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

原理：最近，干细胞（SC）功能中的神经元相关信号传导的关键作用正日益得到认可。尽管在大多数类型的SC中线粒体含量低并且依赖于非依赖于细胞的糖酵解代谢来获得能量，但我们目前的研究已经涉及线粒体在SC活化、命运决定和抵抗衰老（定义为细胞分裂能力的丧失）中的功能（Zhang et al，Science 2016）。与线粒体功能相关，我们以前已经证明了两个大家族的蛋白质，聚（ADPribose）聚合酶（PARP）和Sirtuin（SIRT）酶，平衡细胞应激反应与线粒体稳态，并可能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，作为酶活性的辅因子，因此竞争NAD+的细胞内储存。有趣的是，在细胞应激、DNA损伤或炎症条件下，PARP家族的酶变得高度活化，这导致NAD+储存的耗尽，损害SIRT酶活性，导致线粒体功能障碍。因此，PARP抑制或 例如，小鼠中Parp 1功能的遗传丧失增加了NAD+水平和SIRT指导的线粒体生物发生。与此相关，我们的实验室最近证明，在肌营养不良症导致代谢功能障碍期间，PARP活性在肌肉中过度活跃（Ryu et al，Science Translational Medicine 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活性改变组织再生能力和干细胞功能与损伤，疾病和衰老的确切机制。为了实现这一长期目标，我们的近期目标，这是本NSERC发现补助金的主题，是： (1)描绘运动训练后小鼠肌肉SC中PARP-、NAD+-和β-代谢的交叉点。 (2)在使用心脏毒素和下坡跑或肌肉营养不良小鼠模型的肌肉损伤诱导再生期间，评估PARP激活在肌肉SC中的作用。 (3)确定肌肉SC中的PARP激活是否是运动诱导的肌肉SC增殖/分化或使用PARP抑制剂在体内或细胞培养中损伤后组织再生所必需的。 拟议的实验将结合联合收割机的各种体外和小鼠运动模型已经建立在PI的实验室。我们希望从拟议的实验中产生的数据，以确定新的PARP指导机制负责肌肉干细胞的命运在运动和组织再生。拟议的研究将为感兴趣的分子运动生理学和干细胞生物学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.