Pathomechanism of distal myopathy with rimmed vacuoles and GNE gene aberration

为了了解远端肌病伴边缘空泡的发病机制和治疗进展，我们研究了溶酶体系统在多种人和动物边缘空泡肌病的肌纤维破坏中的作用，Gne在哺乳动物组织和细胞中的定位，以及突变的Gne细胞和GNE击倒细胞的制备。在孤立包涵体肌炎(IBM)中，表现为肌纤维中大量的环状空泡，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运输相关蛋白如甘露糖6-磷酸受体(M6PR)和clathrin，以及自噬相关基因Athg5和Atg12，Atg12与正常和疾病对照组相比，在IBM肌肉中均有表达。我们还用Northern印迹法检测了盐水和氯喹处理的大鼠失神经和神经支配比目鱼肌中溶酶体相关蛋白的蛋白和/或mRNA的表达水平。失神经的m…氯喹处理的大鼠更常被称为人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，生理盐水和氯喹处理的大鼠失神经比目鱼肌与对侧神经支配的比目鱼肌相比，其胞膜蛋白免疫染色和M6PR mRNA显著增加。这些数据表明，在各种边缘空泡肌病如IBM和氯喹诱导的肌病的骨骼肌中，新合成的溶酶体酶通过高尔基体的跨高尔基网络运输(一种内噬途径)以及自噬形成(一种自噬过程)被激活。在自噬小体与早期内吞体融合时或融合后，由于自溶酶体的异常形成或翻转，空泡可能随后积累。Western blotts显示，在HEK293等培养细胞以及各种哺乳动物组织和细胞的粗提物中检测到一条约79 kDa的单一蛋白条带。我们的免疫组织化学和免疫印迹显示在包括肌肉在内的所有组织和细胞中都有普遍表达。GNE在肝脏中表达水平较高，在肌肉中表达水平较低。免疫荧光检测显示GNE在胞浆和胞核呈强阳性颗粒反应，并用突变型GNE蛋白(V572L)制备了肌管细胞。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。较少

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