Cdh1-APC Regulation of Axonal Growth

描述（由申请人提供）：本研究的长期目标是阐明哺乳动物大脑轴突生长和再生的调节机制。我们最近发现，泛素连接酶，即后期促进复合体（Cdh1-APC），在哺乳动物小脑轴突生长和模式的控制中起着关键作用。RNAi敲低Cdh1可显著促进原代大鼠小脑颗粒神经元轴突生长。在小脑切片覆盖实验和出生后大鼠小脑体内敲除实验中，我们发现Cdh1细胞自主控制颗粒神经元轴突的层特异性生长和平行纤维模式。在其他实验中，发现Cdh1敲低显著超越髓鞘对轴突生长的抑制。因此，Cdh1-APC也可能导致哺乳动物中枢神经系统（CNS）中受损神经元无法延伸轴突。我们的发现提出了几个基本问题。神经元中如何调节Cdh1-APC功能？Cdh1-APC控制轴突生长的机制是什么？Cdh1-APC如何参与髓磷脂抑制因子对轴突生长的负面影响？为了解决这些问题，我们提出以下具体目标：(1)表征神经元中Cdh1-APC功能的调节机制。我们将对Cdh1进行结构/功能分析，并表征Cdh1磷酸化和Cdh1相互作用蛋白在Cdh1- apc控制轴突生长中的作用。(2)确定Cdh1-APC控制轴突生长的机制。我们将确定调节轴突生长的Cdh1-APC底物。(3)表征Cdh1-APC在限制轴突生长中的细胞内在作用。我们将描述Cdh1-APC在哺乳动物小脑以外的CNS神经元中的活性和作用，并确定Cdh1-APC如何促进髓鞘抑制轴突生长。这项研究代表了一组重要的实验，它应该解决我们对控制哺乳动物大脑轴突生长的细胞内在机制的理解中的一个主要空白。此外，拟议的研究应该为开发可能最终用于刺激损伤和疾病后轴突再生的药物提供基础。

DESCRIPTION (provided by applicant): The long-term goals of the proposed research are to elucidate the mechanisms regulating axonal growth and regeneration in the mammalian brain. We recently discovered that the ubiquitin ligase, the anaphase promoting complex (Cdh1-APC), plays a critical role in the control of axonal growth and patterning in the mammalian cerebellum. Cdh1 knockdown by RNAi in primary rat cerebellar granule neurons robustly promoted axonal growth. In cerebellar slice overlay assays and by in vivo knockdown in the postnatal rat cerebellum, we found that Cdh1 cell-autonomously controls the layer-specific growth of granule neuron axons and parallel fiber patterning. In other experiments, Cdh1 knockdown was found to remarkably override myelin-inhibition of axonal growth. Thus, Cdh1-APC may also contribute to the inability of injured neurons to extend axons in the mammalian central nervous system (CNS). Our findings have raised several fundamental questions. How is Cdh1-APC function regulated in neurons? What are the mechanisms by which Cdh1-APC controls axonal growth? How does Cdh1-APC contribute to the negative influence of myelin inhibitory factors on axonal growth? To address these questions, we propose the following specific aims: (1) characterize mechanisms regulating Cdh1-APC function in neurons. We will perform structure/function analyses of Cdh1 and characterize the role of Cdh1 phosphorylation and Cdh1-interacting proteins in Cdh1-APC's control of axonal growth. (2) Determine the mechanism by which Cdh1-APC controls axonal growth. We will identify the substrates of Cdh1-APC that regulate axonal growth. (3) Characterize cell intrinsic role of Cdh1-APC in limiting axonal growth. We will characterize the activity and role of Cdh1-APC in mammalian CNS neurons beyond the cerebellum, and determine how Cdh1-APC contributes to the myelin-inhibition of axonal growth. The proposed research represents an important set of experiments that should address a major gap in our understanding of the cell-intrinsic mechanisms controlling axonal growth in the mammalian brain. In addition, the proposed research should provide the foundation for the development of drugs that might ultimately be used to stimulate axonal regeneration following injury and disease.