Pathomechanism and treatment of distal myopathy with rimmed vacuoles and GNE gene aberration

编码UDP-N-乙酰葡萄糖胺-2-表位酶/N-乙酰甘露糖胺激酶的GNE基因突变被认为在远端近视伴边缘空泡（DMRV）的发病中起重要作用。为了解DMRV的发病机制和治疗进展，我们对肌纤维动物边缘空泡性肌病中溶酶体的功能、GNE在哺乳动物组织细胞中的定位和功能、GNE突变体细胞和GNE敲低细胞的制备进行了研究，免疫组化、Western blot和真实的时间RT-PCK研究表明，GNE在小鼠和人组织中广泛表达。在小鼠肌肉中，注射心脏毒素后，GNE在再生纤维，特别是在细胞核中，以及在退变纤维中均被诱导，提示GNE可能在肌纤维再生的细胞核中起特定的功能作用。氯喹处理的大鼠失神经肌肉被称为人DMRV的动物模型，观察到空泡的积累 ...更多信息 仅在氯喹处理的失神经肌肉中。我们发现，微管相关蛋白-1轻链-3（LC 3）蛋白和基因的增加与边缘空泡形成在失神经肌肉氯喹治疗大鼠，这表明在分子水平上的自噬增加。在这种肌病中，边缘空泡的异常积聚似乎不是由自噬体相关基因或GNE基因的抑制介导的。此外，我们证明，增加蛋白酶体，泛素连接酶，和泛素在去神经肌肉氯喹治疗的大鼠，并建议，泛素蛋白酶体蛋白水解途径以及溶酶体蛋白水解途径介导的实验性肌病肌纤维破坏。我们制备了GNE基因突变（C303 stop）的HEK 293细胞和GNE敲低的C2 C4肌管细胞。萎缩和死亡在突变型GNE细胞中比野生型细胞更常见，尽管既没有镶边空泡，也没有溶酶体衍生的空泡，如自溶酶体和自噬体。

Mutation in GNE gene, encoding UDP-N-acetyIglucosamine-2-epiraerase/N-acetylmannosamine kinase, are believed to play a causative role in the onset of distal myopatiiy with rimmed vacuoles (DMRV). Tb understand the pathomechanism and development of the therapy for DMRV, we studied on lysosomal function in the muscle fiber animal rimmed vacuolar myopathies, localization and function of GNE in mammalian tissues and cells, and preparation of mutant GNE cells and GNE knock-down cells.Our immunohistochemical, Western blot, and real time RT-PCK studies showed that GNE is ubiquitously expressed in both murine and human tissues. GNE was induced in regenerating fibers, especially in nuclei, as well as degenerating fibers of murine muscles after injection with cardiotoxin, suggesting that GNE may play a specific functional role in nuclei of regeneration of muscles fibers.The denervated muscle of chloroquine-treated rats is known as animal model of human DMRV Accumulation of vacuoles was observed … More only in chloroquine-treated denervated muscles. We found that microtubule associated protein-1 light chain-3 (LC3)protein and gene increased in association with rimmed vacuolar formation in denervated muscles from chloroquine-treated rata, suggesting an increase in autophagy at the molecular level. Abnormal accumulation of rimmed vacuoles in this myopathy does not appear to be mediated by inhibition of autophagosome-related genes or GNE gene. Furthermore, we demonstrated that increased proteasomes, ubiquitin ligases, and ubiquitin in denervated muscles of chloroquine-treated rats and suggest that the ubiquitin-proteasome proteolytic pathway as well as the lysosomal proteolytic pathway mediate muscle fiber destruction in experimental myopathy. IGF-1 treatment inhibited the overdevelopment of rimmed vacuolar in these muscles.We prepared the HEK293 cells with mutant GNE gene (C303stop) and GNE knocked-down C2C4 myotube cells. Both atrophy and death were more frequent in mutant GNE cells than in wild-type cells, despite of neither rimmed vacuoles nor lysosome-derived vacuoles such as autolysosomes and autophagosomes Less