Role of ERK1/2 in Neuromuscular Synapses and Myofiber Development in vivo

描述(申请人提供)：细胞外信号调节激酶1和2(ERK1/2)是典型的细胞内有丝分裂原激活蛋白激酶(MAPK)。ERK1/2在多种细胞类型中被多种生长因子和细胞因子激活。体外研究表明，ERK1/2参与了细胞增殖、分化和凋亡的多种过程。ERK活性在培养的骨骼肌管分化过程中既有刺激作用，也有抑制作用，这种分化作用随这一漫长过程的不同阶段而异。然而，到目前为止，还没有关于ERK1/2在体内肌肉纤维发育中作用的基因靶向研究。运动神经元释放的Agin诱导和/或维持乙酰胆碱受体(AChR)簇和脊椎动物神经肌肉接头(NMJ)的突触后分化的其他方面。NMJ是脊髓中的运动神经元和骨骼肌纤维之间的突触。AGRIN通过结合和激活包含低密度脂蛋白受体相关蛋白4(Lrp4)和肌肉特异性激酶(Musk)的受体复合体来发挥作用。我们最近报道，在培养的肌管中，集聚蛋白以Lrp4/Musk依赖的方式诱导ERK1/2激活，这对集聚蛋白诱导AChR簇的能力具有负面调节作用。此外，我们的初步数据显示，肌纤维中ERK2的缺失导致了年轻成年小鼠NMJ维持的缺陷。在这些动物中，NMJ表现出与老年小鼠或建立Duchenne肌营养不良模型的MDX小鼠NMJ相似的碎片化。本研究旨在探讨ERK1/2在体内对神经肌肉突触的形成和维持以及骨骼肌纤维的分化和成熟的意义。一种条件基因打靶策略，最近在周围神经的其他细胞中取得了成果 该系统将用于选择性地灭活骨骼肌纤维中的ERK1/2。上述实验的结果将阐明ERK1/2在体内在NMJ的集聚蛋白信号转导中的作用，特别是在骨骼肌纤维的发育中。此外，它们可能为正常衰老、Duchenne肌营养不良以及肌肉中表达的ERK1/2下游底物突变引起的疾病提供新的分子机制，例如与Coffin-Lowry综合征相关的核糖体S6激酶2(RSK2)。 公共卫生相关性：该项目将调查蛋白激酶ERK1/2是否在活体内骨骼肌分化以及神经-肌肉连接的形成和维持中发挥重要作用。这些实验的结果也可能与正常衰老、Duchenne肌营养不良症和Coffin-Lowry综合征有关。

DESCRIPTION (provided by applicant): Extracellular-signal regulated kinases 1 and 2 (ERK1/2) are the prototypical intracellular mitogen activated protein kinases (MAPK). ERK1/2 are activated by multiple growth factors and cytokines in many cell types. Studies in vitro implicate ERK1/2 in a myriad of cellular processes during proliferation, differentiation and apoptosis. ERK activity has both stimulatory and inhibitory roles in the differentiation of culture skeletal myotubes that vary with the stage of this protracted process. However, no gene targeting investigations on the role of ERK1/2 in developing muscle fibers in vivo have been reported to date. Agrin released by motoneurons induces and/or maintains acetylcholine receptor (AChR) clustering and other aspects of postsynaptic differentiation at the vertebrate neuromuscular junction (NMJ), the synapse between a motoneuron in the spinal cord and a skeletal muscle fiber. Agrin acts by binding and activating a receptor complex containing LDL receptor related protein 4 (Lrp4) and muscle specific kinase (MuSK). We reported recently that in cultured myotubes agrin induces ERK1/2 activation in an Lrp4/MuSK-dependent fashion and that this negatively regulates the ability of agrin to induce AChR clusters. Furthermore, our preliminary data show that loss of ERK2 in myofibers leads to defects in NMJ maintenance in young adult mice. In these animals, NMJs display fragmentation similar to than seen in NMJs of old mice or in mdx mice, which model Duchenne muscular dystrophy. The aim of this R21 application is to investigate the significance in vivo of ERK1/2 for neuromuscular synapse formation and maintenance and for skeletal myofiber differentiation and maturation. A strategy of conditional gene targeting that has recently bear fruit in other cells of the peripheral nervous system will be used to selectively inactivate Erk1/2 in skeletal muscle fibers. Results from the above experiments will clarify the in vivo role of ERK1/2 in agrin signaling at the NMJ, in particular, and in the development of skeletal muscle fibers, in general. In addition, they may provide new insights into the molecular mechanisms underlying normal aging, Duchenne muscular dystrophy and diseases caused by mutation of ERK1/2 downstream substrates expressed in muscle, such as ribosomal S6 kinase 2 (Rsk2), which is linked to Coffin-Lowry syndrome. PUBLIC HEALTH RELEVANCE: This project will investigate whether the protein kinases ERK1/2 have important roles in skeletal muscle differentiation and formation and maintenance of nerve-muscle connections in vivo. Results from these experiments may also be relevant for normal aging, Duchenne muscular dystrophy and Coffin-Lowry syndrome.