THE ROLE OF MICROTUBULES IN EXCITOTOXIN-INDUCED AXON DEGENERATION

Abstract

Axon degeneration is a process which occurs in several neurodegenerative diseases, including Alzheimer's disease, although, mechanisms are not well characterized. One potential cause of axon degeneration, excitotoxicity, results in the breakdown of the microtubule cytoskeleton, by unknown mechanisms. Investigating microtubule alterations following excitotoxic insult could provide insight into potential therapeutic treatment with microtubule stabilizing drugs. Therefore, we examined a time course of microtubule modifications after kainic acid-induced excitotoxicity and the potential of stabilizing microtubules with trichostatin A to prevent axon degeneration in vitro. Primary cortical culture of embryonic day 15.5 C57Bl/6 mice were grown to 10 days in vitro (DIV) prior to treatment with 10μM and 25μM concentrations of kainic acid to induce excitotoxicity. Alterations to microtubules, including post-translational modifications acetylation, tyrosination and glutamylation as well as alterations to microtubule associated proteins were assessed at 1, 6 and 18 hours post-treatment using ELISA and immunocytochemical techniques. Cell viability was assessed using alamarBlue® after kainic acid and trichostatin A treatment. Kainic acid-induced excitotoxicity significantly (p<0.05) reduced acetylation of microtubules but significantly (p<0.05) increased tau expression after 6 hours. However, tyrosination of microtubules was unchanged. Cell viability assessed using alamarBlue® was reduced after kainic acid treatment. Microtubule stabilizing agent trichostatin A, increased acetylation of microtubules after 2 hours and was not toxic to neurons. Furthermore, when neurons were pre-treated with the drug prior to kainic acid exposure axon fragmentation was significantly (p<0.05) reduced. These data suggest that excitotoxic insult reduces microtubule acetylation and targeting microtubule post-translational modifications may be a promising potential therapeutic target for axon protection. Tyrosination was unaffected by excitotoxic insult at 10μM and 25μM kainic acid, suggesting it does not play a role in microtubule breakdown.