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.

糖尿病性胃肠病包括不对称延迟胃排空，消化不良，有或没有轻度胃排空延迟或胃乳酸，其特征是更严重的症状和延迟胃排空。糖尿病性胃轻瘫也可能导致葡萄糖控制受损，营养妥协，焦虑和沮丧，生活质量差。糖尿病性胃肠病通常与硝酸盐有关神经病；和硝化神经元的丧失，部分原因是神经元一氧化氮转录的转录合成酶（NOS1）已随便与胃轻瘫有关。但是，药物会增加一氧化氮由于胃和全身副作用，信号传导无益。并破坏组织微环境在糖尿病中可能会限制神经元移植的效率。我们已经确定了身体缺氧（“ Physioxia”），在肠神经元中发音，而缺氧诱导因子1α（HIF1A），转录由分子氧传感器酶调节的因子，作为正常NOS1表达和NOS1的关键因素蛋白质水平。我们还发现，除了刺激RNA聚合酶2暂停释放外，HIF1A还通过重新配置将近端NOS1调节元素与远程超级增强剂。 DM如何干扰NOS1转录和表观遗传调节以及是否是否这些变化在体内是可逆的。我们假设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转录通过丙酰羟化酶结构酶降解HIF1A，并通过阻断HIF1A介导的反式激活。上调HIF1AN的活性（特定目标1）；通过增加三核酸周期的比率增加代谢产物琥珀酸酯：α-酮戊二酸，抑制组蛋白和DNA脱甲基酶并上调反射性染色质（特定目标2）；并通过降低的ATP合成，从而损害染色质循环和重新配置增强剂 - 启动器相互作用（特定目标3）。我们将研究这些机制使用表观基因组学包括染色质免疫沉淀的技术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。机械研究将依赖于体外RNA干扰，条件基因缺失小鼠以及CRISPR-Cas9介导的基因组和表观基因组编辑中的细胞和小鼠的编辑。药理研究将直接或间接地靶向分子O2传感器，包括培养细胞和小鼠，将通过非侵入性功能测定法监测胃功能。促进我们的翻译调查结果，我们将验证人体组织中的关键观察结果，并试图恢复NOS1水平和胃使用在人类中有效的药物在小鼠中的功能。连接与糖尿病相关的线粒体功能障碍，低氧信号传导和胃功能将改变我们对糖尿病性胃肠病的看法。