SnapShot: Lysosomal Storage Diseases.

Lysosomal storage diseases (LSDs) represent a group of monogenic inherited metabolic disorders characterized by the progressive accumulation of undegraded substrates inside lysosomes, resulting in aberrant lysosomal activity and homeostasis. This SnapShot summarizes the intracellular localization and function of proteins implicated in LSDs. Common aspects of LSD pathogenesis and the major current therapeutic approaches are noted. To view this SnapShot, open or download the PDF.

TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress.

In recent years, our vision of lysosomes has drastically changed. Formerly considered to be mere degradative compartments, they are now recognized as key players in many cellular processes. The ability of lysosomes to respond to different stimuli revealed a complex and coordinated regulation of lysosomal gene expression. This review discusses the participation of the transcription factors TFEB and TFE3 in the regulation of lysosomal function and biogenesis, as well as the role of the lysosomal pathway in cellular adaptation to a variety of stress conditions, including nutrient deprivation, mitochondrial dysfunction, protein misfolding, and pathogen infection. We also describe how cancer cells make use of TFEB and TFE3 to promote their own survival and highlight the potential of these transcription factors as therapeutic targets for the treatment of neurological and lysosomal diseases.

Decoding the muscle transcriptome of patients with late onset Pompe disease reveals markers of disease progression.

Late-onset Pompe Disease (LOPD) is a rare genetic disorder caused by the deficiency of acid alpha-glucosidase leading to progressive cellular dysfunction due to the accumulation of glycogen in the lysosome. The mechanism of relentless muscle damage - a classic manifestation of the disease - has been extensively studied by analysing the whole muscle tissue; however, little, if any, is known about transcriptional heterogeneity among nuclei within the multinucleated skeletal muscle cells. This is the first report of application of single nuclei RNA sequencing to uncover changes in the gene expression profile in muscle biopsies from eight patients with LOPD and four muscle samples from age and gender matched healthy controls. We matched these changes with histology findings using GeoMx Spatial Transcriptomics to compare the transcriptome of control myofibers from healthy individuals with non-vacuolated (histologically unaffected) and vacuolated (histologically affected) myofibers of LODP patients. We observed an increase in the proportion of slow and regenerative muscle fibers and macrophages in LOPD muscles. The expression of the genes involved in glycolysis was reduced, whereas the expression of the genes involved in the metabolism of lipids and amino acids was increased in non-vacuolated fibers, indicating early metabolic abnormalities. Additionally, we detected upregulation of autophagy genes, and downregulation of the genes involved in ribosomal and mitochondrial function leading to defective oxidative phosphorylation. The upregulation of the genes associated with inflammation, apoptosis and muscle regeneration was observed only in vacuolated fibers. Notably, enzyme replacement therapy - the only available therapy for the disease - showed a tendency to restore metabolism dysregulation, particularly within slow fibers. A combination of single nuclei RNA sequencing and spatial transcriptomics revealed the landscape of normal and the diseased muscle, and highlighted the early abnormalities associated with the disease progression. Thus, the application of these two new cutting-edge technologies provided insight into the molecular pathophysiology of muscle damage in LOPD and identified potential avenues for therapeutic intervention.

Nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants enhances autophagic clearance in Pompe disease.

Alglucosidase alpha is an orphan drug approved for enzyme replacement therapy (ERT) in Pompe disease (PD); however, its efficacy is limited in skeletal muscle because of a partial blockage of autophagic flux that hinders intracellular trafficking and enzyme delivery. Adjunctive therapies that enhance autophagic flux and protect mitochondrial integrity may alleviate autophagic blockage and oxidative stress and thereby improve ERT efficacy in PD. In this study, we compared the benefits of ERT combined with a ketogenic diet (ERT-KETO), daily administration of an oral ketone precursor (1,3-butanediol; ERT-BD), a multi-ingredient antioxidant diet (ERT-MITO; CoQ10, α-lipoic acid, vitamin E, beetroot extract, HMB, creatine, and citrulline), or co-therapy with the ketone precursor and multi-ingredient antioxidants (ERT-BD-MITO) on skeletal muscle pathology in GAA-KO mice. We found that two months of 1,3-BD administration raised circulatory ketone levels to ≥1.2 mM, attenuated autophagic buildup in type 2 muscle fibers, and preserved muscle strength and function in ERT-treated GAA-KO mice. Collectively, ERT-BD was more effective vs. standard ERT and ERT-KETO in terms of autophagic clearance, dampening of oxidative stress, and muscle maintenance. However, the addition of multi-ingredient antioxidants (ERT-BD-MITO) provided the most consistent benefits across all outcome measures and normalized mitochondrial protein expression in GAA-KO mice. We therefore conclude that nutritional co-therapy with 1,3-butanediol and multi-ingredient antioxidants may provide an alternative to ketogenic diets for inducing ketosis and enhancing autophagic flux in PD patients.

Novel degenerative and developmental defects in a zebrafish model of mucolipidosis type IV.

Mucolipidosis type IV (MLIV) is a lysosomal storage disease characterized by neurologic and ophthalmologic abnormalities. There is currently no effective treatment. MLIV is caused by mutations in MCOLN1, a lysosomal cation channel from the transient receptor potential (TRP) family. In this study, we used genome editing to knockout the two mcoln1 genes present in Danio rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing unprecedented opportunities for therapeutic discovery.

Therapeutic Benefit of Autophagy Modulation in Pompe Disease.

The complexity of the pathogenic cascade in lysosomal storage disorders suggests that combination therapy will be needed to target various aspects of pathogenesis. The standard of care for Pompe disease (glycogen storage disease type II), a deficiency of lysosomal acid alpha glucosidase, is enzyme replacement therapy (ERT). Many patients have poor outcomes due to limited efficacy of the drug in clearing muscle glycogen stores. The resistance to therapy is linked to massive autophagic buildup in the diseased muscle. We have explored two strategies to address the problem. Genetic suppression of autophagy in muscle of knockout mice resulted in the removal of autophagic buildup, increase in muscle force, decrease in glycogen level, and near-complete clearance of lysosomal glycogen following ERT. However, this approach leads to accumulation of ubiquitinated proteins, oxidative stress, and exacerbation of muscle atrophy. Another approach involves AAV-mediated TSC knockdown in knockout muscle leading to upregulation of mTOR, inhibition of autophagy, reversal of atrophy, and efficient cellular clearance on ERT. Importantly, this approach reveals the possibility of reversing already established autophagic buildup, rather than preventing its development.

Failure of Autophagy in Pompe Disease.

Autophagy is an evolutionarily conserved lysosome-dependent degradation of cytoplasmic constituents. The system operates as a critical cellular pro-survival mechanism in response to nutrient deprivation and a variety of stress conditions. On top of that, autophagy is involved in maintaining cellular homeostasis through selective elimination of worn-out or damaged proteins and organelles. The autophagic pathway is largely responsible for the delivery of cytosolic glycogen to the lysosome where it is degraded to glucose via acid α-glucosidase. Although the physiological role of lysosomal glycogenolysis is not fully understood, its significance is highlighted by the manifestations of Pompe disease, which is caused by a deficiency of this lysosomal enzyme. Pompe disease is a severe lysosomal glycogen storage disorder that affects skeletal and cardiac muscles most. In this review, we discuss the basics of autophagy and describe its involvement in the pathogenesis of muscle damage in Pompe disease. Finally, we outline how autophagic pathology in the diseased muscles can be used as a tool to fast track the efficacy of therapeutic interventions.

Acid α-glucosidase (GAA) activity and glycogen content in muscle biopsy specimens of patients with Pompe disease: A systematic review.

Pompe disease is a rare genetic disorder characterized by a deficiency of acid α-glucosidase (GAA), leading to the accumulation of glycogen in various tissues, especially in skeletal muscles. The disease manifests as a large spectrum of phenotypes from infantile-onset Pompe disease (IOPD) to late-onset Pompe disease (LOPD), depending on the age of symptoms onset. Quantifying GAA activity and glycogen content in skeletal muscle provides important information about the disease severity. However, the distribution of GAA and glycogen levels in skeletal muscles from healthy individuals and those impacted by Pompe disease remains poorly understood, and there is currently no universally accepted standard assay for GAA activity measurement. This systematic literature review aims to provide an overview of the available information on GAA activity and glycogen content levels in skeletal muscle biopsies from patients with Pompe disease. A structured review of PubMed and Google Scholar literature (with the latter used to check that no additional publications were identified) was conducted to identify peer-reviewed publications on glycogen storage disease type II [MeSH term] + GAA, protein human (supplementary concept), Pompe, muscle; and muscle, acid alpha-glucosidase. A limit of English language was applied. Results were grouped by methodologies used to quantify GAA activity and glycogen content in skeletal muscle. The search and selection strategy were devised and carried out in line with Preferred Reporting of Items in Systematic Reviews and Meta-Analysis guidelines and documented using a flowchart. Bibliographies of papers included in the analysis were reviewed and applicable publications not already identified in the search were included. Of the 158 articles retrieved, 24 (comprising >100 muscle biopsies from >100 patients) were included in the analysis, with four different assays. Analysis revealed that patients with IOPD exhibited markedly lower GAA activity in skeletal muscles than those with LOPD, regardless of the measurement method employed. Additionally, patients with IOPD had notably higher glycogen content levels in skeletal muscles than those with LOPD. In general, however, it was difficult to fully characterize GAA activity because of the different methods used. The findings underscore the challenges in the interpretation and comparison of the results across studies because of the substantial methodological variations. There is a need to establish standardized reference ranges of GAA activity and glycogen content in healthy individuals and in Pompe disease patients based on globally standardized methods to improve comparability and reliability in assessing this rare disease.

Skeletal muscle effects of antisense oligonucleotides targeting glycogen synthase 1 in a mouse model of Pompe disease.

Pompe disease (PD) is a progressive myopathy caused by the aberrant accumulation of glycogen in skeletal and cardiac muscle resulting from the deficiency of the enzyme acid alpha-glucosidase (GAA). Administration of recombinant human GAA as enzyme replacement therapy (ERT) works well in alleviating the cardiac manifestations of PD but loses sustained benefit in ameliorating the skeletal muscle pathology. The limited efficacy of ERT in skeletal muscle is partially attributable to its inability to curb the accumulation of new glycogen produced by the muscle enzyme glycogen synthase 1 (GYS1). Substrate reduction therapies aimed at knocking down GYS1 expression represent a promising avenue to improve Pompe myopathy. However, finding specific inhibitors for GYS1 is challenging given the presence of the highly homologous GYS2 in the liver. Antisense oligonucleotides (ASOs) are chemically modified oligomers that hybridize to their complementary target RNA to induce their degradation with exquisite specificity. In the present study, we show that ASO-mediated Gys1 knockdown in the Gaa -/- mouse model of PD led to a robust reduction in glycogen accumulation in skeletal and cardiac muscle. In addition, combining Gys1 ASO with ERT further reduced glycogen content in muscle, eliminated autophagic buildup and lysosomal dysfunction, and improved motor function in Gaa -/- mice. Our results provide a strong foundation for further validation of the use of Gys1 ASO, alone or in combination with ERT, as a therapy for PD. We propose that early administration of Gys1 ASO in combination with ERT may be the key to preventative treatment options in PD.

WITHDRAWN: Clearance of lysosomal glycogen accumulation by Transcription factor EB (TFEB) in muscle cells from lysosomal alpha-glucosidase deficient mice.

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AAV-mediated delivery of secreted acid α-glucosidase with enhanced uptake corrects neuromuscular pathology in Pompe mice.

Gene therapy is under advanced clinical development for several lysosomal storage disorders. Pompe disease, a debilitating neuromuscular illness affecting infants, children, and adults with different severity, is caused by a deficiency of lysosomal glycogen-degrading enzyme acid α-glucosidase (GAA). Here, we demonstrated that adeno-associated virus-mediated (AAV-mediated) systemic gene transfer reversed glycogen storage in all key therapeutic targets - skeletal and cardiac muscles, the diaphragm, and the central nervous system - in both young and severely affected old Gaa-knockout mice. Furthermore, the therapy reversed secondary cellular abnormalities in skeletal muscle, such as those in autophagy and mTORC1/AMPK signaling. We used an AAV9 vector encoding a chimeric human GAA protein with enhanced uptake and secretion to facilitate efficient spread of the expressed protein among multiple target tissues. These results lay the groundwork for a future clinical development strategy in Pompe disease.

Autophagy and mitochondria in Pompe disease: nothing is so new as what has long been forgotten.

Macroautophagy (often referred to as autophagy) is an evolutionarily conserved intracellular system by which macromolecules and organelles are delivered to lysosomes for degradation and recycling. Autophagy is robustly induced in response to starvation in order to generate nutrients and energy through the lysosomal degradation of cytoplasmic components. Constitutive, basal autophagy serves as a quality control mechanism for the elimination of aggregated proteins and worn-out or damaged organelles, such as mitochondria. Research during the last decade has made it clear that malfunctioning or failure of this system is associated with a wide range of human pathologies and age-related diseases. Our recent data provide strong evidence for the role of autophagy in the pathogenesis of Pompe disease, a lysosomal glycogen storage disease caused by deficiency of acid alpha-glucosidase (GAA). Large pools of autophagic debris in skeletal muscle cells can be seen in both our GAA knockout model and patients with Pompe disease. In this review, we will focus on these recent data, and comment on the not so recent observations pointing to the involvement of autophagy in skeletal muscle damage in Pompe disease.

Restoration of muscle functionality by genetic suppression of glycogen synthesis in a murine model of Pompe disease.

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by acid alpha-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Affected individuals store glycogen mainly in cardiac and skeletal muscle tissues resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severe infantile form. Enzyme replacement therapy has already proved some efficacy, but results remain variable especially in skeletal muscle. Substrate reduction therapy was successfully used to improve the phenotype in several lysosomal storage disorders. We have recently demonstrated that shRNA-mediated reduction of glycogen synthesis led to a significant reduction of glycogen accumulation in skeletal muscle of GSDII mice. In this paper, we analyzed the effect of a complete genetic elimination of glycogen synthesis in the same GSDII model. GAA and glycogen synthase 1 (GYS1) KO mice were inter-crossed to generate a new double-KO model. GAA/GYS1-KO mice exhibited a profound reduction of the amount of glycogen in the heart and skeletal muscles, a significant decrease in lysosomal swelling and autophagic build-up as well as a complete correction of cardiomegaly. In addition, the abnormalities in glucose metabolism and insulin tolerance observed in the GSDII model were corrected in double-KO mice. Muscle atrophy observed in 11-month-old GSDII mice was less pronounced in GAA/GYS1-KO mice, resulting in improved exercise capacity. These data demonstrate that long-term elimination of muscle glycogen synthesis leads to a significant improvement of structural, metabolic and functional defects in GSDII mice and offers a new perspective for the treatment of Pompe disease.

The role of TRAIL in mediating autophagy in myositis skeletal muscle: a potential nonimmune mechanism of muscle damage.

OBJECTIVE: Multinucleated cells are relatively resistant to classic apoptosis, and the factors initiating cell death and damage in myositis are not well defined. We hypothesized that nonimmune autophagic cell death may play a role in muscle fiber damage. Recent reports indicate that TRAIL may induce both NF-κB activation and autophagic cell death in other systems. We undertook this study to investigate the role of TRAIL in cell death and pathogenesis in vitro and in vivo, using myositis muscle tissues from humans and mice. METHODS: Gene expression profiling was performed in myositis patient and control muscle specimens. Immunohistochemistry analysis was performed to confirm the gene array findings. We also analyzed TRAIL-induced cell death (apoptosis and autophagy) and NF-κB activation in vitro in cultured cells. RESULTS: TRAIL was expressed predominantly in myositis muscle fibers, but not in biopsy specimens from normal or other dystrophic-diseased muscle. Autophagy markers were up-regulated in humans with myositis and in mouse models of myositis. TRAIL expression was restricted to regenerating/atrophic areas of muscle fascicles, blood vessels, and infiltrating lymphocytes. TRAIL induced NF-κB activation and IκB degradation in cultured cells that are resistant to TRAIL-induced apoptosis but that undergo autophagic cell death. CONCLUSION: Our data demonstrate that TRAIL is expressed in myositis muscle and may mediate both activation of NF-κB and autophagic cell death in myositis. Thus, this nonimmune pathway may be an attractive target for therapeutic intervention in myositis.

Impaired autophagy: The collateral damage of lysosomal storage disorders.

Lysosomal storage disorders (LSDs), which number over fifty, are monogenically inherited and caused by mutations in genes encoding proteins that are involved in lysosomal function. Lack of the functional protein results in storage of a distinctive material within the lysosomes, which for years was thought to determine the pathophysiology of the disorder. However, our current view posits that the primary storage material disrupts the normal role of the lysosome in the autophagic pathway resulting in the secondary storage of autophagic debris. It is this "collateral damage" which is common to the LSDs but nonetheless intricately nuanced in each. We have selected five LSDs resulting from defective proteins that govern widely different lysosomal functions including glycogen degradation (Pompe), lysosomal transport (Cystinosis), lysosomal trafficking (Danon), glycolipid degradation (Gaucher) and an unidentified function (Batten) and argue that despite the disparate functions, these proteins, when mutant, all impair the autophagic process uniquely.

Chemoenzymatic glycan-selective remodeling of a therapeutic lysosomal enzyme with high-affinity M6P-glycan ligands. Enzyme substrate specificity is the name of the game.

Functionalization of therapeutic lysosomal enzymes with mannose-6-phosphate (M6P) glycan ligands represents a major strategy for enhancing the cation-independent M6P receptor (CI-MPR)-mediated cellular uptake, thus improving the overall therapeutic efficacy of the enzymes. However, the minimal high-affinity M6P-containing N-glycan ligands remain to be identified and their efficient and site-selective conjugation to therapeutic lysosomal enzymes is a challenging task. We report here the chemical synthesis of truncated M6P-glycan oxazolines and their use for enzymatic glycan remodeling of recombinant human acid α-glucosidase (rhGAA), an enzyme used for treatment of Pompe disease which is a disorder caused by a deficiency of the glycogen-degrading lysosomal enzyme. Structure-activity relationship studies identified M6P tetrasaccharide oxazoline as the minimal substrate for enzymatic transglycosylation yielding high-affinity M6P glycan ligands for the CI-MPR. Taking advantage of the substrate specificity of endoglycosidases Endo-A and Endo-F3, we found that Endo-A and Endo-F3 could efficiently deglycosylate the respective high-mannose and complex type N-glycans in rhGAA and site-selectively transfer the synthetic M6P N-glycan to the deglycosylated rhGAA without product hydrolysis. This discovery enabled a highly efficient one-pot deglycosylation/transglycosylation strategy for site-selective M6P-glycan remodeling of rhGAA to obtain a more homogeneous product. The Endo-A and Endo-F3 remodeled rhGAAs maintained full enzyme activity and demonstrated 6- and 20-fold enhanced binding affinities for CI-MPR receptor, respectively. Using an in vitro cell model system for Pompe disease, we demonstrated that the M6P-glycan remodeled rhGAA greatly outperformed the commercial rhGAA (Lumizyme) and resulted in the reversal of cellular pathology. This study provides a general and efficient method for site-selective M6P-glycan remodeling of recombinant lysosomal enzymes to achieve enhanced M6P receptor binding and cellular uptake, which could lead to improved overall therapeutic efficacy of enzyme replacement therapy.

Histidyl-tRNA synthetase and asparaginyl-tRNA synthetase, autoantigens in myositis, activate chemokine receptors on T lymphocytes and immature dendritic cells.

Autoantibodies to histidyl-tRNA synthetase (HisRS) or to alanyl-, asparaginyl-, glycyl-, isoleucyl-, or threonyl-tRNA synthetase occur in approximately 25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl-tRNA synthetases to induce leukocyte migration. HisRS induced CD4(+) and CD8(+) lymphocytes, interleukin (IL)-2-activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH(2)-terminal domain, 1-48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl-tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl-tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl-tRNA and lysyl-tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl-tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.

Modulation of glycogen synthesis by RNA interference: towards a new therapeutic approach for glycogenosis type II.

Glycogen storage disease type II (GSDII) or Pompe disease is an autosomal recessive disorder caused by defects in the acid alpha-glucosidase gene, which leads to lysosomal glycogen accumulation and enlargement of the lysosomes mainly in cardiac and muscle tissues, resulting in fatal hypertrophic cardiomyopathy and respiratory failure in the most severely affected patients. Enzyme replacement therapy has already proven to be beneficial in this disease, but correction of pathology in skeletal muscle still remains a challenge. As substrate deprivation was successfully used to improve the phenotype in other lysosomal storage disorders, we explore here a novel therapeutic approach for GSDII based on a modulation of muscle glycogen synthesis. Short hairpin ribonucleic acids (shRNAs) targeted to the two major enzymes involved in glycogen synthesis, i.e. glycogenin (shGYG) and glycogen synthase (shGYS), were selected. C2C12 cells and primary myoblasts from GSDII mice were stably transduced with lentiviral vectors expressing both the shRNAs and the enhanced green fluorescent protein (EGFP) reporter gene. Efficient and specific inhibition of GYG and GYS was associated not only with a decrease in cytoplasmic and lysosomal glycogen accumulation in transduced cells, but also with a strong reduction in the lysosomal size, as demonstrated by confocal microscopy analysis. A single intramuscular injection of recombinant AAV-1 (adeno-associated virus-1) vectors expressing shGYS into newborn GSDII mice led to a significant reduction in glycogen accumulation, demonstrating the in vivo therapeutic efficiency. These data offer new perspectives for the treatment of GSDII and could be relevant to other muscle glycogenoses.

Suppression of autophagy in skeletal muscle uncovers the accumulation of ubiquitinated proteins and their potential role in muscle damage in Pompe disease.

The role of autophagy, a catabolic lysosome-dependent pathway, has recently been recognized in a variety of disorders, including Pompe disease, the genetic deficiency of the glycogen-degrading lysosomal enzyme acid-alpha glucosidase. Accumulation of lysosomal glycogen, presumably transported from the cytoplasm by the autophagic pathway, occurs in multiple tissues, but pathology is most severe in skeletal and cardiac muscle. Skeletal muscle pathology also involves massive autophagic buildup in the core of myofibers. To determine if glycogen reaches the lysosome via autophagy and to ascertain whether autophagic buildup in Pompe disease is a consequence of induction of autophagy and/or reduced turnover due to defective fusion with lysosomes, we generated muscle-specific autophagy-deficient Pompe mice. We have demonstrated that autophagy is not required for glycogen transport to lysosomes in skeletal muscle. We have also found that Pompe disease involves induction of autophagy but manifests as a functional deficiency of autophagy because of impaired autophagosomal-lysosomal fusion. As a result, autophagic substrates, including potentially toxic aggregate-prone ubiquitinated proteins, accumulate in Pompe myofibers and may cause profound muscle damage.

Differences in the predominance of lysosomal and autophagic pathologies between infants and adults with Pompe disease: implications for therapy.

Pompe disease is a lysosomal storage disorder caused by the deficiency of acid alpha-glucosidase, the enzyme that degrades glycogen in the lysosomes. The disease manifests as a fatal cardiomyopathy and skeletal muscle myopathy in infants; in milder late-onset forms skeletal muscle is the major tissue affected. We have previously demonstrated that autophagic inclusions in muscle are prominent in adult patients and the mouse model. In this study we have evaluated the contribution of the autophagic pathology in infants before and 6 months after enzyme replacement therapy. Single muscle fibers, isolated from muscle biopsies, were stained for autophagosomal and lysosomal markers and analyzed by confocal microscopy. In addition, unstained bundles of fixed muscles were analyzed by second harmonic imaging. Unexpectedly, the autophagic component which is so prominent in juvenile and adult patients was negligible in infants; instead, the overwhelming characteristic was the presence of hugely expanded lysosomes. After 6 months on therapy, however, the autophagic buildup becomes visible as if unmasked by the clearance of glycogen. In most fibers, the two pathologies did not seem to coexist. These data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults.