Abstract
BackgroundChronic ethanol exposure has been shown to result in changes in neuronal cyto-architecture such as aberrant sprouting and alteration of neurite outgrowth. In PC12 cells, chronic ethanol treatment produces an increase in Nerve Growth Factor (NGF)-induced neurite outgrowth that appears to require the epsilon, but not delta, isoform of Protein Kinase C (PKC). Neurites contain a core of microtubules that are formed from polymerization of free-tubulin. Therefore, it would be expected that an increase in neurite outgrowth would correlate with an increase in microtubule content. We examined the effect of chronic ethanol exposure on microtubule content in PC12 cells and the role of PKC epsilon and delta in ethanol's effect on microtubule levels.ResultsChronic ethanol exposure of wild-type and vector control PC12 cells resulted in a significant increase in microtubule content and a corresponding decrease in free tubulin. There was also a significant increase in microtubule content in PC12 cells expressing a dominate-negative inhibitor of epsilon PKC; cells which have previously been shown to have no ethanol-induced increase in neurite outgrowth. In contrast, ethanol had no effect on microtubule content in PC12 cells expressing a dominate-negative inhibitor of delta PKC.ConclusionThese results suggest that chronic ethanol exposure alters the relative ratio of free tubulin to microtubule-associated tubulin, an important component of the cytoskeleton. Further, the data from the PKC dominant-negative cell lines suggest that the effects of ethanol on microtubule content do not correlate with the effects of ethanol on neurite outgrowth. The delta isoform of PKC appears to be necessary for the ethanol-induced increase in microtubule content. These studies demonstrate an effect of chronic ethanol exposure which may contribute to previously documented alterations of neuronal cyto-architecture.
Highlights
Chronic ethanol exposure has been shown to result in changes in neuronal cytoarchitecture such as aberrant sprouting and alteration of neurite outgrowth
Chronic ethanol exposure increases microtubule content in PC12 cells For the following studies, we used 100 mM ethanol for four days as a chronic exposure; a dose and duration used by previous researchers to demonstrate ethanol's enhancement of neurite outgrowth [10]
Chronic ethanol exposure in Protein Kinase C (PKC) dominant-negative PC12 cells We used PC12 cells which over-express the first variable domain of PKC epsilon or delta, which acts as an isozyme specific inhibitor of PKC epsilon or delta [32], http://www.biomedcentral.com/1471-2202/6/16
Summary
Chronic ethanol exposure has been shown to result in changes in neuronal cytoarchitecture such as aberrant sprouting and alteration of neurite outgrowth. In PC12 cells, chronic ethanol treatment produces an increase in Nerve Growth Factor (NGF)-induced neurite outgrowth that appears to require the epsilon, but not delta, isoform of Protein Kinase C (PKC). Contrary to the enhancement of neurite outgrowth, other studies have shown that chronic ethanol exposure inhibits the growth of dendrites in CA1 hippocampal neurons and cerebellar Purkinje cells in vivo and inhibits chick spinal cord neurite formation in vitro [8,9]. PC12 cells are a rat chromaffin cell line that differentiate into neuronal-like cells in the presence of Nerve Growth Factor (NGF) [12] Using these cells, chronic ethanol has been shown to enhance NGF-induced neurite outgrowth [10,11]. PC12 cells have proven to be a valuable system for studying the mechanisms underlying ethanol-induced enhancement of neurite outgrowth
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