P.1.26 Conditional knockout of mu opioid receptors

Abstract

status within the GLT-1 promoter at single nucleotide resolution. The expression status of the EAAT2 promoter was analysed at the endogenous locus by real-time PCR and additionally evaluated using a reporter plasmid. Results: Among the evaluated drugs, only VPA was capable of inducing hyper-acetylation of H3/K9 and H4 in cortical astrocytes. Concomitantly, VPA treatment caused a pronounced decrease in cell viability as well as changes in cellular morphology resembling astrocytic stellation. The combined treatment of VPA with antidepressants did not alter the level of acetylated histones. Surprisingly however, antidepressants exerted protective activities against VPA mediated toxicity. VPA exposure also resulted in a concentration and time dependent reduction of the global DNA methylation level which was reversible by withdrawal of the drug. At the level of the GLT-1 gene promoter, VPA induced a weak demethylation of some cytosine residues. These changes were paralleled by an increase of GLT-1 mRNA transcription. Moreover, VPA robustly enhanced the activity of an EAAT2/GLT-1 reporter construct. Conclusion: Our results revealed that VPA treatment increased both H3/K9 and H4 acetylation levels in primary astrocytes, as expected. This was accompanied by reversible changes in global DNA methylation. Further, we show for the first time that VPA induced demethylation of distinct GLT-1 promoter CpG sites that was correlated with increase of its mRNA expression level. In contrast, other analyzed drugs did not exhibit measurable changes in global epigenetic marks, indicating that their mode of action is independent of HDAC inhibition, but rather through alteration of neurotransmission and signalling circuits. We suggest that clinical application should take into consideration whether or not a drug has a broad effect on the epigenetic machinery of the cell, particularly when substances are administered over a long period of time.