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Modulation of mTOR signaling as a strategy for the treatment of Pompe disease

2017; Springer Nature; Volume: 9; Issue: 3 Linguagem: Inglês

10.15252/emmm.201606547

1757-4684

Jeong‐A Lim, Lishu Li, Orian S. Shirihai, Kyle Trudeau, Rosa Puertollano, Nina Raben,

Glycogen Storage Diseases and Myoclonus

Research Article27 January 2017Open Access Source DataTransparent process Modulation of mTOR signaling as a strategy for the treatment of Pompe disease Jeong-A Lim Jeong-A Lim Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Lishu Li Corresponding Author Lishu Li [email protected] orcid.org/0000-0003-2133-6702 Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Orian S Shirihai Orian S Shirihai orcid.org/0000-0001-8466-3431 Department of Medicine, Obesity and Nutrition Section, Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Kyle M Trudeau Kyle M Trudeau Department of Medicine, Obesity and Nutrition Section, Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Rosa Puertollano Corresponding Author Rosa Puertollano [email protected] orcid.org/0000-0002-1106-5489 Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Nina Raben Corresponding Author Nina Raben [email protected] orcid.org/0000-0001-9519-3535 Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Jeong-A Lim Jeong-A Lim Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Lishu Li Corresponding Author Lishu Li [email protected] orcid.org/0000-0003-2133-6702 Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Orian S Shirihai Orian S Shirihai orcid.org/0000-0001-8466-3431 Department of Medicine, Obesity and Nutrition Section, Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Kyle M Trudeau Kyle M Trudeau Department of Medicine, Obesity and Nutrition Section, Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Rosa Puertollano Corresponding Author Rosa Puertollano [email protected] orcid.org/0000-0002-1106-5489 Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Nina Raben Corresponding Author Nina Raben [email protected] orcid.org/0000-0001-9519-3535 Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA This article has been contributed to by US Government employees and their work is in the public domain in the USA Search for more papers by this author Author Information Jeong-A Lim1,2,†, Lishu Li *,1,†, Orian S Shirihai3, Kyle M Trudeau3, Rosa Puertollano *,2 and Nina Raben *,1 1Laboratory of Muscle Stem Cells and Gene Regulation, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA 2Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD, USA 3Department of Medicine, Obesity and Nutrition Section, Evans Biomedical Research Center, Boston University School of Medicine, Boston, MA, USA † These authors contributed equally to this work *Corresponding author. E-mail: [email protected] *Corresponding author. Tel: +1 301 451 2361; E-mail: [email protected] *Corresponding author. Tel: +1 301 496 1474; E-mail: [email protected] EMBO Mol Med (2017)9:353-370https://doi.org/10.15252/emmm.201606547 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mechanistic target of rapamycin (mTOR) coordinates biosynthetic and catabolic processes in response to multiple extracellular and intracellular signals including growth factors and nutrients. This serine/threonine kinase has long been known as a critical regulator of muscle mass. The recent finding that the decision regarding its activation/inactivation takes place at the lysosome undeniably brings mTOR into the field of lysosomal storage diseases. In this study, we have examined the involvement of the mTOR pathway in the pathophysiology of a severe muscle wasting condition, Pompe disease, caused by excessive accumulation of lysosomal glycogen. Here, we report the dysregulation of mTOR signaling in the diseased muscle cells, and we focus on potential sites for therapeutic intervention. Reactivation of mTOR in the whole muscle of Pompe mice by TSC knockdown resulted in the reversal of atrophy and a striking removal of autophagic buildup. Of particular interest, we found that the aberrant mTOR signaling can be reversed by arginine. This finding can be translated into the clinic and may become a paradigm for targeted therapy in lysosomal, metabolic, and neuromuscular diseases. Synopsis Muscle loss is a feature of lysosomal glycogen storage disorder Pompe disease, also known as acid maltase deficiency. mTORC1 is a key regulator of protein synthesis in muscle. Myotubes and whole muscle from Pompe mice display aberrant mTOR signaling. mTOR activity is diminished in the diseased muscle cells. mTOR is not fully inactivated in the diseased cells after starvation. mTOR remains at the lysosome irrespective of nutrient availability. Lysosomal acidification defect and activation of the AMPK-tuberous sclerosis complex (TSC) pathway are the major culprits responsible for the defective mTOR signaling. TSC inhibition and l-arginine treatments largely correct the defects. Introduction Mechanistic target of rapamycin (mTOR), a highly conserved serine/threonine kinase, forms two multiprotein complexes, mTOR complex 1 (TORC1) and mTOR complex 2 (TORC2). Rapamycin-sensitive mTORC1 complex responds to multiple signals, and when activated, changes the cell metabolism from catabolic to anabolic program, thus promoting protein synthesis and cell growth while repressing autophagy. The role of lysosome in controlling metabolic programs is emphasized by the discovery that activation of this potent anabolic regulator happens at the lysosome in a process mediated through an amino acid-sensing cascade involving V-ATPase, Ragulator, and Rag GTPases. When cells have sufficient amino acids, V-ATPase promotes the guanine nucleotide exchange factor (GEF) activity of Ragulator leading to the formation of active RagA/B·GTP complex at the lysosome; in this active configuration, Rag binds to and delivers mTORC1 to the lysosome where the kinase is activated by Rheb (Ras homolog enriched in brain), a small GTPase that is fixed to the lysosomal surface (Sancak et al, 2010; Zoncu et al, 2011; reviewed in Bar-Peled & Sabatini, 2014). Rheb is a downstream target of tuberous sclerosis complex (TSC) that functions as a GTPase-activating protein (GAP) and converts active GTP-bound Rheb to inactive GDP-bound form, thus inhibiting mTORC1 activity (Inoki et al, 2003; Demetriades et al, 2014; Menon et al, 2014). The recent view of the lysosomes as a site of the mTORC1 activation, along with the long-established role of this kinase in the control of muscle mass, has made the study of mTORC1 signaling of particular interest to research on Pompe disease, a severe muscle wasting disorder characterized by altered lysosomal function. Profound muscle atrophy is a hallmark of Pompe disease, a rare genetic disorder caused by a deficiency of acid alpha-glucosidase (GAA), the enzyme that breaks down glycogen to glucose within lysosomes. Absence of the enzyme leads to a rapidly fatal cardiomyopathy and skeletal muscle myopathy in infants; low levels of residual enzyme activity are associated with childhood and adult-onset progressive skeletal muscle myopathy usually without cardiac involvement (Van der Ploeg & Reuser, 2008). The introduction of enzyme replacement therapy (ERT) changed the natural course of the infantile form because of the notable effect in cardiac muscle; however, the effect in skeletal muscle has been modest at best (Kishnani et al, 2007; Strothotte et al, 2010; Van der Ploeg et al, 2010; Prater et al, 2012). The pathophysiology of muscle damage involves enlargement and rupture of glycogen-filled lysosomes, disturbance of calcium homeostasis and endocytic trafficking, mitochondrial abnormalities, and autophagic defect (Thurberg et al, 2006; Lim et al, 2014, 2015; Nascimbeni et al, 2015). The search for a more effective therapy is currently underway. However, even if muscles are cleared of glycogen and autophagic debris—the two major pathologies in Pompe disease—profound muscle wasting will persist and will remain a major therapeutic challenge. The signaling pathways responsible for the loss of muscle mass in Pompe disease are largely unknown, and the reported studies on mTOR signaling yielded conflicting results. The temptation to boost protein synthesis by stimulating the mTOR pathway is reflected in recent data showing that leucine supplementation halted the decline in muscle mass and reduced glycogen accumulation in GAA-KO muscle (Shemesh et al, 2014). On the other hand, clearance of the excess muscle glycogen was reported following treatment of GAA-KO mice with mTORC1 inhibitor rapamycin; co-administration of rapamycin with the replacement enzyme (recombinant human GAA; alglucosidase alfa, Myozyme®, Genzyme Corporation, a Sanofi Company) reduced muscle glycogen content more than rhGAA or rapamycin alone (Ashe et al, 2010). This study is the first systematic analysis of the upstream regulators and downstream targets of mTORC1 in Pompe muscle cells. We have found a dysregulation of mTOR signaling in the diseased cells—a diminished basal level of mTOR activity, weakened response to cellular stress, and the failure to reallocate mTOR away from lysosomes upon starvation. We have elucidated the molecular mechanisms underlying mTOR dysregulation in Pompe disease and identified points for therapeutic intervention along the mTOR signaling pathway. Furthermore, we have used targeted approaches to reverse the abnormalities in this prototypical lysosomal storage disorder. Results Perturbed mTOR signaling in cultured Pompe muscle cells To explore the mTOR signaling pathway in Pompe disease, we took advantage of a recently developed in vitro model of the disease—GAA-deficient myotubes. These myotubes are formed from conditionally immortalized myoblasts derived from the GAA-KO mice; differentiated myotubes, but not myoblasts, contain large glycogen-filled lysosomes, thus replicating the disease phenotype (Spampanato et al, 2013). Since mTOR kinase is a principal regulator of protein synthesis, we evaluated the rate of protein synthesis in KO cells by using a surface sensing of translation (SUnSET) method, which relies on the incorporation of puromycin into nascent peptide chains resulting in the termination of their elongation (Goodman et al, 2011). A significant decrease (~60%) in anti-puromycin immunoreactivity was detected in KO myotubes compared to WT controls, a finding consistent with a reduction in protein translation (Fig 1A and B). Figure 1. Decreased protein translation in KO cellsSurface sensing of translation (SUnSET) analysis was used to evaluate the incorporation of puromycin into nascent polypeptides. Representative image of Western blot analysis of WT and KO cells treated with puromycin (1 μM) for 30 min. Western blot with anti-vinculin antibody and Ponceau S staining were used as loading controls. Total intensity of puromycin-labeled polypeptides was quantified. Student's t-test was used for statistical analysis. Data are mean ± SE. ***P < 0.001 (P = 0.0009; n = 4). Source data are available online for this figure. Source Data for Figure 1 [emmm201606547-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint Phosphorylation of the 4E-BP1 repressor protein, a downstream mTOR target (Hay & Sonenberg, 2004) reduces its affinity for eIF4E which can then associate with eIF4G to form active eIF4E·eIF4G complex, thus initiating cap-dependent translation. Unexpectedly, the level of phosphorylated 4E-BP1 (p-4E-BP1T37/46) was significantly higher in KO myotubes compared to WT controls (Fig 2A) but the abundance of total 4E-BP1 followed a similar trend, thus confounding the assessment of mTORC1 involvement in protein translation. Therefore, we compared the abundance of eIF4E bound to 4E-BP1 in WT and KO cells; immunoprecipitation of 4E-BP1 from the WT and KO cell lysates followed by Western blotting with eIF4E (and vice versa) antibodies showed an increased eIF4E/4E-BP1 binding in KO cells (Fig 2B), suggesting insufficient mTORC1 activity and suppression of protein synthesis. Consistent with these data, the abundance of non-phosphorylated 4E-BP1 (active form), which binds to and reduces eIF4E availability, was increased in KO cells (Fig 2A). Figure 2. Dysregulation of mTOR signaling and activation of eIF2α/ATF4 pathway in KO cells Representative Western blot of total lysates of WT and KO myotubes. All three forms of 4E-BP1, phosphorylated (p-4E-BP1T37/46), non-phosphorylated (Non-p-4E-BP1T46), and total, are increased in KO cells. WT and KO cell lysates were immunoprecipitated (IP) with either anti-4E-BP1 (left) or anti-eIF4E (right); the immunoprecipitated proteins were then probed with eIF4E or 4E-BP1, respectively. Increased eIF4E/4E-BP1 binding is seen in KO cells. Immunoprecipitation with IgG was included as negative control. Representative Western blot of total lysates of WT and KO myotubes with the indicated antibodies. The levels of eIF2αS51 and ATF4 are increased in KO cells; graphs represent mean ± SE of p-eIF2α/eIF2α ratios (n = 3) and ATF4 (n = 3) levels. *P < 0.05, Student's t-test. Immunoblot analysis of WT and KO lysates showing an increase in both total and p-4E-BP1S65 and a decrease in p-4E-BP1S65/4E-BP1 ratio in KO cells; graph represents mean ± SE (n = 6). **P < 0.01, Student's t-test. Immunoblot analysis of WT and KO lysates showing a decrease in the p-S6K/S6K (n = 5) and p-S6/S6 (n = 6) ratios in KO cells; graphs represent mean ± SE. *P < 0.05, Student's t-test. Data information: Vinculin was used as a loading control (vinculin and its splice variant are commonly seen in both WT and KO, although the ratio of these forms is different; both bands are used for quantitative analysis). All blots (except for IP) are representative of at least three independent experiments. Source data are available online for this figure. Source Data for Figure 2 [emmm201606547-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint A striking enhancement of 4E-BP1 translation despite the general inhibition of protein synthesis in KO cells prompted us to look at the eIF2α/ATF4 pathway. The phosphorylation of eIF2α represses global translation, but leads to increased translation of ATF4 (activation transcription factor 4), which regulates the transcription of many genes [reviewed in Sonenberg and Hinnebusch (2009)]; 4E-BP1 is a potential target of ATF4 because Eif4ebp1 gene contains the ATF4-responsive elements (Kilberg et al, 2009). Indeed, we have found an increase in the levels of p-eIF2αS51 and ATF4 in the KO (Fig 2C). eIF2α/ATF4 pathway plays an important role in the adaptation to stress caused by the generation of reactive oxygen species (Rajesh et al, 2015)—a condition observed in KO cells (Lim et al, 2015). To better assess mTORC1 activity, we evaluated 4E-BP1S65 phosphorylation which is a more reliable indicator since this site is serum- and rapamycin-sensitive, whereas p-4E-BP1T37/46 is only partially sensitive to these treatments (Gingras et al, 2001). Again, we found an increase in both forms, but the ratio of p-4E-BP1S65/total was decreased in Pompe muscle cells, suggesting a diminished mTOR activity (Fig 2D). We then studied the phosphorylation state of S6K, a direct mTORC1 substrate that phosphorylates the ribosomal protein 6 (S6) of the 40S ribosomal subunit. The ratios of p-S6KT421/S424/total S6K and p-S6S235/236/total were decreased in the KO cells (Fig 2E), again suggesting a compromised mTORC1 activity. Next, we began analysis of the upstream inputs to mTORC1—the phosphorylation status of the major upstream regulators of mTOR, AKT, and the AMP-activated protein kinase (AMPK), which have opposite effect on mTORC1 activity. AKT activates mTORC1 through the inhibitory phosphorylation of the tuberous sclerosis complex 2 (TSC2T1426); AMPK-mediated phosphorylation of TSC2S1387 leads to its activation and suppression of mTORC1 (Inoki et al, 2003; Huang & Manning, 2008; Sengupta et al, 2010). The phosphorylation levels of AKT (p-AKTS473) were similar in WT and KO cells on days 4-5 in differentiation medium (not shown) and were even increased in the KO at a later stage of myotubes differentiation (Fig 3A). Despite this increase, AKT-mediated phosphorylation of TSC2T1426 was decreased in KO cells, suggesting a failure of AKT to inhibit TSC2 (Fig 3A). On the other hand, the level of TSC2, as well as the levels of active phosphorylated form of AMPKα (p-AMPKαT172) and its downstream target, p-ACCS79, was increased in KO cell lysates (Fig 3A). AMPKα is activated under low-energy conditions (Sengupta et al, 2010), and its increased activity in KO cells under basal condition is not unexpected; the failure to digest lysosomal glycogen to glucose may deprive muscle cells of a source of energy (Fukuda et al, 2006). Figure 3. Activation of AMPK-TSC2 signaling pathway in KO cells Immunoblot analysis of the phosphorylation levels of AKTS473, TSCT1462, AMPKαT172, and ACCS79 in WT and KO cell lysates. Graphs represent mean ± SE. n = 6 for p-AMPKα; n = 3 for TSC2; n = 3 for p-ACC/ACC. *P < 0.05, **P < 0.01, Student's t-test. Vinculin was used as a loading control. WT and KO myotubes were lysed and subjected to fractionation to obtain lysosome-enriched fractions. The isolated fractions were then examined by Western blot showing increased levels of total and p-TSC2S1387, total and p-AMPKαT172, and total LKB1 in KO cells. Graphs represent mean ± SE. n = 5 for each, p-AMPKα and TSC2; n = 4 for LKB1. *P < 0.05, **P < 0.01, Student's t-test. RHEB was used as a loading control. The blot for RHEB is a composite image; the samples were run on the same gel. Source data are available online for this figure. Source Data for Figure 3 [emmm201606547-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint Recent data have demonstrated that LKB1-mediated phosphorylation of AMPKα in response to energy stress takes place at the endosomal/lysosomal surface leading to inactivation of mTOR and its dissociation from endosome (Zhang et al, 2014). Therefore, we examined the levels of LKB1, AMPKα, and TSC2 in the lysosome-enriched fraction from KO cells (the purity of this fraction is shown in Appendix Fig S1). Indeed, the levels of all three proteins were elevated in KO cells compared to WT (Fig 3B). Furthermore, the levels of p-AMPKαT172 and active phosphorylated form of TSC2S1387 (AMPK-mediated phosphorylation) were increased in the lysosomal fraction in KO cells (Fig 3B). Since TSC2 inhibits mTOR by inactivating the small GTPase Rheb (Inoki et al, 2003), increased levels of AMPK activated TSC2 at the lysosomal surface may explain diminished basal mTOR activity in KO cells. Of note, AKT-mediated phosphorylated form of TSC2T1426 was not detected in the lysosomal fraction (not shown). In vivo results mirror the findings in cultured cells To validate in vivo the relevance of our in vitro findings, we analyzed mTOR signaling in whole muscle of the GAA-KO mice. For these studies, we have used the white part of the gastrocnemius muscle, which are most resistant to ERT (Lim et al, 2014). Significant portions of 4E-BP1 and S6 remained non-phosphorylated (decreased ratios of p-4E-BP1T37/46/total 4E-BP1 and p-S6S235/236/total S6) in GAA-KO muscle compared to WT, suggesting a decrease in mTORC1 activity (Fig 4A and B). Of note, similar to what was found in cultured KO cells, the levels of both p-4E-BP1 and total 4E-BP1 were increased in GAA-KO muscle, consistent with our previous data (Raben et al, 2010). Figure 4. Disturbance of mTOR signaling in vivo in GAA-KO miceMuscle biopsies (white part of gastrocnemius) were obtained from 4- to 6-month-old WT and GAA-KO (KO) mice. A, B. Western blot analysis of whole muscle lysates from WT and GAA-KO mice with the indicated antibodies. Graphical presentation of the data is shown in (B). Data illustrate the mean ± SE. n = 6 for p-4E-BP1/4E-BP1, p-S6/S6, p-PRAS40/PRAS40, and p-TSC2/TSC2; n = 5 for p-AMPKα; n = 3 for LKB1. *P < 0.05, **P < 0.01, ***P < 0.001, Student's t-test. GAPDH was used as a loading control. C. An increase in ADP/ATP ratio in whole muscle of GAA-KO mice. Data illustrate the mean ± SE. n = 3 for WT; n = 4 for KO. *P < 0.05, Student's t-test. D, E. Muscle tissues derived from WT (n = 3) and GAA-KO mice (n = 4) were homogenized in lysis buffer and subjected to fractionation to obtain lysosome-enriched fractions. The isolated fractions were then examined by Western blot showing increased levels of total mTOR, TSC2, and AMPKα in GAA-KO. Graphical presentation of the data is shown in (E). Graphs represent mean ± SE. **P < 0.01, ***P < 0.001, Student's t-test. RHEB and Ponceau S staining were used to verify equal protein loading. F. Immunostaining of a single fiber from a GAA-KO mouse with anti-LAMP1 (red) and anti-mTOR (green) antibodies showing extensive co-localization of the two stains. Scale bar: 10 μm. Source data are available online for this figure. Source Data for Figure 4 [emmm201606547-sup-0008-SDataFig4.pdf] Download figure Download PowerPoint No significant changes in the level of active p-AKTS473 were seen in GAA-KO muscle (Fig 4A). Furthermore, the level of phosphorylated PRAS40 (proline-rich AKT substrate of 40 kDa; p-PRAS40T246), a downstream target of AKT, was also no different in GAA-KO muscle compared to WT, but the total level of PRAS40 was significantly increased (Fig 4A and B). Because AKT- mediated phosphorylation of PRAS40 is known to relieve the inhibitory effect of PRAS40 on mTORC1 (Sancak et al, 2007), an increase in the amount of hypophosphorylated PRAS40 would lead to the inhibition of mTORC1 activity. As in cultured KO cells, the phosphorylation levels of p-AMPKαT172, the master regulator of cellular energy homeostasis, was increased in GAA-KO compared to those in WT muscle (Fig 4A and B). Furthermore, we have found an elevated ADP/ATP ratio in GAA-KO muscle (Fig 4C), indicating energy deprivation—a condition known to trigger AMPKα activation. The rise in the amount of active p-AMPKαT172 also agrees with both an increase in the level of its upstream activation kinase LKB1 and an increase in the phosphorylation level of its direct downstream target, p-TSC2S1387 (Fig 4A and B). Again, as in cultured KO cells, the abundance of AMPKα and TSC2 was increased in lysosome-enriched fraction from GAA-KO muscle (Fig 4D and E). Unexpectedly, the level of mTOR was also increased in the lysosomal fraction, and immunostaining of isolated muscle fibers with mTORC1 and lysosomal marker LAMP1 confirmed a striking co-localization of the two stains (Fig 4D–F). It appears that the activity of mTOR in GAA-KO muscle is reduced despite its excessive accumulation at the lysosome. Thus, the mechanism of perturbed mTORC1 signaling in GAA-KO muscle and in cultured KO cells is similar in that the suppression of mTORC1 activity is AKT-independent, and that a decrease in the ATP content leads to AMPKα-mediated mTOR inhibition. mTOR is locked on the lysosome under nutrient deprivation in KO cells Recent data have shown that multiple proteins which reside in the cytosol and on the lysosomes are engaged in the recruitment of mTORC1 to the lysosome (activation) and its release from the lysosome (inactivation) (Bar-Peled et al, 2012; Demetriades et al, 2014, 2016; Zhang et al, 2014). We reasoned that lysosomal enlargement and the acidification defect in the diseased muscle cells (Fukuda et al, 2006; Takikita et al, 2009) would affect the interaction of the components of the complex machinery responsible for the proper localization and activation/inactivation of mTORC1. To investigate the relationship between the activity of mTOR and its intracellular localization in Pompe muscle, we again turned to the in vitro model. As expected, by 2 h of starvation 4E-BP1 and S6 were almost completely dephosphorylated in WT cells; in contrast, the degree of dephosphorylation in the KO was less pronounced, particularly when the cells were treated with medium lacking only amino acids in the presence of dialyzed serum containing growth factors (Fig 5A and B). A weakened mTORC1 response in KO cells is also observed after refeeding subsequent to 2 h of starvation. In WT cells, the phosphorylation of 4E-BP1 after 30 min rebounds to a level that is higher than that at the basal level, whereas in the KO it does not, as shown by the abundance of hypophosphorylated forms in the diseased cells; consistent with this, the levels of non-phosphorylated 4E-BP1 in the KO are much higher than those in the WT at both 15 and 30 min after refeeding (Fig 5C). Of note, the levels of S6K and S6 in the KO were similar to those in WT following refeeding, suggesting a differential effect on 4E-BP1 versus S6K (Fig 5C). This contrary activity of mTORC1 toward its substrates has been reported in other systems (Liu et al, 2004; Choo et al, 2008). Figure 5. KO cells exhibit a diminished response to starvation and inadequate activation after refeeding WT and KO myotubes were starved (HBSS) for 0, 1, and 2 h, lysed and subjected to immunoblot analysis with the indicated antibodies. The levels of p-4E-BP1T37/46 and p-S6S235/236 in KO are higher compared to WT at 1 and 2 h of starvation. Graph shows an increase in p-4E-BP1T37/46/total (n = 5) and p-S6S235/236/total (n = 3) ratios in KO compared to WT after 2 h starvation; the data represent mean ± SE. *P < 0.05, **P < 0.01, Student's t-test. Vinculin was used as a loading control. The blots are composite images; the samples were run on the same gel. WT and KO myotubes were incubated in HBSS with or without dialyzed serum for 2 h, lysed, and subjected to immunoblot analysis with the indicated antibodies. Both p-4E-BP1T37/46 and p-4E-BP1S65 antibodies were used for the experiments. The degree of 4E-BP1 dephosphorylation after amino acid starvation (AA) is different from that after HBSS in KO (*P < 0.05) but not in WT cells (n.s., not significant). Graph represents mean ± SE; Student's t-test. n = 3 for each condition. WT and KO myotubes were starved (HBSS) for 2 h, and then refed for 15 and 30 min using differentiation medium as shown schematically. Cell lysates were then subjected to immunoblot analysis with the indicated antibodies. Significant amounts of non-phosphorylated and hypophosphorylated forms of 4E-BP1 are still seen after 15 and 30 min of refeeding in KO cells; the levels of S6K and S6 in the KO were not different from those in controls following refeeding. WT and KO myotubes were starved as in (A). The level of p-ULK1S757 in KO is higher than in WT after 2 h starvation. The p-ULK1/vinculin (n = 4) and LC3-II/total (LC3-I + LC3-II; n = 3) ratios at 2 h after starvation were calculated. Data are mean ± SE; *P < 0.05, ***P < 0.001, Student's t-test. The blots are composite images; the samples were run on the same gel. Source data are available online for this figure. Source Data for Figure 5 [emmm201606547-sup-0009-SDataFig5.pdf]