EPIGENETIC MODIFICATIONS OF GABA NEURONS IN PSYCHOSIS

描述（由申请人提供）：GABA中间神经元在整合皮质输出中起核心作用，并正在成为精神分裂症分子病理学的主要位点。Reelin（NBN）、GAD 67和DNA甲基化酶（DNMT 1）都在GABA中间神经元中表达，并且在精神分裂症脑中各自受到异常调节。此外，DNMT 1是表观遗传调控级联的关键组成部分，现在显示在神经元基因表达中发挥重要作用。我们先前报道了表观遗传修饰剂（DNA甲基化，染色质结构和甲基化DNA结合蛋白）对GAD 67和GAD 67表达水平的影响。这个职业发展奖将使用原代神经元培养物（PNC）来扩展这些观察结果，以描述药物治疗和膜去极化导致的EPORN和GAD 67启动子的表观遗传修饰的变化。支持使用PNC，我们和其他人已经观察到E14小鼠神经元培养物在表型上主要是GABA能的（共表达JNN、GAD和DNMT 1）。我们的第一个目标，使用这些同质的神经元文化，将是分析DNA甲基化，局部染色质结构和启动子DNA-蛋白质相互作用的药理学治疗与已知的表观遗传效应，特别是L-蛋氨酸，一个hypermylating刺激，和组蛋白脱乙酰酶（HDAC）抑制剂，如丙戊酸。支持这第一个目标是我们早期的证明，L-蛋氨酸下调JNK和GAD 67 mRNA表达的结果，表观遗传修饰沿着其启动子，这是逆转的应用丙戊酸。我们的第二个目标将是检查这些GABA能神经元内的去极化诱导型启动子的表观遗传修饰的膜去极化的影响。我们将应用反义寡核苷酸策略下调表观遗传阻遏蛋白，如DNMT 1和MeCP 2，以剖析它们对去极化诱导的基因转录的贡献。支持这第二个目标，我们现在已经表明，反义诱导的DNMT 1 mRNA和蛋白质的下调将导致DNMT 1 mRNA表达的显着增加。此外，我们有初步的数据表明GAD 67和DNMT 1 mRNA响应于去极化的强烈变化。 有了这个KO 1职业发展奖，候选人寻求在表观遗传基因调控的分子研究的重点方法的培训。由于表观遗传机制可以通过药物干预来改变，因此这项工作与新疗法的开发直接相关。

DESCRIPTION (provided by applicant): 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.