EPIGENETIC MODIFICATIONS OF GABA NEURONS IN PSYCHOSIS

精神病中 GABA 神经元的表观遗传修饰

• 批准号: 7234306
• 负责人: Rajiv Pandit Sharma
• 金额: $ 17.41万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7234306
• 关键词: Antisense Oligonucleotides; Binding; Binding Proteins; Biological Assay; Brain; Brain-Derived Neurotrophic Factor; Cell Line; Characteristics; Chromatin; Chromatin Structure; Co-Immunoprecipitations; CpG Islands; DNA; DNA Binding; DNA Methylation; DNA Sequence; DNA-Binding Proteins; DNA-Protein Interaction; Data; Development; Disease; Dose; Down-Regulation; Enzymes; Epigenetic Process; Exons; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Genome; Histone Deacetylase Inhibitor; Histones; Hypermethylation; Interneurons; Intervention; K-Series Research Career Programs; Laboratories; Location; Measures; Membrane; Messenger RNA; Methionine; Methods; Methyl-CpG-Binding Protein 2; Methylation; Mitotic; Modification; Molecular; Mus; Mutation; Neocortex; Neuron-Specific Enolase; Neurons; Output; Patients; Pharmaceutical Preparations; Pharmacological Treatment; Phenotype; Play; Proteins; Psychotic Disorders; Recording of previous events; Reporting; Repressor Proteins; Rett Syndrome; Reverse Transcriptase Polymerase Chain Reaction; Role; Schizophrenia; Site; Standards of Weights and Measures; Stimulus; Training; Valproic Acid; Variant; Western Blotting; Work; bisulfite; chromatin remodeling; gamma-Aminobutyric Acid; inhibitor/antagonist; mRNA Expression; molecular pathology; novel therapeutics; promoter; response; therapeutic target

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.

描述（由申请人提供）：GABA Intemeuron在整合皮质输出中起着核心作用，并且正在成为精神分裂症分子病理的主要基因座。 reelin（reln），GAD67和DNA甲基化酶（DNMT1）均在GABA中间神经元中表达，并且在精神分裂症大脑中均异常调节。此外，DNMT1是表观遗传调节级联反应的关键组成部分，现在证明在神经元基因表达中起着至关重要的作用。我们之前已经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RELN和GAD67表达水平的影响。该职业发展奖将使用原发性神经元培养物（PNC）扩展这些观察结果，以描述由药理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PNC的使用，我们和其他人都观察到EL4小鼠神经元培养物在表型中主要是GABA能的（共表达RELN，GAD和DNMT1）。我们使用这些同质神经元培养物的第一个目标将是分析DNA甲基化，局部染色质结构和启动子DNA-蛋白相互作用，以响应具有已知表观遗传学作用的药理治疗，特异性L-甲基酮，一种特异性L-甲基氨酸，高甲基刺激刺激和组蛋白脱纤维酶（HDAC）（HDAC）抑制剂。支持这个第一个目标的是我们较早的演示，即L-甲硫氨酸下降会根据其启动子的表观遗传修饰而调节Reln和GAD67 mRNA表达，这是由于丙丙酸的应用而逆转。我们的第二个目标是检查膜去极化对这些GABA能神经元中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来降低调节表观遗传阻遏蛋白，例如DNMT1和MECP2，以剖析其对去极化诱导基因转录的贡献。支持第二个目标，我们现在表明反义诱导的调节DNMT1 mRNA和蛋白质将导致RELN mRNA表达显着增加。此外，我们有初步数据，表明GAD67和DNMT1 mRNA的稳健变化响应于去极化。 通过这一KO1职业发展奖，该候选人寻求对表观遗传基因调节分子研究的重点方法进行培训。由于表观遗传机制可以通过药物干预进行修改，因此这项工作与新型治疗剂的发展有关。