EPIGENETIC MODIFICATIONS OF GABA NEURONS IN PSYCHOSIS

描述(由申请人提供)：GABA内神经元在整合皮质输出方面发挥着核心作用，并正在成为精神分裂症分子病理学的一个主要部位。Reelin(RELN)、GAD67和DNA甲基化酶(DNMT1)均在GABA中间神经元中表达，且在精神分裂症脑中均有异常调控。此外，DNMT1是表观遗传调控级联反应的关键组成部分，目前已被证明在神经元基因表达中发挥重要作用。我们早些时候已经报道了表观遗传修饰物(DNA甲基化、染色质结构和甲基化DNA结合蛋白)对RELN和GAD67表达水平的影响。这项职业发展奖将使用原代神经元培养(PNC)来扩展这些观察，以描述由于药物治疗和膜去极化导致的RELN和GAD67启动子的表观遗传修饰的变化。支持使用PNC，我们和其他人观察到EL4小鼠神经元培养的表型主要是GABA能(共表达RELN、GAD和DNMT1)。我们的第一个目标，使用这些同质神经元培养，将分析DNA甲基化，局部染色质结构和启动子DNA-蛋白质的相互作用，以响应已知的表观遗传效应的药物治疗，特别是L蛋氨酸，一种超甲基化刺激，和组蛋白脱乙酰酶抑制剂，如丙戊酸。支持第一个目标的是我们早期的证明，即L蛋氨酸下调RELN和GAD67mRNA的表达是沿着其启动子的表观遗传修饰的结果，而丙戊酸的应用逆转了这一结果。我们的第二个目标将是检查膜去极化对这些GABA能神经元内去极化诱导启动子的表观遗传修饰的影响。我们将应用反义寡核苷酸策略下调表观遗传抑制蛋白，如DNMT1和MeCP2，以分析它们在去极化诱导基因转录中的作用。支持这第二个目标，我们现在已经证明了反义诱导DNMT1mRNA和蛋白的下调将导致RELN mRNA表达的显著增加。此外，我们有初步数据表明，GAD67和DNMT1的mRNA在去极化时发生了强烈的变化。 凭借这个KO1职业发展奖，候选人寻求在表观基因调控的分子研究方面进行重点方法的培训。由于表观遗传机制可以通过药物干预来改变，这项工作与新疗法的发展直接相关。

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.