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

研究人员推测，伴有边缘空泡的远端肌病(DMRV)的退变过程主要涉及溶酶体系统。为了研究DMRV感染肌肉中可能存在的与细胞内溶酶体蛋白分解途径有关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，发现DMRV肌肉中的网状蛋白、适配素(AP-1和AP-2)、高尔基带蛋白与正常对照组和疾病对照组相比异常增加。这些蛋白常见于空泡内或空泡边缘、空泡纤维的细胞质以及萎缩纤维和坏死纤维中。有边框的空泡的边缘不表达内质网结合蛋白Kinectin，但在受DMRV影响的纤维中，有时在空泡内可见该蛋白的阳性染色。这些结果表明E-…升高。在DMRV中，质膜上有更多的核细胞以及涉及高尔基体跨高尔基网络运输的分泌物。在有边框的空泡中，各种溶酶体相关蛋白的积累表明，至少有一些这些空泡可能是自溶体。与正常和疾病对照相比，DMRV肌肉中的clathrin、Adaptin(AP-1和AP-2)和高尔基带蛋白异常增加。这些蛋白常见于空泡内或空泡边缘、空泡纤维的细胞质以及萎缩纤维和坏死纤维中。有边框的空泡边缘的内质网结合蛋白--激动素呈阴性。然而，这种蛋白的阳性染色有时出现在受DMRV影响的纤维的液泡内。这些结果表明，在DMRV中，质膜上的内吞作用以及涉及高尔基体跨高尔基网络运输的分泌增加。在有边框的空泡中积累各种溶酶体相关蛋白表明，这些空泡中至少有一些可能是自溶体。我们还用Northern印迹法检测了去神经和神经支配的比目鱼肌中溶酶体相关蛋白的mRNA水平。氯喹处理的大鼠失神经肌肉是人类DMRV的动物模型，与实验大鼠的失神经肌肉和生理盐水处理的失神经肌肉相比，氯喹处理大鼠失神经肌肉中这些蛋白的mRNA水平，特别是甘露糖-6-磷酸受体mRNA的水平显著升高。在去神经支配后，仅氯喹处理组肌肉中有明显的边缘空泡样自噬空泡的积累，尤以第8天更为明显。这一结果表明，在有边缘空泡或自噬空泡的肌病患者中，溶酶体系统、来自内质网的自噬空泡的形成和来自反高尔基网络的早期内体(初级溶酶体)的形成明显激活。较少

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