Supplement to existing R01 to increase diversity in health-related science

 描述(申请人提供)：端粒缩短会导致部分端粒酶突变患者的肝硬变，并在后天条件下容易发展为肝硬变和肝功能衰竭。端粒领域的一个主要空白是我们对短端粒激活并驱动疾病过程的机制缺乏了解。因此，由于对端粒缩短如何损害细胞健康的了解不足，目前还不存在预防端粒相关肝病的治疗干预措施。我们最近发现端粒酶基因敲除小鼠(TKO)的端粒缩短影响细胞代谢，部分是通过P53依赖下调调节线粒体生物发生/功能的共激活因子PGC1a/？这会导致线粒体数量和功能的减少。然而，端粒介导的P53激活是否导致正常衰老过程中的代谢变化，P53影响代谢的确切途径，以及这些代谢变化与端粒相关疾病(如肝硬变)发病机制的相关性仍有待确定。在这项拨款提案中，我们通过在肝脏中进行的一系列实验来解决这些关键问题。这项建议是基于我们最近的观察，即TKO和老年野生型小鼠的肝组织中所有七种sirtuin的水平都降低了，这是一种与疾病高度相关的酶。这些酶是如何调节的还不清楚，然而，人们很好地理解它们的下调会导致许多疾病。在TKO小鼠中，sirtuins的下调部分依赖于P53。此外，我们的初步研究表明，p53在转录水平上以PGC1a依赖的方式调控线粒体sirtuins(Sirtuin 3，4，5)，在翻译水平上通过miRNAs调控非线粒体sirtuins(Sirtuin 1，2，6，7)。我们的假设是，端粒缩短诱导的P53下调sirtuins，这种Sirtuin抑制有助于端粒依赖性肝病。我们将通过三种方法验证这一假说：首先，为了确定衰老过程中P53和Sirtuin表达之间的因果关系，我们将删除老年小鼠肝组织中的P53，使用P53+/m突变小鼠(P53+/m突变小鼠)导致的提前衰老模型，然后检测Sirtuin水平和功能的变化(目标1)。其次，我们将重点研究P53活性的改变如何影响翻译调控的sirtuins。我们已经在TKO小鼠的肝组织中发现了四个依赖于P53的miRNAs，它们与sirtuins结合。我们将使用功能获得/功能丧失研究来测试这些候选miRNAs是否抑制单个非线粒体sirtuins，并确定它们缺失对肝功能的影响(目标2)。最后，为了研究Sirtuin下调对肝硬变的致病作用，我们将过度表达Sirt1，并确定Sirt1的恢复是否足以改善由短端粒引起的肝硬变。总之，这些实验将为端粒缩短如何影响新陈代谢和易患肝病提供新的见解。

 DESCRIPTION (provided by applicant): Telomere shortening causes liver cirrhosis in a subset of patients with telomerase mutations and predisposes to the development of liver cirrhosis and liver failure in acquired conditions. A major gap in the telomere field is our poor understanding of the mechanisms that are activated by short telomeres and drive the disease process. Consequently, no therapeutic interventions exist currently to prevent telomere-related liver disease due to an insufficient understanding how telomere shortening compromises cellular health. We have recently discovered that telomere shortening in telomerase knockout mice (TKO) impacts cellular metabolism in part through a p53-dependent down-regulation of PGC1a/ß, co-activators that regulate mitochondrial biogenesis/function. This leads to a decrease in mitochondrial numbers and function. However, whether telomere-mediated p53 activation leads to metabolic changes during normal aging, the precise pathways by which p53 impacts metabolism, and the relevance of these metabolic changes for the pathogenesis of telomere-related diseases such as liver cirrhosis remains to be defined. In this grant proposal, we address these critical questions experimentally through a series in experiments in the liver. The proposal is based on our recent observation that TKO and old wild-type mice liver tissues have reduced levels of all seven Sirtuins, a class of enzymes highly implicated in diseases. How these enzymes are regulated is not known, however, their down-regulation is well understood to lead to many disorders. In TKO mice, the down-regulation of Sirtuins is partially p53-dependent. Furthermore, our preliminary studies indicate that p53 regulates the mitochondrial Sirtuins (Sirtuin 3, 4, 5) in a PGC1a-dependent manner at the transcriptional level, and the non- mitochondrial Sirtuins (Sirtuin 1, 2, 6, 7) through miRNAs at the translational level. Our hypothesis is that telomere shortening-induced p53 down-regulates Sirtuins and that this Sirtuin repression contributes to telomere-dependent liver disease. We will test this hypothesis through three approaches: First, to determine the causal relationship between p53 and Sirtuin expression during aging, we will delete p53 in liver tissue of old mice, use a mouse model of premature aging due to hyper-active p53 (p53+/m mutant mice) and then assay changes in Sirtuin levels and function (Aim 1). Second, we will focus on how altered p53 activity impacts the translationally regulated Sirtuins. We have identified four p53-dependent miRNAs in liver tissue of TKO mice that bind to Sirtuins. We will use gain/loss-of-function studies to test whether these candidate miRNAs repress individual non-mitochondrial Sirtuins and determine the consequence of their deletion for liver function (Aim 2). Finally, to examine the pathogenic impact of Sirtuin down-regulation on liver cirrhosis, we will overexpress Sirt1 and determine whether restoration of Sirt1 is sufficient to improve liver cirrhosis induced by short telomeres. Together, these experiments will provide new insights in how telomere shortening impacts metabolism and predisposes to liver disease.