Pathomechanism of distal myopathy with rimmed vacuoles and GNE gene aberration

为了解边缘空泡型远端肌病（DMRV）的发病机制和治疗进展，我们研究了溶酶体系统在各种人和动物边缘空泡型肌病肌纤维破坏中的作用，GNE在哺乳动物组织和细胞中的定位，以及GNE突变细胞和GNE敲除细胞的制备。在肌纤维中显示出大量有边空泡，溶酶体相关蛋白和基因异常增加，特别是受体介导的细胞内转运相关蛋白如甘露糖6-磷酸受体（M6 PR）和网格蛋白，以及自噬相关基因如hAtg 5和hAtg 12，与正常和患病对照相比，在IBM肌肉中得到证实。我们还测量了蛋白质和/或溶酶体相关蛋白的mRNA的表达水平在失神经支配和神经支配的比目鱼肌从盐水和氯喹处理的大鼠使用北方印迹。失去神经的M ...更多信息 氯喹处理的大鼠的肌肉被称为人DMRV的动物模型。仅在氯喹处理的失神经肌肉中观察到空泡的积累。此外，网格蛋白免疫染色和M6 PR mRNA显着增加，在失神经比目鱼肌从生理盐水和氯喹治疗的大鼠相比，对侧，神经支配的肌肉。这些数据表明，运输新合成的溶酶体酶从分泌途径通过高尔基体的trans-Golgi网络（内体途径）以及自噬体形成（自噬过程）被激活的各种镶边空泡性肌病，如IBM和氯喹诱导的肌病的骨骼肌。自噬体与早期核内体融合时或融合后，自溶酶体的异常形成或转换可导致胞内醇的积累。蛋白质印迹，在培养细胞如HEK 293和各种哺乳动物组织和细胞的粗提物中检测到约79 kDa的单一蛋白质条带。我们的免疫组织化学和Western印迹显示，在所有组织和细胞，包括肌肉中普遍表达。GNE在肝脏中的表达量较高，而在肌肉中的表达量较低。免疫荧光检测显示GNE在细胞质和细胞核中呈强阳性颗粒反应。我们正在研究突变型GNE细胞和野生型细胞在病理和功能上的差异。少

To understand the pathomechanism and development of the therapy for distal myopathy with rimmed vacuoles (DMRV), we studied on the role of lysosomal system in the muscle fiber destruction of various human and animal rimmed vacuolar myopathies, localization of GNE in mammalian tissues and cells, and preparation of mutant GNE cells and GNE knock-down cells.In solitary inclusion body myositis (IBM), showing numerous rimmed vacuoles in muscle fibers, abnormal increases in the lysosome- related proteins and genes, especially receptor mediated intracellular transport-related proteins such as mannose 6-phosphate receptor (M6PR) and clathrin, and autophagy-related genes such as hAtg5 and hAtg12, were demonstrated in IBM muscles as compared with normal and diseased controls. We also measured the expression levels of protein and/or mRNA for lysosome-related proteins in the denervated and innervated soleus muscles from saline- and chloroquine-treated rats using the Northern blot. The denervated m … More uscle of chloroquine-treated rats is known as animal model of human DMRV. Accumulation of vacuoles was observed only in chloroquine-treated denervated muscles. Further, clathrin immunostaining and M6PR mRNA were significantly increased in denervated soleus muscle from saline and chloroquine-treated rats compared to contralateral, innervated muscles. These data suggest that the transport of newly synthesized lysosomal enzymes from the secretory pathway via the trans-Golgi network of the Golgi apparatus (an endosomal pathway) as well as autophagosome formation (an autophagic process) were activated in the skeletal muscles of various rimmed vacuolar myopathy such as IBM and chloroquine-induced myopathy. Vacuoles may subsequently accumulate secondary to abnormal formation or turnover of autolysosomes at or after fusion of autophagosomes with early endosomes.Two polyclonal UDP-N-acetylglucosamine-2-epimerase/ N-acetylmannosamine kinase (GNE) antisera were raised in rabbit against recombinant GNE proteins. Western blots, a single protein band of about 79 kDa is detected in crude extracts of culture cells such as HEK293, and various mammalian tissues and cells. Our immunohistochemistry and Western blots demonstrated ubiquitously expression in all tissues and cells including muscle. The expression level of GNE was high in liver, whereas it was low in muscles. The immunofluorescence study showed strongly positive granular reaction for GNE in the cytoplasm and nucleus.Furthermore, we prepared the myotube cells with mutant GNE protein (V572L). We are studying the difference in the pathology and function between both mutant GNE cell and wild-type cell. Less