; D006575:Heterocyclic Compounds, 3-Ring; D007093:Imidazoles / Q000494:pharmacology; D007608:Kainic Acid / Q000494:pharmacology; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D016472:Motor Neuron Disease / Q000378:metabolism; D009046:Motor Neurons / Q000187:drug effects* / Q000473:pathology; D016202:N-Methylaspartate / Q000494:pharmacology; D009419:Nerve Tissue Proteins / Q000378:metabolism; D009474:Neurons / Q000187:drug effects; D009498:Neurotoxins / Q000494:pharmacology / Q000633:toxicity*; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052248:Nitric Oxide Synthase Type I; D018384:Oxidative Stress; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species; D018091:Receptors, AMPA / Q000187:drug effects; D018092:Kainic Acid Receptors / Q000187:drug effects; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology*; D012235:Rhodamines; D013116:Spinal Cord / Q000166:cytology / Q000196:embryology; D013482:Superoxide Dismutase / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid / Q000494:pharmacology
D019297:2,4-Dinitrophenol; D018750:6-Cyano-7-nitroquinoxaline-2,3-dione; D002118:Calcium; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002259:Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; D016572:Cyclosporine; D003992:Dibucaine; D016291:Dizocilpine Maleate; D004791:Enzyme Inhibitors; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018698:Glutamic Acid; D006575:Heterocyclic Compounds, 3-Ring; D007093:Imidazoles; D007608:Kainic Acid; D016202:N-Methylaspartate; D009419:Nerve Tissue Proteins; D009498:Neurotoxins; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D017382:Reactive Oxygen Species; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D016194:Receptors, N-Methyl-D-Aspartate; D012235:Rhodamines; D013482:Superoxide Dismutase; D014475:Uncoupling Agents; D018350:alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; C109098:2,7-dihydrodichlorofluorescein diacetate; C058319:dihydrorhodamine 123; C068483:rhod-2; C031938:calmidazolium; C496322:Nos1 protein, rat