Expression of lysosomal-related proteins and their gene in the skeletal muscles of distal myopathy with rimmed vacuoles

研究者推测，远端肌病变伴边缘空泡（DMRV）的退行性过程主要涉及溶酶体系统。为了研究DMRV感染肌肉中与细胞内溶酶体蛋白水解途径相关的可能的蛋白质或基因异常，我们对包括DMRV在内的各种神经肌肉疾病患者的肌肉活检标本中的某些蛋白质进行了免疫组织化学分析。与正常和患病对照相比，DMRV肌肉中网格蛋白、适应蛋白（AP-1和AP-2）、高尔基带蛋白异常升高。这些蛋白常在液泡内或液泡边缘以及液泡化纤维、萎缩纤维和坏死纤维的细胞质中被观察到。运动蛋白（一种内质网结合蛋白）在边缘液泡的边缘呈阴性，然而，在dmrv感染的纤维的液泡内，这种蛋白有时呈阳性染色。这些结果表明，在DMRV中，质膜上有更多的胞吞作用以及涉及从高尔基体的反式高尔基网络运输的分泌。各种溶酶体相关蛋白在边缘液泡内的积累表明这些液泡中至少有一些可能是自溶酶体。与正常和患病对照相比，DMRV肌肉中网格蛋白、适应蛋白（AP-1和AP-2）、高尔基带蛋白异常升高。这些蛋白常在液泡内或液泡边缘以及液泡化纤维、萎缩纤维和坏死纤维的细胞质中被观察到。边缘液泡的边缘是阴性的运动蛋白，一种内质网结合蛋白。然而，这种蛋白的阳性染色有时在dmrv感染纤维的液泡内可见。这些结果表明，在DMRV中，质膜上的内吞作用以及涉及从高尔基体的反式高尔基网络运输的分泌增加。各种溶酶体相关蛋白在边缘液泡内的积累表明这些液泡中至少有一些可能是自溶酶体。我们还使用Northern blot方法测量了生理盐水和氯喹处理大鼠去神经和神经支配比目鱼肌中溶酶体相关蛋白的mRNA水平。使用氯喹治疗的大鼠的失神经肌肉被认为是人类DMRV的动物模型。这些蛋白mRNA水平，尤其是甘露糖-6-磷酸受体mRNA，在氯喹处理的大鼠去神经肌肉中，与实验大鼠的神经肌肉和盐水处理的大鼠去神经肌肉相比，显著升高。这些水平在第4天高于第1、2和8天。在去神经支配后，仅在氯喹处理的肌肉中，特别是在第8天，有边缘空泡样自噬空泡的积累。这一结果表明，溶酶体系统，源自内质网的自噬液泡形成和来自反式高尔基网络的早期内体（原发性溶酶体）形成，在具有边缘液泡或自噬液泡的肌病中被显著激活，如DMRV。少

Investigators have speculated that the degenerative process in distal myopathy with rimmed vacuoles (DMRV) mainly involves the lysosomal system. To investigate possible protein or gene abnormalities related to intracellular lysosomal proteolytic pathways in DMRV-affected muscles, we performed immunohistochemical analyses of certain proteins in muscle biopsy specimens obtained from patients with various neuromuscular diseases including DMRV.Abnormal increases in clathrin, adaptin (AP-1 and AP-2), Golgi zone protein were demonstrated in DMRV muscles as compared with normal and diseased controls. These proteins were often observed inside or on the rims of vacuoles and in the cytoplasm of vacuolated fibers as well as atrophic fibers and necrotic fibers. The rims of rimmed vacuoles were negative for kinectin, an endoplasmic reticulum-binding protein, Positive staining for this protein, however, was sometimes seen inside the vacuoles in DMRV-affected fibers. These results suggest increased e … More ndocytosis at the plasma membrane as well as secretion involving transport from the trans-Golgi network of the Golgi apparatus in DMRV.Accumulation of various lysosome-related proteins within the rimnmed vacuoles indicates at least some of these vacuoles may be autolysosomes.Abnormal increases in clathrin, adaptin (AP-1 and AP-2), Golgi zone protein were demonstrated in DMRV muscles as compared with normal and diseased controls. These proteins were often observed inside or on the rims of vacuoles and in the cytoplasm of vacuolated fibers as well as atrophic fibers and necrotic fibers. The rims of rimmed vacuoles were negative for kinectin, an endoplasmic reticulum-binding protein. Positive staining for this protein, however, was sometimes seen inside the vacuoles in DMRV-affected fibers. These results suggest increased endocytosis at the plasma membrane as well as secretion involving transport from the trans-Golgi network of the Golgi apparatus in DMRV.Accumulation of various lysosome-related proteins within the rimmed vacuoles indicates at least some of these vacuoles may be autolysosomes.We also measured the levels of mRNA for lysosome-related proteins in the denervated and innervated soleus muscles from saline- and chloroquine-treated rats using the Northern blot. The denervated muscle of chloroquine-treated rats is known as an animal model of human DMRV.These protein mRNA levels, especially mannose-6-phospate receptor mRNA, were significantly elevated in denervated muscles from chloroquine-treated rats as compared with innervated muscles of both experimental rats and denervated muscle from saline-treated rats. These levels were higher on day 4 than on days 1,2, and 8. The accumulation of rimmed vacuoles-like autophagic vacuoles was marked only in chloroquine-treated muscles after denervation, especially on day 8. This result suggests that lysosomal system, autophagic vacuole formation derived from endoplasmic reticulum and early endosome (primary lysosome) formation from trans-Golgi network, is markedly activated in the myopathies with rimmed vacuoles or autophagic vacuoles such as DMRV. Less