Elucidation of the Molecular Mechanisms of Werner Syndrome with Stem Cell Models

Werner Syndrome (WS) is an autosomal recessive disease which is characterized by accelerated aging phenotypes, such as short stature, dyslipidemia, the loss of subcutaneous fat, and premature atherosclerosis. The mutations of WRN gene are currently the only causes reported associated with WS. However, precisely how do WRN mutations cause the WS pathologies are still unclear. Here we used stem cell models to investigate the pathobiology of two WS dysregulations that we are interested in: short stature and metabolic dysfunction.Stem cells make ideal laboratory models for investigating the development of diseases; they help us pinpoint and track the very beginning of disease-causing event in cells. With this tool, the early events of development could be recaptured and how different are these events in the diseases could be observed. Therefore, two stem cell models were chosen for our study, namely, human embryonic stem cells (hESCs) and mesenchymal stem cells (hMSCs), respectively.A direct chondrocyte differentiation protocol was followed to investigate why WS patients usually have a short stature. The impairment of chondrogenesis was speculated to be the cause. Thus, chondrocyte formation was examined in both hESCs and hMSCs. Depletion of WS was observed to lead to the failure of chondrogenesis. The RecQ helicase family includes WRN. WRN helicase unwind and separate certain double-stranded DNA generally. Before DNA can be duplicated in preparation for cell division, certain actions are required, such as DNA replication. WRN helicase is also vital for the transcription process, which creates a blueprint of gene for protein production. More intriguingly, WRN helicase was found to play a pivotal role in chondrogenesis. Combined analyses of RNA-sequencing and ChIP-sequencing led to the identification of a novel gene SHOX which was found to interact directly with WRN to modulate chondrogenesis. Loss of SHOX is one of the major causes leading to skeletal dysplasia and short stature, such as Tuner syndrome, Léri-Weill dyschondrosteosis, and idiopathic short stature. SHOX is an important transcriptional regulator in chondrocyte proliferation and differentiation, bone formation and chondrogenic growth arrest. Moreover, we revealed that WRN could regulate SHOX expression through unwinding its G-quadruplexes (G4s). G-quadruplex DNA is a stable structure with G-G base pairs that hold it together. G4s are thought to be common in human gene promoters, with one or more quadruplex motifs found in roughly 40% of all human gene promoters. Because of the important function it plays in DNA repair, many genome-regulating activities have been reported to be associated with the development of G4s. We reported that WRN helicase could open SHOX G4s and promoted its expression during chondrogenesis.Additionally, the cause of metabolic dysfunction and NAD+ depletion occurred in WS was studied. Circadian rhythms are important for health, and disturbances to the clock have been linked to unfavorable health outcomes. The circadian system undergoes considerable alternations as people age, affecting rhythms of behaviors. Numerous metabolic rhythms show gradual dampening with age. We firstly reported that depletion of WRN causes accelerated premature onset of adipogenesis in both WS mesenchymal stem cells (hMSCs) and in a zebrafish model of WS. Nicotinamide adenine dinucleotide (NAD+) is a crucial metabolite and coenzyme for a cell's redox status and various metabolic energy balances. Nicotinamide riboside (NR) is the precursor of NAD+. NAD+ levels decrease with age, and various enzymes involved in NAD+ degradation and biosynthesis are changed. Mechanistically, we revealed that the life essential molecule NAD+ is reduced in WS MSCs, leading to accelerated adipogenesis; NAD+ repletion normalizes pathways related to metabolism and mitophagy, and normalizes adipogenesis in the MSCs and zebrafish models of WS. Mitochondria plays an important role in metabolism, as well as the process of ageing and age-related diseases. Damaged mitochondria with low ATP synthesis and high reactive oxygen species (ROS) release are linked to ageing and age-associated diseases in which mitochondrial dynamics and turnover are disrupted. We reported that NAD+ repletion counteracts WS phenotypes, improves mitochondrial quality and adipogenic circadian rhythm both in vivo and in vitro. Collectively, WRN mutation causes accelerated adipogenesis, at least partially, due to NAD+ depletion-related dysfunctional control of gene transcription or chromatin organization via the transcription factors such as SMCA5 and MSX2. Since there is no cure for WS, our findings shed light on novel therapeutics against WS by targeting on the regulation of NAD+, mitochondrial homeostasis, and circadian rhythm.Understanding how WRN led to these dysregulations has vital implications for manifesting the mechanisms of premature aging in WS and for therapeutic trials development.Werner綜合征（WS）是常染色體隱性遺傳疾病，其特徵是衰老表型加快，例如身材矮小，血脂異常，皮下脂肪減少和動脈粥樣硬化。 WRN基因的突變是目前報導與WS相關的唯一原因。然而，確切地講WRN突變導致WS的病態仍不清楚。在這裡，我們使用幹細胞模型進一步研究了我們感興趣的兩種WS功能異常：身材矮小和代謝功能異常。幹細胞是研究疾病發展的理想實驗室模型，這有助於我們查明並追蹤細胞中致病事件的最開始。通過這種方式，我們可以重新捕捉發育的早期起源，並觀察其在疾病中的不同之處。因此，我們選擇了兩種幹細胞模型進行進一步研究：人胚胎幹細胞（hESCs）和間充質幹細胞（hMSCs）。我们采用将人胚胎干细胞直接诱导分化为软骨细胞的方法来研究WS 患者身材矮小的机理。我们观察到 WRN基因的缺失是导致软骨形成失败的主要原因。WRN属于RecQ 解旋酶家族。 WRN 解旋酶一般会解开和分离某些双链 DNA。WRN 解旋酶对于转录过程也至关重要，转录过程为蛋白质生产创造了基因蓝图。更有趣的是，发现 WRN 解旋酶在软骨形成中起着关键作用。 RNA 测序和 ChIP 测序的组合分析鉴定出一种新基因 SHOX，该基因被发现可以通过直接与 WRN 相互作用以调节软骨形成。 SHOX 的丧失是导致骨骼发育不良和身材矮小的主要原因之一，例如 Tuner 综合征、Léri-Weill 软骨骨病和特发性身材矮小。 SHOX 在软骨细胞增殖和分化、骨形成和软骨形成生长停滞发挥重要调节作用。此外，我们还发现 WRN 可以通过解开其 G-四链体 (G4s) 来调节 SHOX 的表达。 G-四链体 DNA 是一种稳定的结构，具有将其固定在一起的 G-G 碱基对。 G4 被认为在人类基因启动子中很常见，在大约 40% 的人类基因启动子中发现了一个或多个四链体基序。由于它在 DNA 修复中发挥重要作用，据报道许多基因组调节活动与 G4 的发育有关。我们报道 WRN 解旋酶可以打开 SHOX G4s 并在软骨形成过程中促进其表达。此外，研究了 WS 中发生代谢功能障碍和 NAD+ 耗竭的原因。昼夜节律对健康很重要，时钟紊乱导致身体健康出现问题。随着人们年龄的增长，昼夜节律系统会发生相当大的变化，并且影响人体戒律健康。许多代谢节律随着年龄的增长而逐渐减弱。我们首先报道了在WS 间充质干细胞 (hMSC) 和 WS 斑马鱼模型中， WRN 的缺失导致脂肪生成的过早发生。烟酰胺腺嘌呤二核苷酸 (NAD+) 是细胞氧化还原状态和各种代谢能量平衡的关键代谢物和辅酶。烟酰胺核苷 (NR) 是 NAD+ 的前体。 NAD+ 水平随着年龄的增长而降低，参与 NAD+ 降解和生物合成的各种酶也会发生变化。从机制上讲，我们发现 WS MSC 中生命必需分子 NAD+ 减少，导致脂肪生成加速； NAD+ 补充使与代谢和线粒体自噬相关的通路正常化，并使 WS 的 MSC 和斑马鱼模型中的脂肪生成正常化。线粒体在新陈代谢以及衰老和年龄相关疾病的过程中起着重要作用。具有低 ATP 合成和高活性氧 (ROS) 释放的受损线粒体与衰老和与年龄相关的疾病有关，其中线粒体动力学和更新被破坏。我们报道了 NAD+ 补充抵消了 WS 表型，改善了体内和体外的线粒体质量和脂肪生成昼夜节律。总的来说，WRN 突变导致脂肪生成加速，至少部分是由于 NAD+ 耗竭相关的基因转录或染色质组织通过转录因子（如 SMCA5 和 MSX2）的功能失调控制。由于 WS 目前尚无较好的治疗方法，我们的研究结果通过针对 NAD+、线粒体稳态和昼夜节律的调节，揭示了针对 WS 的新疗法。了解 WRN 如何导致这些失调对于揭示 WS 过早衰老的机制和开发治疗试验具有重要意义。TIAN, Yuyao.Ph.D. Chinese University of Hong Kong 2021.Includes bibliographical references (leaves )Abstracts in English and Chinese.Title from PDF title page (viewed on ...