Progranulin and zebrafish motor neuron development

背景技术背景：单个人颗粒蛋白前体基因编码携带多个颗粒蛋白结构域的生长因子，其在许多癌症中相对于正常组织上调。颗粒蛋白前体基因内的常染色体显性突变已被证明会导致额颞叶痴呆的早期发作形式。最近，颗粒蛋白前体已被证明是一种神经营养因子，并调节神经突起的生长和增强神经元的存活。 研究依据：尽管越来越多的赞赏颗粒蛋白前体的生物学特性，我们没有充分了解其生理功能和作用模式，特别是膜受体，共受体或可溶性结合蛋白尚未完全确定。在这个建议中概述的是一个程序，使用斑马鱼来表征颗粒蛋白前体介导其作用的方式：1）解剖其神经营养特性的机制，使用反义吗啉代基因敲低技术和2）通过筛选组织提取物与颗粒蛋白前体相互作用的蛋白质，以高亲和力和特异性识别候选受体和/或结合蛋白。 初步结果：我们已经表明，颗粒蛋白前体在小鼠脊髓内的运动神经元和小鼠胚胎脊髓神经元培养物中高度表达。在NSC-34神经元细胞系中颗粒蛋白前体的过表达刺激树突状结构的出现，并赋予在培养物中自主存活非常长时间的能力。分析斑马鱼初级运动神经元的发育和命运揭示了前颗粒蛋白-A表达的敲低导致发育轴突的过早终止和不适当的分支。一个假定的可溶性结合蛋白颗粒蛋白前体，C型凝集素家族的成员进行了鉴定。 未来的研究：我们将明确颗粒蛋白前体神经营养作用的机制。我们将确定与颗粒蛋白前体相互作用的蛋白质在介导其生物活性中的作用。

BACKGROUND: The single human progranulin gene encodes a growth factor bearing multiple granulin domains that is up-regulated in many cancers relative to normal tissues. Autosomal dominant mutations within the progranulin gene have been shown to cause an early onset form of frontotemporal dementia. Recently progranulin has been shown to be a neurotrophic factor and to regulate neurite outgrowth and enhance neuronal survival. RATIONALE FOR STUDY: Despite the increasing appreciation of the biological properties of progranulin, we do not have a full understanding of its physiological functions and mode of action; in particular a membrane receptor, co-receptor or soluble binding protein has yet to be fully defined. Outlined in this proposal is a program to use the zebrafish to characterize the manner in which progranulin mediates its actions by: 1) dissecting the mechanism underlying its neurotrophic properties using antisense morpholino gene knockdown technology and 2) identifying candidate receptors and/or binding proteins by screening tissue extracts for proteins that interact with progranulin with high affinity and specificity. PRELIMINARY RESULTS: We have shown that progranulin is highly expressed in motor neurons within the mouse spinal cord and cultures of mouse embryonic spinal cord neurons. Over-expression of progranulin in the NSC-34 neuronal cell line stimulates the appearance of dendritic structures and imparts the ability to survive autonomously for very long periods in culture. Analysing the development and fate of zebrafish primary motor neurons has revealed that knockdown of progranulin-A expression leads to premature termination and inappropriate branching of developing axons. A putative soluble binding protein for progranulin, a member of the C-type lectin family was identified. FUTURE STUDIES: We will define the mechanism underlying the neurotrophic actions of progranulin. We will determine the role that proteins that interact with progranulin play in mediating its biological activities.