Epigenetic dysregulation in diabetic enteric neuropathy

糖尿病肠神经病变的表观遗传失调

基本信息

批准号：
10321223
负责人：
Tamas Ordog
金额：
$ 56.57万
依托单位：
MAYO CLINIC ROCHESTER
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10321223
关键词：
ATP Synthesis Pathway Anxiety Binding Biological Assay CRISPR/Cas technology Cells ChIP-seq Chromatin Chromatin Loop Citric Acid Cycle Clinical Trials Confocal Microscopy Cultured Cells DNA DNA-Directed RNA Polymerase Data Diabetes Mellitus Drug usage Dyspepsia Enhancers Enteral Enzymes Epigenetic Process Gastric Emptying Gastroparesis Gene Deletion Genes Genetic Transcription HIF1A gene Healthcare Histones Human Hypoxia Impairment In Vitro Label Link Mediating Mental Depression Mitochondria Molecular Molecular Target Mus NOS1 gene NOS2A gene Neurons Neuropathy Neurotransmitters Nitrergic Neurons Nitric Oxide Nitric Oxide Synthase Nitric Oxide Synthase Type I Nuclear Translocation Nutritional Oxygen Patients Pharmaceutical Preparations Pharmacology Pharmacology Study Physiological Procollagen-Proline Dioxygenase Quality of life RNA Interference Regulatory Element Research Role Sampling Signal Transduction Stomach Succinates Symptoms Techniques Tissues Transactivation Transcriptional Regulation Translations Transplantation Upstream Enhancer Western Blotting alpha ketoglutarate base blood glucose regulation care burden chromosome conformation capture cohesin curative treatments diabetic diabetic gastroparesis effective therapy enteric neuropathy epigenetic regulation epigenome editing epigenomics gene repression genome editing genome-wide histone demethylase histone methylation human tissue hypoxia inducible factor 1 in vivo inhibitor metabolomics mitochondrial dysfunction mitochondrial metabolism motor disorder mouse model non-invasive monitor promoter recruit restoration sensor side effect transcription factor transcriptome sequencing

项目摘要

Diabetic gastroenteropathy includes asymptomatic delayed gastric emptying, dyspepsia with or without mildly delayed gastric emptying, or gastroparesis, which is characterized by more severe symptoms and delayed gastric emptying. Diabetic gastroparesis may also result in impaired glucose control, nutritional compromise, anxiety and depression, and poor quality of life. Diabetic gastroenteropathy is often associated with nitrergic neuropathy; and loss of nitrergic neurons due in part to repressed transcription of neuronal nitric oxide synthase (Nos1) has been causally linked to gastroparesis. However, pharmacologically increasing nitric oxide signaling is not beneficial due to gastric and systemic side effects; and compromised tissue microenvironment in diabetes may limit the efficacy of transplantation of neurons. We have identified physiological hypoxia (“physioxia”), which is pronounced in enteric neurons, and hypoxia-inducible factor 1 α (HIF1A), a transcription factor regulated by molecular oxygen sensor enzymes, as key factors of normal Nos1 expression and NOS1 protein levels. We have also found that HIF1A, in addition to stimulating RNA polymerase 2 pause-release, increases Nos1 transcription by reconfiguring chromatin loops linking proximal Nos1 regulatory elements to remote super-enhancers. How DM interferes with Nos1 transcription and epigenetic regulation and whether these changes are reversible in vivo is unclear. We hypothesize that mitochondrial dysfunction in diabetes interferes with HIF1A-inducible Nos1 transcription via increased intracellular O2 levels that facilitate the degradation of HIF1A by prolyl hydroxylase domain enzymes and block HIF1A-mediated transactivation by upregulating the activity of HIF1AN (Specific Aim 1); via increased ratio of the tricarboxylic acid cycle metabolites succinate:α-ketoglutarate that inhibits histone and DNA demethylases and upregulates repressive chromatin (Specific Aim 2); and via reduced ATP synthesis, which impairs chromatin looping and reconfigures enhancer–promoter interactions (Specific Aim 3). We will study these mechanisms in cultured and freshly isolated, genetically labeled nitrergic neurons, mouse models and patient samples using epigenomic techniques including chromatin immunoprecipitation-sequencing and genome-wide chromosome conformation capture, metabolomics, in-vivo analysis of intracellular O2 levels, RNA sequencing, Western blots, and confocal microscopy. Mechanistic studies will rely on in-vitro RNA interference, conditional gene deletions in mice, and CRISPR-Cas9-mediated genome and epigenome editing in cells and mice. Pharmacological studies will target molecular O2 sensors directly or indirectly including via mitochondrial targets in cultured cells and mice, where gastric functions will be monitored by noninvasive functional assays. To facilitate translation of our findings, we will validate key observations in human tissues and attempt to restore NOS1 levels and gastric functions in mice using drugs with proven efficacy in humans. Linking diabetes-associated mitochondrial dysfunction, hypoxic signaling and gastric functions will change how we think about diabetic gastroenteropathy.

糖尿病胃肠病包括无症状的胃排空延迟、伴或不伴轻度消化不良胃排空延迟或胃轻瘫，其特征是更严重的症状和延迟糖尿病胃轻瘫还可能导致血糖控制受损、营养不良、焦虑和抑郁以及生活质量差通常与氮能有关。神经病；以及氮能神经元的丧失，部分原因是神经元一氧化氮的转录受到抑制然而，合酶（Nos1）与胃轻瘫有因果关系，但从药理上讲，一氧化氮会增加。由于胃和全身副作用以及组织微环境受损，信号传导并不有益；我们已经确定，在糖尿病中，生理性缺氧可能会限制神经元移植的功效。（“生理氧”），在肠神经元中很明显，缺氧诱导因子 1 α (HIF1A) 是一种转录受分子氧传感器酶调节的因子，是Nos1正常表达和NOS1的关键因子我们还发现 HIF1A 除了刺激 RNA 聚合酶 2 暂停释放之外，通过重新配置连接近端 Nos1 调控元件的染色质环来增加 Nos1 转录远程超级增强子如何干扰 Nos1 转录和表观遗传调控？这些变化在体内是否可逆尚不清楚。我们一直在努力解决糖尿病中的线粒体功能障碍。通过增加细胞内 O2 水平来干扰 HIF1A 诱导的 Nos1 转录，从而促进通过脯氨酰羟化酶结构域酶降解 HIF1A，并通过以下方式阻断 HIF1A 介导的反式激活通过增加三羧酸循环的比例上调 HIF1AN 的活性（具体目标 1）；代谢物琥珀酸：α-酮戊二酸，抑制组蛋白和 DNA 去甲基化酶并上调抑制性染色质（具体目标 2）；以及通过减少 ATP 合成，从而损害染色质循环和重新配置增强子-启动子相互作用（具体目标 3）我们将在培养物和新鲜物中研究这些机制。使用表观基因组分离、基因标记的氮能神经元、小鼠模型和患者样本技术包括染色质免疫沉淀测序和全基因组染色体构象捕获、代谢组学、细胞内 O2 水平的体内分析、RNA 测序、蛋白质印迹和共聚焦显微镜的机制研究将依赖于体外RNA干扰、条件基因缺失。小鼠，以及 CRISPR-Cas9 介导的细胞和小鼠基因组和表观基因组编辑的药理学研究。将直接或间接靶向分子 O2 传感器，包括通过培养细胞中的线粒体靶标，小鼠，胃功能将通过非侵入性功能测定进行监测，以促进我们的翻译。研究结果，我们将验证人体组织中的关键观察结果，并尝试恢复 NOS1 水平和胃使用在人类中已被证明有效的药物在小鼠中发挥作用，将与糖尿病相关的线粒体联系起来。功能障碍、缺氧信号传导和胃功能将改变我们对糖尿病胃肠病的看法。