Pathomechanism of distal myopathy with rimmed vacuoles and GNE gene aberration

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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