tetrodotoxin and Amyotrophic-Lateral-Sclerosis

Voltage-gated Ca(2+) channels (VGCCs) mediate calcium entry into neuronal cells in response to membrane depolarisation and play an essential role in a variety of physiological processes. In Amyotrophic Lateral Sclerosis (ALS), a fatal neurodegenerative disease caused by motor neuron degeneration in the brain and spinal cord, intracellular calcium dysregulation has been shown, while no studies have been carried out on VGCCs. Here we show that the subtype N-type Ca(2+) channels are over expressed in G93A cultured cortical neurons and in motor cortex of G93A mice compared to Controls. In fact, by western blotting, immunocytochemical and electrophysiological experiments, we observe higher membrane expression of N-type Ca(2+) channels in G93A neurons compared to Controls. G93A cortical neurons filled with calcium-sensitive dye Fura-2, show a net calcium entry during membrane depolarization that is significantly higher compared to Control. Analysis of neuronal vitality following the exposure of neurons to a high K(+) concentration (25 mM, 5h), shows a significant reduction of G93A cellular survival compared to Controls. N-type channels are involved in the G93A higher mortality because ω-conotoxin GVIA (1 μM), which selectively blocks these channels, is able to abolish the higher G93A mortality when added to the external medium. These data provide robust evidence for an excess of N-type Ca(2+) expression in G93A cortical neurons which induces a higher mortality following membrane depolarization. These results may be central to the understanding of pathogenic pathways in ALS and provide novel molecular targets for the design of rational therapies for the ALS disorder.

Topics: Amyotrophic Lateral Sclerosis; Animals; Animals, Newborn; Calcium Channel Blockers; Calcium Channels, N-Type; Cell Survival; Cells, Cultured; Cerebral Cortex; Cytophotometry; Disease Models, Animal; Electric Stimulation; Gene Expression Regulation; Humans; Membrane Potentials; Mice; Mice, Transgenic; Motor Neurons; omega-Agatoxin IVA; omega-Conotoxin GVIA; Patch-Clamp Techniques; Sodium Channel Blockers; Superoxide Dismutase; Tetrodotoxin

Cortical hyperexcitability has been observed in Amyotrophic Lateral Sclerosis (ALS) patients. Familial ALS accounts for 10% of all cases and mutations of the Cu,Zn superoxide dismutase (SOD1) gene have been identified in about 20% of the familial cases. The aim of this study was to investigate whether in a mouse model of ALS the cortical neurons developed hyperexcitability due to intrinsic properties of the single cell. We first examined the passive membrane properties and the pattern of repetitive firing in cultured cortical neurons from Control mice and transgenic mice expressing high levels of the human mutated protein (Gly(93)-->Ala, G93A). The former did not display significantly differing values between Control and G93A cortical neurons. However, the threshold potential and time of the first action potential decreased significantly and the firing frequency increased significantly in the G93A compared to Control neurons. The analysis of the voltage-dependent sodium currents revealed that the fast transient sodium current was unaffected by the SOD1 mutation whereas the persistent sodium current was significantly higher in the mutated neurons. Finally, Riluzole, a selective blocker of the persistent sodium current at low concentrations, decreased the firing frequency in G93A neurons, strongly indicating an involvement of this current in the observed hyperexcitability. These are the first data that demonstrate an intrinsic hyperexcitability in the G93A cortical neurons due to a higher current density of the persistent sodium current in the mutated neurons and open up new prospects of understanding ALS disease etiopathology.

Topics: Amyotrophic Lateral Sclerosis; Animals; Biophysical Phenomena; Cells, Cultured; Cerebral Cortex; Disease Models, Animal; Electric Stimulation; Excitatory Amino Acid Antagonists; Humans; Ion Channel Gating; Membrane Potentials; Mice; Mice, Transgenic; Neurons; Patch-Clamp Techniques; Riluzole; Sodium Channel Blockers; Sodium Channels; Superoxide Dismutase; Tetrodotoxin

The neurotoxicity of the AMPA/kainate receptor agonist kainate was investigated in motor and cortical neurones from mice over-expressing the wild-type and G93A mutant form of Cu/Zn superoxide dismutase (SOD1) human gene, a mouse model of familial amyotrophic lateral sclerosis. G93A mutant motor neurones were more vulnerable and wild-type SOD1 motor neurones were more resistant to kainate toxicity than were controls. Voltage-gated Na channels blockage prevented G93A mutant SOD1 motor neurone death. Cortical cultures exhibited fewer differences in their vulnerability to kainate toxicity. These results demonstrate that SOD1 over-expression selectively affects the sensitivity to kainate excitotoxicity of motor neurones but not neocortical neurones, and that wild-type SOD1 expression increases the resistance to excitotoxicity of motor neurones.

Topics: Amyotrophic Lateral Sclerosis; Analysis of Variance; Animals; Asparagine; Calcium Channel Blockers; Cell Count; Cell Survival; Cells, Cultured; Cerebral Cortex; Cobalt; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Female; Glial Fibrillary Acidic Protein; Glutamic Acid; Immunohistochemistry; Kainic Acid; Male; Mice; Mice, Transgenic; Motor Neurons; Neurotoxins; Nifedipine; Phosphopyruvate Hydratase; Sodium Channel Blockers; Spinal Cord; Superoxide Dismutase; Tetrodotoxin; Time Factors

The effect of the IgG from amyotrophic lateral sclerosis (ALS) patients was tested on the voltage-dependent barium currents (IBa) in mammalian dissociated Purkinje cells and in isolated P-type calcium channels in lipid bilayers. Whole cell clamp of Purkinje cells demonstrates that ALS IgG increases the amplitude of IBa without modifying their voltage kinetics. This increased IBa could be blocked by a purified nonpeptide toxin from Agelenopsis aperta venom (purified funnel-web spider toxin) or by a synthetic polyamine analog (synthetic funnel-web spider toxin) and by a peptide toxin from the same spider venom, omega-Aga-IVA. Similar results were obtained on single-channel recordings from purified P channel protein. The addition of ALS IgG increased single-channel IBa open time without affecting slope conductance. The results described above were not seen with normal human IgG nor with boiled ALS IgG. It is concluded that ALS IgG enhances inward current through P-type calcium channels. Since P-type Ca2+ channels are present in motoneuron axon terminals, we propose that the enhanced calcium current triggered by ALS IgG may contribute to neuronal damage in ALS.

Topics: Amyotrophic Lateral Sclerosis; Animals; Calcium Channels; Cerebellar Cortex; Guinea Pigs; Humans; Immunoglobulin G; In Vitro Techniques; Lipid Bilayers; Membrane Potentials; Neurotoxins; Polyamines; Purkinje Cells; Tetrodotoxin

Embryonic human spinal cord cells have been grown in dissociated monolayer cultures for 1 to 7 weeks. Using cell type specific markers, it was possible to show that the cultures contain neurons, astrocytes and fibroblasts. Electrical membrane properties were studied with patch electrodes using the whole cell recording technique. Neurons had short duration action potentials that could be blocked by tetrodotoxin. The membrane currents in these neurons were studied in voltage clamp experiments. Three types of voltage-dependent currents were observed: a sodium current; a potassium current made up of two components, IA and IK; and a calcium current. Both cholinergic and GABAergic neurons are present in the cultures. There is more choline acetyltransferase activity in cultures prepared from the anterior as compared to the posterior part of the spinal cord, suggesting that the cultures contain motoneurons. This tissue culture preparation was developed for the study of amyotrophic lateral sclerosis; we have been unable to detect the presence of any toxic agent from the serum of these patients on the cultured cells. Experiments are in progress to purify the motoneurons using Percoll gradients.

Topics: Acetylcholine; Amyotrophic Lateral Sclerosis; Blood Proteins; Cell Survival; Cells, Cultured; Choline O-Acetyltransferase; Cholinergic Fibers; Electric Stimulation; Embryo, Mammalian; gamma-Aminobutyric Acid; Humans; Membrane Potentials; Spinal Cord; Tetraethylammonium Compounds; Tetrodotoxin