Validation and characterization of the identified variants associated with human longevity in mouse models

摘要 尽管衰老是极其复杂的，但人类中发生的对寿命有积极影响的基因变异 和健康为系统识别干预措施的分子靶点提供了一个理想的起点 可以延长人类的健康寿命。通过对德系犹太人百岁老人资源的遗传分析 阿尔伯特·爱因斯坦医学院的这一U19发现了多种罕见的、显著的功能性遗传变异 与健康长寿有关，这是药物发现的潜在目标。其中包括编码 IGF1R、SIRT6、ATM、USP35、UBE3C和BLM中的变体，以及FOXO3a中的功能性非编码变体， SIRT6、Smad3，以及IKK/NF-kB通路的组成成分，包括p65、NFKB1/p50和NFKB1a(IkBA)。 然而，这些变异基因产物延长健康和寿命的机制仍需进一步研究 在模型系统中发现并验证。在之前的资助期间，我们使用了ERCC1-/∆鼠标 加速衰老和衰老野生型小鼠模型，证明ATM和NF-kB活性降低， 通过ATM和p65/relA单倍体不足和hSIRT6的过表达减少细胞衰老 延长小鼠的健康寿命。此外，对ERCC1/∆小鼠的治疗(与项目4合作) 新的IKK/NF-kB抑制剂(SR12343)，已知的ATM抑制剂(KU-55933)，以及一种被证明能刺激 SIRT6(岩藻糖胶)的单腺苷二磷酸核糖转移酶(MADPRT)活性降低 促进小鼠衰老和改善与年龄相关的表型。我们还培育了携带稀有百岁老人的老鼠 IGF-1R变异(Igf1rhR407H)，Smad3，Foxo3a和Usp35杂合子小鼠，以及携带 SIRT6突变导致脱乙酰酶或mADPRT活性丧失，目前正在分析中。我们的 数据表明，这些罕见的变体中有许多可以减少DNA损伤和/或细胞衰老。在这里，我们 建议扩大这些努力，以验证项目1和项目2确定的其他稀有变种在 他们使用我们的人类早衰症小鼠模型来加速分析，从而延长健康寿命的能力。 我们还将继续测试针对提供的变异基因产物和相关途径的化合物 项目4在孕期和老年野生型小鼠中都进行了研究。总体而言，项目3的目标是使用遗传方法 验证项目1和2中确定的影响小鼠健康寿命的途径。我们将确定 单倍体不足、过度表达或特定突变对动物健康、分子终点的影响 与小鼠的衰老、韧性和老年病理学的特征有关。我们还会在药理上 与项目4合作，在老鼠身上确定这些途径的目标，以确定对相同终点的影响。 该项目的成功完成将验证已识别的人类变种对健康衰老的重要性 并确定具有延长健康寿命潜力的新治疗策略。

Abstract Although aging is extremely complex, the occurrence of genetic variants in humans that positively affect longevity and health offer an ideal starting point for the systematic identification of molecular targets for interventions that could extend human healthspan. Through genetic analysis of the Ashkenazi Jewish centenarian resource at the Albert Einstein College of Medicine, this U19 identified multiple rare, functional genetic variants significantly associated with healthy longevity, which represent potential targets for drug discovery. These include coding variants in IGF1R, SIRT6, ATM, USP35, UBE3C, and BLM, as well as functional non-coding variants in FOXO3A, SIRT6, SMAD3, and components of the IKK/NF-kB pathway, including p65, NFKB1/p50 and NFKB1a (IkBa). However, the mechanism by which these variant gene products extend healthspan and lifespan still need to be discovered and validated in model systems. During the previous funding period, we used the Ercc1-/∆ mouse model of accelerated aging and aged wild-type mice to demonstrate that reduction in ATM and NF-kB activity, achieved by ATM and p65/RelA haploinsufficiency, and overexpression of hSIRT6 reduced cellular senescence and extended healthspan of mice. In addition, treatment of Ercc1-/∆ mice (in collaboration with Project 4) with a novel IKK/NF-kB inhibitor (SR12343), a known ATM inhibitor (KU-55933), and a compound shown to stimulate the mono–adenosine 5′-diphosphate (ADP)–ribosyltransferase (mADPRT) activity of SIRT6 (fucoidan) reduce senescence and improve age-related phenotypes in mice. We also generated mice carrying a centenarian rare variant in IGF-1R (Igf1rhR407H), mice heterozygous for Smad3, Foxo3a, and Usp35, as well as mice carrying mutations in Sirt6 conferring loss of deacetylase or mADPRT activity, which are currently being analyzed. Our data suggests that many of these rare variants reduce DNA damage and/or cellular senescence. Here, we propose to expand these efforts to validate the role of additional rare variants identified by Projects 1 and 2 for their ability to extend healthspan using our murine model of a human progeroid syndrome to accelerate analyses. We also will continue to test compounds targeting the variant gene products and associated pathways provided by Project 4 in both progeroid and aged wild-type mice. Overall, the aim of Project 3 is to use genetic approaches to validate the pathways identified in Projects 1 and 2 as impacting healthspan in mice. We will determine the impact of haploinsufficiency, overexpression, or specific mutations on animal health, molecular endpoints associated with the hallmarks of aging, resilience, and geropathology in mice. We will also pharmacologically target these pathways in mice, in collaboration with Project 4, to determine the impact on the same endpoints. The successful completion of this project will validate the identified human variants as important for healthy aging and identify novel therapeutic strategies with the potential to extend healthspan.