tetrodotoxin and Mitochondrial-Diseases

Axonal degeneration occurs in multiple neurodegenerative disorders of the central and peripheral nervous system. Although the underlying molecular pathways leading to axonal degeneration are incompletely understood, accumulating evidence suggests contributions of impaired mitochondrial function, disrupted axonal transport, and/or dysfunctional intracellular Ca(2+)-homeostasis in the injurious cascade associated with axonal degeneration. Utilizing an in vitro model of axonal degeneration, we studied a subset of mouse peripheral sensory neurons in which neurites were exposed selectively to conditions associated with the pathogenesis of axonal neuropathies in vivo. Rotenone-induced mitochondrial dysfunction resulted in neurite degeneration accompanied by reduced ATP levels and increased ROS levels in neurites. Blockade of voltage-gated sodium channels with TTX and reverse (Ca(2+)-importing) mode of the sodium-calcium exchanger (NCX) with KB-R7943 partially protected rotenone-treated neurites from degeneration, suggesting a contribution of sodium channels and reverse NCX activity to the degeneration of neurites resulting from impaired mitochondrial function. Pharmacological inhibition of the Na(+)/K(+)-ATPase with ouabain induced neurite degeneration, which was attenuated by TTX and KB-R7943, supporting a contribution of sodium channels in axonal degenerative pathways accompanying impaired Na(+)/K(+)-ATPase activity. Conversely, oxidant stress (H2O2)-induced neurite degeneration was not attenuated by TTX. Our results demonstrate that both energetic and oxidative stress targeted selectively to neurites induces neurite degeneration and that blockade of sodium channels and of reverse NCX activity blockade partially protects neurites from injury due to energetic stress, but not from oxidative stress induced by H2O2.

Topics: Animals; Axons; Axotomy; Cell Death; Cells, Cultured; Ganglia, Spinal; Humans; Hydrogen Peroxide; Immunohistochemistry; Mice; Mice, Transgenic; Microtubules; Mitochondrial Diseases; Nerve Degeneration; Neurites; Oxidants; Rotenone; Sodium Channel Blockers; Sodium Channels; Sodium-Calcium Exchanger; Sodium-Potassium-Exchanging ATPase; Tetrodotoxin; Thiourea; Uncoupling Agents

Networks of neurons express persistent spontaneous network activity when maintained in dissociated cultures. Prolonged blockade of the spontaneous activity with tetrodotoxin (TTX) causes the eventual death of the neurons. In this study, we investigated some molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 3-4 days of exposure to TTX, well before the neurons die, they began to express markers that lead to their eventual death, 7-10 days later. There was a reduction in glutamate receptor (GluR2) expression, a persistent increase in intracellular calcium concentration, activation of calpain, and an increase in spectrin breakdown products. At this point, blockade of GluR2-lacking GluR1 or calpain (either with a selective antagonist or through the natural regulator of calpain, calpastatin), protected cells from the toxic action of TTX. Subsequently, mitochondria lost their normal elongated shape as well as their membrane potential. Eventually, neurons activated caspase 3 and PUMA (p53 up-regulated modulator of apoptosis), hallmarks of neuronal apoptosis, and died. These experiments will lead to a better understanding of slow neuronal death, typical of neurodegenerative diseases.

Topics: Action Potentials; Animals; Animals, Newborn; Apoptosis; Calcium; Calcium Signaling; Calpain; Cells, Cultured; Cerebral Cortex; Energy Metabolism; Mitochondria; Mitochondrial Diseases; Nerve Degeneration; Nerve Net; Neural Pathways; Neurons; Rats; Rats, Wistar; Signal Transduction; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin