Elucidation of molecular signaling mechanism of axonal guidance

阐明轴突引导的分子信号机制

基本信息

批准号：
15590247
负责人：
YANAGI Shigeru
金额：
$ 2.3万
依托单位：
Kobe University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15590247/
关键词：
CRAM CRMP-5 Semaphorin Axon Pruning Filopodia セマホリン

项目摘要

Collapsin response mediator proteins (CRMPs) have been implicated in signaling of axonal guidance, including semaphorins. I have previously identified a novel CRMP-associated protein, designated CRAM for CRMP-Associated Molecule that belongs to the unc-33 gene family. The deduced amino acid sequence reveals that the CRAM gene encodes a protein of 563 amino acids and shows 57 % identity with dihydropyrimidinase (DHPase) and 50-51 % identity with CRMPs. A phylogenetic tree analysis indicates that CRAM sequence shows the greatest similarity with DHPase and divergence from the four CRMPs, indicating that CRAM is more closely related to DHPase than to the four CRMPs. The expression of CRAM is remarkable for brain, and especially high in fetal and neonatal, but decreases to very low levels in adult brain. Thus, CRAM is a unique new member of this gene family and may play a role in axonal guidance signaling distinct from other four CRMPs. Furthermore I have recently reported that CRAM was associated with several cellular proteins including tyrosine kinase Fes/Fps and mitochondrial septin.In this study, I have examined the distribution and function of CRAM in developing neurons. Immunohistochemical analysis showed accumulation of CRAMin the filopodia of growth cones. Experiments using cytochalasin D indicated that filopodial localization of CRAM was independent of filamentous actin. Overexpression of CRAM in neuronal cells significantly promoted filopodial growth and led to the formation of supernumerary growth cones, which acquired resistance to semaphorin-3A stimulation. Finally, knockdown of CRAM by using RNA interference blocked filopodial formation and revealed an aberrant morphology of growth cones. I propose that CRAM regulates filopodial dynamics and growth cone development, thereby restricting the response of growth cone to repulsive guidance cues.

崩解蛋白反应介导蛋白 (CRMP) 与轴突引导信号传导有关，包括信号蛋白。我之前发现了一种新的 CRMP 相关蛋白，命名为 CRAM（CRMP 相关分子），属于 unc-33 基因家族。推导的氨基酸序列显示，CRAM基因编码563个氨基酸的蛋白质，与二氢嘧啶酶(DHPase)有57%的同一性，与CRMP有50-51%的同一性。系统发育树分析表明，CRAM 序列与 DHPase 具有最大的相似性，但与四个 CRMP 的差异最大，表明 CRAM 与 DHPase 的相关性比与四个 CRMP 的相关性更密切。 CRAM 在大脑中的表达非常显着，在胎儿和新生儿中尤其高，但在成人大脑中降低至非常低的水平。因此，CRAM 是该基因家族的独特新成员，可能在轴突引导信号传导中发挥与其他四种 CRMP 不同的作用。此外，我最近报道了 CRAM 与几种细胞蛋白相关，包括酪氨酸激酶 Fes/Fps 和线粒体隔膜。在这项研究中，我检查了 CRAM 在发育神经元中的分布和功能。免疫组织化学分析显示 CRAM 在生长锥的丝状伪足中积累。使用细胞松弛素 D 的实验表明，CRAM 的丝状定位不依赖于丝状肌动蛋白。神经元细胞中 CRAM 的过度表达显着促进丝状伪足生长，并导致多余生长锥的形成，从而获得对 semaphorin-3A 刺激的抵抗力。最后，使用 RNA 干扰敲低 CRAM 阻断了丝状伪足的形成，并揭示了生长锥的异常形态。我认为 CRAM 调节丝状伪足动力学和生长锥发育，从而限制生长锥对排斥性引导信号的反应。