Mechanisms of telomere-induced disease: Role of intestinal malabsorption, barrier dysfunction and dsybiosis.

尽管人们越来越认识到饮食和肠道中宿主特定遗传因素的相互作用在肠道和全身疾病中发挥着重要作用，但我们对这个新兴领域的理解仍然有限，这代表了一个重要的知识差距。在这里，我们建议通过提高我们对肠道端粒缩短如何影响肠上皮细胞成熟的了解来弥补这一差距，肠上皮细胞是屏障维持和吸收所必需的终末分化细胞。端粒对于干细胞依赖性组织（例如肠道）的再生很重要。端粒缩短发生在正常衰老过程中，并且在端粒酶突变的患者或在溃疡性结肠炎和克罗恩病等高细胞周转情况下会加速。端粒缩短会导致肠道中的多种病变，包括萎缩、炎症以及患者结肠炎进展为癌症，所有这些病变都在端粒酶敲除小鼠 (TKO) 中忠实地重现。从机制上讲，短端粒被认为是通过持续凋亡介导的肠道干细胞耗竭来驱动这些病理的。除了干细胞耗竭之外，其他致病机制尚不清楚。特别是，尚不清楚端粒缩短是否会损害肠道中的分化细胞谱系，其中肠细胞占绝大多数。在这里，我们发现端粒缩短会损害肠上皮细胞谱系的成熟，导致不成熟和功能受损的肠上皮细胞的产生。这得到了包括 RNAseq、蛋白质组学、透射和扫描电子显微镜在内的多水平分析的支持，证明消化酶、转运蛋白和微绒毛结构成分的表达在 TKO 小鼠的近端肠道中受到抑制。重要的是，初步研究表明，这种肠上皮细胞妥协保留在端粒较短的人类肠上皮细胞中。从机制上讲，TKO 上皮细胞中 p53 的缺失可以挽救肠上皮细胞的缺陷。此外，这些肠上皮细胞的特点是线粒体功能障碍并且 ATP 水平较低。当接触富含果糖的饮食时，它们表现出明显的 ATP 水平急剧下降和随后的细胞凋亡的倾向，从而加剧屏障缺陷和吸收不良。在这里，我们建议建立 p53 导致分化缺陷的机制（目标 1），通过灭活 ATP 消耗酶果糖激酶或通过补充 NAD 改善线粒体功能来增加 ATP 水平，以确定低 ATP 水平是否是果糖毒性的驱动源。在目标 3 中，我们使用具有不同端粒长度的细胞系、类器官和肠类，确定了短端粒与引起人类肠上皮细胞缺陷的相关性。

Although it is increasingly recognized that the interaction of the diet and host specific genetic factors in the gut play an important role in intestinal and systemic disease, our understanding of in this emerging field is still limited and this represents an important knowledge gap. Here we propose to bridge this gap by advancing our knowledge how telomere shortening in the gut impacts the maturation of enterocytes, the terminally differentiated cells that are essential for barrier maintenance and absorption. Telomeres are important for the regeneration of stem cell-dependent tissues such as the intestine. Telomere shortening occurs during normal aging and is accelerated in patients with mutations in telomerase or in high cell turnover conditions such as ulcerative colitis and Crohn’s disease. Telomere shortening causes several pathologies in the intestine including atrophy, inflammation, and progression of colitis to cancer in patients and all these pathologies are faithfully recapitulated in telomerase knockout mice (TKO). Mechanistically, it is believed that short telomeres drive these pathologies through continuous apoptosis-mediated depletion of intestinal stem cells. Beyond stem cell- depletion, other pathogenic mechanisms are not known. In particular, it is not known whether telomere shortening compromises differentiated cell lineages in the gut, of which enterocytes represent the vast majority. Here we have found that telomere shortening impairs the maturation towards the enterocyte lineage leading to the generation of immature and functionally compromised enterocytes. This is supported by a multi- level analysis including RNAseq, proteomics, transmission and scanning electron microscopy demonstrating that the expression of digestive enzymes, transporters and structural components of microvilli are repressed in the proximal intestine in TKO mice. Importantly, preliminary studies indicate that this enterocyte compromise is preserved in human enterocytes with short telomeres. Mechanistically, deletion of p53 in TKO epithelium rescues enterocyte defects. Furthermore, these enterocytes are characterized by mitochondrial dysfunction and have low ATP levels. When exposed to a fructose-rich diet, they show pronounced propensity to steep decline in ATP levels and subsequent apoptosis, which exacerbates the barrier defect and malabsorption. Here we propose to establish the mechanisms through which p53 causes differentiation defects (Aim 1), establish whether the low ATP levels are the driving source for fructose toxicity by increasing ATP levels either through inactivation of the ATP depleting enzyme fructokinase or improving mitochondrial function through NAD supplementation. In Aim 3 we establish the relevance of short telomeres in causing enterocyte defects in humans using cell lines, organoids and enteroids with various telomere length.