Interstitial cells of Cajal in diabetic gastropathy

平滑肌功能控制紊乱在胃肠道疾病中很常见，这些疾病造成数十亿美元的损失 每年的医疗保健支出。胃瘫是胃瘫最显著的表现之一。 胃肠动力障碍，尤其是糖尿病患者。然而，治疗选择是有限的，反映出 对神经肌肉间隔内的细胞动力学的不完全理解。之前的研究已经 确定Cajal间质细胞(ICC)是胃轻瘫最常见的细胞类型。国际刑事法院作为 时相收缩活动和介导胆碱能和硝能神经肌肉的生理性起搏器 神经传递。胰岛素/IGF1信号转导和巨噬细胞减少可能导致糖尿病ICC耗竭 作用，干扰受体酪氨酸激酶试剂盒的表达。我们知识中的一个关键差距是 由于这些侮辱，对国际刑事法院的命运缺乏了解。已经观察到细胞死亡，但没有 一以贯之，促使研究人员假设ICC转分化，这可能是可逆的。 然而，Kit和其他生物标志物丢失后ICC存活的证据一直难以捉摸。因此， 这个项目的主要目标是确定ICC的命运，它们的机制和糖尿病的可逆性。我们的中央 假说是，由于Kit信号的减少，ICC转录因子ETV1的耗尽，以及 由于线粒体代谢紊乱而抑制抑制性表观遗传标记的“擦除器”，导致 正常驱动基因转录的超级增强子的退役和最终沉默 对包括套件在内的ICC身份至关重要。这些变化伴随着密切相关的 基因PDGFRA，赋予幸存的ICC“成纤维样细胞”(FLC)的身份和功能， 介导嘌呤能神经肌肉神经传递。具体目标1是提供国际刑事法院的确凿证据- 利用体内遗传谱系追踪和新细胞系结合表型的TO-FLC表型转换 用流式细胞术、免疫荧光、Western blotting和转录组测序 经荧光激活细胞分选纯化。具体目标2是建立表观遗传机制 潜在的ICC去分化和表型转化为FLC。我们将分析组蛋白和DNA标记， ETV1结合和染色质构象在特定的基因座和全基因组。我们将表演机械化 利用RNA干扰(RNAi)以及体外CRISPR-Cas9介导和体内CRE介导的研究 基因组编辑结合胃排空纵向分析。具体目标3是确定角色 利用RNAi、体内基因组编辑等技术研究琥珀酸对ICC基因的表观遗传抑制 表观基因组学和表型分析方法。为了便于翻译我们的调查结果，我们将验证关键机制 并试图使用被批准用于人类的表观遗传药物在小鼠身上恢复ICC。这个 通过对持续改变的细胞进行表观遗传重新编程来操纵间质细胞的概念 转录程序将改变我们对健康和疾病中神经肌肉可塑性的看法。

Disordered control of smooth muscle function is common in gastrointestinal diseases, which cost billions of dollars in health care spending each year. Gastroparesis is one of the most significant manifestations of gastrointestinal dysmotility, particularly in diabetes mellitus. However, therapeutic options are limited reflecting incomplete understanding of cellular dynamics within the neuromuscular compartment. Previous research has identified interstitial cells of Cajal (ICC) as the cell type most commonly affected in gastroparesis. ICC serve as the physiological pacemaker for phasic contractile activity and mediate cholinergic and nitrergic neuromuscular neurotransmission. Diabetic ICC depletion may arise from reduced insulin/IGF1 signaling and macrophage action, which interfere with expression of the receptor tyrosine kinase Kit. A critical gap in our knowledge is the lack of understanding of the fate of ICC lost from these insults. Cell death has been observed but not consistently, prompting investigators to hypothesize ICC transdifferentiation, which could be reversible. However, evidence of ICC survival following loss of Kit and other biomarkers has been elusive. Therefore, the main objective of this project is to identify ICC fates, their mechanisms and reversibility in diabetes. Our central hypothesis is that depletion of the ICC transcription factor Etv1 from reduced Kit signaling, together with inhibition of “erasers” of repressive epigenetic marks due to disturbed mitochondrial metabolism, lead to decommissioning and eventual silencing of super-enhancers that normally drive the transcription of genes critical for ICC identity including Kit. These changes are accompanied by the upregulation of the closely related gene Pdgfra, conferring on the surviving ICC the identity and function of “fibroblast-like cells” (FLC), which mediate purinergic neuromuscular neurotransmission. Specific Aim 1 is to provide definitive evidence of ICC- to-FLC phenotypic switch using in-vivo genetic lineage tracing and novel cell lines combined with phenotyping by flow cytometry, immunofluorescence, Western blotting and transcriptome sequencing in cell populations purified by fluorescence-activated cell sorting. Specific Aim 2 is to establish the epigenetic mechanisms underlying ICC dedifferentiation and phenotypic conversion into FLC. We will analyze histone and DNA marks, Etv1 binding and chromatin conformation in specific loci and genome-wide. We will perform mechanistic studies using RNA interference (RNAi), as well as in-vitro CRISPR-Cas9-mediated and in-vivo Cre-mediated genome editing combined with longitudinal analysis of gastric emptying. Specific Aim 3 is to determine the role of succinate in the epigenetic repression of ICC genes using RNAi, in-vivo genome editing and the above epigenomic and phenotyping methods. To facilitate translation of our findings, we will validate key mechanisms in human tissues and attempt to restore ICC in mice using epigenetic drugs approved for use in humans. The concept of manipulating interstitial cells via epigenetic reprogramming of cells with persistently altered transcriptional programs will change how we think about neuromuscular plasticity in health and disease.