权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

Study of disfunctions of axons and abnormal phosphorylation of neurofilament in Alzheimer barins

阿尔茨海默病轴突功能障碍和神经丝异常磷酸化的研究

基本信息

批准号：
09670988
负责人：
NAKAMURA Yu
金额：
$ 2.18万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09670988/
关键词：
neurofilament Alzheimer PKA PKO phosphorylation A Kinase C Kinase

项目摘要

Ser-51 and Ser-55 are the phosphorylation site of PKC and PKA, respectively, in he N-terminal head region of 68 kDa neurofilament subunit protein (NF-L). Site-specific phosphorylation-dependent antibodies (abNFL51 and abNFL55) against those phosphorylation sites were prepared by using affinity-column. The in vitro specificity of those antibodies were appreciated by dot and western blot analyses. Thereafter, The in vivo specificity was studied in rat primary cultured neurons. A little amount of phosphorylated NF-L recognized by abNFL55 was found in them, and the amount was increased by okadaic acid treatment. However, forskolin did not increase the amount, while simultaneous treatment of forskolin and okadaic acid dramatically increased it. These findings suggest that a small part of NF-L is phosphorylated by PKA and that the amount of the phosphorylated NF-L is regulated by okadaic acid-sensitive phosphatase, implying that NF-L dynamics in neurons is regulated by kinases and phosphatases. On the other hand, no signal was found by abNFL51 in cultured neurons, indicating that NF-L is not phosphorylated by PKC in neurons.15 micrometer thick free-floating sections of Alzheimer and control brains were immunohistochemically studied by abNFL51 and abNFL55. abNFL51 stained neurofibrillary structures, and abNFL55 stained neuronal perikarya and neurites in both brains.

Ser-51 和 Ser-55 分别是 68 kDa 神经丝亚基蛋白 (NF-L) N 末端头部区域中 PKC 和 PKA 的磷酸化位点。使用亲和柱制备针对这些磷酸化位点的位点特异性磷酸化依赖性抗体（abNFL51和abNFL55）。通过斑点和蛋白质印迹分析评估了这些抗体的体外特异性。此后，在大鼠原代培养的神经元中研究了体内特异性。其中发现少量被abNFL55识别的磷酸化NF-L，并且经冈田酸处理后其量增加。然而，毛喉素并没有增加该量，而同时使用毛喉素和冈田酸则显着增加该量。这些发现表明，一小部分 NF-L 被 PKA 磷酸化，并且磷酸化 NF-L 的量受到冈田酸敏感磷酸酶的调节，这意味着神经元中 NF-L 的动力学受到激酶和磷酸酶的调节。另一方面，在培养的神经元中没有发现abNFL51的信号，这表明神经元中的NF-L不被PKC磷酸化。使用abNFL51和abNFL55对阿尔茨海默病和对照脑的15微米厚的自由漂浮切片进行免疫组织化学研究。 abNFL51 对神经原纤维结构进行染色，abNFL55 对两个大脑中的神经元周核和神经突进行染色。