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

理论基础：最近，线粒体相关信号在干细胞（SC）功能中的关键作用越来越被认识到。尽管在大多数类型的SC中，线粒体含量较低，并且依赖于线粒体独立的糖酵解代谢来获取能量，但我们目前的研究表明，线粒体在SC激活、命运决定和抵御衰老（定义为细胞分裂能力的丧失）中的功能（Zhang等人，Science 2016）。与线粒体功能相关，我们之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，通过线粒体稳态平衡细胞应激反应，并且可以对组织或全身代谢产生重大影响。有趣的是，这些蛋白家族中的每一个都需要NAD+作为酶活性的辅助因子，NAD+是一种有限的关键能量代谢物，因此它们会竞争细胞内储存的NAD+。有趣的是，在细胞应激、DNA损伤或炎症条件下，PARP酶家族变得高度激活，这导致NAD+储存的消耗，SIRT酶活性受损，导致线粒体功能障碍。因此，在小鼠中，PARP抑制或Parp1功能的遗传丧失，例如，增加NAD+水平和sirt引导的线粒体生物发生。与此相关，我们的实验室最近证明，在肌肉营养不良导致代谢功能障碍的过程中，肌肉中的PARP活性过度活跃（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活性在损伤、疾病和衰老时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（NSERC Discovery Grant的主题）是：(1)描述运动训练后小鼠肌肉SCs中PARP-， NAD+-和线粒体代谢的交叉点。(2)评估心肌毒素和下坡跑步诱导的肌肉损伤再生或肌营养不良小鼠模型中PARP激活在肌肉SCs中的作用。(3)确定肌肉SCs中PARP的激活对于运动诱导的肌肉SC增殖/分化或使用PARP抑制剂在体内或细胞培养中损伤后的组织再生是否必要。拟议的实验将结合PI实验室已经建立的各种体外和小鼠运动模型。我们期望从拟议的实验中产生的数据能够确定在运动和组织再生过程中负责肌肉干细胞命运的新的PARP指导机制。本课程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训练机会。

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 orgenetic 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.