Autophagy and lysosomes in Pompe disease

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, intralysosomal glycogen accumulates in multiple tissues, with skeletal and cardiac muscle most severely affected.(1) Complete enzyme deficiency results in rapidly progressive infantile cardiomyopathy and skeletal muscle myopathy that is fatal within the first two years of life. Patients with partial enzyme deficiency suffer from skeletal muscle myopathy and experience shortened lifespan due to respiratory failure. The major advance has been the development of enzyme replacement therapy, which recently became available for Pompe patients. However, the effective clearance of skeletal muscle glycogen, as shown by both clinical and preclinical studies, has proven more difficult than anticipated.(2-4) Our recent work published in Annals of Neurology(5) was designed to cast light on the problem, and was an attempt to look beyond the lysosomes by analyzing the downstream events affected by the accumulation of undigested substrate in lysosomes. We have found that the cellular pathology in Pompe disease spreads to affect both endocytic (the route of the therapeutic enzyme) and autophagic (the route of glycogen) pathways, leading to excessive autophagic buildup in therapy-resistant skeletal muscle fibers of the knockout mice.     

Autophagy and Lysosomes in Pompe Disease

In Pompe disease, a deficiency of lysosomal acid alpha-glucosidase, intralysosomal glycogen accumulates in multiple tissues, with skeletal and cardiac muscle most severely affected.¹ Complete enzyme deficiency results in rapidly progressive infantile cardiomyopathy and skeletal muscle myopathy that is fatal within the first two years of life. Patients with partial enzyme deficiency suffer from skeletal muscle myopathy and experience shortened lifespan due to respiratory failure. The major advance has been the development of enzyme replacement therapy, which recently became available for Pompe patients. However, the effective clearance of skeletal muscle glycogen, as shown by both clinical and pre-clinical studies, has proven more difficult than anticipated.^2-4 The work published in Annals of Neurology⁵ was designed to cast light on the problem, and was an attempt to look beyond the lysosomes by analyzing the downstream events affected by the accumulation of undigested substrate in lysosomes. We have found thatthe cellular pathology in Pompe disease spreads to affect both endocytic (the route of the therapeutic enzyme) and autophagic (the route of glycogen) pathways, leading to excessive autophagic buildup in therapy-resistant skeletal muscle fibers of the knockout mice.

Addendum to:

Dysfunction of Endocytic and Autophagic Pathways in a Lysosomal Storage Disease

Tokiko Fukuda, Lindsay Ewan, Martina Bauer, Robert J. Mattaliano, Kristien Zaal,Evelyn Ralston, Paul H. Plotz and Nina Raben

Ann Neurol 2006; 59:700-8     

Eccentric exercise-induced muscle damage impairs muscle glycogen repletion

Five healthy untrained young male subjects were studied before, immediately after, and 10 days after a 45-min bout of eccentric exercise on a cycle ergometer (201 W). The subjects were sedentary at all other times and consumed a eucaloric meat-free diet. Needle biopsies of the vastus lateralis muscle were examined for intracellular damage and glycogen content. Immediately after exercise, muscle samples showed myofibrillar tearing and edema. At 10 days, there was myofibrillar necrosis, inflammatory cell infiltration, and no evidence of myofibrillar regeneration. Glycogen utilization during the exercise bout was 33 mmol glycosyl units/kg muscle, consistent with the metabolic intensity of 44% of maximal O2 uptake; however, the significant glycogen use by type II fibers contrasted with concentric exercise performed at this intensity. At 10 days after exercise, muscle glycogen was still depleted, in both type I and II fibers. It is possible that the alterations in muscle ultrastructures were related to the lack of repletion of muscle glycogen. Damage produced by eccentric exercise was more persistent than previously reported, indicating that more than 10 days may be necessary for recovery of muscle ultrastructure and carbohydrate reserves.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alpha-glucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.Key words: Pompe disease, lysosomal glycogen storage, myopathy, Atg7, enzyme replacement therapy     

Deconstructing Pompe disease by analyzing single muscle fibers: to see a world in a grain of sand..

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogenfilled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.     

Deconstructing Pompe Disease by Analyzing Single Muscle Fibers: “To See a World in a Grain of Sand…”

Autophagy is a major pathway for delivery of proteins and organelles to lysosomes where they are degraded and recycled. We have previously shown excessive autophagy in a mouse model of Pompe disease (glycogen storage disease type II), a devastating myopathy caused by a deficiency of the glycogen-degrading lysosomal enzyme, acid alpha-glucosidase. The autophagic buildup constituted a major pathological component in skeletal muscle and interfered with delivery of the therapeutic enzyme. To assess the role of autophagy in the pathogenesis of the human disease, we have analyzed vesicles of the lysosomal-degradative pathway in isolated single muscle fibers from Pompe patients. Human myofibers showed abundant autophagosome formation and areas of autophagic buildup of a wide range of sizes. In patients, as in the mouse model, the enormous autophagic buildup causes greater skeletal muscle damage than the enlarged, glycogen-filled lysosomes outside the autophagic regions. Clearing or preventing autophagic buildup seems, therefore, a necessary target of Pompe disease therapy.     

Suppression of autophagy permits successful enzyme replacement therapy in a lysosomal storage disorder—murine Pompe disease

Autophagy, an intracellular system for delivering portions of cytoplasm and damaged organelles to lysosomes for degradation/recycling, plays a role in many physiological processes and is disturbed in many diseases. We recently provided evidence for the role of autophagy in Pompe disease, a lysosomal storage disorder in which acid alphaglucosidase, the enzyme involved in the breakdown of glycogen, is deficient or absent. Clinically the disease manifests as a cardiac and skeletal muscle myopathy. The current enzyme replacement therapy (ERT) clears lysosomal glycogen effectively from the heart but less so from skeletal muscle. In our Pompe model, the poor muscle response to therapy is associated with the presence of pools of autophagic debris. To clear the fibers of the autophagic debris, we have generated a Pompe model in which an autophagy gene, Atg7, is inactivated in muscle. Suppression of autophagy alone reduced the glycogen level by 50–60%. Following ERT, muscle glycogen was reduced to normal levels, an outcome not observed in Pompe mice with genetically intact autophagy. The suppression of autophagy, which has proven successful in the Pompe model, is a novel therapeutic approach that may be useful in other diseases with disturbed autophagy.     

Lysosomal dysfunction in muscle with special reference to glycogen storage disease type II

The importance of proper lysosomal activity in cell and tissue homeostasis is underlined by "experiments of nature", i.e. genetic defects in one of the at least 40 lysosomal enzymes/proteins present in the human cell. The complete lack of 1-4 alpha-glucosidase (glycogen storage disease type II (GSD II) or Pompe disease) is life-threatening. Patients suffering from GSD II commonly die before the age of 2 years because of cardiorespiratory insufficiency. Striated muscle cells appear to be particularly vulnerable in GSD II. The high cytoplasmic glycogen content in muscle cells most likely gives rise to a high rate of glycogen engulfment by the lysosomes. The polysaccharides become subsequently trapped in these organelles when 1-4 alpha-glucosidase activity is absent. During the course of the disease, muscle wasting occurs. It is hypothesised that the gradual loss of muscle mass is caused by a combination of disuse atrophy and lipofuscine-mediated apoptosis of myocytes. Moreover, we hypothesise that in the remaining skeletal muscle cells, longitudinal transmission of force is hampered by swollen lysosomes, clustering of non-contractile material and focal regions with degraded contractile proteins, which results in muscle weakness.     

Detection and imaging of non-contractile inclusions and sarcomeric anomalies in skeletal muscle by second harmonic generation combined with two-photon excited fluorescence

The large size of the multinucleated muscle fibers of skeletal muscle makes their examination for structural and pathological defects a challenge. Sections and single fibers are accessible to antibodies and other markers but imaging of such samples does not provide a three-dimensional view of the muscle. Regrettably, bundles of fibers cannot be stained or imaged easily. Two-photon microscopy techniques overcome these obstacles. Second harmonic generation (SHG) by myosin filaments and two-photon excited fluorescence (2PEF) of mitochondrial and lysosomal components provides detailed structural information on unstained tissue. Furthermore, the infrared exciting light can penetrate several layers of muscle fibers and the minimal processing is particularly valuable for fragile biopsies. Here we demonstrate the usefulness of SHG, combined with 2PEF, to reveal enlarged lysosomes and accumulations of non-contractile material in muscles from the mouse model for the lysosomal storage disorder Pompe disease (PD), and in biopsies from adult and infant PD patients. SHG and 2PEF also detect sarcomeric defects that may presage the loss of myofibrils in atrophying muscle and signify loss of elasticity. The combination of SHG and 2PEF should be useful in the analysis and diagnosis of a wide range of skeletal muscle pathologies.     

AUTOPHAGIC DEGRADATION OF GLYCOGEN IN SKELETAL MUSCLES OF THE NEWBORN RAT

Large amounts of glycogen accumulate in rat skeletal muscle fibers during the late fetal stages and are mobilized in the first postnatal days. This glycogen depletion is relatively slow in the immature leg muscles, in which extensive deposits are still found 24 hr after birth and, to some extent, persist until the 3rd day. In the more differentiated psoas muscle and especially in the diaphragm, the glycogen stores are completely mobilized already during the early hours. Section of the sciatic nerve 3 days before birth or within the first 2 hr after delivery does not affect glycogen depletion in the leg muscles. Neonatal glycogenolysis in rat muscle fibers takes place largely by segregation and digestion of glycogen particles in autophagic vacuoles. These vacuoles: (a) are not seen in fetal muscle fibers or at later postnatal stages, but appear concomitantly with the process of glycogen depletion and disappear shortly afterwards; (b) are prematurely formed in skeletal muscles of fetuses at term treated with glucagon; (c) contain almost exclusively glycogen particles and no other recognizable cell constituents; (d) have a double or, more often, single limiting membrane and originate apparently from flattened sacs sequestering glycogen masses; (e) are generally found to contain reaction product in preparations incubated from demonstration of acid phosphatase activity. The findings emphasize the role of the lysosomal system in the physiological process of postnatal glycogen mobilization and appear relevant in the interpretation of type II glycogen storage disease.     

New insights into therapeutic options for Pompe disease

Glycogen storage disease type II or Pompe disease (GSD II, MIM 232300) is a rare inherited metabolic myopathy caused by a deficiency of lysosomal acid α-glucosidase or acid maltase (GAA; EC 3.2.1.20), resulting in a massive lysosomal glycogen accumulation in cardiac and skeletal muscles. Affected individuals exhibit either severe hypotonia associated with hypertrophic cardiomyopathy (infantile forms) or progressive muscle weakness (late-onset forms). Even if enzyme replacement therapy has recently become a standard treatment, it suffers from several limitations. This review will present the main results of enzyme replacement therapy and the recent findings concerning alternative treatments for Pompe disease, such as gene therapy, enzyme enhancement therapy, and substrate reduction therapy.     

Correction of glycogenosis type 2 by muscle-specific lentiviral vector

Glycogen storage disease type II (GSDII) or Pompe disease is an inherited disease of glycogen metabolism caused by a lack of functional lysosomal acid α-glucosidase (GAA). Affected individuals store glycogen in lysosomes resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe form. We investigated for the first time the use of lentiviral vectors to correct the GSDII phenotype in human and murine GAA-deficient cells. Fibroblasts from infantile and adult GSDII patients were efficiently transduced by a GAA-expressing lentiviral vector placed under the control of the strong MND promoter, leading to a complete restoration of enzymatic activity. We also developed a muscle-specific lentiviral vector based on the synthetic C5–12 promoter and tested it on deficient myogenic satellite cells derived from a GSDII mouse model. GAA was expressed as a correctly processed protein allowing a complete enzymatic and metabolic correction in myoblasts and differentiated myotubes, as well as a significant mannose-6-phosphate (M6P)-dependent secretion reuptake by naive cells. Transduced cells showed lysosomal glycogen clearance, as demonstrated by electron microscopy. These results form the basis for a therapeutic approach of GSDII using lentiviral vector-mediated gene transfer into muscle stem cells.     

Evidence that lysosomes are not involved in the degradation of myofibrillar proteins in rat skeletal muscle.

To examine the role of lysosomes in the degradation of skeletal-muscle myofibrillar proteins, we measured the release of N tau-methylhistidine from perfused muscle of starved and fed rats in the presence or absence of agents that inhibit lysosomal proteinase activity. After 1 day of starvation, the release of N tau-methylhistidine by perfused muscle of 4-, 8- and 24-week-old rats increased by 322, 159 and 134% respectively. On the other hand, total protein breakdown, assessed by tyrosine release, increased by 62, 20 and 20% respectively. Inhibitors of lysosomal proteinases as well as high concentrations of insulin or amino acids failed to diminish the release of N tau-methylhistidine by perfused muscle of starved and fed rats, despite a 25-35% inhibition of total protein breakdown. The data strongly suggest that the complete breakdown of myofibrillar proteins occurs via a non-lysosomal pathway. They also suggest that total proteolysis, which primarily reflects non-myofibrillar protein breakdown, occurs at least in part within lysosomes.     

Muscle degeneration in neuraminidase 1-deficient mice results from infiltration of the muscle fibers by expanded connective tissue

Neuraminidase 1 (NEU1) regulates the catabolism of sialoglycoconjugates in lysosomes. Congenital NEU1 deficiency in children is the basis of sialidosis, a severe neurosomatic disorder in which patients experience a broad spectrum of clinical manifestations varying in the age of onset and severity. Osteoskeletal deformities and muscle hypotonia have been described in patients with sialidosis. Here we present the first comprehensive analysis of the skeletal muscle pathology associated with loss of Neu1 function in mice. In this animal model, skeletal muscles showed an expansion of the epimysial and perimysial spaces, associated with proliferation of fibroblast-like cells and abnormal deposition of collagens. Muscle fibers located adjacent to the expanded connective tissue underwent extensive invagination of their sarcolemma, which resulted in the infiltration of the fibers by fibroblast-like cells and extracellular matrix, and in their progressive cytosolic fragmentation. Both the expanded connective tissue and the juxtaposed infiltrated muscle fibers were strongly positive for lysosomal markers and displayed increased proteolytic activity of lysosomal cathepsins and metalloproteinases. These combined features could lead to abnormal remodeling of the extracellular matrix that could be responsible for sarcolemmal invagination and progressive muscle fiber degeneration, ultimately resulting in an overt atrophic phenotype. This unique pattern of muscle damage, which has never been described in any myopathy, might explain the neuromuscular manifestations reported in patients with the type II severe form of sialidosis. More broadly, these findings point to a potential role of NEU1 in cell proliferation and extracellular matrix remodeling.     

The values and limits of an in vitro model of Pompe disease: The best laid schemes o’ mice an’ men ……..†

In Pompe disease, a lysosomal glycogen storage disorder, cardiac and skeletal muscle abnormalities are responsible for premature death and severe weakness. Swollen glycogen-filled lysosomes, the expected pathology, are accompanied in skeletal muscle by a secondary pathology – massive accumulation of autophagic debris – that appears to contribute greatly to the weakness. We have tried to reproduce these defects in murine, Pompe myotubes derived from either primary myoblasts or myoblasts with extended proliferative capacity. The cells accumulated large lysosomes filled with glycogen, but, to our disappointment, did not have autophagic buildup even though basal autophagy was intact. When we suppressed autophagy by knocking down Atg7, we found that glycogen uptake by lysosomes was not affected, suggesting that macroautophagy is not the major pathway for glycogen delivery to lysosomes. But two apparently incidental observations – a peculiar distribution of both microinjected dextran and of small acidic structures adjacent to the interior membrane of large alkalinized glycogen-containing lysosomes – raised the possibility that glycogen traffics to the lysosomes by microautophagy or/and by the engulfment of small lysosomes by large ones. The cultured myotubes, therefore, appear to be a useful model for studying the mechanisms involved in glycogen accumulation in Pompe disease and to test substrate deprivation approaches.

^?From To a Mouse, Robert Burns, 1785     

Rescue of enzyme deficiency in embryonic diaphragm in a mouse model of metabolic myopathy: Pompe disease

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid alpha-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.     

Effects of Ca2+(-)binding agent on unloaded rat soleus: muscle morphology and sarcomeric titin content

It had been hypothesized and recently shown that the exposure to gravitational unloading brought out to sufficient accumulation of Ca2+ in the myoplasm of soleus muscle fibers. Some authors believe that this dramatic Ca2+ accumulation induces the muscle protein degradation (including cytoskeletal proteins) by means of Ca 2(+)-activated proteases. For instance, the loss of giant sarcomeric cytoskeletal protein titin which is believed to determine the elasticity properties of muscle fibers, may contribute to the fiber stiffness decrease under unloading conditions. The study was designed to test the hypothesis suggesting that intracellular Ca2+ binding by means of EDTA administration would allow to attenuate hypogravity-induced atrophic changes including changes in myofibrillar proteins of skeletal muscle fibers.     

Studies of lysosomal sialic acid metabolism: retention of sialic acid by Salla disease lysosomes

Purified rat liver lysosomes were incubated in 0.2 M sialic acid resulting in an increase in lysosomal free sialic acid of 3.8 +/- 1.5 nmol/unit beta hexosaminidase. Sialic acid loss by these lysosomes was stimulated 2-3 fold by 25 mM sodium phosphate. Loss of sialic acid by lysosomes from cultured human diploid fibroblasts was similar to that observed in rat liver lysosomes while loss of sialic acid by lysosomes from cultured fibroblasts from a patient with infantile Salla disease occurred much more slowly. Salla disease appears to be the consequence of defective lysosomal transport of sialic acid and is analogous to cystinosis, a disorder of lysosomal amino acid transport.     

Ultrastructure of human dermal mast cells in 29 different lysosomal storage diseases

The effect of lysosomal storage diseases on the ultrastructure of human mast cells has not previously been reported. Indeed, there has been little published evidence indicating that mast cells contain typical lysosomes. However, mast cell cytoplasmic granules contain hydrolases similar to those found in lysosomes, but which differ from lysosomal hydrolases in exhibiting optimal activity at higher pH. We therefore examined by transmission electron microscopy the dermal mast cells in 58 biopsies of patients exhibiting 1 of 29 different lysosomal storage diseases. We found mast cells containing abnormal lysosomes in 16 of these disorders. In 6 of these 16 diseases, the mast cells' cytoplasmic granules appeared normal. These observations indicate that human mast cells can contain lysosomes, and provide evidence that the enzymes affected by lysosomal storage diseases are active in mast cells.