Studies on the tau lysosomal proteolytic pathway

tau溶酶体蛋白水解途径的研究

• 批准号: 11680734
• 负责人: OYAMA Fumitaka
• 金额: $ 2.37万
• 依托单位: University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2000
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680734/
• 关键词: Alzheimer's disease; tau; lysosome; chloroquine myopathy; knockin mouse

Tau is the major component of paired helical filaments (PHF) in Alzheimer's disease (AD) brain. Our results suggested that tau is degraded in lysosomes or their functional equivalents. To confirm the tau degradation pathway in lysosome we studied the binding between tau and lysosome membrane. Tau bound the lysosomes with the dissociation constant or 8 x 10^<-7> M.This observations prompted us to search for a receptor for tau in rat brain lysosomes. Rat brain lysosomal membranes were separated by SDS polyacrylamide gel electrophoresis, transferred to nitrocellulose membrane, and incubated with tau. We found that the proteins with the molecular weight of 130,100, and 76 kDa specifically bound tau. The C-termtinal regions of major lysosomal proteins were expressed as fusion proteins with GST and tested its binding with tau using BIAcore system. We found that both LAMP-1 and LAMP-2 bound tau. On the other hand, we identified glutamate dehydrogenase in the lysosomal fraction as a possible t … More au-binding protein by use of tau affinity chromatography. These results strongly suggest that these proteins are candidate for tau receptors on the lysosomes. Next we overexpressed the tau binding protein in CHO and neuro-2a cells and studied the turnover of tau by pulse chase labelling using the ^<35>S-methionine. We found that the tau levels in the cells are not largely changed for 3 days. In addition, the levels of tau in the cells were unaffected even after overexpressing these receptor proteins. These results suggest the possibility that tau is resistant against proteolysis in the cells and the difficulty in studying on the tau lysosomal proteolytic pathway in the cultured cells.Missense mutations were identified in the family of FTDP-17 patients. This clearly shows that tau and/or its proteolytic products cause the neuronal cell death. To study the effect of the mutations on the tau proteolytic pathway, we have generated knockin mouse lines expressing wild-type or mutant human tau under transcriptional control of the mouse tau promoter. In the brains of knockin mice, the levels of human tau mRNA and protein were similar to those of endogenous mouse tau. We will study the tau lysosomal proteolytic pathway using this mouse model. Less

Tau是阿尔茨海默病(AD)脑内成对螺旋细丝(PHF)的主要成分。我们的结果表明，tau在溶酶体或其功能等价物中被降解。为了确定tau在溶酶体内的降解途径，我们研究了tau与溶酶体膜的结合。Tau以解离常数或8×10^-7&gt；M与溶酶体结合。这一观察结果促使我们在大鼠脑溶酶体中寻找tau的受体。用SDS-聚丙烯酰胺凝胶电泳法分离大鼠脑溶酶体膜，将其转移到硝酸纤维素膜上，与tau孵育。我们发现，相对分子质量分别为130、100和76 kDa的蛋白质可以与tau特异结合。将主要溶酶体蛋白的C末端区域表达为与GST的融合蛋白，并用Biacore系统检测其与tau的结合。我们发现LAMP-1和LAMP-2都与tau结合。另一方面，我们鉴定了溶酶体组分中的谷氨酸脱氢酶可能是t…。利用tau亲和层析获得更多的Au结合蛋白。这些结果有力地表明，这些蛋白是溶酶体上tau受体的候选蛋白。接下来，我们在CHO和Neuro-2a细胞中过表达了tau结合蛋白，并用脉冲追逐标记的方法研究了tau的周转。我们发现，细胞内tau的水平在3天内没有很大变化。此外，即使在过度表达这些受体蛋白后，细胞中tau的水平也没有受到影响。这些结果提示tau可能对细胞内的蛋白分解具有抵抗力，以及在培养细胞中研究tau溶酶体蛋白分解途径的难度。在FTDP-17患者家族中发现了错义突变。这清楚地表明，tau和/或其蛋白分解产物导致神经细胞死亡。为了研究突变对tau蛋白分解途径的影响，我们建立了在小鼠tau启动子的转录控制下表达野生型或突变型人tau的敲打小鼠系。在敲击小鼠的脑中，人tau的mRNA和蛋白水平与内源性小鼠的tau相似。我们将使用这个小鼠模型来研究tau溶酶体的蛋白分解途径。较少

项目成果

Oyama, F: "The tau lysosomal proteolytic pathway."Dementia Japan. 13. 35-43 (1999)

Oyama, F：“tau 溶酶体蛋白水解途径。”日本痴呆症。