Abstract
We previously found that glial cell line-derived neurotrophic factor (GDNF) in the midbrain ventral tegmental area (VTA) negatively regulates alcohol drinking (He, D. Y., McGough, N. N., Ravindranathan, A., Jeanblanc, J., Logrip, M. L., Phamluong, K., Janak, P. H., and Ron, D. (2005) J. Neurosci. 25, 619-628). Several studies suggest a role for GDNF in the regulation of tyrosine hydroxylase (TH) levels in the midbrain (Georgievska, B., Kirik, D., and Bjorklund, A. (2004) J. Neurosci. 24, 6437-6445). Up-regulation of TH levels has been reported as a hallmark of biochemical adaptations to in vivo chronic exposure to drugs of abuse, including ethanol (Ortiz, J., Fitzgerald, L. W., Charlton, M., Lane, S., Trevisan, L., Guitart, X., Shoemaker, W., Duman, R. S., and Nestler, E. J. (1995) Synapse 21, 289-298). We hypothesized that GDNF plays an important role in regulating prolonged ethanol-mediated increases in TH protein levels. Using the SH-SY5Y dopaminergic-like cell line, we found that the increase in TH levels in the presence of ethanol required the activation of the cAMP/PKA pathway and was reversed by GDNF. Ethanol treatment did not alter the mRNA level or protein translation of TH, but enhanced the stability of the protein that was decreased by GDNF. Interestingly, we observed that ethanol treatment resulted in an increase in TH association with the chaperone heat shock protein (HSP90) that was mediated by the cAMP/PKA pathway and inhibited by GDNF. Taken together, these data suggest that prolonged ethanol exposure leads to increased association of TH and HSP90 via the cAMP/PKA pathway, resulting in the stabilization and subsequent accumulation of TH. GDNF reverses this ethanol-mediated adaptation by inhibiting the interaction of TH with HSP90.
Highlights
Limiting step in the biosynthesis of catecholamine neurotransmitters, including dopamine [6, 7]
The ethanol-induced increase in tyrosine hydroxylase (TH) protein levels was long-lasting and could be detected 24 h after ethanol washout (Fig. 1C). These results indicate that the increase in TH levels is a longlasting adaptation to ethanol exposure, which can be detected in SH-SY5Y cells
Cyclic AMP-dependent Protein Kinase A (PKA) Is Required for Ethanol Induction of TH Protein Levels—we set out to identify the mechanism underlying the action of ethanol on TH protein levels. Because it is well-established that several signaling pathways are altered in response to ethanol, including those mediated by protein kinase A (PKA), Fyn, and protein kinase C (PKC) [32, 33], we investigated the possibility that kinase-mediated signaling pathways may be required for ethanol to increase TH protein levels by using inhibitors of these kinases in conjunction with chronic ethanol treatment
Summary
Limiting step in the biosynthesis of catecholamine neurotransmitters, including dopamine [6, 7]. We found that prolonged ethanol treatment resulted in an increase in TH protein levels after 24 and 48 h of exposure to ethanol (Fig. 1A). We found that exposure of cells to ethanol resulted in an increase in PKA-mediated phosphorylation of TH, which protein levels that were expressed as ratios to those of actin.
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