Unfolded Protein Response And Endoplasmic Reticulum Stress In Hereditary Inclusion Body Myopathy

Objective:Hereditary inclusion body myopathy is a pathological term which was suggested by Askanas in 1993 for a group of hereditary muscle diseases characterized by intranuclear and cytoplasmic tubufilamentous inclusions. Many dominant and recessive hereditary inclusion body myopathies with variant clinical manifestations have been found recently, and their genes and proteinic nature have been illuminated gradually. The recessive types of hereditary inclusion body myopathy include GNE disease and oculopharyngo distal myopathy, and the dominant types include Welander distal myopathy , Tintin disease and Valosin disease. GNE disease is the prototype of hereditary inclusion body myopathy including VMSQ in Jewish of Middle East and DMRV in Japanese, characterized by young adult onset, frontier muscle weakness and atrophy in lower legs with quadriceps sparing. The disease is caused by the mutation of GNE gene which located in 9p13.2.UDP-N-acetylglucosamine-2-epimerase/ N-acetylmannosamine kinas is a dual functional limiting enzyme in the sialic acid synthesis pathway. The GNE mutation associated with hereditary inclusion body myopathy caused reduction of activity of both enzymes which induced sialic metabolic disorder and the degeneration of muscle fibres. The definite pathogenesy is still unknown. We chose 8 HIBM patients diagnosed by clinical and muscle pathologic manifestations to study the existence,distribution and nature of abnormal proteins and expression of chaperon proteins with immunofiuorescence and west blotting methods and to investigate the pathogenesy of HIBM.Material and MethodsWe examined a total of 8 HIBM patients who come from Institute of peripheral neuropathy and muscular diaeases of Qilu hospital of Shandong University diagnosed clinically and histochemically. There were 2 females and 6 males.As a control we selected 2 normal muscle specimens confirmed by pathologic profiles. The biopsied skeletal muscle specimens were flash-forzen in isopentane chilled with liquid nitrogen,and then were made into 4μm section. We used antibodies forβ-amyloid protein precursor(β-APP), phosphorylated tau protein (P-tau), glucose regulated protein(GRP94), exceptional reticulum protein72 (ERp72) and calreticulin. We extracted total protein from the muscle tissue of 6 patients and performed WB analysis.Results:There were various accumulation ofβ-APP and P-tau observed in all HIBM patients. Someβ-APP accumulation distributied in patching and grid shape in muscle fibres especially atrophic ones, the others were in round shape in normal size fibres. P-tau accumulation distributed in patching and grid shape in atrophic fibres. The accumulation of abnormal proteins were located besides rimmed vacuoles. Endomysium and perimysium were nonspecific stained. There was only nonspecific staining of endomysium and perimysium seen in muscle fibres of normal controls.GRP94 and calreticulin expressed in fibres of HIBM patients especially the atrophic ones. Double immunofluorescence revealed the physical interaction ofβ-APP and GRP94 which indicates GRP94 expressed in the location ofβ-APP accumulation. There were no GRP94 and calreticulin expressed in fibres of normal controlled specimens. The expression of ERp72 in HIBM was increased out of rimmed vacuoles and enhanced in the periphery of rimmed vacuoles. There were obvious differences between 2 types fibres in normal controlled specimens.For 8 specimens of HIBM patients, to extract the total proteins from the muscle and detect the density for the WB analysis of 5 proteins.β-APP WB analysis was performed in 3 HIBM patients. It was significantly higher expressed in all HIBM patients than in normal control. P-tau WB analysis was performed in 7 patients. It was significantly higher expressed in 5 HIBM patients than in normal control. Accrescence in 1 patient was slight and meaningless. It was decreasing in 1 patient. GRP94 WB analysis performed in 3 HIMB patients showed significantly higher expression than in normal control.ERp72 WB analysis performed in 4 patients showed significantly higer expression in all except one. Calreticulin WB analysis was performed in 4 patients. It was significantly higher expressed in 2 patients. And it was slight higher in other 2 patients. Conclusion:1 .The accumulation ofβ-APP and P-tau in atrophic muscle fibres suggest the reason for degeneration of muscle fibres may due to the accumulation of abnormal disfold or unfold proteins. The accumulation of proteins of round shape in normal size muscle fibre may be in the primary stage of accumulation.2.The strong immunostaing of GRP94, ERp72 and calreticulin may indicate the up-regulation of molecule chaperons and possible endoplasmic reticulum stress. The double IF showed GRP94 expressed in the place whereβ-APP accumulated manifest a physical correlation between them.3.We had used two kinds of immunologic skills in the research, IF and WB.We found that the patients who had expression ofβ-APP,P-tau,GRP94,ERp72 and calreticulin in IF would had the same results in WB. Both skills showed a high coincidence in the situation.