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

尽管人们越来越认识到肠道中饮食和宿主特异性遗传因子的相互作用在肠道和全身性疾病中起着重要作用，但我们对这一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，我们建议通过推进我们的知识来弥合这一差距，即肠道中的端粒缩短如何影响肠上皮细胞的成熟，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如肠的再生很重要。端粒缩短发生在正常衰老过程中，并且在端粒酶突变或高细胞更新条件（如溃疡性结肠炎和克罗恩病）的患者中加速。端粒缩短导致肠中的几种病理，包括萎缩、炎症和患者中结肠炎向癌症的进展，并且所有这些病理在端粒酶敲除小鼠（TKO）中如实地再现。从机制上讲，据信短端粒通过肠干细胞的连续变性介导的耗竭来驱动这些病理。除了干细胞耗竭，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，我们发现端粒缩短损害肠上皮细胞谱系的成熟，导致产生不成熟和功能受损的肠上皮细胞。这得到了多水平分析的支持，包括RNAseq、蛋白质组学、透射和扫描电子显微镜，证明消化酶、转运蛋白和微绒毛结构组分的表达在TKO小鼠的近端肠中受到抑制。重要的是，初步研究表明，这种肠细胞妥协是保存在人类肠细胞与短端粒。从机制上讲，TKO上皮中p53的缺失挽救了肠上皮细胞缺陷。此外，这些肠上皮细胞的特征在于线粒体功能障碍，并且具有低ATP水平。当暴露于富含果糖的饮食时，它们表现出ATP水平急剧下降和随后的细胞凋亡的明显倾向，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（目的1），通过ATP消耗酶果糖激酶的失活或通过NAD补充改善线粒体功能来增加ATP水平，从而确定低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、类器官和类肠细胞建立了短端粒在引起人类肠上皮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.