The GABA intemeuron plays a central role in integrating cortical output and is emerging as a major locus of schizophrenia molecular pathology. Reelin (RELN), GAD67 and DNA methylating enzyme (DNMT1) are all expressed in GABA interneurons, and are each abnormally regulated in schizophrenic brain. Further, DNMT1 is a critical component of the epigenetic regulatory cascade, now shown to play an essential role in neuronal gene expression. We have earlier reported on the effects of epigenetic modifiers (DNA methylation, chromatin structure, and methylated-DNA binding proteins) on the levels of RELN and GAD67 expression. This career development award will extend these observations using primary neuronal cultures (PNC) to describe variations in epigenetic modifications of the RELN and GAD67 promoters resulting from pharmacological treatment and membrane depolarization. Supporting the use of PNCs, we and others have observed El4 mouse neuron cultures to be predominantly GABAergic in phenotype (co-expressing RELN, GAD and DNMT1). Our first objective, using these homogenous neuronal cultures, will be to analyze DNA methylation, local chromatin structure and promoter DNA-protein interactions in response to pharmacological treatments with known epigenetic effects, specifically L-methionine, a hypermethylating stimulus, and histone deactylase (HDAC) inhibitors such as valproic acid. Supporting this first objective is our earlier demonstration that L-methionine down regulates RELN and GAD67 mRNA expression as a consequence of epigenetic modifications along their promoters, and this is reversed by the application of valproic acid. Our second objective will be to examine the effects of membrane depolarization on epigenetic modification of depolarization inducible promoters within these GABAergic neurons. We will apply antisense oligonucleotide strategies to down regulate epigenetic repressor proteins such as DNMT1 and MeCP2 to dissect their contribution to depolarization induced gene transcription. Supporting this second objective, we have now shown that antisense induced down regulation of DNMT1 mRNA and protein will result in significant increases in RELN mRNA expression. Also, we have preliminary data indicating robust changes in GAD67 and DNMT1 mRNA in response to depolarization. With this KO1 career development award, the candidate seeks training in focused methods in the molecular study of epigenetic gene regulation. Because epigenetic mechanisms are modifiable by drug interventions, this work has immediate relevance to the development of novel therapeutics.
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