Expression and Methylation of HPA Axis Genes in Adult Suicide Brain

 自杀是一个主要的公共卫生问题，因为每年约有35，000人死于自杀。最近对自杀行为患者和自杀受害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，如血清素功能异常。研究还发现，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，自杀中HPA轴异常的分子机制和原因尚不清楚。认为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）过度分泌有关。本研究的主要目的是检测HPA轴基因在自杀者的前额叶皮层（PFC）、前扣带回皮层/脑回（CG）、海马和杏仁核中是否异常表达。为了实现这一特定的目的，我们将确定HPA轴基因、糖皮质激素受体的蛋白和mRNA表达，（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的靶基因，即PFC、CG、海马中的GILZ和FKBP，和杏仁核的不同诊断的成年自杀受害者（抑郁症，精神分裂症，其他自杀）和非自杀患者的不同诊断（抑郁症，精神分裂症）以及正常对照。表观遗传调控的缺失与HPA轴基因的调控有关，特别是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。我们建议确定蛋白质和mRNA的表达酶，甲基化DNA（DNA甲基转移酶1 [DNMT 1]，DNMT 3a，DNMT 3b）和羟甲基化DNA（TET 1，TET 2，TET 3）在PFC，CG和海马的成年自杀受害者，非自杀受试者和正常对照组。我们还建议确定DNA甲基化和羟甲基化的HPA轴基因启动子近端区域的PFC，CG和成年自杀受害者，非自杀患者和对照组的海马。我们将使用甲基DNA免疫沉淀的DNA（MeDIP）和羟甲基DIP（hMeDIP），以确定差异乙基化/羟甲基化的利益区域在HPA轴基因的成人自杀队列。我们将使用从每个死后区域分离的DNA的焦磷酸测序来验证识别区域中的差异。总之，这些研究将提供重要的信息，以支持我们的假设，自杀是与一些HPA轴相关基因的异常表达，并与HPA轴基因的异常甲基化/羟甲基化，这种压力反应的下游效应的关键。这些研究不仅将增强我们对自杀的病理生理学的认识，特别是HPA轴在自杀中的作用，而且还可能导致确定重要的位点，这些位点可用于开发治疗自杀行为的适当治疗药物，并作为自杀的生物标志物。

 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.