6-cyano-7-nitroquinoxaline-2-3-dione and Motor-Neuron-Disease

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.

Topics: 2,4-Dinitrophenol; 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Calcium; Calcium Signaling; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cells, Cultured; Cyclosporine; Dibucaine; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluoresceins; Fluorescent Dyes; Glutamic Acid; Heterocyclic Compounds, 3-Ring; Imidazoles; Kainic Acid; Mitochondria; Motor Neuron Disease; Motor Neurons; N-Methylaspartate; Nerve Tissue Proteins; Neurons; Neurotoxins; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Receptors, AMPA; Receptors, Kainic Acid; Receptors, N-Methyl-D-Aspartate; Rhodamines; Spinal Cord; Superoxide Dismutase; Uncoupling Agents

We employed a mouse model of ALS, in which overexpression of a familial ALS-linked Cu/Zn-SOD mutation leads to progressive MN loss and a clinical phenotype remarkably similar to that of human ALS patients, to directly test the excitotoxicity hypothesis of ALS. Under basal culture conditions, MNs in mixed spinal cord cultures from the Cu/Zn-SOD mutant mice exhibited enhanced oxyradical production, lipid peroxidation, increased intracellular calcium levels, decreased intramitochondrial calcium levels, and mitochondrial dysfunction. MNs from the Cu/Zn-SOD mutant mice exhibited greatly increased vulnerability to glutamate toxicity mediated by alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors. The increased vulnerability of MNs from Cu/Zn-SOD mutant mice to glutamate toxicity was associated with enhanced oxyradical production, sustained elevations of intracellular calcium levels, and mitochondrial dysfunction. Pretreatment of cultures with vitamin E, nitric oxide-suppressing agents, peroxynitrite scavengers, and estrogen protected MNs from Cu/Zn-SOD mutant mice against excitotoxicity. Excitotoxin-induced degeneration of spinal cord MNs in adult mice was more extensive in Cu/Zn-SOD mutant mice than in wild-type mice. The mitochondrial dysfunction associated with Cu/Zn-SOD mutations may play an important role in disturbing calcium homeostasis and increasing oxyradical production, thereby increasing the vulnerability of MNs to excitotoxicity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Antioxidants; Calcium; Cells, Cultured; Cyclic N-Oxides; Estradiol; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Free Radicals; Glutamic Acid; Homeostasis; Humans; Imidazoles; Lipid Peroxidation; Mice; Mice, Transgenic; Mitochondria; Motor Neuron Disease; Motor Neurons; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitrates; Oxidative Stress; Rhodamine 123; Spinal Cord; Superoxide Dismutase; Superoxides; Vitamin E

The distribution and density of non-NMDA receptors in spinal cord and motor cortex was compared in 10 cases of motor neuron disease (MND) and 8 neurologically normal controls by quantitative autoradiography using [3H]CNQX and [3H]kainate. In the motor cortex of MND cases, an increased density of [3H]kainate binding sites was observed which was most marked in the deep layers. No significant differences were observed in [3H]CNQX binding in the motor cortex between MND and control cases. In the spinal cord significantly increased densities of both [3H]CNQX and [3]kainate binding sites were found in the substantia gelatinosa and the intermediate grey matter in the MND group. The changes in [3H]kainate binding were observed only in the amyotrophic lateral sclerosis (ALS) subgroup of MND, while the changes in [3H]CNQX binding in the spinal cord were more marked in ALS compared to progressive muscular atrophy (PMA) cases. These findings provide evidence in support of a disturbance of glutamatergic neurotransmission in MND and suggest that there may be an increased excitatory drive to motor neurons via non-NMDA receptors. It is unclear at present whether the changes observed represent a compensatory response to loss of motor neurons in MND or a pathophysiological phenomenon contributing to motor neuron degeneration. Modulation of non-NMDA receptor activity may represent a possible target for therapeutic intervention in this disease.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Adult; Aged; Autoradiography; Female; Humans; Kainic Acid; Male; Middle Aged; Motor Cortex; Motor Neuron Disease; Nerve Degeneration; Receptors, AMPA; Receptors, Glutamate; Receptors, Kainic Acid; Spinal Cord