Neural deficits contribute to respiratory insufficiency in Pompe disease.
ABSTRACT: Pompe disease is a severe form of muscular dystrophy due to glycogen accumulation in all tissues, especially striated muscle. Disease severity is directly related to the deficiency of acid alpha-glucosidase (GAA), which degrades glycogen in the lysosome. Respiratory dysfunction is a hallmark of the disease, muscle weakness has been viewed as the underlying cause, and the possibility of an associated neural contribution has not been evaluated previously. Therefore, we examined behavioral and neurophysiological aspects of breathing in 2 animal models of Pompe disease--the Gaa(-/-) mouse and a transgenic line (MTP) expressing GAA only in skeletal muscle, as well as a detailed analysis of the CNS in a Pompe disease patient. Glycogen content was elevated in the Gaa(-/-) mouse cervical spinal cord. Retrograde labeling of phrenic motoneurons showed significantly greater soma size in Gaa(-/-) mice vs. isogenic controls, and glycogen was observed in Gaa(-/-) phrenic motoneurons. Ventilation, assessed via plethysmography, was attenuated during quiet breathing and hypercapnic challenge in Gaa(-/-) mice (6 to >21 months of age) vs. controls. We confirmed that MTP mice had normal diaphragmatic contractile properties; however, MTP mice had ventilation similar to the Gaa(-/-) mice during quiet breathing. Neurophysiological recordings indicated that efferent phrenic nerve inspiratory burst amplitudes were substantially lower in Gaa(-/-) and MTP mice vs. controls. In human samples, we demonstrated similar pathology in the cervical spinal cord and greater accumulation of glycogen in spinal cord compared with brain. We conclude that neural output to the diaphragm is deficient in Gaa(-/-) mice, and therapies targeting muscle alone may be ineffective in Pompe disease.
Project description:Pompe disease is a form of muscular dystrophy due to lysosomal storage of glycogen caused by deficiency of acid ?-glucosidase (GAA). Respiratory failure in Pompe disease has been attributed to respiratory muscle dysfunction. However, evaluation of spinal tissue from Pompe patients and animal models indicates glycogen accumulation and lower motoneuron pathology. We hypothesized that restoring GAA enzyme activity in the region of the phrenic motor nucleus could lead to improved breathing in a murine Pompe model (the Gaa(-/-) mouse). Adeno-associated virus serotype 5 (AAV5), encoding either GAA or green fluorescent protein (GFP), was delivered at the C(3)-C(4) spinal level of adult Gaa(-/-) mice and the spinal cords were harvested 4 weeks later. AAV5-GAA injection restored spinal GAA enzyme activity and GAA immunostaining was evident throughout the cervical ventral horn. The periodic acid Schiff (PAS) method was used to examine neuronal glycogen accumulation, and spinal PAS staining was attenuated after AAV5-GAA injection. Lastly, plethysmography revealed that minute ventilation was greater in unanesthetized AAV5-GAA versus AAV5-GFP treated Gaa(-/-) mice at 1-4 months postinjection. These results support the hypothesis that spinal cord pathology substantially contributes to ventilatory dysfunction in Gaa(-/-) mice and therefore requires further detailed evaluation in patients with Pompe disease.
Project description:Pompe disease is an autosomal recessive genetic disorder characterized by a deficiency of the enzyme responsible for degradation of lysosomal glycogen (acid α-glucosidase (GAA)). Cardiac dysfunction and respiratory muscle weakness are primary features of this disorder. To attenuate the progressive and rapid accumulation of glycogen resulting in cardiorespiratory dysfunction, adult Gaa (-/-) mice were administered a single systemic injection of rAAV2/9-DES-hGAA (AAV9-DES) or bimonthly injections of recombinant human GAA (enzyme replacement therapy (ERT)). Assessment of cardiac function and morphology was measured 1 and 3 months after initiation of treatment while whole-body plethysmography and diaphragmatic contractile function was evaluated at 3 months post-treatment in all groups. Gaa (-/-) animals receiving either AAV9-DES or ERT demonstrated a significant improvement in cardiac function and diaphragmatic contractile function as compared to control animals. AAV9-DES treatment resulted in a significant reduction in cardiac dimension (end diastolic left ventricular mass/gram wet weight; EDMc) at 3 months postinjection. Neither AAV nor ERT therapy altered minute ventilation during quiet breathing (eupnea). However, breathing frequency and expiratory time were significantly improved in AAV9-DES animals. These results indicate systemic delivery of either strategy improves cardiac function but AAV9-DES alone improves respiratory parameters at 3 months post-treatment in a murine model of Pompe disease.
Project description:Pompe disease is a systemic metabolic disorder characterized by lack of acid-alpha glucosidase (GAA) resulting in ubiquitous lysosomal glycogen accumulation. Respiratory and ambulatory dysfunction are prominent features in patients with Pompe yet the mechanism defining the development of muscle weakness is currently unclear. Transgenic animal models of Pompe disease mirroring the patient phenotype have been invaluable in mechanistic and therapeutic study. Here, we demonstrate significant pathological alterations at neuromuscular junctions (NMJs) of the diaphragm and tibialis anterior muscle as prominent features of disease pathology in Gaa knockout mice. Postsynaptic defects including increased motor endplate area and fragmentation were readily observed in Gaa(-/-) but not wild-type mice. Presynaptic neuropathic changes were also evident, as demonstrated by significant reduction in the levels of neurofilament proteins, and alterations in axonal fiber diameter and myelin thickness within the sciatic and phrenic nerves. Our data suggest the loss of NMJ integrity is a primary contributor to the decline in respiratory and ambulatory function in Pompe and arises from both pre- and postsynaptic pathology. These observations highlight the importance of systemic phenotype correction, specifically restoration of GAA to skeletal muscle and the nervous system for treatment of Pompe disease.
Project description:Lysosomal storage disorders such as Pompe disease can be more effectively treated, if immune tolerance to enzyme or gene replacement therapy can be achieved. Alternatively, immune responses against acid ?-glucosidase (GAA) might be evaded in Pompe disease through muscle-specific expression of GAA with adeno-associated virus (AAV) vectors.An AAV vector containing the MHCK7 regulatory cassette to drive muscle-specific GAA expression was administered to GAA knockout (KO) mice, immune tolerant GAA-KO mice and mannose-6-phosphate deficient GAA-KO mice. GAA activity and glycogen content were analyzed in striated muscle to determine biochemical efficacy.The biochemical efficacy from GAA expression was slightly reduced in GAA-KO mice, as demonstrated by higher residual glycogen content in skeletal muscles. Next, immune tolerance to GAA was induced in GAA-KO mice by co-administration of a second AAV vector encoding liver-specific GAA along with the AAV vector encoding muscle-specific GAA. Antibody formation was prevented by liver-specific GAA, and the biochemical efficacy of GAA expression was improved in the absence of antibodies, as demonstrated by significantly reduced glycogen content in the diaphragm. Efficacy was reduced in old GAA-KO mice despite the absence of antibodies. The greatest impact upon gene therapy was observed in GAA-KO mice lacking the mannose-6-phosphate receptor in muscle. The clearance of stored glycogen was markedly impaired despite high GAA expression in receptor-deficient Pompe disease mice.Overall, antibody formation had a subtle effect upon efficacy, whereas the absence of mannose-6-phosphate receptors markedly impaired muscle-targeted gene therapy in murine Pompe disease.
Project description:Due to the lack of acid alpha-glucosidase (GAA) activity, Pompe mice develop glycogen storage pathology and progressive skeletal muscle dysfunction with age. Applying either gene or enzyme therapy to reconstitute GAA levels in older, symptomatic Pompe mice effectively reduces glycogen storage in skeletal muscle but provides only modest improvements in motor function. As strategies to stimulate muscle hypertrophy, such as by myostatin inhibition, have been shown to improve muscle pathology and strength in mouse models of muscular dystrophy, we sought to determine whether these benefits might be similarly realized in Pompe mice. Administration of a recombinant adeno-associated virus serotype 8 vector encoding follistatin, an inhibitor of myostatin, increased muscle mass and strength but only in Pompe mice that were treated before 10 months of age. Younger Pompe mice showed significant muscle fiber hypertrophy in response to treatment with follistatin, but maximal gains in muscle strength were achieved only when concomitant GAA administration reduced glycogen storage in the affected muscles. Despite increased grip strength, follistatin treatment failed to improve rotarod performance. These findings highlight the importance of treating Pompe skeletal muscle before pathology becomes irreversible, and suggest that adjunctive therapies may not be effective without first clearing skeletal muscle glycogen storage with GAA.
Project description:Glycogen storage disease type II or Pompe disease is a severe neuromuscular disorder caused by mutations in the lysosomal enzyme, acid ?-glucosidase (GAA), which result in pathological accumulation of glycogen throughout the body. Enzyme replacement therapy is available for Pompe disease; however, it has limited efficacy, has high immunogenicity, and fails to correct pathological glycogen accumulation in nervous tissue and skeletal muscle. Using bioinformatics analysis and protein engineering, we developed transgenes encoding GAA that could be expressed and secreted by hepatocytes. Then, we used adeno-associated virus (AAV) vectors optimized for hepatic expression to deliver the GAA transgenes to Gaa knockout (Gaa-/-) mice, a model of Pompe disease. Therapeutic gene transfer to the liver rescued glycogen accumulation in muscle and the central nervous system, and ameliorated cardiac hypertrophy as well as muscle and respiratory dysfunction in the Gaa-/- mice; mouse survival was also increased. Secretable GAA showed improved therapeutic efficacy and lower immunogenicity compared to nonengineered GAA. Scale-up to nonhuman primates, and modeling of GAA expression in primary human hepatocytes using hepatotropic AAV vectors, demonstrated the therapeutic potential of AAV vector-mediated liver expression of secretable GAA for treating pathological glycogen accumulation in multiple tissues in Pompe disease.
Project description:Pompe disease is a neuromuscular disease resulting from deficiency in acid ?-glucosidase (GAA), results in cardiac, skeletal muscle, and central nervous system (CNS) pathology. Enzyme replacement therapy (ERT) has been shown to partially correct cardiac and skeletal muscle dysfunction. However, ERT does not cross the blood-brain barrier and progressive CNS pathology ensues. We tested the hypothesis that intrapleural administration of recombinant adeno-associated virus (rAAV9)-GAA driven by a cytomegalovirus (CMV) or desmin (DES) promoter would improve cardiac and respiratory function in Gaa(-/-) mice through a direct effect and retrograde transport to motoneurons. Cardiac magnetic resonance imaging revealed significant improvement in ejection fraction in rAAV9-GAA-treated animals. Inspiratory phrenic and diaphragm activity was examined at baseline and during hypercapnic respiratory challenge. Mice treated with AAV9 had greater relative inspiratory burst amplitude during baseline conditions when compared with Gaa(-/-). In addition, efferent phrenic burst amplitude was significantly correlated with diaphragm activity in both AAV9-DES and AAV9-CMV groups but not in Gaa(-/-). This is the first study to indicate improvements in cardiac, skeletal muscle, and respiratory neural output following rAAV administration in Pompe disease. These results further implicate a role for the CNS in Pompe disease pathology and the critical need to target the neurologic aspects in developing therapeutic strategies.
Project description:Pompe disease is due to a deficiency in acid-?-glucosidase (GAA) and results in debilitating skeletal muscle wasting, characterized by the accumulation of glycogen and autophagic vesicles. Given the role of lysosomes as a platform for mTORC1 activation, we examined mTORC1 activity in models of Pompe disease. GAA-knockdown C2C12 myoblasts and GAA-deficient human skin fibroblasts of infantile Pompe patients were found to have decreased mTORC1 activation. Treatment with the cell-permeable leucine analog L-leucyl-L-leucine methyl ester restored mTORC1 activation. In vivo, Pompe mice also displayed reduced basal and leucine-stimulated mTORC1 activation in skeletal muscle, whereas treatment with a combination of insulin and leucine normalized mTORC1 activation. Chronic leucine feeding restored basal and leucine-stimulated mTORC1 activation, while partially protecting Pompe mice from developing kyphosis and the decline in muscle mass. Leucine-treated Pompe mice showed increased spontaneous activity and running capacity, with reduced muscle protein breakdown and glycogen accumulation. Together, these data demonstrate that GAA deficiency results in reduced mTORC1 activation that is partly responsible for the skeletal muscle wasting phenotype. Moreover, mTORC1 stimulation by dietary leucine supplementation prevented some of the detrimental skeletal muscle dysfunction that occurs in the Pompe disease mouse model.
Project description:Pompe disease, a severe and often fatal neuromuscular disorder, is caused by a deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). The disease is characterized by the accumulation of excess glycogen in the heart, skeletal muscle, and CNS. Currently approved enzyme replacement therapy or experimental adeno-associated virus (AAV)-mediated gene therapy has little effect on CNS correction. Here we demonstrate that a newly developed AAV-PHP.B vector can robustly transduce both the CNS and skeletal muscles in GAA-knockout (GAAKO) mice. A single intravenous injection of an AAV-PHP.B vector expressing human GAA under the control of cytomegalovirus (CMV) enhancer-chicken ?-actin (CB) promoter into 2-week-old GAAKO mice resulted in widespread GAA expression in the affected tissues. Glycogen contents were reduced to wild-type levels in the brain and heart, and they were significantly decreased in skeletal muscle by the AAV treatment. The histological assay showed no visible glycogen in any region of the brain and spinal cord of AAV-treated mice. In this study, we describe a set of behavioral tests that can detect early neurological deficits linked to extensive lysosomal glycogen accumulation in the CNS of untreated GAAKO mice. Furthermore, we demonstrate that the therapy can help prevent the development of these abnormalities.
Project description:We have recently reported on the pathology of the neuromuscular junction (NMJ) in Pompe disease, reflecting disruption of neuronal and muscle homeostasis as a result of glycogen accumulation. The aim of this study was to examine how the alteration of NMJ physiology contributes to Pompe disease pathology; we performed molecular, physiological, and histochemical analyses of NMJ-related measures of the tibialis anterior muscles of young-, mid-, and late-stage alpha-glucosidase (GAA)-deficient mice.We performed intramuscular injection of an adeno-associated virus (AAV)9 vector expressing GAA (AAV9-hGAA) into the tibialis anterior muscle of Gaa(-/-) mice at early, mid, and severe pathological time points. We analyzed expression of NMJ-related genes, in situ muscle force production, and clearance of glycogen in conjunction with histological assessment of the NMJ.Our data demonstrate that AAV9-hGAA is able to replace GAA to the affected tissue and modify AChR mRNA expression, muscle force production, motor endplate area, and innervation status. Importantly, the degree of restoration for these outcomes is limited by severity of disease. Early restoration of GAA activity was most effective, whereas late correction of GAA expression was not effective in modifying parameters reflecting NMJ structure and function nor in force restoration despite resolution of glycogen storage in muscle.Our data provide new mechanistic insight into the pathology of Pompe disease and suggest that early systemic correction to both neural and muscle tissues may be essential for successful correction of neuromuscular function in Pompe disease. Ann Neurol 2015;78:222-234.