Epigenetic dysregulation in diabetic enteric neuropathy

糖尿病肠神经病变的表观遗传失调

• 批准号: 10542375
• 负责人: Tamas Ordog
• 金额: $ 54.25万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 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/10542375
• 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 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; 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; pharmacologic; promoter; recruit; restoration; sensor; side effect; transcription factor; transcriptome sequencing

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无症状的胃排空延迟，伴有或不伴有轻度消化不良， 胃排空延迟或胃轻瘫，其特征在于症状更严重， 胃排空糖尿病性胃轻瘫还可能导致血糖控制受损、营养不良， 焦虑和抑郁，生活质量差。糖尿病胃肠病常与氮能 神经病;以及部分由于神经元型一氧化氮的转录受抑制而导致的氮能神经元的损失 合成酶（Nos 1）与胃轻瘫有因果关系。然而，不断增加的一氧化氮 由于胃和全身副作用，信号传导是无益的; 可能限制神经元移植的功效。我们发现生理性缺氧 （“生理氧”），这是在肠神经元明显，和缺氧诱导因子1 α（HIF 1A），转录 分子氧传感器酶调控因子，作为正常Nos 1表达和NOS 1 蛋白质水平我们还发现，HIF 1A除了刺激RNA聚合酶2的暂停释放外， 通过重新配置连接近端Nos 1调节元件的染色质环来增加Nos 1转录， 远程超级增强器DM如何干扰Nos 1转录和表观遗传调控以及是否 这些变化在体内是否可逆尚不清楚。我们假设糖尿病中的线粒体功能障碍 通过增加细胞内O2水平干扰HIF 1A诱导的Nos 1转录， 通过脯氨酰羟化酶结构域酶降解HIF 1A，并通过 上调HIF 1AN的活性（特异性目标1）;通过增加三羧酸循环的比率 代谢物琥珀酸：α-酮戊二酸，抑制组蛋白和DNA脱甲基酶，上调抑制性 染色质（特异性目的2）;并通过减少ATP合成，这会损害染色质循环和重新配置 增强子-启动子相互作用（特异性目的3）。我们将研究这些机制，在培养和新鲜 分离的、遗传标记的氮能神经元、小鼠模型和患者样品， 包括染色质免疫沉淀测序和全基因组染色体构象的技术 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 共聚焦显微镜机制研究将依赖于体外RNA干扰，条件性基因缺失， 小鼠，以及细胞和小鼠中CRISPR-Cas9介导的基因组和表观基因组编辑。药理研究 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向， 小鼠，其中胃功能将通过非侵入性功能测定来监测。为了便于翻译我们的 研究结果，我们将验证在人体组织中的关键观察结果，并试图恢复NOS 1水平和胃粘膜。 使用在人类中已证实有效的药物在小鼠中发挥作用。连接糖尿病相关的线粒体 功能障碍、缺氧信号和胃功能将改变我们对糖尿病胃肠病的看法。