Agrin/alpha 3 Na,K-ATPase signaling at the neuromuscular junction

描述(申请人提供)：所有的协调运动，如运动、呼吸和吞咽，都依赖于运动神经元和它们所支配的骨骼肌纤维之间的快速信息传递，这种信息传递发生在一个称为神经肌肉连接的特殊接触处。疾病破坏这一系统，如各种肌无力障碍、脊髓性肌萎缩症和肌萎缩侧索硬化症、肉毒杆菌毒素等毒素或创伤，会导致肌肉无力甚至死亡。因此，通过确定影响这一重要突触功能的一系列疾病的新的治疗靶点，了解神经肌肉连接的形成和维持的机制可能会对公众健康产生显著的好处。该领域的一项重大成功是阐明了一种名为集聚蛋白的蛋白质在介导运动神经元诱导的由肌肉纤维形成的突触后装置分化中的作用。AGRIN通过肌肉纤维膜受体复合体发挥作用，该复合体由低密度脂蛋白受体相关蛋白LRP4和肌肉特异性酪氨酸激酶穆斯克组成。然而，奇怪的是，并不是神经肌肉接头上的所有形式的集聚蛋白都能激活LRP4/穆斯克受体，一些研究表明，这些不活跃的集聚蛋白物种在调节运动轴突和轴突终末的生长和分化方面发挥着作用。可能介导这些突触前效应的受体尚不清楚，但一个很好的候选是A3Na，K-ATPase(NKA)，它是一种神经元特异性的钠-钾泵亚型，可以结合所有形式的集聚蛋白。A3泵的农业依赖性调节中枢神经系统神经元的兴奋性，以及与调节突触功能、生长和成熟有关的胞浆钙和其他第二信使的水平。A3NKA也存在于运动轴突终末，这增加了集聚蛋白/A3NKA通路在神经肌肉接头处发挥相关作用的可能性。为了验证这一假说，我们将考察敲除A3NKA对神经肌肉接头发育的影响，A3NKA在调节运动轴和树突生长中的作用，以及集聚蛋白/A3NKA在调节神经肌肉突触传递中的作用。以前对神经肌肉接头集聚蛋白功能的研究主要集中在它与突触后LRP4/Musk受体的相互作用上。这将是第一次研究在这个重要的突触上另一条集聚蛋白信号通路的意义。神经肌肉疾病，如肌无力障碍、脊髓性肌萎缩症、肌萎缩侧索硬化症，其特征是严重的肌肉无力与神经肌肉突触传递的下降有关。我们相信，agrin/A3NKA通路将被证明是一个新的治疗靶点，可以用于治疗这些和相关的神经肌肉疾病。 公共卫生相关性：神经肌肉接头是负责呼吸和自主运动的神经和肌肉纤维之间的重要连接。在许多神经肌肉疾病中，如脊髓性肌萎缩症、重症肌无力和先天性肌无力综合征，神经肌肉接头功能受损，导致严重的肌肉无力和死亡。在这里，我们研究了一种新的机制，即集聚蛋白，一种以指导神经肌肉连接的突触后装置的形成而闻名的蛋白质，它影响突触前运动轴突终末的生长和功能。这一新的信号通路为神经肌肉疾病的治疗提供了新的靶点。

DESCRIPTION (provided by applicant): All coordinated movements such as locomotion, breathing, and swallowing, depend on the rapid transfer of information between motor neurons and the skeletal muscle fibers they innervate that takes place at a specialized contact called the neuromuscular junction. Disruption of this system by disease, as occurs in various myasthenic disorders, spinal muscular atrophy, and amyotrophic lateral sclerosis, toxins such as botulinum toxin, or traumatic injury, results in muscle weakness and even death. Thus, understanding the mechanisms responsible for the formation and maintenance of the neuromuscular junction has the potential for significant public health benefits by identifying new therapeutic targets for a wide range of disorders that impact the function of this important synapse. A major success in the field has been the elucidation of the role of a protein called agrin in mediating the motor neuron-induced differentiation of the postsynaptic apparatus formed by the muscle fiber. Agrin exerts its effects through a muscle fiber membrane receptor complex consisting of LRP4, a low-density lipoprotein receptor-related protein, and MuSK, a muscle-specific tyrosine kinase. Curiously, however, not all forms of agrin at the neuromuscular junction are competent to activate the LRP4/MuSK receptor and several studies have suggested these "inactive" agrin species play a role regulating growth and differentiation of motor axons and axon terminals. The receptor that might mediate these presynaptic effects is unknown but a good candidate is the a3 Na,K-ATPase (NKA), a neuron-specific isoform of the sodium-potassium pump that binds all forms of agrin. Agrin-dependent regulation of the a3 pump modulates excitability of central nervous system neurons, as well as the levels of cytoplasmic calcium and other second messengers implicated in regulating synaptic function, growth and maturation. The a3 NKA is also present on motor axon terminals, raising the possibility that the agrin/a3 NKA pathway plays a related role at the neuromuscular junction. To test this hypothesis we will examine the effect of knocking out the a3 NKA on development of the neuromuscular junction, the role of the a3 NKA in regulating growth of motor axons and dendrites, and the role of agrin/a3 NKA in modulating neuromuscular synaptic transmission. Previous studies of agrin function at the neuromuscular junction have focused on its interaction with the postsynaptic LRP4/MuSK receptor. This would be the first to examine the significance of an alternate agrin signaling pathway at this important synapse. Neuromuscular diseases such as the myasthenic disorders, spinal muscular atrophy, amyotrophic lateral sclerosis are characterized by profound muscle weakness linked to a decline in neuromuscular synaptic transmission. We believe the agrin/a3 NKA pathway will prove to be a novel therapeutic target that can be exploited for the treatment of these and related neuromuscular disorders. PUBLIC HEALTH RELEVANCE: The neuromuscular junction is the vital connection between the nerves and muscle fibers responsible for respiratory and voluntary movements. In many neuromuscular diseases, such as spinal muscular atrophy, myasthenia gravis and congenital myasthenic syndrome, the function of the neuromuscular junction is compromised resulting in profound muscle weakness and death. Here we examine a novel mechanism whereby agrin, a protein known for its role in directing the formation of the post-synaptic apparatus of the neuromuscular junction, influences growth and function of the pre-synaptic motor axon terminal. This novel signal pathway represents a new therapeutic target for the treatment of neuromuscular disease.