The Role Of Glycophagy In The Progression And Pathological Mechanism Of Late Onset Pompe Disease(LOPD)

Background:Pompe disease,GSD?(glycogen storage disease type ?),or acid ?-glucosidase deficiency(OMIM database number 232300)is a metabolic myopathy,produced by mutations in the GAA gene and inheriting in an autosomal recessive manner.Pompe disease was included in<China's first catalogue of rare diseases>in 2018.Although the root genetic basis of Pompe disease is known,the broader pathophysiology and metabolic consequences and effective therapy remain incompletely understood for broader clinical symptoms.Excessive accumulation of glycogen in lysosome is an important pathological change of Pompe disease,and with the progression of the disease,it will further lead to secondary damage such as lysosomal dysfunction,autophagic build-up and other waterfall pathological changes.So reducing the accumulation of glycogen is important for alleviating the disease.The degradation pathway of glycogen through lysosomes is called "glycophagy",however,its physiological and pathological significance is unclear.What's more,glycophagy in LOPD patients of different disease stages have also been poorly studied.Therefore,it is of great significance to explore the role of glycophagy in the progression and pathogenesis of LOPD for finding potential targets to reduce the accumulation of lysosomal glycogen.Objective:Analyze the expression characteristics of glycophagy marker protein Stbdl in muscle tissue.Explore the role of glycophagy in progression and pathogenesis of LOPD disease combing with possible regulatory mechanismsMethod:In this retrospective case-control study,we collected 18 muscle samples from patients who were diagnosed as late onset Pompe disease(LOPD)by genetic test and enzymology test.LOPD were divided into three groups according to pathological severity,i.e.whether the affected muscle fibers were>50%and whether muscle fibers with large vacuoles or basophilic deposition were>50%.At the same time,3 patients with roughly normal muscle fibers were matched as the control group.HE staining was performed on all muscle specimens.4 cases of LOPD patients with different pathological degrees in HE staining were selected for Stbdl immunohistochemical staining and the differential expression of Stbd1 in muscle tissues at different pathological stages was quantitatively detected by Western-blot.9 cases of LOPD were selected to divide into two groups according to the pathological degree and course of disease,i.e.whether light or moderate-severe pathology,whether less than 5 years or more than 5 years.At the same time,4 cases of muscle fibers were matched as the control group.The quantitative analysis of Stbd1,autophagy flow protein,glycogen synthesis and catabolism enzyme and energy metabolism regulatory protein was performed by Western-blot.Results:1.Extensive and continuous "pathological spectrum" was observed in 18 LOPD muscle tissues.A total of 7 cases,called LOPD-1,showed light pathological changes with small vacuolar muscle fibers occasionally and no obvious abnormalities muscle fibers in HE staining.A total of 5 cases,called LOPD-2,showed moderate pathological changes with interphase distribution of small muscle fiber and large vacuolar muscle fiber or basophilic granule deposition muscle fiber.A total of 6 cases,called LOPD-3,showed several pathological changes with more diffuse distribution of large vacuolar muscle fibers or basophilic granule deposition,resulting in some muscle fibers degenerated and ruptured due to excessive accumulation.In fact,most LOPD can be roughly classified as having a positive correlation between the severity of the pathology and the course of the disease.Therefore,it can be roughly divided into E-LOPD(early LOPD,mild pathological changes,course<5 years)and L-LOPD(late LOPD,moderate and severe pathological changes,course 5 years).Pathological changes of 18 cases of 18 LOPD formed a continuous"pathological spectrum" in proportion to the type of muscle fibers involved and the type of vacuolar muscle fibers.2.Immunohistochemical and Western-blot results of Stbd1 showed positive Stbd1 expression in LOPD,especially in muscle fibers which were mainly vacuolar muscle fibers,and Stbd1 distributed around the vacuoles or basophilic granules.The expression of Stbdl in LOPD was significantly higher when the pathology was severe than in the control group(p<0.05),suggesting that glycophagy was enhanced in LOPD and the up-regulated level was related to the degree of pathological changes.3.Other Western blot results showed that autophagy flow LC3 ?/? expressed in L-LOPD group is significantly higher than control group and E-LOPD group(p<0.05).The expression of glycogen synthase GS in the L-LOPD group was significantly higher than that in the NC group(p<0.05),while there was no difference in the expression of glycogen cytoplasmic degrading enzyme GP and AGL among the three groups(p>0.05).Among the energy metabolism regulation protein,AMPK activation level(p-AMPK/AMPK)was significantly higher in the E-LOPD group than in the NC group(p<0.05),while AKT and mTORC1 activation levels showed no difference among the three groups(p>0.05).Conclusion:On the one hand,the expression level of Stbdl in LOPD muscle tissue was up-regulated especially in the late stage of the disease,which suggested that glycophagy must be involved in the pathological changes of Pompe disease.On the other hand,combined with its distribution characteristics and the expression of related proteins,it suggested that the up-regulation of glycophagy may be related to autophagy,glycogen synthesis and energy regulation mechanism.Reducing glycogen reserve(especially in the early stage of disease)may slow down the glycophagy,thus reducing glycogen accumulation.Significance:Early diagnosis and treatment is still the key to determine the progress and prognosis of patients for individual variation of glycophagy in different pathological processes.It is worth mentioning that guiding patients on a low-carbohydrate diet is likely to reduce the raw materials and reserves of glycogen in muscle tissue,maybe reduce glycogen accumulation by reducing glycophagy,which is a potential therapeutic target for Pompe disease.