Our previously reported experience of treating Pompe mice (an acid alpha glucosidase knockout strain) and the experience in trials in humans with enzyme replacement therapy (ERT), demonstrated that the drug is highly effective in clearing glycogen stored in cardiac muscle cells and poorly effective for skeletal muscle cells. This failure of skeletal muscle response to recombinant enzyme in Pompe disease has become a major focus of our attention and has led us into active new areas of cell biology that in themselves add great interest to our activities. In Pompe fast fibers there are large areas of autophagic accumulation, which may occupy close to half the diameter of a fiber. Massive autophagic buildup in Pompe myofibers results not only from inefficient delivery of autophagic cargo to the lysosomes but also from the induction of autophagy. We have shown that this induction may be an unintended consequence of a cellular attempt to down-regulate glycogen synthesis by activating glycogen synthase kinase (GSK), a recently appreciated regulator of autophagy. We have also shown that GSK up-regulates autophagy in C2C12 muscle cell line and in primary Pompe myoblasts. The autophagic inclusions disrupt the microtubular network and the contractile apparatus of muscle fibers. Furthermore, autophagic buildup poses a problem for the lysosomal delivery of the therapeutic enzyme which traffics to its destination via mannose-6-phosphate receptor mediated endocytosis. Since autophagic and endocytic pathways converge at several steps along the way it is not surprising that the drug ends up the autophagic area. Therefore, the removal of autophagic buildup seemed a reasonable approach to improve the therapy. To this end we have made Pompe mouse strains in which a critical autophagic gene, Atg5 or Atg7, is inactivated specifically in skeletal muscle. The tissue-specific inactivation of autophagic genes is warranted, because it was shown that suppression of autophagy in the whole body in mice leads to early post-natal lethality. As expected, autophagy was suppressed in muscles in both (Atg5 and Atg7) autophagy-deficient Pompe mice as shown by the absence of LC3II, a highly specific marker for autophagic vesicles (autophagosomes). Expansion of lysosomes, a hallmark of Pompe disease, persisted, but autophagic accumulation was absent. ERT in autophagy-deficient Pompe mice resulted in a dramatic reduction in the glycogen level approaching wild type levels. Glycogen clearance was also demonstrated by PAS staining of muscle biopsies and by immunostaining of isolated single fibers for autophagosomal and lysosomal markers. This outcome was never seen in Pompe mice in which autophagy was not tampered with. Of note, ERT plus suppression of autophagy converts Pompe mice into muscle-specific autophagy-deficient wild type mice;the health (longevity, mobility, and single fiber contractility) of these mice is far better than the health of Pompe mice. The accumulation of dysfunctional mitochondria, mild atrophy and age-dependent decrease in force have been reported in muscle-specific autophagy-deficient wild type mice, but these abnormalities seem to be a reasonable price to pay for the reversal of pathology in Pompe disease. To facilitate the development of autophagy inhibitors we have developed a Pompe mouse model, in which autophagosomes are labeled with LC3. These mice are ideally suited for in vivo testing autophagy inhibitors. We have also used these green Pompe mice for the generation of immortalized myoblast cell lines;these cell lines are being used to modulate autophagy in vitro. In yet another approach to rendering Pompe skeletal fast muscle fibers responsive to therapy, we have used the over-expression in muscle of the major regulatory protein, PGC1alpha, which changes some of the characteristics of fast muscle fibers into those of slow muscle fibers. To turn the therapy-resistant fibers into fibers amenable to therapy, we generated transgenic Pompe mice, which express PGC1 alpha in muscle. We have shown that, indeed, PGC1alpha expression resulted in a successful switch from fast to slow fibers, but they still responded poorly to therapy. Although our attempt to improve therapy by transgenic expression of PGC-1 failed, we thought that fiber-type conversion as a way to increase responsiveness to ERT is worth exploring further. The failure of PGC-1 experiments resulted from the effect of this protein on multiple aspects of energy metabolism, including glycogen metabolism. Therefore we looked for a different way to convert fiber types. We have made transgenic Pompe mice, which express myosin-encoded miR-499 under the control of the MCK promoter. The reprogramming of fast myofibers to a slower myofiber type in MCK-miR-499:WT mice is achieved by a change in the contractile rather than the metabolic properties. We are currently analyzing these mice. In a study of single muscle fibers from infants with Pompe disease obtained before and after ERT, we have noted that the dominant pathology before therapy is the presence of huge, glycogen-filled lysosome but the autophagic buildup, which is so prominent in the milder adult form of the disease, is negligible. However, in infants on ERT, as the glycogen-filled lysosomes are shrunk, autophagic buildup becomes visible. The data point to the possibility of differences in the pathogenesis of Pompe disease in infants and adults, with possible implications for the design of therapy. We have also shown that autophagic buildup in muscle fibers persists after years on therapy, and it may be a reason for unsatisfactory response to therapy. We are now conducting a long-term study and a larger number of samples to evaluate the fate of autophagic buildup in ERT-treated patients.

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Raben, Nina; Puertollano, Rosa (2016) TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress. Annu Rev Cell Dev Biol :
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Spampanato, Carmine; Feeney, Erin; Li, Lishu et al. (2013) Transcription factor EB (TFEB) is a new therapeutic target for Pompe disease. EMBO Mol Med 5:691-706

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