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。据报道，包涵体肌炎（IBM）影响的肌肉中存在tau免疫反应性，phf样原纤维，最近在氯醌肌病（CM） （IBM的实验模型）中发现无定形tau沉积物。我们研究CM以深入了解AD大脑中PHF的形成。在CM中，tau mRNA水平在早期短暂上调，而tau自身在晚期缓慢积累。tau mRNA水平及其积累的时间谱与β -淀粉样蛋白前体（APP）及其羧基末端片段的时间谱非常相似，两者都已知在溶酶体中被降解。免疫细胞化学显示，随着酸性磷酸酶活性的增加，tau逐渐积聚在边缘液泡中，免疫电镜显示tau位于液泡内。这些结果表明，tau蛋白在溶酶体或其在肌肉中的功能等同物中被降解。为了证实tau蛋白在溶酶体中的降解途径，我们分析了鸡β -肌动蛋白启动子转录控制下过表达最短形式人类tau蛋白的转基因大鼠肝脏自噬溶酶体。我们发现，在经白细胞介素处理的肝脏的自噬溶酶体中存在tau蛋白，而在未经白细胞介素处理的肝脏中未检测到tau蛋白。这些结果表明tau蛋白也在肝脏溶酶体中被降解。肌肉和肝脏的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。我们发现分子量分别为130、100和76 kDa的蛋白特异性结合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mp50（谷氨酸脱氢酶）可能是tau结合蛋白。少

Tau is the major component of paired helical filaments (PHF) in Alzheimer's disease (AD) brain. The presence of tau-immunoreactive, PHF-like fibrils was reported in the muscle affected by inclusion body myositis (IBM), and amorphous tau deposits were recently found in chloroquine myopathy (CM), an experimental model for IBM.We investigated CM to obtain insight into PHF formation in AD brain. In CM tau mRNA level was transiently upregulated in the early phase, while tau itself slowly accumulated in the late phase. The temporal profiles of tau mRNA levels and its accumulation were very similar to those of beta-amyloid protein precursor (APP) and its carboxyl-terminal fragments, both of which have been known to be degraded in lysosomes. Immunocytochemistry showed that tau progressively accumulated in the rimmed vacuoles with increased acid phosphatase activities, and immunoelectron microscopy demonstrated that tau was located within the vacuoles. These results suggest that tau is degraded … More in lysosomes or their functional equivalents in the muscle.To confirm the tau degradation pathway in lysosome we analyzed the autophagolysosomes from liver of the transgenic rat overexpressing the shortest form of human tau under transcriptional control of the chicken beta-actin promoter. We found that tau was present in autophagolysosomes from leupeptin-treated liver, whereas it was not detected in those from leupeptin-untreated liver. These results suggest that tau is also degraded in lysosomes in liver. The observations in both muscle and liver 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. On the other hand, we identified MP5O (glutamate dehydrogenase) in the lysosomal fraction as a possible tau-binding protein by use of tau affinity chromatography. Less