Studies on the tau lysosomal proteolytic pathway

Tau蛋白是阿尔茨海默病（AD）大脑成对螺旋丝（PHF）的主要成分。我们的研究结果表明，tau蛋白在溶酶体或其功能等同物中被降解。为了确定tau蛋白在溶酶体中的降解途径，我们研究了tau蛋白与溶酶体膜的结合。Tau与溶酶体结合的解离常数为8 × 10^<-7> m .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分离大鼠脑溶酶体膜，转移到硝化纤维素膜上，与tau蛋白孵育。我们发现分子量为130,100和76 kDa的蛋白特异性结合tau。将主要溶酶体蛋白的c端表达为GST融合蛋白，并利用BIAcore系统检测其与tau蛋白的结合。我们发现LAMP-1和LAMP-2结合tau蛋白。另一方面，我们利用tau亲和层析鉴定了溶酶体片段中的谷氨酸脱氢酶可能是一个更多的au结合蛋白。这些结果强烈提示这些蛋白是溶酶体上tau受体的候选蛋白。接下来，我们在CHO和神经-2a细胞中过表达tau结合蛋白，并使用^<35> s -蛋氨酸脉冲追踪标记研究tau的周转。我们发现细胞中的tau水平在3天内没有很大的变化。此外，即使在过度表达这些受体蛋白后，细胞中的tau水平也不受影响。这些结果提示了tau在细胞中抵抗蛋白水解的可能性，以及在培养细胞中研究tau溶酶体蛋白水解途径的困难。在FTDP-17患者家族中发现错义突变。这清楚地表明tau和/或其蛋白水解产物导致神经元细胞死亡。为了研究突变对tau蛋白水解途径的影响，我们在小鼠tau启动子的转录控制下产生了表达野生型或突变型人类tau的敲入小鼠系。在敲入小鼠的大脑中，人tau mRNA和蛋白的水平与内源性小鼠tau相似。我们将使用该小鼠模型研究tau溶酶体蛋白水解途径。少

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