Epigenetic dysregulation in diabetic enteric neuropathy

糖尿病胃肠病变包括无症状的胃排空延迟、轻度或不伴轻度消化不良。 胃排空延迟，或胃轻瘫，其特征是症状更严重和延迟 胃排空。糖尿病胃轻瘫还可能导致血糖控制受损，营养受损， 焦虑和抑郁，以及糟糕的生活质量。糖尿病胃肠病常与氮能有关 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转录受到抑制 合成酶(NOS1)与胃轻瘫有因果关系。然而，药理学上增加的一氧化氮 由于胃部和全身副作用，以及组织微环境受损，信号传递无益。 糖尿病患者可能会限制神经元移植的疗效。我们已经确认了生理性缺氧 (“Physioxia”)和低氧诱导因子1α(HIF1a)，HIF1a是一种转录 受分子氧感受器酶调控的因子，作为NOS1正常表达和NOS1的关键因子 蛋白质水平。我们还发现，HIF1a除了刺激RNA聚合酶2的暂停释放外， 通过重新配置连接NOS1近端调控元件的染色质环来增加NOS1的转录 远程超级增强器。糖尿病是如何干扰NOS1转录和表观遗传调控的 这些变化在体内是可逆的，目前还不清楚。我们假设糖尿病患者线粒体功能障碍 通过增加细胞内O2水平来干扰HIF1a诱导的NOS1转录，从而促进 Pro羟基酶对HIF1a的降解及阻断HIF1a介导的反式激活 上调HIF1AN的活性(特定目标1)；通过增加三元酸循环的比例 代谢物琥珀酸：α-酮戊二酸抑制组蛋白和dna去甲基酶并上调抑制作用 染色质(特异性目标2)；以及通过减少ATP合成，从而削弱染色质环路和重新配置 增强子-启动子相互作用(特定目标3)。我们将在培养的和新鲜的 利用表观基因组学分离、基因标记的氮能神经元、小鼠模型和患者样本 染色质免疫沉淀测序和全基因组染色体构象等技术 捕获、代谢组学、细胞内O2水平的体内分析、RNA测序、Western blotts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CRISPR-Cas9介导的细胞和小鼠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感受器，包括通过培养细胞的线粒体靶标和 小鼠的胃功能将通过非侵入性功能分析进行监测。为了方便翻译我们的 我们将验证在人体组织中的关键观察结果，并尝试恢复NOS1水平和胃 使用在人类上已被证明有效的药物对小鼠的作用。链接糖尿病相关线粒体 功能障碍、低氧信号和胃功能将改变我们对糖尿病胃肠病的看法。

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.