In the course of studying inflammatory muscle diseases, we have encountered patients with other muscle diseases. We have studied patients with two genetic metabolic myopathies in detail: phosphofructokinase (PFK) deficiency, and acid maltase (acid alpha-glucosidase) deficiency (also known as Glycogen Deficiency Type II or GSD II or Pompe Syndrome). Our experience of treating Pompe mice (a knockout strain tolerant to the human enzyme developed here) and the experience in trials in humans, carried out mainly under the aegis the company producing the recombinant drug, demonstrated that the drug is highly effective in clearing glycogen stored in cardiac muscle cells and only poorly effective for skeletal muscle cells. This failure of skeletal muscle response to recombinant enzyme in Pompe Syndrome has become a major focus of our attention. This work has led the lab into very active new areas of cell biology that in themselves add great interest to our activities. The recombinant human enzyme itself (produced by Genzyme, very recently approved by the FDA, and supplied to us through a CRADA that also supports the salary of a fellow) appears to be structurally adequate as established by glycogen clearance in the heart and partial clearance in some skeletal muscle cells. In Pompe mice, only Type I, slow-twitch, oxidative skeletal muscle cells are cleared. Furthermore, endogenously produced enzyme is effective for treating mouse skeletal muscle cells by metabolic cross-correction only if the transgene is switched on in the liver before significant glycogen accumulation. Even transgenic synthesis of enzyme made under a skeletal muscle specific promoter is ineffective if it is switched on after significant glycogen accumulation. Thus the problem is intracellular and dependent on glycogen accumulation or on another as yet unidentified action of the enzyme that is missing. We had concentrated on exploring differences between type I and type II (fast-twitch, glycolytic) cells in both normal and Pompe mice that might point to an explanation. Previously, we had shown that although Pompe mice had more early endosomal Rab5 protein and late endosomal/lysosomal LAMP-2 protein than WT mice in all tissues, Type I and II fibers did not differ. But three proteins involved in endocytosis the cation-independent mannose-6-phosphate receptor, clathrin, and the adapter protein complex AP-2 were markedly lower in Type II muscles, and the ratio of AP-2 isotypes differed. More recent experiments have shown diminished levels of other trafficking proteins in Type II fibers: TfR, GGA2, and AP-1. Most strikingly, large autophagic areas were present uniformly in type II fibers in Pompe mice but present at the same low level in Pompe Type I fibers as in WT mice. We moved to studies of myoblasts and to studies of single fibers taken from Type I predominant (soleus) or Type II predominant (gastrocnemius) muscles. This allowed us to observe the various vacuolar compartments within single cells by confocal microscopy, using suitably labeled markers either with fixed or living cells. With markers for early (Rab5and EEA1) and late (Lamp1 and CI-MPR) endosomes, lysosomes (Lamp1) and autophagic vacuoles (LC3), we found a significant expansion of all of these vacuolar elements in Pompe myoblasts. Furthermore, vesicular movement monitored by a novel technique devised by Dr. Fukuda using GFP-Lamp1 transfected myoblasts was markedly reduced in Pompe compared to WT myoblasts. Again using a novel technique devised by Dr. Fukuda, pH within individual lysosomes was monitored spectrscopically in the confocal microscope. We found that there was a reduced number of lysosomes or late endosomes with a normal pH and the presence of an alkaline endosomal population. Remarkable differences between Type I & II Pompe muscle cells were found when we examined single fibers: in Type I fibers, lysosomes are arranged in long stretches leading to a tube-like structure, and Golgi markers neighbor lysosomes in Pompe as in WT fibers. By contrast, in Type II Pompe fibers, lysosomes are distributed throughout the fiber, and the Golgi markers are less regular and less likely to neighbor lysosomes. Autophagic build-up was present and striking in Type II but not Type I fibers. These huge areas disrupt the microtubular network and the contractile apparatus itself. Also noted was that Type I fibers are enlarged compared to WT and Type II fibers are atrophic. Experiments to measure contractility directly are planned with Drs. Leepo Yu and Robert Horowits. The autophagic areas in Type II fibers may occupy close to half the diameter of a fiber, and are almost always centrally located. When labeled recombinant enzyme or another endocytosed marker is incubated with single fibers, most is found in lysosomes of normal fibers but in the autophagic lagoon in Type II fibers. This provides direct evidence of the transport of endocytosed molecules to autophagic vacuoles as a an apparently final destination. Furthermore, metabolic processing of the recombinant enzyme injected into KO mice was impaired. We are exploring possible reasons for the abnormalities in vesicular trafficking and autophagy. So far, we have noted early accumulation of lipofuscin in autophagic areas, presumably reflecting oxidative stress. In order to study the development of the abnormalities we have observed so far, neither primary myoblast cultures , which cannot be passaged, nor live fibers, which live only briefly in culture is ideal. Thus Drs. Raben, Takikita, and Myerowitz, with the help of the two post-bac students, are currently making a major effort to develop a system for isolating and to establish immortalized myoblast cell lines from WT and KO muscle satellite cells and then inducing myotube formation. Such a system will allow us to follow dynamically the development of autophagy. We can explore the effects of metabolic interventions; of drugs that up- or down-regulate autophagy; the pH question in cells at various stages. We have also begun to do studies on human muscle using lightly fixed human muscle cells sent by investigators from which single fibers are prepared. We expect to attempt to make transformed human satellite cells obtained by biopsies done here. Currently underway are proteomic studies and the analysis and follow-up of expression studies gathered over the past couple of years. We plan to breed several informative strains of mice with disruptions in the autophagic or glycogen synthesis pathways are being planned. Collaborative studies with several investigators at other institutions are underway or under discussion. In the past year, we have successfully developed long-term lines of Pompe myoblasts transfected with CDK4 that retain the ability to mature and fuse into myotubes. These myotubes develop huge glycogen-filled lysosomes but a) they do not develop autophagic accumulation, and b)their lysosomes will shrink and lose accumulated glycogen if given recombinant acid alpha-glucosidase. These observations establish both that - contrary to established belief - macroautophagy is not the route by which glycogen enters lysosomes and that the autophagic accumulation is probably responsible for the failure of enzyme replacement therapy in skeletal muscles. In addition, we have collaborated with Emmanuelle Richard and Gaelle Drouillard in France to explore the consequences of substrate deprivation by using siRNA for glycogenin in culture or breeding glycogen synthase knockout mice with Pompe mice. Ms. Drouillard spent the summer in our lab working with Dr. Raben and Dr. Takikita on these experiments. In our own lab, we have recently successfully developed double KO mice lacking both the essential autophagy gene ATG5 and GAA and have just begun to study the progeny.

Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2007
Total Cost
$1,543,256
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Raben, N; Roberts, A; Plotz, P H (2007) Role of autophagy in the pathogenesis of Pompe disease. Acta Myol 26:45-8
Raben, Nina; Takikita, Shoichi; Pittis, Maria G et al. (2007) Deconstructing Pompe disease by analyzing single muscle fibers: to see a world in a grain of sand... Autophagy 3:546-52
Fukuda, Tokiko; Roberts, Ashley; Plotz, Paul H et al. (2007) Acid alpha-glucosidase deficiency (Pompe disease). Curr Neurol Neurosci Rep 7:71-7
Fukuda, Tokiko; Ewan, Lindsay; Bauer, Martina et al. (2006) Dysfunction of endocytic and autophagic pathways in a lysosomal storage disease. Ann Neurol 59:700-8
Wang, Wei; Parker, Gretchen E; Skurat, Alexander V et al. (2006) Relationship between glycogen accumulation and the laforin dual specificity phosphatase. Biochem Biophys Res Commun 350:588-92
Fukuda, Tokiko; Ahearn, Meghan; Roberts, Ashley et al. (2006) Autophagy and mistargeting of therapeutic enzyme in skeletal muscle in Pompe disease. Mol Ther 14:831-9
Fukuda, Tokiko; Roberts, Ashley; Ahearn, Meghan et al. (2006) Autophagy and lysosomes in Pompe disease. Autophagy 2:318-20
Raben, Nina; Fukuda, Tokiko; Gilbert, Abigail L et al. (2005) Replacing acid alpha-glucosidase in Pompe disease: recombinant and transgenic enzymes are equipotent, but neither completely clears glycogen from type II muscle fibers. Mol Ther 11:48-56
Isenberg, D A; Allen, E; Farewell, V et al. (2004) International consensus outcome measures for patients with idiopathic inflammatory myopathies. Development and initial validation of myositis activity and damage indices in patients with adult onset disease. Rheumatology (Oxford) 43:49-54
Raben, Nina; Nagaraju, Kanneboyina; Lee, Alicia et al. (2003) Induction of tolerance to a recombinant human enzyme, acid alpha-glucosidase, in enzyme deficient knockout mice. Transgenic Res 12:171-8

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