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Study of Molecular Organization of Mouse Cerebellum

小鼠小脑分子组织的研究

基本信息

批准号：
13308047
负责人：
FURUICHI Teiichi
金额：
$ 29.7万
依托单位：
The Institute of Physical and Chemical Research (RIKEN)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

项目摘要

1.By the differential display, microarray, and GeneChip analysis approaches, we explored the gene expression profiles specific to the postnatal developmental stages of mouse cerebellum on a genome-wide basis. In addition, we revealed the spatio-temporal expression patterns of specific genes by RT-PCR and in situ hybridization methods. Integrating all these lines of gene expression information, we have generated the "Cerebellar Development Transcriptome database". By this study, we have succeeded in cloning of many candidates for novel genes responsible for brain development as follows.2.We showed that dendritic clustering and synaptic targeting of Cupidin/Homer, an adaptor protein at postsynaptic density (PSD), coincides those of glutamate receptor-related proteins NR2B and PSD-95 during development of hippocampal neurons, that Cupidin act s as a mobile adaptor in cerebellar granule cells in an activity-dependent manner, and that the Homer family members have differential cellular distributions in developing mouse brains.3.We cloned CAPS2, a paralog of CAPS that regulates Ca2+-dependent exocytosis of secretory granules. CAPS2 was localized to vesicular structures, in which neurotrophins BDNF and NT-3 are included, in parallel fiber terminals of cerebellar granule cells. The overexpression of exogenous CAPS2 in primary cultured granule cells augmented NT-3 release in a depolarization-dependent manner, resulting in promoting survival of Purkinje cells. These data indicate that CAPS2 is a molecule that regulates activity-dependent release of neurotrophins that are indispensable for differentiation and survival of cerebellar neurons.4.We analyzed the structure, function, and brain expression of three genes related to protein phsophorylation (apoptosis activity-related tyrosine kinase AATYK, novel Ser/Thr kinase Ebr, and tyrosine kinase adaptor Cas) and a gene encoded a novel myelin paranodal loop protein.

1.通过差异显示、微阵列和基因芯片分析方法，在全基因组基础上探索了小鼠小脑出生后发育阶段特异性基因表达谱。此外，我们还通过RT-PCR和原位杂交方法揭示了特定基因的时空表达模式。整合所有这些基因表达信息，我们已经产生了“小脑发育转录组数据库”。通过本研究，我们成功地克隆了许多与脑发育相关的新基因候选基因，具体如下：2.我们发现，在海马神经元发育过程中，突触后致密区（postsynaptic density，PSD）的衔接蛋白Cupidin/Homer与谷氨酸受体相关蛋白NR 2B和PSD-95的树突簇集和突触靶向作用是一致的，Cupidin在小脑颗粒细胞中以活性依赖的方式作为移动的适配器，Homer家族成员在发育中的小鼠脑中具有不同的细胞分布。CAPS 2定位于小脑颗粒细胞平行纤维终末的泡状结构，其中包括神经营养因子BDNF和NT-3。在原代培养的颗粒细胞中过表达外源性CAPS 2以去极化依赖的方式增加NT-3的释放，从而促进浦肯野细胞的存活。这些数据表明CAPS 2是一种调节神经营养因子活性依赖性释放的分子，这些神经营养因子对于小脑神经元的分化和存活是必不可少的。4.我们分析了与蛋白磷酸化相关的三个基因的结构、功能和脑表达（凋亡活性相关的酪氨酸激酶AATYK，新的Ser/Thr激酶Ebr，和酪氨酸激酶接头Cas）和编码新的髓磷脂结旁环蛋白的基因。