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

描述（由申请人提供）：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活化蛋白激酶（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在增殖、分化和凋亡过程中参与了无数细胞过程。ERK活性在培养骨骼肌管的分化中具有刺激和抑制作用，随着这一漫长过程的阶段而变化。然而，到目前为止，还没有关于ERK1/2在体内肌肉纤维发育中的作用的基因靶向研究报道。运动神经元释放的Agrin诱导和/或维持脊椎动物神经肌肉连接（NMJ）的乙酰胆碱受体（AChR）聚集和其他方面的突触后分化，NMJ是脊髓运动神经元和骨骼肌纤维之间的突触。Agrin通过结合和激活含有LDL受体相关蛋白4 （Lrp4）和肌肉特异性激酶（MuSK）的受体复合物而起作用。我们最近报道了在培养的肌管中，agrin以Lrp4/麝香依赖的方式诱导ERK1/2激活，这负调控了agrin诱导AChR簇的能力。此外，我们的初步数据表明，肌纤维中ERK2的缺失导致年轻成年小鼠NMJ维持缺陷。在这些动物中，NMJs显示出与老年小鼠或mdx小鼠NMJs相似的碎片，mdx小鼠是杜氏肌营养不良的模型。本R21应用的目的是研究ERK1/2在体内对神经肌肉突触形成和维持以及骨骼肌纤维分化和成熟的意义。一种有条件的基因靶向策略，最近在周围神经的其他细胞中取得了成果

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.