The role of Usp 14 in regulating neuronal function

描述（由申请人提供）：最近在运动神经元疾病（如脊髓性肌萎缩症（SMA））中报告了神经肌肉接头（NMJ）的改变;然而，对调节运动神经元中突触活动和发育的途径知之甚少。虽然转录机制已被证明可以调节神经系统发育中的关键步骤，但最近的研究强调了泛素蛋白酶体系统（UPS）在突触连接的发育和维持中的重要性。通过调节泛素信号通路，如激酶活化和细胞蛋白质的运输和丰度，UPS可以控制神经系统成熟过程中的发育过渡点。然而，尚不清楚细胞如何调节这些过程所需的可用泛素库。鉴于运动神经元细胞体和终板之间的距离，专门的机制必须确保轴突路径发现、突触靶向和运动终板成熟所必需的泛素的稳定表达。我们的研究表明，蛋白酶体去泛素化酶Usp 14是运动神经元终板出生后发育所必需的。Usp 14缺陷的纯合子axJ小鼠表现出静息性震颤、后肢僵硬、肌肉质量减少，并在8周龄时死亡。这些小鼠没有泛素化的蛋白质聚集体或加速的神经元细胞死亡，而是在出生后发育的前两周显示与受损的运动终板成熟相关的泛素损失。axJ小鼠中泛素水平的恢复增加了体重和运动功能，并防止了出生后的死亡，这表明泛素丢失可能是神经肌肉疾病的主要原因。我们最近的研究还证明了SMA小鼠模型中的泛素损失，该模型显示出与axJ小鼠相似的NMJ成熟和功能受损，验证了鉴定由泛素调节的发育途径的重要性。我们的工作假设是，Usp 14的功能，以维持哺乳动物突触的发育和活动所需的泛素水平。该提议的第一个目的是确定axJ小鼠中泛素丢失对NMJ发育和活性的贡献。在第二个目标中，我们将研究一个新提出的催化剂独立的功能的Usp 14的蛋白酶体，并确定它是否需要在NMJ的发展和突触传递。第三个目的是确定运动神经元和运动终板在axJ小鼠疾病过程中的作用。最终的目标将检查控制突触成熟和NMJ功能的泛素依赖性通路。该提案将使用遗传学和生物化学的组合来研究Usp 14对蛋白酶体的基本酶功能，并确定Usp 14活性的变化如何改变突触发育和功能所需的信号通路。

DESCRIPTION (provided by applicant): Alterations in the neuromuscular junction (NMJ) have recently been reported in motor neuron diseases such as Spinal muscular atrophy (SMA); however, little is known about the pathways that regulate synaptic activity and development in motor neurons. Although transcriptional mechanisms have been shown to regulate critical steps in the development of the nervous system, recent studies have highlighted the importance of the ubiquitin proteasome system (UPS) in the development and maintenance of synaptic connections. By regulating ubiquitin signaling pathways, such as kinase activation and the trafficking and abundance of cellular proteins, the UPS can control developmental transition points during the maturation of the nervous system. However, it is not known how the cell regulates available ubiquitin pools required for these processes. Given the distance that separates the motor neuron cell body and endplate, specialized mechanisms must ensure the stable expression of ubiquitin necessary for axon path finding, synaptic targeting and motor endplate maturation. Our studies now demonstrate that the proteasomal deubiquitinating enzyme Usp14 is required for the postnatal development of the motor neuron endplate. Homozygous axJ mice, which are deficient for Usp14, display a resting tremor, hind limb rigidity, reduced muscle mass and die by 8 weeks of age. These mice do not have ubiquitinated protein aggregates or accelerated neuronal cell death, but instead show ubiquitin loss that correlates with impaired motor endplate maturation during the first two weeks of postnatal development. Restoration of ubiquitin levels in the axJ mice increases body mass and motor function and prevents postnatal lethality, indicating that ubiquitin loss can be a major contributor to neuromuscular disease. Our recent studies also demonstrate ubiquitin loss in a mouse model of SMA, which displays impaired NMJ maturation and function similar to the axJ mice, validating the importance of identifying the developmental pathways regulated by ubiquitin. Our working hypothesis is that Usp14 functions to maintain ubiquitin levels required for the development and activity of mammalian synapses. The first aim of this proposal will determine the contribution of ubiquitin loss in the axJ mice to the development and activity of the NMJ. In the second aim, we will investigate a newly proposed catalytic-independent function of Usp14 on the proteasome and determine if it is required for development and synaptic transmission at the NMJ. The third aim is designed to determine the role of motor neurons and motor endplates in the disease process in the axJ mice. The final aim will examine the ubiquitin-dependent pathways that control synaptic maturation and function of the NMJ. This proposal will use a combination of genetics and biochemistry to investigate the essential enzymatic functions of Usp14 on the proteasome and determine how changes in the activity of Usp14 alter signaling pathways required for synaptic development and function.