Expression and Methylation of HPA Axis Genes in Adult Suicide Brain

 描述(由申请人提供)：自杀是一个主要的公共卫生问题，因为在美国每年约有3.5万人死于自杀。最近对有自杀行为的患者和自杀者死后大脑样本的研究强烈表明，自杀与神经生物学异常有关，如5-羟色胺功能异常。还观察到自杀和自杀行为与下丘脑-垂体-肾上腺(HPA)轴功能异常有关。然而，自杀的HPA轴异常的分子机制和原因还不是很清楚。HPA轴异常可能与脑内反馈机制异常和促肾上腺皮质激素释放因子(CRF)分泌过多有关。我们建议的主要目的是检查HPA轴基因是否在自杀者的前额叶皮质(PFC)、前扣带回(CG)、海马体和杏仁核异常表达。为了达到这一特定的目的，我们将检测HPA轴基因、糖皮质激素受体(GR-α和GR-β)、盐皮质激素受体(MR)、CRF、CRF受体(CRF-R1和CRF-R2)、CRF结合蛋白(CRF-BP)和GR靶基因(GILZ和FKBP)在不同诊断(抑郁症、精神分裂症和其他自杀)的成年自杀患者以及不同诊断的非自杀患者(抑郁症、精神分裂症)和正常对照组中的蛋白和基因表达。表观遗传调控异常与某些HPA轴基因的调控有关，尤其是GR。因此，我们建议通过检测DNA甲基化对HPA轴基因调控的影响来研究这些基因的表观遗传调控。我们建议测定成年自杀者、非自杀者和正常对照组的PFC、CG和海马区DNA甲基化酶(DNA甲基转移酶1[DNMT1]、DNMT3a和DNMT3b)和羟薄荷酸DNA(TET1、TET2和TET3)的蛋白质和mRNA的表达。我们还建议检测成年自杀受害者、非自杀患者和对照组的PFC、CG和海马区HPA轴基因启动子近端区域的DNA甲基化和羟甲基化。我们将使用DNA的甲基DNA免疫沉淀(MeDIP)和羟甲基DIP(HMeDIP)来识别成人自杀队列中HPA轴基因的差异乙基化/羟甲基化感兴趣区域。我们将通过对从每个死后区域分离的DNA进行焦磷酸测序来验证识别出的区域的差异。总之，这些研究将提供重要的信息来支持我们的假设，即自杀与一些HPA轴相关基因的异常表达以及HPA轴基因的异常甲基化/羟甲基化有关，这些基因对这种应激反应的下游效应至关重要。这些研究不仅将提高我们对自杀的一般病理生理学，特别是HPA轴在自杀中的作用的认识，而且还可能导致识别重要的位点，这些位点可能被用于开发合适的治疗药物来治疗自杀行为，并作为自杀的生物标记物。

 DESCRIPTION (provided by applicant): Suicide is a major public health concern as about 35,000 people die of suicide in the U.S. every year. Recent studies of patients with suicidal behavior and of postmortem brain samples of suicide victims strongly suggest that suicide is associated with neurobiological abnormalities, such as abnormalities in serotonin function. It has also been observed that suicide and suicidal behavior are associated with abnormal hypothalamic-pituitary- adrenal (HPA) axis function. The molecular mechanisms and causes of HPA axis abnormalities in suicide, however, are not well understood. It is believed that HPA axis abnormalities may be related to an abnormal feedback mechanism and excessive secretion of corticotropin releasing factor (CRF) in the brain. The main objective of our proposal is to examine if HPA axis genes are abnormally expressed in the prefrontal cortex (PFC), anterior cingulate cortex/gyrus (CG), hippocampus, and amygdala of suicide victims. To achieve this specific aim, we will determine the protein and mRNA expression of the HPA axis genes, glucocorticoid receptors (GR-α and GR-β), mineralocorticoid receptors (MR), CRF, the receptors for CRF (CRF-R1 and CRF- R2), CRF binding protein (CRF-BP), and the target genes for GR, which are GILZ and FKBP in the PFC, CG, hippocampus, and amygdala of adult suicide victims of different diagnoses (depression, schizophrenia, other suicide) and in non-suicidal patients with different diagnoses (depression, schizophrenia) as well as normal controls. Abnormalities of epigenetic regulation have been implicated in the regulation of some of the HPA axis genes, especially the GR. We therefore propose to study epigenetic regulation of these genes by examining the effects of DNA methylation on the regulation of HPA axis genes. We propose to determine the protein and mRNA expression of enzymes that methylate DNA (DNA methyl transferase 1 [DNMT1], DNMT3a, and DNMT3b) and hydroxmenthylate DNA (TET1, TET2, and TET3) in the PFC, CG and hippocampus of adult suicide victims, non-suicidal subjects and normal control subjects. We also propose to determine the DNA methylation and hydroxymethylation of promoter proximal regions of HPA axis genes in the PFC, CG and hippocampus of adult suicide victims, non-suicidal patients and controls. We will use methyl DNA immunoprecipitation of DNA (MeDIP) and hydroxymethyl DIP (hMeDIP) to identify differentially ethylated/hydroxymethylated regions of interest in HPA axis genes of adult suicide cohorts. We will validate differences in identified regions using pyrosequencing of DNA isolated from each postmortem region. Together these studies will provide important information to support our hypothesis that suicide is associated with abnormal expression of some of the HPA axis related genes and with abnormal methylation/hydroxymethylation of HPA axis genes critical to the downstream effects of this stress response. These studies will not only enhance our knowledge of the pathophysiology of suicide in general and the role of HPA axis in suicide in particular, but also may result in identifying important sites that may be used for the development of appropriate therapeutic agents for treatment of suicidal behavior and as biomarkers for suicide.