Novel E3 Ligases in Nerve Degeneration and Regeneration

描述(申请人提供)：在神经退行性疾病中，轴突变性通常先于神经元胞体死亡，因此抑制轴突退化是对抗这些疾病的一个潜在的新干预点。轴突变性现在被认为是一种主动的、自毁的过程，泛素蛋白小体途径在其中扮演着重要的角色。我们已经鉴定出一种新的E3泛素连接酶ZNRF1，它在神经损伤后在雪旺细胞中被诱导。在轴突损伤后，ZNRF1与泛素一起在神经元中特异性上调。ZNRF1和另一个相关的E3连接酶ZNRF2在发育和成年期的神经系统中都有高表达。在体外实验中，我们发现ZNRF蛋白活性是正常轴突变性所必需的。在这项提案中，我们概述了定义ZNRF蛋白如何影响轴突变性的实验。首先，我们将分析ZNRF基因缺陷小鼠的表型，以确定这些蛋白的生理作用。特别是，我们将重点分析机械性和代谢性损伤后神经退化/再生的过程以及突触形成。我们的目标也是识别ZNRF介导的泛素化的靶向蛋白质。将进行蛋白质组学分析，从缺乏ZNRF蛋白的小鼠脑组织中或从含有显性负ZNRF突变体和ZNRF靶标的亲和纯化复合体中鉴定ZNRF靶标。这些研究将提高我们对轴索病变和神经元变性的认识，并为开发治疗神经退行性疾病的新方法提供基础。

DESCRIPTION (provided by applicant): Axonal degeneration often precedes the death of neuronal cell bodies in neurodegenerative diseases, thus inhibiting axonal degeneration represents a potential novel point of intervention in combating these diseases. Axonal degeneration is now regarded as an active, self-destructive program in which the ubiquitin proteosome pathway plays an important role. We have identified a novel E3 ubiquitin ligase, ZNRF1 that is induced in Schwann cells after nerve injury. ZNRF1 is also specifically upregulated in neurons, along with ubiquitin, after axonal damage. ZNRF1 and another related E3 ligase, ZNRF2, are both highly expressed throughout the nervous system during development and in adulthood. Using in vitro assays we have found that ZNRF protein activity is required for normal axonal degeneration. In this proposal, we have outlined experiments to define how ZNRF proteins influence axonal degeneration. First, we will analyze the phenotypes of ZNRF-deficient mice in order to determine physiological roles of these proteins. In particular, the will focus our analysis on the processes of nerve degeneration/regeneration after mechanical and metabolic injury as well as synaptogenesis. We also aim to identify proteins targeted by ZNRF-mediated ubiquitination. A proteomic analysis will be performed to identify ZNRF targets either from brain tissues of mice deficient for ZNRF proteins or from affinity-purified complex containing dominant negative ZNRF mutants and ZNRF targets. These studies will improve our understanding of axonopathy and neuronal degeneration, and provide a basis for developing novel treatments for neurodegenerative diseases