The role of carboxypeptidase A6 in development

描述（由申请人提供）：在发育期间和整个成年生活中，某些神经元群体正在增殖，迁移到它们的作用部位，并向适当的目标延伸轴突。这些过程受到许多翻译后修饰的调节，包括蛋白质水解。这些过程的缺陷已被发现导致神经系统疾病，如精神分裂症和卡尔曼综合征。一种蛋白酶，羧基肽酶A6 (CPA6)，参与c端疏水氨基酸的裂解，在脑神经发育和轴突向目标肌肉的引导机制中有一定的作用。具体来说，一名CPA6基因突变的患者被诊断为Duane综合征，这是一种外展神经支配外直肌的缺陷。CPA6在发育期间的眼外侧直肌和成人嗅球中表达。这种在成人嗅球中的限制性表达进一步暗示了CPA6在神经元迁移中的作用，因为嗅球是成人神经元迁移的少数位置之一。本项目的目的是研究CPA6在大脑发育过程中调节神经元迁移和轴突引导中的作用。首先，将评估纯化的CPA6的酶学特性，并通过已建立的肽组学技术鉴定CPA6的底物。CPA6的分布将在斑马鱼的整个发育过程中使用原位杂交技术进行详细表征，而CPA6在小鼠中的分布将由合作者进行研究。最后，我们将利用斑马鱼系统来评估CPA6在体内的作用。CPA6的特异性抑制剂以及morpholinos介导的敲低和基于质粒的过表达，随后进行显微镜和生理分析，将用于评估CPA6在体内神经元迁移和轴突引导中的作用。本研究将增加我们对发育过程中神经元引导机制的翻译后调控的理解。它还将揭示导致杜安综合征的分子缺陷，这种综合征影响多达0.1%的人口。

DESCRIPTION (provided by applicant): During development and throughout adult life certain populations of neurons are proliferating, migrating to their sites of action, and extending axons towards appropriate targets. These processes are regulated by many post-translational modifications, including proteolysis. Defects in these processes have been found to result in neurological disorders such as schizophrenia and Kallmann syndrome. A protease, carboxypeptidase A6 (CPA6), involved in cleavage of C-terminal hydrophobic amino acids, has been implicated in the mechanisms of cranial nerve development and axon guidance towards its target muscle. Specifically, a patient with a mutation disrupting CPA6 has been diagnosed with Duane syndrome, a defect in the innervation of the lateral rectus eye muscle by the abducens nerve. CPA6 is expressed in the lateral rectus eye muscle during development as well as in the adult olfactory bulb. This restricted expression in the adult olfactory bulb further implicates CPA6 in neuronal migration, as the olfactory bulb is one of the few locations of adult neuronal migration. The goal of this project is to investigate the role of CPA6 in regulating neuronal migration and axon guidance in the developing brain. First, the enzymatic properties of purified CPA6 will be assessed and substrates of CPA6 will be identified through established peptidomics techniques. The distribution of CPA6 will be characterized in detail throughout the development of the zebrafish using in situ hybridization, while the distribution of CPA6 in the mouse will be pursued by a collaborator. Finally, the genetically and microscopically tractable zebrafish system will be employed to assess the in vivo role of CPA6. Specific inhibitors as well as morpholino-mediated knockdown and plasmid- based overexpression of CPA6, followed by microscopic and physiological analysis, will be used to assess the role of CPA6 in neuronal migration and axonal guidance in vivo. This research will increase our understanding of post-translational regulation of neuronal guidance mechanisms during development. It will also shed light on the molecular defects leading to Duane syndrome, a syndrome affecting as much as 0.1% of the population.