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Regulation of spinal neurons through proteolytic actions by serine proteases.

通过丝氨酸蛋白酶的蛋白水解作用调节脊髓神经元。

基本信息

批准号：
14370471
负责人：
MITSUI Shinichi
金额：
$ 9.22万
依托单位：
Kochi University (2003-2004)Kyoto Prefectural University of Medicine (2002)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

项目摘要

In order to understand physiological function of a serine protease, motopsin/neurotrypsin (PRSS12), which we previously isolated from brain cDNA, mice lacking motopsin gene were prepared. We also characterized enzymatic property of recombinant motopsin and analyzed the localization of motopsin in the central nervous system. After mutation of motopsin gene has been reported to cause mental retardation just on starting this project, we focused motopsin in the cerebral cortex.1)Preparation of motopsin targeting miceThe first exon of mouse motopsin gene was replaced to pGKneo cassette. Gene targeting was confirmed by Southern hybridization, Northern hybridization and immunohistochemistry using anti-motopsin IgG. The mutant mice showed no deficit on motor on beam walking and foot print tests. Light-dark box test and elevated plus maze showed tendency that mutant mice had less anxiety than wild type. Interestingly, dendritic spine was decreased at pyramidal neurons of cingulate cortex and hi … More ppocampal CA1 region, at which motopsin was preferentially expressed. Our results suggest that motopsin have significant function for the development of emotion rather than motor behavior.2)Characterization of motopsinWe found that motopsin activates tissue plasminogen activator (WA) in vitro last year. The co-localization of motopsin and tPA. When motopsin and tPA were co-expressed in HEK293 cells, both proteases were localized in secretary vesides. Immunohistochemical technique dearly showed oo-localiation of motopsin and tPA at pyramidal neurons of cingulate cortex and hippcampus at postnatal day 10. Further, 12 candidate genes which interact with motopsin were isolated by yeast two hybrid system. Among them, mRNAs for a few genes were detected at motopsin-expressing cells.3)Regulation of the expression of spinesinFour types of spinesin isoforms were generated by alternative splicing. Type 1 and 2 lack the transmembrane domain, whereas type 3 and 4 are type II transmembrane proteins. Interestingly, only type 3 and 4 mRNAs were detected in spinal cord although type 1 and 2 mRNAs were expressed in various tissues including the CNS. Less

为了了解从脑cDNA中分离到的丝氨酸蛋白酶motopsin/neurotrypsin （PRSS12）的生理功能，我们制备了缺乏motopsin基因的小鼠。我们还对重组motopsin的酶学性质进行了表征，并分析了motopsin在中枢神经系统中的定位。motopsin基因突变引起智力迟钝的报道刚开始这个项目，我们就把注意力集中在大脑皮层。1)小鼠motopsin的制备将小鼠motopsin基因的第一个外显子替换为pGKneo卡带。经Southern杂交、Northern杂交和抗motopsin IgG免疫组化证实基因靶向。突变小鼠在梁行走和足印测试中没有表现出运动缺陷。光暗箱试验和升高加迷宫试验显示突变型小鼠的焦虑倾向低于野生型。有趣的是，树突棘在扣带皮层锥体神经元和hi . More海马CA1区，其中motopsin优先表达。我们的研究结果表明，运动蛋白对情绪的发展有重要的作用，而不是运动行为的发展。我们去年在体外实验中发现motopsin可以激活组织型纤溶酶原激活物（tissue plasminogen activator， WA）。胞浆酶和tPA的共定位。当motopsin和tPA在HEK293细胞中共表达时，这两种蛋白酶都定位于秘书侧。免疫组化技术显示，在出生后第10天，鼠扣带皮层和海马锥体神经元的motopsin和tPA明显定位。利用酵母双杂交系统分离到12个与胞浆酶相互作用的候选基因。其中，在表达motopsin的细胞中检测到少数基因的mrna。3)脊柱的表达调控通过选择性剪接产生了四种类型的脊柱蛋白同工型。1型和2型缺乏跨膜结构域，而3型和4型是II型跨膜蛋白。有趣的是，尽管1型和2型mrna在包括中枢神经系统在内的各种组织中表达，但在脊髓中仅检测到3型和4型mrna。少