The role of poly(ADP- ribose)polymerases in muscle stem cell fate and function

理论基础：最近，线粒体连接信号在干细胞(SC)功能中的关键作用越来越被认识到。尽管大多数类型的干细胞线粒体含量低，且依赖于线粒体非依赖的糖酵解代谢来获得能量，但我们目前的研究表明，线粒体在SC激活、命运决定和抗衰老方面发挥了作用，衰老的定义是细胞分裂能力的丧失(Zhang等人，2016)。关于线粒体的功能，我们以前已经证明了两个蛋白质家族，聚(ADPribose)聚合酶(PARP)和Sirtuin(SIRT)酶，平衡细胞应激反应和线粒体的动态平衡，并可以对组织或全身代谢产生重大影响。有趣的是，这些蛋白质家族中的每一个都需要NAD+，一种有限的和关键的能量代谢物，作为酶活性的辅助因子，因此竞争NAD+的细胞内存储。有趣的是，在细胞应激、DNA损伤或炎症条件下，PARP家族酶高度激活，导致NAD+储存枯竭，SIRT酶活性受损，导致线粒体功能障碍。因此，例如，在小鼠中抑制PARP或*遗传丧失PARP1的功能，会增加NAD+水平和SIRT指导的线粒体生物发生。与此相关的是，我们的实验室最近证明，在肌肉营养不良导致代谢功能障碍的过程中，PARP活性在肌肉中过度活跃(Ryu等人，科学转化医学2016)。然而，PARP蛋白在干细胞命运、代谢或功能中的作用尚未被揭示。我们的长期目标是研究PARP活性通过损伤、疾病和衰老改变组织再生能力和干细胞功能的精确机制。为了实现这一长期目标，我们的近期目标是：*(1)描述PARP-的交叉点，*(2)在使用心脏毒素和下坡跑或在肌肉营养不良的小鼠模型中，评估肌肉干细胞中PARP激活的作用。*(3)确定肌肉干细胞中的PARP激活对于运动诱导的肌肉干细胞增殖/分化或使用PARP抑制剂在体内或细胞培养中损伤后的组织再生是必要的。*拟议的实验将结合已经在Pi的实验室中建立的各种体外和小鼠运动模型。我们希望从拟议的实验中产生的数据能够确定PARP指导的新机制，这些机制负责运动和组织再生过程中肌肉干细胞的命运。拟议的研究将为对分子运动生理学和干细胞生物学感兴趣的学生提供独特的培训机会。

Rationale: Recently, the critical roles for mitochondrial-linked signalling in stem cell (SC) function are becoming increasingly recognized. Despite low mitochondrial content and a reliance on mitochondrial-independent glycolytic metabolism for energy in most types of SCs, our current research has implicated mitochondrial function in SC activation, fate decisions and for defense against senescence, defined as the loss of a cells ability to divide (Zhang et al, Science 2016). Related to mitochondrial function, we have previously demonstrated that two large families of proteins, poly(ADPribose) polymerases (PARP) and Sirtuin (SIRT) enzymes, balance cell stress responses with mitochondrial homeostasis, and can have a large impact on tissue or whole body metabolism. Interestingly, each of these family of proteins require NAD+, a limited and key energy metabolite, as a cofactor for enzymatic activity and therefore compete for intracellular stores of NAD+. Interestingly, with cell stress, DNA damage, or inflammatory conditions, the PARP family of enzymes become highly activated, which results in depletion of NAD+ stores, compromised SIRT enzymatic activity leading to mitochondrial dysfunction. As a result, PARP inhibition or***genetic loss of function of Parp1 in mice, for example, increases NAD+ levels and SIRT-directed mitochondrial biogenesis. Related to this, our lab recently demonstrated that PARP activity is overactive in muscle during muscular dystrophy causing metabolic dysfunction (Ryu et al, Science Translational Medicine 2016). However, the role of PARP proteins in stem cell fate, metabolism or function has yet to be revealed. Our long-term goal is to examine the precise mechanisms by which PARP activity alter tissue regenerative capacity and stem cell function with damage, disease and aging. To achieve this long-term objective, our immediate goals, which are the subject of this NSERC Discovery Grant, are to:***(1) Profile the intersections of PARP-, NAD+- and mitochondrial-metabolism in muscle SCs in mice following exercise training.***(2) Evaluate the role of PARP activation in muscle SCs during muscle damage-induced regeneration using cardiotoxin and downhill running or in a mouse model of muscular dystrophy.***(3) Determine if PARP activation in muscle SCs is necessary for exercise-induced muscle SC proliferation/differentiation or for tissue regeneration following damage using PARP inhibitors in vivo or in cell culture.***The proposed experiments will combine a variety of in vitro and mouse exercise models already established in the PI's lab. We expect data generated from the proposed experiments to identify novel PARP directed mechanisms responsible muscle stem cell fate during exercise and tissue regeneration. The proposed studies will provide unique training opportunities for students interested molecular exercise physiology and stem cell biology.