Redox and Ca2+ signaling regulation of enamel mineralization

项目摘要：牙釉质的适当矿化可以防止细菌的攻击， 龋齿（Caries）成釉细胞负责产生和分泌大量的 蛋白质（为釉质生长奠定基础）以及参与主动矿物质运输（钙化 搪瓷）。这些功能是阶段依赖性的，釉质形成分为分泌型和分泌型。 （蛋白质合成）和成熟阶段（矿化）。然而，提供代谢的机制 对这些过程的支持以及线粒体功能障碍如何改变它们还知之甚少。 线粒体是细胞内的细胞器，通过产生ATP将营养物质转化为能量， 并且线粒体功能的缺陷导致人类疾病。线粒体扮演着重要的角色 线粒体DNA缺陷导致釉质发育异常。 因此，这一竞争性更新提案的目标是调查 线粒体功能和釉质形成在环境损伤，并确定这是否是 在患有影响线粒体功能的遗传疾病的患者中改变。我们将特别针对 这在唐氏综合征（DS）或21三体的背景下，以及在氟斑牙期间， 在发育过程中暴露于过量的氟化物会削弱牙釉质。DS患者存在 釉质缺陷包括钙化不足和发育不全，两者都是由发育缺陷引起的， 釉质形成线粒体功能障碍在DS中被广泛报道。最重要的假设是 这一假设是DS成釉细胞中线粒体功能的改变了釉质晶体 形成并影响对氟中毒的敏感性。在强有力的支持下，我们的初步数据显示，DP- 16只小鼠（已建立的DS小鼠模型）具有机械虚弱和形态异常 搪瓷。此外，RCAN 1（一种与DS病理生理学相关的基因， 在成釉细胞中表达），显著损害线粒体功能。我们有 也报道了氟暴露的釉细胞显着影响的蛋白质的生物合成 负责维持ATP产生的电子传递链（ETC），但不在其他细胞中 测试，表明釉细胞对氟化物的独特敏感性，可能与较高的ROS有关 程度.在所提出的研究中，我们将使用DS小鼠模型（Dp 16小鼠、Rcan 1-KO小鼠、Dp 16 x小鼠、Dp 16 x小鼠、Dp 16 x小鼠）。 Rcan 1-KO小鼠），以解决线粒体在这些小鼠的成釉细胞中的作用。我们还将使用 最近开发的表达荧光标记的分泌和成熟阶段的报告小鼠 成釉细胞诱导氟化物和研究线粒体缺陷使用单细胞RNASeq和批量 RNAseq比较成釉细胞与其他组织。为了解决DS模型的成釉细胞是否更多 对氟化物敏感，我们将用氟化物处理细胞并分析线粒体功能。

Project Summary: Proper mineralization of dental enamel protects against bacterial attack that causes tooth decay (caries). The ameloblasts cells are responsible for producing and secreting an abundance of proteins (laying a foundation for enamel growth) as well as engaging in active mineral transport (calcifying the enamel). These functions are stage dependent with amelogenesis being divided into the secretory (protein synthesis) and maturation stage (mineralization). However, the mechanisms providing metabolic support for these processes and how mitochondrial dysfunction might alter them is poorly understood. Mitochondria are organelles within the cells that convert nutrients into energy via the production of ATP, and deficiency in mitochondrial function results in human diseases. Mitochondria play an important role in enamel as evidenced by defects in mitochondrial DNA causing abnormal enamel development. Therefore, the goal in this competing renewal proposal is to investigate the interplay between mitochondrial function and enamel formation during environmental insults and determine whether this is altered in patients with genetic disorders that effect mitochondrial function. We will specifically address this in the context of Down syndrome (DS), or Trisomy 21, and during dental fluorosis, a process in which exposure to excess of fluoride during development weakens tooth enamel. DS patients present with enamel defects including hypocalcification and hypoplasia, both caused by developmental defects in enamel formation. Mitochondrial dysfunction is widely reported in DS. The overarching hypothesis of this proposal is that altered mitochondrial function in DS ameloblasts alters enamel crystal formation and impacts sensitivity to fluorosis. In strong support, our preliminary data show that Dp- 16 mice (an established mouse model of DS) have mechanically weak and morphologically abnormal enamel. Moreover, overexpression of RCAN1 (a gene associated with DS pathophysiology that is expressed in ameloblasts) in enamel cell lines, significantly impaired mitochondrial function. We have also reported that fluoride exposure of enamel cells significantly affected the biosynthesis of the proteins of the electron transport chain (ETC) responsible for maintaining ATP production, but not in other cells tested, suggesting unique sensitivity of enamel cells to fluoride, possibly associated with higher ROS levels. In the proposed studies we will use DS mouse models (Dp16 mice, Rcan1-KO mice, Dp16 x Rcan1-KO mice) to address the role of mitochondria in the ameloblasts of these mice. We will also use recently developed reporter mice expressing fluorescently labelled secretory and maturation stage ameloblasts to induce fluoride and investigate mitochondrial defects using single cell RNASeq and bulk RNAseq to compare ameloblasts with other tissues. To address if ameloblasts of DS models are more sensitive to fluoride, we will treat the cells with fluoride and analyze mitochondrial function.