The Role Of Autophagy And Apoptosis In The Pathogenesis Of Late-onset Pompe Disease

BackgroundPompe disease(Glycogen storage disease type?,GSDII)is a rare hereditary disease caused by genetic defects of GAA(acid alpha-glucosidase)which is a lysosomal enzyme responsible for the degradation of glycogen to glucose.Pompe disease can occur at any age,and is classified into infantile and late-onset forms based on onset age,organ involvement,and disease progression.For a long time the pathogenesis of GSDII is thought to be associated with the accumulation of glycogen in lysosomes leading to lysosome rupture eventually.But in recent years,with the development of the application of animal experiments and enzyme replacement therapy and the development of electron microscopy,the role of autophagy in the pathogenesis has been paid more and more attention.A large number of experiments have proved that the theory of autophagic aggregation and lysosomal swelling and rupture has a place in the pathogenesis of Pompe disease,but the two do not seem to happen simultaneously.Pompe disease is no longer simply considered a lysosomal glycogen storage disease.The accumulation of glycogen and autophagic vacuoles leads to muscle cell transport dysfunction,sustained autophagy aggregation and cytoskeletal structure abnormal,all of changes will enhance autophagy activity and affect the contraction of muscle fiber.Autophagy is proposed by Ashford and Porten as early as 1962,which is a highly conserved evolutionarily degradation process by lysosomes.Autophagy is a self protective mechanism of cells.But it has a perplexing relationship with apoptosis,necrosis and cell death.It is unclear how autophagy induced apoptosis.The main morphological characteristics of apoptosis which is also called type I programmed cell death is nuclear chromatin condensation.The relationship between autophagy and apoptosis is unclear.A large number of experiments confirmed that some common regulatory molecules exist in two pathways.Under certain conditions,autophagy can act as the upstream of apoptosis or trigger cell death.It has been proved that autophagy and apoptosis play an important role in aging muscle atrophy,but there is no report about the mechanism of in the pathogenesis of Pompe disease.In our study,we investigated the mechanism of autophagy and apoptosis in the pathogenesis of Pompe by immunohistochemistry and double immunofluorescence for the first time.Part ?:The pathological characteristics of autophagy and apoptosis in muscle tissue of late-onset Pompe patientsObjectiveTo study the pathological characteristics of LC3B,LAMP-2,VMP1,p62,Bcl-2,cathepsin B in muscle tissue of late-onset Pompe patients.MethodMuscle biopsies were taken from 10 late-onset Pompe patients and 3 controls.We summarized the clinical data and pathological characteristics in 10 Pompe patients.All the biopsy specimens were processed with hematoxylin-eosin,acid phosphatase staining and immunohistochemical staining for anti-LAMP-2,anti-p62,anti-LC3B,anti-VMP1,anti-Bcl-2 and anti-cathepsin B.Result1.Pathological changes of skeletal muscle in 10 patients with Pompe diseaseThe results showed that the size of muscle fiber was different,and there were a large number of vacuoles in it.The shape of vacuoles was irregular.The necrosis and regeneration fibers were seen occasionally,the proliferation of the endomysium was different,the disordered structure of the myofibrils and the glycogen content increased.Pathological manifestations of individual differences,even in the same skeletal muscle of one patient.2.Acid phosphatase stainingACP staining showed weak positive staining in obvious vacuolar region in HE.ACP staining showed strong positive in purple grain regions.ACP staining was weak in the area of incomplete vacuolization,this positive expression lied in the core area of muscle fibers.3.Immunohistochemical stainingThe expression of LAMP-2,VMP1 in 10 cases was strongly positive,but there was significant variability in the expression of p62,LC3B,Bcl-2 and cathepsin B.Generally the expression of p62,LC3B,Bcl-2 and cathepsin B were positive in non-vacuolated or incomplete vacuolated fibers and negative in vacuolated fibers.ConclusionLAMP-2,LC3B,p62,VMP1,Bcl-2,cathepsin B have different levels of expression and distribution in different course of Pompe,which is related to the degree of destruction of skeletal muscle fibers.It is suggested that autophagy and apoptosis may be involved in the destruction of Pompe muscle fiber structure and play different roles in different stages of development.Part ?:The role of autophagy and apoptosis in the pathogenesis of 1 ate-onset Pompe patientsObjectiveTo demonstrate the mechanism of autophagy and apoptosis in the pathogenesis of late-onset Pompe by detecting the expression of caspase-8 and the co-expression of LAMP-2 and cathepsin B,LC3B and VMPland p62,respectively.MethodMuscle specimens were obtained retrospectively from 10 Pompe patients and 6 controls,of whom,3 had LSM,and 3 non-myopathic patients.All the samples were stained with anti-LAMP-2,anti-LC3B,anti-caspase-8(p 18),anti-cathepsin B,anti-VMP1,anti-p62,and double immunofluorescence staining was used to detect the co-expression of them.ResultImmunofluorescence staining showed that LAMP-2 and p62 were strong expressed in vacuolated muscle fibers of Pompe and also had different degrees of expression in muscle of patients with lipid storage disease.The expression of LC3B,cathepsinB,VMP1 and caspase-8(p18)staining was weak or not expressed in the muscle fibers with obvious vacuolization.The non-vacuolated or only dot-like changed fibers showed obvious positive expression of these staining.Co-expression of VMP1 and p62,LAMP-2 and cathepsin B were found in the muscle fibers which had not yet been vacuolated completely,and the expression was mainly in the core region of muscle fibers.However,no obvious co-expression was found in the muscle fibers with obvious vacuolization.In contrast,muscle biopsies from 3 non-disease controls showed negative caspase-8(pl8),LAMP-2,cathepsin B,LC3B,VMP1 and p62 staining.Conclusion The apoptosis pathway mediated by caspase-8 may plays an important role in the pathophysiology of late-onset Pompe muscle tissue.VMP1 mediated selective autophagy pathway may play a protective role in late-onset Pompe muscle tissue.Autophagy and apoptosis related proteins may play different roles in different developmental stages.of late-onset Pompe disease.