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

虽然人们越来越认识到肠道中饮食和宿主特异性遗传因素的相互作用在肠道和系统性疾病中发挥着重要作用，但我们对这一新兴领域的了解仍然有限，这是一个重要的知识鸿沟。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细胞成熟的知识来弥合这一差距，肠细胞是终末分化的细胞，对于屏障的维持和吸收是必不可少的。端粒对于肠等依赖干细胞的组织的再生非常重要。端粒缩短发生在正常衰老过程中，在端粒酶突变的患者或在细胞周转率高的情况下，如溃疡性结肠炎和克罗恩病，端粒缩短会加速。端粒缩短引起肠道的几种病理，包括萎缩、炎症和结肠炎进展为癌症，所有这些病理在端粒酶基因敲除小鼠(TKO)中都得到了忠实的概括。从机制上讲，短端粒被认为是通过持续的凋亡介导的肠道干细胞耗尽来驱动这些病理过程。除了干细胞枯竭，其他致病机制尚不清楚。特别是，目前尚不清楚端粒缩短是否会影响肠道中的分化细胞系，其中肠上皮细胞占绝大多数。在这里，我们发现端粒缩短损害了对肠细胞谱系的成熟，导致了未成熟和功能受损的肠细胞的产生。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表明，TKO小鼠近端肠道中消化酶、转运蛋白和微绒毛结构成分的表达受到抑制。重要的是，初步研究表明，这种肠细胞妥协在端粒较短的人肠细胞中保留。从机制上讲，TKO上皮中p53的缺失可以挽救肠上皮细胞的缺陷。此外，这些肠细胞的特点是线粒体功能障碍，且ATP水平较低。当暴露在富含果糖的饮食中时，他们表现出明显的ATP水平急剧下降和随后的细胞凋亡的倾向，这加剧了屏障缺陷和吸收不良。在这里，我们建议建立p53引起分化缺陷的机制(目标1)，确定低水平的ATP是否是果糖毒性的驱动力来源，通过使ATP消耗酶果糖激酶失活或通过补充NAD改善线粒体功能来增加ATP水平。在目标3中，我们使用不同端粒长度的细胞系、细胞器和肠样细胞，建立了短端粒在导致人类肠道细胞缺陷中的相关性。

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.