We have recently used a new therapeutic approach - modulation of transcription factor EB (TFEB). Overexpression of TFEB in Pompe muscle cell cultures and in mice stimulated lysosomal fusion with plasma membrane leading to a discharge of lysosomal content outside the cell (a process known as lysosomal exocytosis), thus providing clearance of the stored material. In all tested Pompe model systems (developed by our group) conditionally immortalized muscle cells, isolated live muscle fibers, and the whole muscle of Pompe mice revealed that the number of large glycogen-filled lysosomes and the amount of accumulated glycogen were significantly reduced. Thus, this provides strong evidence of TFEBs potential as a therapeutic target in this disorder. Furthermore, unlike the currently available therapy, TFEB overexpression significantly relieved autophagic buildup by stimulating the formation and secretion of autophagolysosomes - a product of lysosomal-autophagosomal fusion. These data suggested that functional autophagy is a prerequisite for efficient TFEB- mediated lysosomal exocytosis. Indeed, the effects of TFEB on lysosomal clearance were much weakened in Pompe mice, in which autophagy was genetically inactivated in skeletal muscle. Thus, while searching for a better treatment for Pompe disease, we have uncovered a previously unrecognized role of autophagy in TFEB-mediated cellular clearance. We have now demonstrated that another closely related transcription factor E3 (TFE3) can also stimulate lysosomal clearance in Pompe muscle cells. Metabolic labeling and confocal microscopy of TFE3-treated immortalized Pompe myoblasts showed an impressive reduction of lysosomal size and alleviation of glycogen burden. Therefore, our data indicate that TFE3 promotes cellular clearance in Pompe myotubes and may be considered as a viable alternative to TFEB for the treatment of this and other LSDs. Of note, in muscle cells, TFE3 appears to be much more abundant compared to TFEB. Until now, TFEB was considered to be the only transcription factor involved in promoting lysosomal/autophagosomal biogenesis. Our studies (in collaboration with Dr. Rosa Puertollano, Laboratory of Cell Biology, NHLBI, NIH) identify TFE3 as a novel major regulator of lysosomal homeostasis. We have generated a new Pompe muscle cell line, which stably expresses mCherry-LAMP1 (lysosomal marker);this cell line will allow us to monitor the effect of TFE3 in live cells. We are also exploring ways to modulate TFE3 activity by pharmacological agents in skeletal muscle. Two kinases, mTORC1 and ERK, have been implicated in TFEB regulation in different cells;however, the regulation of TFE3 in skeletal muscle remains an open question. We are using mTORC1 and ERK inhibitors in Pompe cell cultures to see whether they induce nuclear translocation (activation) of endogenous TFE3. By using CHIP-sequencing method we have shown that in muscle cells (C2C12) TFE3 binds to the promoter region of many lysosomal and autophagic genes. These experiments are done in collaboration with Dr. Sartorelli (NIAMS, NIH). In addition to autophagic dysfunction, which is a major component of pathology in muscle fibers from Pompe mice, we identified yet another downstream secondary abnormality resulting from excessive lysosomal glycogen accumulation. The diseased muscle cells have a significantly increased intracellular Ca2+ concentration and calcium flux compared to those in the controls. The increase in the intracellular Ca2+ is associated with oxidative stress manifested by elevated oxidant activity (ROS). Also, Pompe myotubes exhibit increased susceptibility to H2O2-induced oxidative stress as indicated by increased cell death. In agreement with these in vitro data, we have found a dramatic increase in the levels of isoforms of the L-type calcium channel, which mediate influx of extracellular Ca2+ in skeletal muscle of Pompe mice. Activation of L-type calcium channels, as well as mitochondrial ROS production due to disturbed autophagy, may be responsible for the excessive production of oxidants in Pompe muscle cells, suggesting the potential benefits of calcium channel blockers. These findings may have important implications for designing a second generation of therapeutic drugs for Pompe disease. We have continued to analyze muscle biopsies from Pompe patients who receive enzyme replacement therapy (ERT). This project, designed to investigate the long-term impact of ERT in both infantile (IPD) and late-onset patients (LOPD), is now close to completion. Muscle biopsies from a group of patients with a confirmed diagnosis of classic infantile form of the disease (IPD), including some of the oldest survivors, were evaluated by standard histopathological approaches and by immunostaining for lysosomal and autophagosomal markers. At the time of muscle biopsy, ERT duration ranged from 0 (i.e., baseline, pre-ERT) to 8 years. The response to therapy varied considerably among the patients: some demonstrated motor gains while others experienced deterioration of motor function, either with or without a period of initial clinical benefit. Skeletal muscle pathology included fiber destruction, lysosomal vacuolation, and autophagic abnormalities (i.e., buildup), particularly in fibers with minimal lysosomal enlargement. The findings indicate that ERT does not fully halt or reverse the underlying skeletal muscle pathology in IPD. The balance between glycogen transport to lysosomes and its degradation by the drug tips in favor of lysosomal accumulation of the substrate as patients with IPD become older. The best outcomes were observed in patients who began therapy earliest - within days rather than months after birth. An early start of therapy can be better achieved with the establishment of a newborn screening (NBS) program;our results and those obtained in Taiwanese patients (Chien et al, 2008, 2009) strongly argue in favor of such a program. A group of ERT-treated LOPD patients including adult- and juvenile-onset patients, and those identified through NBS had muscle biopsies analyzed by LAMP-LC3 immunostaining, by staining for lipids, and by time-resolved Fluorescence Lifetime Imaging (FLIM);analysis was done in collaboration with Dr. Evelyn Ralston. The study revealed yet another pathological feature in Pompe skeletal muscle: widespread accumulation of autofluorescent lipofuscin-like inclusions typically associated with ageing. These inclusions were a prominent feature in muscle biopsies from all three patient groups. In many fibers, they were the major pathology remaining after ERT. Numerous lysosomes and autolysosomes loaded with lipofuscin appear to be a hallmark of LOPD skeletal muscle. Lipofuscin accumulation - a result of inefficient lysosomal degradation - may in turn exacerbate both lysosomal and autophagic abnormalities. The utility of muscle biopsies in LOPD is rightly questioned in the Pompe community. From the perspective of a clinician, muscle biopsies are not reliable for diagnostic purposes, do not always serve as a prognostic tool, and expose patients to further discomfort and anesthesia risk. Considering the shortcomings of the muscle biopsy, there is a growing tendency to avoid this procedure. However, the muscle biopsy remains invaluable in at least one regard - understanding the pathogenesis of PD and the mechanisms of skeletal muscle damage. Just as the muscle biopsy has previously enabled us to uncover autophagic defects, the technique has now facilitated the identification of a related pathological feature, namely lipofuscin accumulation. The disease may in fact be characterized as a muscle lipofuscinosis, requiring a new approach to therapy. Muscle biopsies remain an invaluable material for further analysis of molecular composition of lipofuscin inclusions and their fate 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 :
Pastore, Nunzia; Brady, Owen A; Diab, Heba I et al. (2016) TFEB and TFE3 cooperate in the regulation of the innate immune response in activated macrophages. Autophagy 12:1240-58
Lim, Jeong-A; Li, Lishu; Kakhlon, Or et al. (2015) Defects in calcium homeostasis and mitochondria can be reversed in Pompe disease. Autophagy 11:385-402
Lim, Jeong-A; Kakhlon, Or; Li, Lishu et al. (2015) Pompe disease: Shared and unshared features of lysosomal storage disorders. Rare Dis 3:e1068978
Martina, José A; Diab, Heba I; Lishu, Li et al. (2014) The nutrient-responsive transcription factor TFE3 promotes autophagy, lysosomal biogenesis, and clearance of cellular debris. Sci Signal 7:ra9
Lim, Jeong-A; Li, Lishu; Raben, Nina (2014) Pompe disease: from pathophysiology to therapy and back again. Front Aging Neurosci 6:177
Feeney, Erin J; Austin, Stephanie; Chien, Yin-Hsiu et al. (2014) The value of muscle biopsies in Pompe disease: identifying lipofuscin inclusions in juvenile- and adult-onset patients. Acta Neuropathol Commun 2:2
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
Feeney, Erin J; Spampanato, Carmine; Puertollano, Rosa et al. (2013) What else is in store for autophagy? Exocytosis of autolysosomes as a mechanism of TFEB-mediated cellular clearance in Pompe disease. Autophagy 9:1117-8
Prater, Sean N; Patel, Trusha T; Buckley, Anne F et al. (2013) Skeletal muscle pathology of infantile Pompe disease during long-term enzyme replacement therapy. Orphanet J Rare Dis 8:90

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