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

编码UDP-N-acetyIglucosamine-2-epiraerase/N-acetylmannosamine激酶的GNE基因突变被认为在远端伴有边缘空泡的近视眼的发病中起着重要作用。为了了解DMRV的发病机制和治疗进展，我们研究了GNE在肌纤维动物空泡性肌病中的溶酶体功能，GNE在哺乳动物组织和细胞中的定位和功能，以及突变的GNE细胞和GNE敲除细胞的制备。我们的免疫组织化学、Western印迹和实时RT-PCK研究表明，GNE在小鼠和人类组织中普遍表达。小鼠肌肉注射心脏毒素后，在再生纤维尤其是细胞核以及变性纤维中均可诱导出GNE，提示GNE可能在肌肉纤维再生的细胞核中起着特定的功能作用。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的是在氯喹处理的失神经肌肉中。我们发现氯喹处理的大鼠失神经肌肉中微管相关蛋白-1轻链-3(Lc3)蛋白和基因表达增加，并伴随着边缘空泡的形成，提示在分子水平上自噬增加。这种肌病中边缘空泡的异常堆积似乎不是由自噬相关基因或GNE基因的抑制所介导的。此外，我们还证实了氯喹处理的大鼠失神经肌肉中蛋白酶体、泛素连接酶和泛素的增加，并提示泛素-蛋白酶体蛋白分解途径以及溶酶体蛋白分解途径介导了实验性肌病的肌肉纤维破坏。IGF-1处理抑制了这些肌肉中有边框的空泡的过度发育。我们制备了突变的GNE基因(C303STOP)的HEK293细胞和GNE基因敲除的C2C4肌管细胞。突变的gne细胞的萎缩和死亡都比野生型细胞更常见，尽管边缘空泡和溶酶体衍生的空泡(如自溶酶体和自噬小体)都较少。

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