2-3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline and Amyotrophic-Lateral-Sclerosis

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Amyotrophic Lateral Sclerosis; Animals; Drug Design; Humans; Quinoxalines; Receptors, AMPA

The toxin beta-methylamino-l-alanine (BMAA) has been proposed to contribute to amyotrophic lateral sclerosis-Parkinsonism Dementia Complex of Guam (ALS/PDC) based on its ability to induce a similar disease phenotype in primates and its presence in cycad seeds, which constituted a dietary item in afflicted populations. Concerns about the apparent low potency of this toxin in relation to estimated levels of human ingestion led to a slowing of BMAA research. However, recent reports identifying potential new routes of exposure compel a re-examination of the BMAA/cycad hypothesis. BMAA was found to induce selective motor neuron (MN) loss in dissociated mixed spinal cord cultures at concentrations ( approximately 30 muM) significantly lower than those previously found to induce widespread neuronal degeneration. The glutamate receptor antagonist NBQX prevented BMAA-induced death, implicating excitotoxic activation of AMPA/kainate receptors. Using microfluorimetric techniques, we further found that BMAA induced preferential [Ca(2+)](i) rises and selective reactive oxygen species (ROS) generation in MNs with minimal effect on other spinal neurons. Cycad seed extracts also triggered preferential AMPA/kainate-receptor-dependent MN injury, consistent with the idea that BMAA is a crucial toxic component in this plant. Present findings support the hypothesis that BMAA may contribute to the selective MN loss in ALS/PDC.

Topics: Amino Acids, Diamino; Amyotrophic Lateral Sclerosis; Animals; Calcium; Cell Survival; Cells, Cultured; Cyanobacteria Toxins; Cycas; Dose-Response Relationship, Drug; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Humans; Mice; Motor Neurons; Neurotoxins; Plant Extracts; Plant Poisoning; Quinoxalines; Reactive Oxygen Species; Receptors, AMPA; Receptors, Kainic Acid; Seeds; Spinal Cord

Several lines of evidence in the literature purport the contribution of glutamate mediated excitotoxicity in the etiology of amyotrophic lateral sclerosis (ALS) but the cellular mechanisms responsible for selective loss of motor neurons are still obscure. Elevation of intracellular Ca(2+) is considered as the early event in glutamate mediated cell injury. We have studied the changes in [Ca(2+)](i) and cytotoxicity in motor neurons and other spinal neurons in culture upon exposure to cerebrospinal fluid (CSF) from ALS patients. CSFs from 20 ALS patients and 20 disease control patients were examined. Eighteen out of twenty (90%) ALS-CSF samples induced a transient but pronounced elevation of [Ca(2+)](i) in neurons, whereas only 1/20 (5%) sample from disease control patients induced a marginal elevation of [Ca(2+)](i). Strikingly the [Ca(2+)](i) rise was 2-3-fold higher and longer lasting in motor neurons in comparison to the other spinal neurons. Exposure of cells to ALS-CSF drastically decreased the survival rate of motor neurons to 32.26+/-2.06% whereas a moderate decrease was observed in case of other spinal neurons (67.90+/-2.04%). In cultures treated with disease control CSF, a small decrease was observed in the survival rate with 80.14+/-2.00% and 90.07+/-1.37% survival of motor neuron and other spinal neurons respectively. The AMPA/kainate receptor antagonist NBQX rendered significant protection against the ALS-CSF induced Ca(2+) influx and neurotoxicity while the NMDA receptor antagonist APV showed a mild effect. Our data demonstrate that the exposure of spinal cord neurons to ALS-CSF differentially elevates [Ca(2+)](i) and neurotoxicity in motor neurons by activation of glutamate receptors, the AMPA/kainate receptor playing the major role.

Topics: 2-Amino-5-phosphonovalerate; Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Calcium; Case-Control Studies; Cell Survival; Cells, Cultured; Cerebrospinal Fluid; Diagnostic Imaging; Drug Interactions; Embryo, Mammalian; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Female; Humans; Intracellular Fluid; Male; Motor Neurons; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, Kainic Acid; Spinal Cord; Time Factors

alpha-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor-mediated excitotoxicity has been implicated in the selective motor neuron loss in amyotrophic lateral sclerosis (ALS). The extent to which excitotoxicity contributes to motor neuron death remains incompletely understood. We therefore tested the potent and selective AMPA/kainate receptor antagonist 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide (NBQX) on motor neurons in culture and in the G93A mouse model for familial ALS. Kainate-induced currents and changes in intracellular Ca(2+) concentration were measured with the perforated patch clamp technique combined with Ca(2+) imaging, motor neuron death was quantified by counting experiments and G93A mice were treated with saline or 8 mg/kg NBQX. NBQX blocked kainate-induced currents and concomitant changes in intracellular Ca(2+), prevented the kainate-induced motor neuron death, and prolonged survival of G93A mice.

Topics: Age Factors; Alanine; Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Behavior, Animal; Calcium; Cells, Cultured; Disease Models, Animal; Drug Interactions; Embryo, Mammalian; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Forelimb; Glycine; Hand Strength; Kainic Acid; Membrane Potentials; Mice; Mice, Inbred C57BL; Mice, Transgenic; Motor Neurons; Psychomotor Performance; Quinoxalines; Receptors, AMPA

We studied ionotropic glutamate receptor subtypes and the effect of chronic treatment with NBQX [6-nitro-7-sulphamoyl-benzo(F)quinoxaline-2,3-dione], a selective (rs)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor antagonist, in the spinal cord of mnd mice. NBQX (8 mg/kg daily i.p. for 3 weeks starting from 24 weeks old) significantly improved the behavioural scores (hind leg extension reflex, cage rung grasping and gait) in mnd mice, measured after the last drug injection, and increased the number of mice with 'normal' gait (from 50% to 90%, P < 0.05). Receptor binding autoradiography of the competitive N-methyl-D-aspartate (NMDA) antagonist, [3H]CGP 39653, of [3H]AMPA and [3H]kainic acid in spinal cord sections, measured after 1 week of drug washout, were not significantly different in control and mnd mice, and were not modified by NBQX. GluR2/3 immunoreactivity, assessed using Western blotting, was significantly enhanced (by 59%, P < 0.01) in the spinal cord but not in the brain of 28-week-old mnd mice compared to age-matched control mice. NBQX treatment increased GluR2/3 immunoreactivity in the spinal cord of control mice and mnd mice by 327 +/- 74% (P < 0.01) and 212 +/- 52% (P < 0.01), respectively. The changes in GluR2/3 subunits may involve adaptive mechanisms of the receptor and play some role in the protective effect of NBQX. These findings suggest that selective antagonism of ionotropic non-NMDA receptors may be of value in the treatment of motor neuron disease.

Topics: 2-Amino-5-phosphonovalerate; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Amyotrophic Lateral Sclerosis; Animals; Antibodies; Autoradiography; Blotting, Western; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Male; Mice; Mice, Mutant Strains; Motor Neurons; Nerve Degeneration; Neurotoxins; Quinoxalines; Receptors, AMPA; Spinal Cord; Tritium