extfieldsofstudy: Biology Medicine

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Bmi1 enhances skeletal muscle regeneration through MT1-mediated oxidative stress protection in a mouse model of dystrophinopathy

Enhanced polycomb complex protein Bmi1 expression in adult stem cells of the skeletal muscle leads to improved muscle function in a model of Duchenne Muscular Dystrophy via metallothionein1-mediated protection from oxidative stress.

Skeletal muscle is characterized by a remarkable capacity to regenerate after injury, mainly due to the function of satellite cells, the main skeletal muscle stem cells (Brack and Rando, 2012;Wang and Rudnicki, 2012). Polycomb group (PcG) proteins are essential regulators of stem cell function during normal development and in adult organs. They form multi-protein chromatin-associated complexes that play an essential role in the genome-wide epigeneticmediated remodeling of gene expression during myogenic differentiation of satellite cells, mainly through posttranslational modifications of histones (Asp et al., 2011). Ezh2 and Bmi1 are required for adult satellite cell homeostasis and proliferation in response to muscle injury, an effect mediated at least in part by repression of the ink4a locus (Juan et al., 2011;Robson et al., 2011). Importantly, although Bmi1 is expressed in several types of cancer and its mechanism of action may be similar in a non-neoplastic and neoplastic context, its overexpression does not initiate tumorigenesis (He et al., 2009;Yadirgi et al., 2011).
An emerging role for PcG proteins is their involvement in DNA repair (Liu et al., 2009;Facchino et al., 2010;Ismail et al., 2010;Ginjala et al., 2011;Pan et al., 2011). Bmi1 / -derived cells show significant mitochondrial dysfunction accompanied by sustained increase in reactive oxygen species (ROS) production that are sufficient to engage the DNA repair pathway (Liu et al., 2009), which is in turn impaired, thus leading to a magnified cellular damage. The balance between intracellular ROS and antioxidant molecules is vital in determining the rate of oxidative damage accumulation and the impaired function of satellite cells in aging and in myopathies, in which decreased anti-oxidative capacity has been documented (Fulle et al., 2005;Whitehead et al., 2006;Tidball and Wehling-Henricks, 2007). X-linked Duchenne muscular dystrophy (DMD) is the most common primary myopathy caused by the loss of the dystrophin protein from the plasma membrane, which causes loss of its integrity and fiber damage during repeated cycles of muscle degeneration and regeneration (Duncan, 1989). The proliferative capacity of myogenic cells was reported to be rapidly exhausted in dystrophin-deficient muscle, also because they are more sensitive to oxidative stress injury, leading to reduced and defective regeneration of the muscle as the disease progresses (Blau et al., 1983(Blau et al., , 1985Disatnik et al., 1998). Moreover, enzymatic adaptations to exercise-induced production of ROS and free radical damage are significantly decreased in dystrophic compared with normal muscles (Faist et al., 1998(Faist et al., , 2001. Overall, an impaired protection against ROS in dystrophic muscle appears to contribute to disease progression as also indicated by the beneficial, albeit transient, effect of antioxidants in ameliorating the skeletal muscle pathophysiology of DMD patients (Whitehead et al., 2008).
Metallothionein 1 (MT1) and MT2 are ubiquitously expressed (Kägi and Hunziker, 1989) low molecular weight, cysteine-rich zinc binding proteins. Although the role of MT1 in promoting cell proliferation is controversial (Smith et al., 2008), studies on MT-null liver cells showed their failure to regenerate after oxidative stress injury (Oliver et al., 2006).
Here, we show that overexpression of Bmi1 in the satellite cells significantly improves muscle strength through enhanced MT1-mediated protection of these cells from oxidative stress in a mouse model of dystrophinopathies but not after acute traumatic injury.

Bmi1 expression in mouse models of acute traumatic and chronic degenerative skeletal muscle injuries
To understand the potential impact of fine tuning Bmi1 expression in muscle injury, we characterized its expression profile in satellite cells at representative time points (3 and 10 d after i. and 10 d.a.i. (mean ± SEM from three independent experiments with n = 3 uninjured, n = 3 3 d.a.i., and n = 2 10 d.a.i.; ***, P < 0.001). (c) Immunofluorescence for Pax7 and Bmi1 on muscle sections from uninjured mice, injured mice (10 d.a.i.), and mdx mice (representative results from n = 4 uninjured, n = 2 3 d.a.i., n = 2 10 d.a.i., and n = 4 mdx). Arrows indicate Bmi1 staining intensity (expression level) in Pax7 +ve satellite cells in three different conditions (uninjured, 10 d.a.i., and mdx). (d) Quantification of Bmi1 staining of Pax7 +ve cells with pixel intensity above 400 at 3 and 10 d.a.i. compared with uninjured controls (mean ± SEM from two independent experiments with n = 4 uninjured, n = 2 3 d.a.i., n = 2 10 d.a.i., and n = 4 mdx; *, P < 0.05). (e) Bmi1 expression in satellite cells obtained from mdx single fibers after switching from growth to differentiation-inducing culturing conditions (48 h in differentiation conditions) compared with control satellite cells (mean ± SEM from two independent experiments with n = 3 for each condition; *, P < 0.05). Bar, 50 µm. (Fig. 1,c and d [quantification]). Interestingly, although Bmi1 expression increased significantly in control satellite cell cultures obtained from single fibers after switching from growth to differentiation-inducing conditions, no significant change was observed in mdx satellite cells (Fig. 1 e). These results show that the levels of Bmi1 expression are differentially regulated during the regenerative process in a pathological specific context.

Conditional Bmi1 expression increases the satellite cell pool and does not prevent myogenic differentiation in vitro
To explore the biological effect of enhancing Bmi1 expression in the satellite cells, we used a mouse line where Bmi1 expression can be activated in a cell-and time-specific fashion upon Cre-mediated recombination (Yadirgi et al., 2011). Transduction of satellite cell cultures derived from STOP-FloxBmi1 muscles with Adeno-Cre (A-Cre) virus induced efficient recombination and increased Bmi1 expression at protein injury [d.a.i.]) in a well-established model of acute traumatic muscle injury: the freeze injury model (Gayraud-Morel et al., 2007). Satellite cells were isolated 3 and 10 d.a.i. by magnetic activated cell sorting using SM/C-2.6 antibody (Fukada et al., 2004; Fig. 1 a). qRT-PCR analysis revealed significant increase in the expression of Bmi1 in these cells at both time points (Fig.1 b), a result which was confirmed by an increased percentage of Pax7 +ve cells expressing high level of Bmi1 in the muscle tissue, as assessed by double immunolabeling for Bmi1 and Pax7 and quantification of staining intensity (van der Laak et al., 2000;Gavet and Pines, 2010;Fig. 1, c and d [quantification]).
The mdx mouse is a well characterized model of chronic degenerative muscle pathology (Bulfield et al., 1984), which is genetically and biochemically similar to the human DMD (Collins and Morgan, 2003). We observed down-regulation of Bmi1 expression in Pax7 +ve satellite cells in the muscle tissue STOPFloxBmi1 single fibers, infected with A-GFP or A-Cre viruses and immunostained for Bmi1 (representative results from three independent experiments with n = 3 for each condition). (c and d) Levels of expression of Bmi1 are analyzed with WB (c; representative blot from 2 independent experiments with n = 4 biological samples) and qRT-PCR on the same samples (d; mean ± SEM from three independent experiments with n = 3 for each condition; ***, P < 0.001).
(e and f) Representative images (e; 6 independent experiments with n = 8 for each condition) of satellite cell cultures infected with either A-GFP or A-Cre viruses (Bmi1 Over cells; f) and stained for Pax7, MyoD, and MyHC, 2 and 5 d.a.i. in differentiation conditions. (g and h) Quantification of the findings in normal (g; mean ± SEM from six independent experiments with n = 8 for each condition; *, P < 0.05) and dystrophic context (h; mean ± SEM from 4 independent experiments with n = 4 for each condition; **, P < 0.01). (i and j) Representative images of the BrdU staining on A-GFP (i) and A-Cre-infected satellite cells (j) in four different conditions (six independent experiments with n = 8 for each condition): growth, 2 d differentiation, 5 d differentiation, and restimulation (i and j). (k) Quantification of BrdU +ve cells in growth, differentiation, and upon high serum restimulation conditions compared with A-GFPinfected satellite cells (mean ± SEM from six independent experiments with n = 8 for each condition; *, P < 0.05; ***, P < 0.001). (l) Quantification of Pax7 +ve satellite cells in Bmi1 Over satellite cells compared with A-GFP-infected satellite cells in growth, differentiation, and upon high serum restimulation conditions (mean ± SEM from four independent experiments with n = 4 for each condition; *, P < 0.05; **, P < 0.01). Bars, 62.5 µm. Bmi1 Over : Bmi1-overexpressing cells.

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To investigate whether these results could be explained by modulation of satellite cell proliferation by Bmi1, satellite cells were treated with BrdU in four different culturing conditions: growth (proliferation medium), early differentiation (2 d in differentiation medium), late differentiation (5 d in differentiation medium), and restimulation (2 d in proliferation medium after 5 d of differentiation, Fig. 2, i and j). Under growth conditions, 59% of Bmi1 Over satellite cells had incorporated BrdU compared with 44% of the controls, and in early and late differentiation, 40% as compared with 5% and 8% as compared with 2%, respectively (Fig. 2,i,j,and k [quantification]). These results indicate that Bmi1 overexpression prolongs proliferation even in differentiation-inducing conditions. When satellite cell cultures were returned to high serum conditions after 5 d of differentiation (restimulation), Next, satellite cell cultures derived from single fibers isolated from the SOL muscle of postnatal day 60 (P60) STOPFloxBmi1 mice were infected with A-GFP or A-Cre virus and induced to differentiate by switching to low serum conditions for 2 d (Fig. 2, e-g). The number of Pax7 +ve cells was increased by 14% in cultures overexpressing Bmi1 (Bmi1 Over ), whereas no alteration in the proportion of MyoD +ve cells (29% in both conditions) was found (Fig. 2,e,f,and g [quantification]). The higher number of Pax7 +ve ;MyoD -ve cells in Bmi1 Over cultures suggested that the stem cell pool of satellite cells was increased (Fig. 2 g). Importantly, Bmi1 overexpression in satellite cells derived from P60 mice did not inhibit differentiation and the percentage of MyHC +ve cells remained constant as compared with controls ( Fig. 2, e, f, and g [quantification]). Similar results were obtained also when (a) Schematic temporal summary of the experiment. (b and c) Quantification of Bmi1 expression upon Tamoxifen injections in mdx;Pax7 Bmi1 mice compared with controls at RNA (b, mean ± SEM from four independent experiments with n = 12 mdx;Pax7 Bmi1 and n = 12 mdx; ***, P < 0.001) and at protein level (c; arrowheads indicate a Pax7 +ve cell then included in the insert, representative results from two independent experiments with n = 4 mdx, n = 3 mdx;Pax7 Bmi1 , n = 12 fields for mdx, and n = 11 fields for mdx;Pax7 Bmi1 ). (d) Quantification of Bmi1 pixel intensity in mdx;Pax7 Bmi1 mice compared with controls (mean ± SEM from two independent experiments with n = 4 mdx, n = 3 mdx;Pax7 Bmi1 , n = 12 fields for mdx, and n = 11 fields for mdx;Pax7 Bmi1 ; ***, P < 0.001). (e) The progression of the muscle pathology is followed by the forelimb grip strength test before each treadmill session. The table reports the statistical significant differences between groups and the coefficient representing the improving (if positive) or worsening (if negative) of the phenotype (four independent experiments with n = 5 controls, n = 9 mdx, n = 11 mdx;Pax7 Bmi1 ; mdx vs. mdx;Pax7 Bmi1 : **, P < 0.01; mdx vs. control: ***, P < 0.001; and control vs. mdx;Pax7 Bmi1 : *, P < 0.05). (f and g) Quantification of Pax7 +ve cells in mdx;Pax7 Bmi1 and mdx control (f) and quantification of double-positive cells Pax7 +ve /Ki67 +ve in mdx;Pax7 Bmi1 and mdx control (g; mean ± SEM from n = 3 mdx, n = 7 mdx;Pax7 Bmi1 ; *, P < 0.05). (h) Representative images of muscle sections stained for Pax7 and Ki67 in mdx;Pax7 Bmi1 as compared with mdx mice (n = 3 mdx, n = 7 mdx;Pax7 Bmi1 ; *, P < 0.05). Mdx;Pax7CreER T2 ;STOPFloxBmi1: mdx;Pax7 Bmi1 (Bmi1 overexpression in satellite cells in a dystrophic environment). Bars: (c) 125 µm; (h) 62.5 µm.
staining, revealed activation of the construct in 75% of the Pax7CreER T2 ;R26R injected mice (unpublished data). LacZ/Laminin double labeling confirmed the specificity of the recombination in satellite cells (unpublished data). FACS analysis revealed that at least 65% of the satellite cells isolated from Pax7CreER T2 ;R26R muscles by means of M-Cadherin labeling (Cooper et al., 1999) were -galactosidase positive, hence confirming high recombination efficiency (unpublished data).
To test the hypothesis that Bmi1 overexpression in satellite cells could enhance regeneration in a chronic injury model, triple transgenic mice mdx;Pax7CreER T2 ;STOPFloxBmi1, here called mdx;Pax7 Bmi1 , were generated. Mdx mice are more susceptible to exercise-induced injury, and a cycle of forced exercise on a treadmill has often been applied to accelerate muscle pathology and to test the efficacy of novel treatments in preclinical trials (De Luca et al., 2003;Whitehead et al., 2006). incorporation of BrdU was found in 12% of the cells, whereas almost 70% of the Bmi1 Over satellite cells incorporated BrdU after 2 d of growth restimulation (Fig. 2,i,j,and k [quantification]). Importantly, at this time point a higher number of Pax7 +ve cells was observed, in keeping with an increase in the pool of satellite cells even after a prolonged induction of differentiation ( Fig. 2 l).

Conditional activation of Bmi1 expression in satellite cells improves muscle strength in dystrophic muscles but not after traumatic injury in vivo
To assess the impact of Bmi1 overexpression on muscle regeneration in vivo, we used an inducible CreER line targeting satellite cells (Pax7CreER T2 ; Mourikis et al., 2012). Crosses with the R26R reporter mouse (Soriano, 1999), followed by Tamoxifen-mediated activation of Cre expression in double transgenic mice and whole mount LacZ mice compared with controls at RNA (b; mean ± SEM from two independent experiments with n = 5 controls and n = 3 Pax7 Bmi1 ; *, P < 0.05) and at protein level (via double immunolabeling for Pax7 and Bmi1; c; arrows indicate a Pax7 +ve cell that is included in the insert, representative results from one experiment with n = 3 controls and n = 3 Pax7 Bmi1 ); quantification of the average pixel intensity of Bmi1 staining in Pax7 +ve cells (d; mean ± SEM with n = 3 controls and n = 3 Pax7 Bmi1 ; ***, P < 0.001). (e) H&E staining of hind limbs 10 d.a.i. in injured controls and injured Pax7 Bmi1 mice (top, representative results from two independent experiments with n = 6 controls and n = 3 Pax7 Bmi1 ). Gomori Trichrome staining for endomysial fibrosis in the same samples of H&E (injured controls and injured Pax7 Bmi1 mice; bottom; representative pictures from one experiment with n = 3 controls and n = 3 Pax7 Bmi1 ). (f) Quantification of the ratio of regeneration in Pax7 Bmi1 mice compared with controls (f, mean ± SEM from two independent experiments with n = 6 controls and n = 3 Pax7 Bmi1 ). (g) Quantification of the CSA distribution of CNF in injured Pax7 Bmi1 and injured controls: the percentage of CNF of control mice (gray), Pax7 Bmi1 mice (blue), and the distribution of mature fibers (striped gray) is shown (mean ± SEM from two independent experiments and five independent experiments with n = 8 injured controls, n = 3 Pax7 Bmi1 , and n = 15 uninjured controls; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (h) Treadmill performance in terms of the time run (h, mean ± SEM from three independent experiments with n = 12 uninjured controls, n = 7 injured controls and n = 3 Pax7 Bmi1 . **, P < 0.01) in injured control mice (gray) as compared with uninjured mice (striped), as well as in the distances run (i; mean ± SEM from three independent experiments with n = 7 injured controls, n = 3 Pax7 Bmi1 , and n = 12 uninjured controls; *, P < 0.05; **, P < 0.01). Bars, 125 µm.
was up-regulated (5.73 ± 2.03-fold) also in Bmi1 Over cultures upon restimulation (Fig. 6 a, last two bars on the right). IPA system biology analysis on all differentially expressed genes Triple transgenic mice, mdx, and nontransgenic littermates were injected with Tamoxifen, followed by a 6-wk period of running on a treadmill for 30 min twice weekly (Fig. 3 a). Bmi1 overexpression was confirmed both at RNA level in whole muscle tissue (Fig. 3 b) and at protein level specifically in the targeted satellite cells by double immunolabeling for Pax7/Bmi1 (Fig. 3, c and d [quantification]). Before each treadmill session, a forelimb grip test was performed to follow the progression of the disease. The forelimb strength of mdx mice was clearly compromised compared with controls ( Fig. 3 e) but the strength of both groups (mdx and control littermates) was stable throughout the duration of the experiment. Strikingly, mdx;Pax7 Bmi1 mice showed a significant improvement in muscle strength from the fourth week onwards (Fig. 3 e).
Histological examination of the muscle did not reveal morphological differences between mdx and mdx;Pax7 Bmi1 , in particular an impact on the degree of endomysial fibrosis or on adipose tissue dedifferentiation was excluded (unpublished data). Immunolabeling for Pax7 showed an increased percentage of Pax7 +ve cells in mdx;Pax7 Bmi1 mice as compared with mdx controls (Fig. 3, h and f [quantification]). Double labeling for Pax7 and the proliferation marker Ki67 revealed that it was the Ki67 ve component of the Pax7 +ve population which was increased in mdx;Pax7 Bmi1 (Fig. 3, h and g [quantification]), suggesting that at this time point overexpression of Bmi1 in Pax7 +ve cells had increased the pool of quiescent satellite cells in a dystrophic environment. Interestingly, when a muscle freeze injury (FZ) was performed on the TA of hind limbs of Pax7CreER T2 ;STOPFloxBmi1 compound mutant mice-here called Pax7 Bmi1 -after Tamoxifen injection ( Fig. 4 a) to induce Bmi1 overexpression ( Fig. 4, b-d), no significant differences were found morphologically (Fig. 4, e-g) or functionally (Fig. 4, h and i) in injured Pax7 Bmi1 as compared with injured controls.

Transcriptome analysis reveals increased expression of MT1 in satellite cells upon activation of Bmi1 expression
To gain insights into the downstream mechanisms mediating the observed phenotype, we first analyzed the canonical Bmi1 downstream target genes p16 ink4a , p19 Arf , and p21 waf1/cip1 . No changes were found in the expression level of these genes in Bmi1 over satellite cells cultured under differentiation-inducing conditions or upon restimulation (unpublished data). Alternative ink4a/p21-independent mechanisms were then explored by whole-genome transcriptome analysis on the same cultures (Illumina platform mouse v2). A set of 254 genes was identified (Table S1), the expression of which was deregulated in satellite cell cultures overexpressing Bmi1 compared with controls (P < 0.05 and absolute fold change > 1.9). Out of 10 genes (Ahnak, Bmi1, DNAjb6, Ehd1, Hspd1, Itgb1, Klf6, MT1, p21 waf1/cip1 , and Tpm4) selected for validation analysis, all but one (p21 waf1/cip1 ) were confirmed to be differentially expressed (Figs. 5 a and 6 a; and not depicted). MT1 was the most significantly up-regulated gene (5.27 ± 0.99-fold) in these cultures (Fig. 6, a and b) and importantly, its expression cell cultures compared with A-GFP-infected controls (mean ± SEM from two independent experiments with n = 3 for each condition; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (b and c) System biology analysis using the Ingenuity platform reporting genes in the top canonical pathways (b) and the top tox lists (c; n = 3 for each condition). (d and e) qRT-PCR analysis of expression of redox homeostasis genes in Bmi1 Over satellite cell cultures compared with A-GFP-infected controls (mean ± SEM from three independent experiments with n = 5 for each condition; *, P < 0.05; **, P < 0.01) and in Bmi1 / satellite cell cultures as compared with controls (e; mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

MT1 is a crucial mediator of the enhanced satellite cell pool elicited by Bmi1 overexpression in vitro
Because the DMD pathology is linked to accumulation of ROS in satellite cells with a consequent increase in the oxidative stress induced cellular damage Zhuang et al., 2001;Wozniak and Anderson, 2007), we analyzed whether Bmi1 overexpression would exert a protective role against oxidative stress-induced cellular damage in satellite cells. We observed a decrease in ROS production in Bmi1 Over satellite cell cultures incubated with the fluorescent dye H 2 DCFDA, followed by flow cytometry (Fig. 6 c, white and black bars), which led to decreased oxidative damage to the cells as assessed by 8-hydroxydeoxyguanosine (8-OHdG) staining, a marker of oxidative stress-dependent DNA damage (Won et al., 1999;Fig. 6, e and d [quantification]).
showed hypoxia signaling in the cardiovascular system, p53 signaling, and protein ubiquitination pathway (Fig. 5 b) to be the most significantly deregulated top canonical pathways. Hypoxia-inducible factor signaling and NRF2-mediated oxidative stress response were most significantly affected among the top tox lists (Fig. 5 c). To further extend and validate these findings, the expression of a selection of genes involved in Bmi1-mediated control of redox homeostasis through generation of ROS (Liu et al., 2009) was analyzed. We found a significant repression of Alox15, Bnip3L, Cdo1, and Duox2 (Fig. 5 d). Down-regulation of MT1 expression (Fig. 6 a, first two bars; and Fig. 7 a) and increased expression of Alox5, Bnip3L, Cdo1, Duox1, and Duox2 in Bmi1 / satellite cells confirmed the specificity of these associations (Fig. 5 e). (a) Relative expression level of MT1 in Bmi1 / , Bmi1 Over , and control satellite cells (mean ± SEM from two independent experiments with n = 3 for each condition; *, P < 0.05; **, P < 0.01). (b) Expression levels of MT1 are also analyzed at the protein level in Bmi1 Over satellite cells both in growth and differentiation-inducing conditions (left, three independent experiments with n = 3 for each condition). Efficient knock down of MT1 is also shown in proliferation (P) and differentiation (D) conditions (b, right; representative results from three independent experiments with n = 3 for each condition). WB analysis of the levels of 3-NT modification in Bmi1 Over satellite cells both in proliferation and differentiation-inducing conditions (b, left; three independent experiments with n = 3 for each condition). (c) The amount of ROS production is measured via DCFDA relative fluorescence intensity in Bmi1 Over satellite cells compared with controls and treated with either sh Scramble or knockdown of MT1 (mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05; ***, P < 0.001). (d and e) Quantification of the percentage of strongly 8OHdG +ve satellite cells in Bmi1 Over cultures with and without knockdown of MT1 (mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05) and representative examples of immunolabeling (e) are shown. (f) Representative examples of immunolabeling for Pax7 and Edu in Bmi1 Over satellite cell cultures upon MT1 knockdown (two independent experiments with n = 4 for each condition). (g) Quantification of the percentage of Edu +ve or Pax7 +ve or Edu +ve /Pax7 +ve cells is shown (mean ± SEM from two independent experiments with n = 4 for each condition; *, P < 0.05). (h) Total concentration of nitrosylated proteins (ng/µl) in Bmi1 Over mdx;Bmi1 Over satellite cells compared with A-GFP-infected controls, with and without Mt1 knockdown (mean ± SEM from two independent experiments with n = 4; ***, P < 0.001; *, P < 0.05). (i and j) The level of SNO-Mef2c is shown in mdx;Bmi1 Over satellite cells compared with controls A-GFP-infected satellite cells as assessed by a modified biotin switch assay, followed by immunoblot (i) and quantification (j; mean ± SEM from two independent experiments with n = 4; *, P < 0.05). cells isolated from Bmi1 / mice showed increased ROS production ( Fig. 7 b), increased number of 8-OHdG +ve cells (Fig. 7, c and d [quantification]), and increased levels of 3-NT in Bmi1 / muscles and satellite cell cultures (Fig. 7 a), hence supporting the interpretation that the observed protection from oxidative stress is a Bmi1-dependent effect.

Bmi1-driven improved muscle strength in dystrophic mice is mediated by MT1 up-regulation
Next, we assessed whether up-regulation of MT1 mediates the improved muscle strength observed in the mdx model upon Bmi1 overexpression in satellite cells. MT1 up-regulation was detected in 33% of mdx;Pax7 Bmi1 (Fig. 8 a). Importantly, no MT1 up-regulation was ever detected in mdx control mice (Fig. 8 a), in keeping with a Bmi1-induced effect. Reevaluation of the muscle strength data with focus on MT1 expression revealed that mdx;Pax7 Bmi1 mice with MT1 upregulation were the animals with the best improvement in forelimb grip strength (Fig. 8 b) as compared with mdx control mice. mdx;Pax7 Bmi1 mice up-regulating MT1 showed a behavior similar to that of unaffected control mice, whereas mdx;Pax7 Bmi1 with no MT1 up-regulation showed a performance comparable to mdx control mice (Fig. 8 b). These findings show that Bmi1-induced MT1 up-regulation molecularly mediates the impact of Bmi1 overexpression on muscle strength. Furthermore, centrally nucleated fiber (CNF) percentage was significantly reduced in mdx;Pax7 Bmi1 with MT1 up-regulation compared with mdx control mice and to mdx;Pax7 Bmi1 mice without MT1 up-regulation (Fig. 8,c and d [quantification]), suggesting that muscle fibers were more resistant to exercise-induced damage in mdx;Pax7 Bmi1 with MT1 up-regulation. The reduced number of H2AX +ve (a marker of DNA double-strand break) satellite cells detected in these mice (double immunofluorescence for Pax7 and H2AX; Fig. 8, e and f [quantification]) is in keeping with the protective effect having been exerted specifically on the satellite cells. We did not observe differences in the number of apoptotic These findings suggest that the amount of oxidative damage incurred by satellite cells in vitro depends on Bmi1 expression levels and raise the possibility that this effect is mediated by MT1. To test this hypothesis functionally, we set out to knock down MT1 expression (MT1kd) in both Bmi1 Over and mdx;Bmi1 Over cultures and we analyzed ROS production, the percentage of 8-OHdG +ve cells, and the number of proliferating satellite cells and/or Pax7 +ve cells. Western blot analysis confirmed efficient knockdown of MT1 in mdx;Bmi1 Over cultures in growth and in differentiation conditions (Fig. 6 b). A significant increase in ROS production was observed upon MT1kd in Bmi1 Over and in mdx;Bmi1 Over satellite cells (Fig. 6 c and not depicted) with concomitant increase in the percentage of 8-OHdG +ve cells (Fig. 6, d and e) and significantly fewer EdU +ve , Pax7 +ve and EdU +ve ;Pax7 +ve cells (Fig. 6, f and g [quantification]), compared with controls treated with scrambled sequences.

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satellite cells or in the total number of apoptotic nuclei (double immunolabeling for Pax7 and cCaspase3; Fig. 8 h and not depicted), or in the percentage of necrotic fibers (not depicted), but a generalized decrease in mitochondrial oxidative enzyme activities was detected by means of in vitro biochemical analysis in mdx;Pax7 Bmi1 with MT1 up-regulation (Fig. 8 g). To functionally validate these findings, we transplanted satellite cells isolated from mdx;STOPFloxBmi1 mice induced to overexpress Bmi1 in vitro and either up-regulating MT1 (Bmi1-induced) or not (ShRNA-MT1 mediated knockdown) into the TA muscle of young mdx mice. We show that the contribution of the cells to the regenerative process was more efficient when MT1 was up-regulated, as demonstrated by the reduced number of donor satellite cell-derived GFP-positive fibers upon MT1 knockdown (Fig. 9, a and b [quantification]).

Bmi1 expression is reduced in patients affected by DMD
To translate the significance of these findings to human pathology, we analyzed the expression of Bmi1 in DMD muscles and in morphologically normal controls. Four DMD patients aged 2.2, 3.2, 6.1, and 9.5 yr and six age-matched controls were used. Reduction of Bmi1 expression was found in DMD muscle biopsies at all ages (Fig. 10 a). Immunolabeling for Bmi1 performed on adjacent sections confirmed a reduction in the number of Bmi1 +ve cells in DMD cases (Fig. 10, a and b) and double labeling for Bmi1 and Pax7 revealed lower levels of Bmi1 expression in Pax7 +ve satellite cells in Figure 9. MT1 up-regulation is essential for the efficient contribution of Bmi1 Over satellite cells to muscle regeneration upon transplantation in the mdx mouse. (a) Satellite cells are isolated from mdx;STOPFloxBmi1 mice, cultured for 14 d, infected with A-Cre virus to activate Bmi1 expression, and treated either with Sh scramble and Mt1 knockdown, both viruses expressing GFP in infected cells. 6 d after treatment, the cells are transplanted in the TA muscles of mdx recipient mice. The transplanted muscles are collected after 27 d. Representative pictures of mdx muscles transplanted with A-Cre; mdx;STOPFloxBmi1 + Sh Scr satellite cells (a, left) or A-Cre; mdx;STOPFloxBmi1 + Sh MT1 satellite cells (a, right) are shown (a, representative results from n = 4 for each condition). (b) Quantification of GFP +ve fibers in A-Cre; mdx;STOPFloxBmi1 + Sh MT1 transplanted satellite as compared with A-Cre; mdx;STOPFloxBmi1 + Sh MT1 transplanted satellite (mean ± SEM from one experiment with n = 4 A-Cre;Mdx;STOPFloxBmi1+Sh Scr and n = 4 Mt1 A-Cre; Mdx;STOPFloxBmi1+ShMt1; *, P < 0.05). Bar, 125 µm.

Figure 10. Bmi1 expression is reduced in the muscle of human DMD cases. (a) H&E
staining of a DMD case as compared with an age-matched morphologically normal control. The same muscles are double stained for Bmi1 and Pax7 (representative results from two different cases). (b) Quantification of Bmi1 +ve cells in DMD muscle biopsies and age matched controls (mean ± SEM from two independent experiments with n = 2 control and DMD in both age groups, five high power fields/case; *, P < 0.05; **, P < 0.01). (c) Levels of expression of Bmi1 are analyzed by qRT-PCR in the muscle biopsies of DMD patients as compared with age matched unaffected muscles (mean ± SEM from two independent experiments, n = 3 control and n = 2 DMD in both age groups, two technical replicas for each case; *, P < 0.05; **, P < 0.01). (d) Expression levels of MT1 are measured by qRT-PCR in the muscle biopsies of DMD patients >5 yr compared with age-matched unaffected muscles (mean ± SEM from two independent experiments with n = 3 control and n = 2 DMD in both age groups, two technical replicas for each case; *, P < 0.05; **, P < 0.01). Bar: (H&E) 62.5 µm; (immunofluorescence) 31 µm. pool seen in dystrophic muscle conditions after repeated degeneration/regeneration cycles. Genome-wide transcriptome analysis of satellite cells overexpressing Bmi1 revealed significantly increased expression of MT1, which has been shown to be necessary to maintain proliferation and normal cellular functions under stress conditions, including when cells reenter the cell cycle from a quiescent state (Zbinden et al., 2010). In keeping with this notion, improvement of muscle strength is seen only in those mdx mice in which up-regulation of MT1 occurred concomitantly with overexpression of Bmi1 in satellite cells. The reduced contribution to regeneration after transplantation in a dystrophic environment of Bmi1 Over satellite cells upon MT1 knockdown lends additional support to this conclusion. At present it is unclear why up-regulation of MT1 occurs only in a proportion of mdx mice overexpressing Bmi1 and it will be important to investigate this fully in future studies. However, the correlation of muscle strength and MT1 up-regulation suggests that Bmi1 exerts its effect through protection against oxidative stress-induced cellular damage in vitro and in vivo, hence delaying muscle wasting through reduction of ROS-induced oxidative stress. The reduction of H2AX +ve satellite cells observed in the mdx;Pax7 Bmi1 with concomitant MT1 up-regulation is in keeping with this conclusion, as it is the MT1-dependent reduction of 3-NT levels in mdx;Pax7 Bmi1 satellite cell cultures and the reduced levels of nitrosylated Mef2c, a transcription factor with an essential role in muscle regeneration (Liu et al., 2014;Panda et al., 2014). We also observed a decrease in metabolic and mitochondrial oxidative enzyme activities in these mice, raising the possibility that because satellite cells are better protected against exercise-induced oxidative stress in a dystrophic environment, metabolic adaptation is less prominent. Although additional studies will be required to fully elucidate the impact of Bmi1 overexpression on metabolism, it is intriguing that decreased mitochondrial oxidative phosphorylation activities have been shown to increase resistance to metabolic diseases through prevention of ROS accumulation (Pospisilik et al., 2007) and to be associated with better tolerance to oxidative stress (Patel et al., 2013). It is currently unclear what mediates Bmi1-induced up-regulation of MT1 expression in the skeletal muscle. As the main mode of function of PcG genes is through transcriptional repression, it is possible to speculate that a MT1 repressor may be involved in this process.
Finally, we show here an important correspondence of Bmi1 expression between mice and humans, including its depletion in human DMD muscles. Interestingly there is evidence that MT1 expression increases in human skeletal muscle 24 h after intense exercise (Urso et al., 2006); here, we show decreased expression of MT1 in human DMD muscle, raising the possibility that manipulation of Bmi1 expression could be a useful approach to enhance satellite cell-driven regeneration also in human primary myopathies. Identifying novel pharmacological agents to enhance and sustain muscle regeneration in DMD is highly desirable as they could have a tremendous impact on DMD therapy either alone or, most DMD patients (Fig. 10 a). Importantly, reduced expression of Bmi1 (Fig. 10 c) and MT1 (Fig. 10 d) was detected also in DMD patients at the RNA level.

DISCUSSION
Here, we show for the first time that mild overexpression of a chromatin modifier gene has a functionally relevant impact on muscle regeneration. Bmi1 overexpression in satellite cells confers enhanced protection from exercise-induced injury and improves muscle strength in a mouse model of DMD. We also demonstrate that the mechanism underlying the effect of enhanced physiological function of Bmi1 in the satellite cells is mediated by MT1-driven modulation of resistance against oxidative stress.
Skeletal muscle is an important and highly relevant platform to assess how manipulation of stem cell properties may enhance a functionally significant regenerative process in vivo. We show that the expression level of Bmi1 is modulated in response to pathological conditions in the satellite cells. Indeed, Bmi1 is up-regulated upon freeze injury in wild-type mice, whereas reduction of the proportion of Pax7 +ve cells with high Bmi1 expression is found in the mdx mouse, with the latter finding corroborated by the lack of Bmi1 up-regulation in mdx satellite cell cultures upon induction of differentiation. In keeping with these findings, overexpression of Bmi1 in satellite cells has a beneficial effect solely in the mdx mouse, where downregulation of its expression was observed.
The best characterized molecular mechanisms mediating Bmi1 function relies on transcriptional repression of the ink4a locus, encoding for p16 ink4a and p19 arf ( Jacobs et al., 1999), as well as of p21 waf1/cip1 . However, we found no change in the expression levels of these genes upon activation of Bmi1 expression raising the intriguing possibility that a novel mechanism may mediate the observed phenotype. The generation of free radicals and ROS/reactive nitrogen species (RNS) is a physiological, continuous process in aerobic organisms. When the production of ROS/RNS exceeds the endogenous antioxidant buffering capacity, oxidative stress-induced cellular damage occurs. There is increasing evidence that oxidative stress level is elevated in diseases such as DMD and in normal aging (Fulle et al., 2004;Whitehead et al., 2006;Jackson, 2009;Cozzoli et al., 2011;Spassov et al., 2011). Bmi1 has an important role in mitochondrial function and redox homeostasis in thymocytes and in neurons (Liu et al., 2009;Abdouh et al., 2012); however, the mechanisms mediating this role of Bmi1 are currently unclear. Up-regulation of Bmi1 expression in Pax7 +ve satellite cells increases the pool of quiescent Ki67 ve cells in vivo after 6 wk of training on a treadmill, indicating that Bmi1 is involved in the regulation of self-renewal of these cells. To explain these results, two scenarios can be envisaged: it is possible that a phase of increased proliferation has also occurred in vivo, perhaps immediately after induction of Bmi1 overexpression in a similar fashion as we observed in vitro. Alternatively, satellite cells overexpressing Bmi1 are better able to reenter quiescence, hence preventing the typical exhaustion of the satellite cell 2628 Bmi1 overexpression in skeletal muscle regeneration | Di Foggia et al.

Microscopy and quantification.
Fluorescent and bright field image capture was performed using an epifluorescent (Leica) or confocal (Meta 510 LSM or LSM 710; Zeiss) microscope. In myoblast cultures, all DAPIpositive cells and antigen-positive cells were counted and the percentage of Antigen +ve cells was calculated. Data from three or more cultures were pooled to give a population mean ± SEM.
For the quantification of the intensity staining, all samples were stained simultaneously and the pictures were acquired with an LSM 710 confocal microscope at the same ratio of emissions. For the analysis with ImageJ software (National Institutes of Health), the shape of each Pax7 +ve cell was drawn around the nucleus and the intensity of Bmi1 staining calculated for each cell as mean of integrated density (mean intensity × area). The mean integrated density was calculated among all cells per each field acquired. The percentage of cells above or below certain pixel intensity (determined by the mean integrated density between all measured cells) was calculated out of the total number of measured cells per each muscle section analyzed. The method was also validated using Definiens Tissue Studio (IF) 2.1.1, action library 3.6.1, and Developer XD 2.1.1 (Definiens AG). In particular for the validation on sections stained for Pax7 and Bmi1, an initial analysis was performed using Tissue Studio to identify the nuclei based on DAPI stain and Pax7. Nuclei were identified using a stain threshold of 1.62 (RGB space converted to HSD model to give a range of 0-2; van der Laak et al., 2000) and typical size of 18 µm 2 . Pax7 was processed twice using thresholds of 0 and 25 (RGB space) to classify all nonnuclear tissue (Marker 1) and areas with significant Pax7 staining (Marker 2), respectively. These are overlaid to give classifications of marker 1 and markers 1 and 2. Developer was used to perform post-processing on the classification resultant from Tissue Studio. Before classification of nuclei as positive or negative, nuclei with area <5 µm 2 were merged with connecting nuclei. In cases where these small nuclei did not border another nucleus, no change occurred. In cases where a small nucleus borders multiple nuclei, the merge resulting in the lowest elliptical fit was selected. Several dynamic thresholds were calculated for each image: the mean Pax7 stain intensity for each area classified as marker 1 was used to give Pax7 Background; areas of marker 1 <10 µm 2 were excluded from this calculation to prevent skewing of resultant threshold toward the upper limit of 24 because these are generally isolated areas within the intensely stained regions (markers 1 and 2) just below the threshold of 25. The threshold for classifying nuclei as positive Pax7 threshold was calculated as: maximum Pax7 pixel value  (2 × Pax7 Background). A second set of thresholds were calculated based on the Bmi1 stain to segment positive nuclei staining as below mean, low intensity, and high intensity. The mean Bmi1 stain intensity in all pixels with intensity <50 (RGB space) gives mean Bmi1, and the range between mean Bmi1 and the maximum Bmi1 value was divided by two to give low Bmi1 and high Bmi1. Nuclei with more than five pixels with Pax7 greater than Pax7 threshold were classified as positive nuclei. Positive nuclei were then segmented based on BMI1 stain as below mean, low intensity, and high intensity. To compare this analysis with the manual analysis with ImageJ, the mean Bmi1 intensity for each single slice of the stack where Bmi1 was on focus was multiplied to the area (in pixel) so to obtain integrated density values. The percentage of cells above or below certain pixel intensity (determined by the mean integrated importantly, in combination with genetic therapies aiming at reconstituting dystrophin expression.

Satellite cell culture.
Single fibers from the soleus (SOL) and the extensor digitorum longus (EDL) muscles of adult Bmi1 / , STOPFloxBmi1, mdx;STOPFloxBmi1, and nontransgenic littermates were isolated and cultured to obtain pure satellite cell cultures according to standard protocols. In brief, the muscles were dissected from P60 mice being careful not to stretch the muscles. Tendon and other connective tissues were carefully removed and the muscles incubated in 0.2% Collagenase type 1 (Worthington Biochemical Corporation) at 37°C for 2 h. Fibers were liberated by gentle trituration in DMEM. Single fibers were placed in a 24-well plate (BD) coated with 1mg/ml Geltrex Reduced Growth Factor Basement Membrane Matrix in DMEM (Invitrogen) and allowed to adhere for 5 min at 37°C before 500 µl of plating medium was added (DMEM supplemented with 10% horse serum [PAA] and 0.5% chick embryo extract [Gibco] with antibiotics) and incubated at 37°C in 5% CO 2 . After 3 d, the media was changed to proliferation media (DMEM supplemented with 20% FCS [PAA] and 2% chick embryo extract [Gibco] plus antibiotics). To induce differentiation, satellite cells were plated at 2,000 cells per well in an 8-well chambered slides (BD) and cultured in differentiation media (DMEM supplemented with 2% horse serum and antibiotics) for 2 or 5 d. Satellite cells derived from the same fiber of STOPFloxBmi1 and mdx;STOPFloxBmi1 mice were set up in parallel wells and treated with either A-GFP or A-Cre (Akagi et al., 1997) at the time of plating in the differentiating media. To control for nonspecific effects of viral treatment and overexpression of any exogenous proteins, infection with A-GFP was used as control for all experiments where A-Cre infection was performed.
Magnetic activated satellite cells sorting. TA muscles were isolated from C57BL/6J mice at time zero (uninjured controls), and 3 and 10 d after freeze injury. The muscles were dissected and finely chopped with scissors and incubated with 0.2% Collagenase in DMEM for 30 min at 37°C while gently shaking (250 rpm). This step of the procedure was performed twice. The cells were then filtered with 30-µm filters (Miltenyi Biotec). 1 ml of serum was added and the suspension was centrifuged for 20 min at 1,200 rpm at 4°C. The supernatant was decanted and the staining was performed with the SM/C-2.6 antibody (Fukada et al., 2004;1:200 for 40 min on ice) and with anti-Biotin MicroBeads (Miltenyi Biotec) as secondary antibody according to manufacturer's protocol. After magnetic sorting the cells were centrifuged 20 min at 2,500 rpm and the pellet was snap frozen on dry ice.
When satellite cells were analyzed, cell pellets were collected from different culture conditions and lysed with RIPA buffer as mentioned above. 40 µg of protein extracted from cell samples was loaded per lane on the Nu-Page 4-12% Bis-Tris gel.
Production of shRNA-Mt1 lentiviral vector. A pGIPZ-lentiviral shRNAmir vector containing a hairpin sequence targeting MT1 is commercially available from Open Biosystems. The shRNA-containing lentiviral vector was cotransfected with lentiviral packaging, using the calcium phosphate method into 293T cells. The medium was refreshed and viruses were harvested after 48 h of transfection, passed through 0.45-µm filters, concentrated by PEG precipitation, and stored at 80°C. The infectious titer was determined by FACS analysis of GFP-positive 293T cells.
Lentiviral-mediated Mt1 knockdown. Satellite cells were seeded in multiple-well plates and transduced with concentrated lentivirus containing shRNA that targets MT1, along with A-Cre (to induce Bmi1 overexpression) in proliferation medium. The medium was replaced on the second day. 96 h after transduction, cells were switched to differentiation medium for 48 h and the transduction efficiency was analyzed. Cells were collected for Western blot, immunocytochemistry, or ROS assay.

ROS assay.
Satellite cells were treated with 40 µM H 2 DCFDA in differentiation medium for 1 h at 37°C before being harvested and resuspended in 500 µl of PBS. Cells were then immediately processed for flow cytometry analysis using an LSRII instrument (BD) and the relative fluorescence intensity was calculated.

ELISA for 3-NT detection. Satellite cells prepared from mdx;STOPFloxBmi1
EDL muscles were transfected with A-Cre to overexpress Bmi1 and a proportion then treated with LV-shMt1. Cells infected with A-GFP were used as controls. Proteins were extracted from the cells and concentration was measured by BCA protein assay kit (Thermo Fisher Scientific). The amount of 3-NT protein in the lysates was determined by the Nitrotyrosine ELISA kit (Abcam) according to the manufacturer's protocol. The OD was recorded at 450 nm and the data were analyzed with the program XLfit 5.3.1.3 (IBDS). density between all measured cells) was calculated out of the total number of measured cells per each muscle section analyzed.
For the analysis of satellite cells in vitro, satellite cells were stained for Bmi1 and counterstained with DAPI. Immunofluorescent images taken at 16-bit were analyzed using ImageJ and Developer XD (2.1.1; Definiens AG). The analysis in ImageJ was conducted using the same settings as above. For the analysis in Definiens, pixels with intensity above 30 (RGB space) were classified as nucleus, those below 30 remained unclassified; and small unclassified areas bordering only nucleus were reclassified as nucleus and merged in. The distance of each pixel classified as nucleus to unclassified was then calculated to facilitate the splitting of connected nuclei. Nuclei with irregular shapes (roundness >0.5; roundness is based on the difference between the largest enclosing ellipse and the smallest enclosed ellipse) were then segmented based on these distances; local minima provide seeds for nuclei. These seeds were then grown to fill the space originally classified as nuclei, with separate nuclei growing to share a border rather than joining, thus splitting connected nuclei. Nuclei with area < 100 µm 2 were then removed. For each nucleus, the sum of the Bmi1 staining for each pixel was exported and this value was multiplied for the area of each single nucleus. For both the analyses, the fold change of increased Bmi1 staining was calculated using the average of Bmi1 staining of A-GFP-infected cells as one per each coverslip analyzed.

RNA extraction and qPCR analysis.
Satellite cell cultures were harvested after 48 h of either differentiation or restimulation. The cell pellets were lysed in RLT buffer from RNeasy Micro purification kit (QIAGEN). Total RNA was isolated using the same kit. Digestion with DNase (on column) was performed to remove genomic DNA. cDNA synthesis was performed with SuperScript III Reverse transcription kit (Invitrogen). For the sorted satellite cells, the retrotranscription was performed with QuantiTect Whole Transcriptome kit (QIAGEN) according to manufacturer's protocols. Analysis of gene expression was performed with a Rotor-Gene apparatus (Corbett Life Science) using SYBR Green JumpStart TaqReadyMix (Sigma-Aldrich) according to the manufacturer's instruction. A list of the primers used is reported. The Ct values of all the genes analyzed were normalized with GAPDH and fold changes were calculated. qRT-PCR analysis for Bmi1 was performed with TaqMan assays (Applied Biosystems) with FAM-labeled probes in 96-well plates using the 7500 RT-PCR machine (Applied Biosystems) according to the manufacturer's instructions (Bmi1: Mm 00776122_gH). The cDNA content was normalized against the expression of the housekeeping gene -actin (Mm 00607939_s1). Technical duplicates for each culture or muscle tissue and three or more independently derived cultures or biological samples were analyzed. The following primers were used in qPCR experiments: Alox5 sense, 5-CATGTTACCGC-

2630
Bmi1 overexpression in skeletal muscle regeneration | Di Foggia et al.
2008a). The researcher who performed all the analysis was blinded to the animal's genotype.
Enforced treadmill exercise and histological analysis. The dystrophic animals mdx;Pax7 Bmi1 , mdx control littermates and C57BL/6J-BL/10 mice (2-4 mo) were used in this experiment. Mice were injected with Tamoxifen. Starting from 3 d after the last injection, one mouse was put in each lane of the treadmill and forced to run at the speed of 12 m/min for 30 min, twice a week for 6 wk. This protocol was not exhausting and generally all the mice were able to perform the exercise. Whenever an avoiding behavior was observed, the mice were stimulated to run by a gentle touch of the tail or with the presence of a soft barrier at the end of the treadmill lane (De Luca, 2008b). The mice were sacrificed 6 d after the last treadmill session. One forelimb was embedded in OCT and frozen in isopentane cooled at the liquid nitrogen temperature. The other forelimb was dissected and the muscles snap-frozen in liquid nitrogen to perform RNA extraction and to check Bmi1 expression levels.
The frozen muscles were serially sectioned at 8 µm and processed for H&E staining and/or immunohistochemistry. The percentage of CNF was counted in the injured area of three different sections along the length of the leg for the same animal. The researcher who performed all the analysis was blinded to the animal's genotype.
Assessment of muscle strength. The dystrophic animals mdx;Pax7 Bmi1 , mdx control littermates and C57BL/6J-BL/10 mice (2-4 mo) performing the enforced treadmill exercise were used in this experiment. Before each treadmill session, a forelimb grip test was performed (De Luca, 2008c) to assess the course of the disease. The test was performed using a horizontal grip strength meter (Linton). In brief, each mouse was lifted by the tail toward the metal pull bar. Once the mouse had grasped the bar with the forelimbs, the mouse was pulled back in a horizontal plane. The protocol was repeated three consecutive times within 30 s. The grip strength was normalized against the body weight.
Cell transplantation experiments. Satellite cells isolated from mdx;STOPFloxBmi1 were treated as for the MT1 kd experiments. 6 d later, cells were harvested and 20,000 cells were transplanted into surgically exposed TA muscles of 3-wk-old mdx mice. After 27 d, the muscle tissues were fixed in 4% PFA for 6 h, left on in 30% sucrose, and embedded in OCT.
Serial sections (8 µm thick) were cut, mounted, and analyzed at the microscope for GFP positivity (two sections/mouse; three fields/section). The same settings were applied to take pictures of MT1 kd and MT1 SCr transplanted mice. For the analysis of the strong GFP positivity a defined threshold was applied to all pictures and the GFP +ve fibers were counted out of the total number of fibers/microscopic field. The percentage of strong GFP +ve fibers was calculated out of the total number of GFP +ve fibers/field. The researcher who performed all the analysis was blinded to the different treatments of the transplanted satellite cells.

Microarray analysis.
Three independently prepared satellite cell cultures isolated from STOPFloxBmi1 mice, treated with A-Cre infection and induced to differentiate for 2 d were analyzed by whole-genome Illumina platform mouse v2 as compared with the same cells treated with A-GFP. Integrity of starting total RNA was evaluated using capillary electrophoresis (Bioanalyzer 2100; Agilent Technologies) and quantified using a NanoDrop 1000 (NanoDrop). Aliquots of RNA (250 ng) samples were amplified according to the specifications of the Illumina TotalPrep RNA Amplification kit (Ambion). The cRNA samples obtained were applied to the MouseWG-6 v2.0 Expression BeadChip arrays (Illumina) and hybridized according to the manufacturer's specification. Each array on the BeadChip targets >45,200 transcripts using 3-µm beads bearing covalently attached 50-base oligonucleotide probes. The BeadChips were scanned with the BeadSrray system scanner (Illumina). The hybridization images signal intensity was extracted and background subtracted using Bead-Studio software (version 3.3.7 Illumina). The data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus and are accessible through GEO Series accession no. GSE41878.
Modified biotin switch assay to detect SNO-MEF2c. The biotinswitch assay (BST) is a modified immunoblot analysis used to detect the levels of S-nitrosylated proteins. Here, we used a modified version of this assay. In brief, cell lysates were prepared from A-Cre-or A-GFP-treated mdx;STOPFloxBmi1 satellite cell cultures in HENS Buffer (Pierce S-Nitrosylation Western Blot kit; Thermo Fisher Scientific) and the BCA Protein Assay was used (Thermo Fisher Scientific) to determine the protein concentration. 300 µg of protein from each sample was mixed with 20 mM methyl methane thiosulfonate (MMTS) for 30 min at room temperature to block free thiol groups. After removing excess MMTS by acetone precipitation (1 h at 20°C), S-nitrosothiols were reduced to thiols with 20 mM sodium ascorbate. In the negative control, ultrapure water was used instead of sodium ascorbate. Labeling reagent containing tandem mass tag (TMT) was added to label the newly formed thiols. After acetone precipitation, samples were resuspended into HENS buffer. TMT labeled proteins were loaded onto spin columns with pre-added immobilized anti-TMT resin (Thermo Fisher Scientific) and eluted. The eluted proteins were concentrated using Pierce Concentrator (Thermo Fisher Scientific) and further analyzed by immunoblotting. Input was used for the control blot.
Pax7CreERT2 T2 tamoxifen injection. Tg:Pax7CreER T2 mice were crossed with R26R to optimize the injections protocol. 2-mo-old littermate Pax7CreER T2 ;R26R and R26R (as controls) were injected with Tamoxifen (Sigma-Aldrich) 20 mg/ml suspended in corn oil (Sigma-Aldrich; 4.5 mg/40 g of body weight) twice a day for five consecutive days. 5 d after the last injection the mice were culled and the efficiency of recombination was assessed. Pax7CreER T2 mice were bred with the STOPFloxBmi1 mice to overexpress Bmi1 in a cell type-specific fashion and the mdx mice for the experiments in a dystrophic model. In particular, the following compound mutant mice were generated: Pax7CreER T2 ;STOPFloxBmi1 (Pax7 Bmi1 ) and mdx;Pax7CreER T2 ;STOPFloxBmi1 (mdx;Pax7 Bmi1 ).
Acute muscle injury and histological analysis. Pax7 Bmi1 and littermate control mice (2-4 mo) were used in this experiment. Mice were injected with Tamoxifen (as described above) to induce Bmi1 overexpression in satellite cells. 10 d after the last injection, muscle freeze injury (FZ) was performed on these mice. 2% isoflurane inhalation was used to anesthetize the mice. The left hind limb was shaved and then wet with 70% ethanol to remove hair. To induce the damage to the Tibialis Anterior (TA) muscle, an incision of 5 mm was done in the skin above the muscle; then, a dry ice precooled probe was applied on the muscle for 10 s. The incision was closed by veterinary glue (VETBOND Tissue Adhesive). The mice were either sacrificed or used in a test for treadmill performance 10 d.a.i. Both the injured and contralateral hind limbs were collected. The injured leg was embedded in OCT and frozen in liquid nitrogen-precooled isopentane, whereas the contralateral muscles were snap-frozen in liquid nitrogen and processed for RNA extraction to check Bmi1 expression levels.
The frozen muscles were serially sectioned at 8 µm (with a step of 120 µm) and processed for H&E staining and/or immunohistochemistry. The percentage of CNF was counted in the injured area of three different sections along the length of the leg for each animal. In the same sections, the crosssectional areas (CSAs) of the fibers in regenerating and uninjured areas (as control) were measured using the MetaMorph software (Life Sciences Research Imaging Systems). The researcher who performed all the analysis was blinded to the animal's genotype.
Treadmill performance test after FZ injury. Tamoxifen-injected Pax7 Bmi1 and littermate controls mice (2-4 mo) were used in this experiment. 10 d after the muscle freeze injury mice performed a functional test running on a treadmill (LE8710M, Panlab Harvard Apparatus). 3 d before the test, mice were trained for each day for 5 min, at the speed of 10 m/min, with an inclination of 5°. The day of the test, the mice started running for 4 min (speed 10 m/min) and then every 4 min the speed was increased of 2 m/min until the mice were tired. Tiredness was defined as the inability of the mouse to go back to the treadmill belt despite five mechanical stimulations (De Luca,


s2fieldsofstudy: Biology Chemistry

text:
The pleiotropic roles of sphingolipid signaling in autophagy

The autophagic process involves encompassing damaged proteins and organelles within double- or multi-membraned structures and delivering these molecules to the lytic compartments of vacuoles. Sphingolipids (SLs), which are ubiquitous membrane lipids in eukaryotes, participate in the generation of various membrane structures, including rafts, caveolae, and cytosolic vesicles. SLs are a complex family of molecules that have a growing number of members, including ceramide, sphingosine-1-phosphate, and dihydroceramide, which have been associated with the essential cellular process of autophagy. This review highlights recent studies focusing on the regulation and function of SL-associated autophagy and its role in cell fate, diseases, and therapeutic interventions.

Sphingolipid-mediated signals that have long been known to induce apoptosis are now also recognized to cause autophagy. Are sphingolipid-associated apoptosis and autophagy interlinked? How does sphingolipid-associated autophagy occur and to what extent do alterations in autophagy contribute to disease phenotype? What drugs or compounds that target sphingolipid metabolism are attractive candidates for therapeutic development?
Autophagy is generally considered to be a cell survival mechanism, and this process also contributes to cell death in several situations. 1 Sphingolipids (SLs) are ubiquitous components of membrane structures, and renewed interest in SLs has focused on SL-induced intracellular and extracellular signaling. 2 Accumulating evidence indicates that a direct link exists between SL metabolism and autophagy. Moreover, studies have uncovered that a dynamic balance among SL metabolites is significant in cell fate determination. 3,4 Here we review the key aspects of bioactive SLs that have emerged as important effectors in regulating the autophagic pathway, mediating the cross talk between apoptosis and autophagy, and determining the associated cell fate. A deeper understanding of the relationship between SL metabolism and autophagy explains how intact or impaired SL-related autophagic pathways are involved in the malfunctions associated with neurodegeneration, cancer, and other diseases. We also discuss how autophagy-regulating drugs work via cells' SL metabolism and the methods that could be used to monitor SL-related autophagy. Present and future investigations regarding SL-related autophagy will help to develop novel treatment strategies to control autophagy-related diseases.

The SL Family
SLs represent a significant class of lipids that contain a backbone of sphingoid bases and are ubiquitous constituents of membranes in eukaryotes. SLs were first discovered in brain extracts in 1876, 3 and within a century of intensive research, the chemical structures of thousands of individual SLs had been elucidated. 4 The SL metabolic pathway displays an intricate network of reactions that result in the formation of multiple SLs, including ceramide (Cer), dihydroceramide (dhCer), and sphingosine-1-phosphate (S1P). 5 SLs are produced in at least three distinct ways ( Figure 1). First, a substantial portion of SLs are derived from de novo biosynthesis in most organisms, 2,6 with the condensation of serine and palmitoyl-CoA catalyzed by serine palmitoyl transferase to generate dehydrosphinganine. Dehydrosphinganine is subsequently reduced to form dihydrosphingosine (sphinganine), which is then N-acylated by dhCer synthase to produce dhCer or Cer. Second, through hydrolysis via a complex lipid-turnover pathway, 2,6 several specific hydrolases are involved in the turnover process, including sphingomyelinases (SMases) and glucocerebrosidase (GCase). In addition to the above two sources, Cer is also recycled. 6,7 In this pathway, sphingosine is recycled into Cer via Cer synthases (CerS); therefore, this pathway is also called the salvage pathway. The cellular metabolic homeostasis of the sphingolipidome is often achieved through the coordination of the biosynthesis and removal of SL species, which requires a delicate balance between de novo biosynthesis, turnover, and recycling. 8 SLs are found in cellular membranes, lipoproteins, and other lipid-rich structures; however, they are synthesized in the endoplasmic reticulum (ER) and Golgi apparatus. The scheme in Figure 1 depicts the subcellular localization of SL biosynthesis, turnover, and recycling. In addition, SLs may be incorporated into other intracellular compartments, such as mitochondria or autophagosomes. 3 The turnover and recycling of SLs also occur at several intracellular locations, including the endosome, phagosome, and lysosome. 3 Thus, SLs travel between organelles, and this transport occurs via either transport vesicles or transfer proteins. 9 CERT (Cer transfer protein) and FAPP2 (four-phosphate adaptor protein 2) have proved to be two critical SL-trafficking proteins that regulate the trafficking of SLs to specific compartments within cells. 10,11 Lipids are increasingly implicated in the control of the membrane remodeling and vesicle transport that underlie the biogenesis of autophagosomes. 12 However, our knowledge of whether and how SL trafficking is associated with the autophagic process remains incomplete.

Figure 1
Cer formation through the de novo, turnover, or salvage pathway. Red arrows: de novo pathway; green arrows: turnover pathway; blue arrows: salvage pathway. PM, plasma membrane SLs are considered to primarily have roles as components of membranes and other biological structures. However, several SL metabolites, including Cer, dhCer, and S1P, have drawn attention as bioactive signaling molecules that mediate cell growth, differentiation, senescence, apoptosis, and autophagy. 4,5 SLs Involved in the Autophagic Pathway 'Autophagy,' which is derived from Greek and means 'to eat oneself,' ensures the synthesis, degradation, and recycling of cellular components in eukaryotic cells ranging from yeasts to mammals. [13][14][15] At present, the following three modes of autophagy have been identified: macroautophagy (which is commonly called 'autophagy,' including in this review), microautophagy, and chaperone-mediated autophagy. During the autophagic process, nonspecific or targeted cytoplasmic constituents are delivered to and degraded in the lysosome via these autophagic pathways. 16,17 The formation of the autophagosome is a multistep process that includes the biogenesis of the isolation membrane, followed by its elongation and closure. 18 Most autophagy-related genes (Atgs) contribute to autophagosome formation, and many are well conserved from yeasts to mammals. 19,20 SLs are abundant lipid components of eukaryotic plasma membranes that function in a wide range of biological processes, 21,22 which make SLs strong candidates as possible autophagosomal lipids. Several studies have implied that SLs, and especially Cer, are autophagosomal membrane components. 23,24 In addition, SLs formed by de novo biosynthesis in the ER might be a driving force for the formation of the autophagosomal vacuole, in what has been referred to as the 'membrane extension' step, 25 which occurs after many of the associated autophagosomal proteins have been recruited. Because the enzymes required for de novo Cer biosynthesis reside in the ER, 26 it is possible that they might be recruited into autophagosomes and perhaps continue to produce SLs there.
The best-characterized pathway regulating autophagy includes a class I phosphatidyl inositol 3-kinase (PI3K) and mammalian target of rapamycin (mTOR), which act to inhibit autophagy. Although how autophagy is suppressed by these molecular signals remains to be revealed, mTOR complex 1 (mTORC1) is known to phosphorylate the autophagy regulatory complex, ULK1 (containing unc-51-like kinase 1), the mammalian Atg13 protein, and focal adhesion kinase-interacting protein of 200 kD (FIP200). 27,28 A class III PI3K is needed for the activation of autophagy. 4,16 SLs participate in cell survival and cell death signaling pathways. 29,30 Complex autophagy regulation might be mediated by SLs or SL metabolism. Indeed, different bioactive SL species have been shown to mediate distinct autophagic pathways, described as protective autophagy and autophagy-associated cell death (Figure 2), which have opposing functions in cellular life-ordeath decisions. 3,4,31 Cer, which is known to induce cell cycle arrest and has been implicated in important physiological roles in cell differentiation, senescence, migration, adhesion, and inflammatory responses, is the key intermediate in SL metabolism. 6,32 Exogenous Cer-related autophagy was shown to trigger autophagy-associated cell death, which occurs in several malignant cell types, including colon cancer, breast cancer, cervical cancer, nasopharyngeal cancer, and glioma cells. 3,[33][34][35][36][37] In addition to exogenous Cer, endogenous Cer species are critical for the induction of autophagy. Tamoxifen, an estrogen receptor antagonist used to treat several types of breast cancer, has been shown to induce autophagyassociated cell death by increasing endogenous Cer levels. 33 Exogenous or endogenous Cer induces autophagy through several mechanisms. For example, Cer has been found to stimulate autophagy by regulating classic or atypical autophagic pathways and signals. 4 Via these signals, class I PI3K and Akt negatively regulate autophagy, but a class III PI3K is needed for the activation of autophagy. Cer was found to promote the interaction of class III PI3K with other regulators of autophagy 38 and to inhibit Akt by activating phosphoprotein phosphatase 2A. 33,39,40 Moreover, exogenously added Cer and the accumulation of endogenous Cer due to treatment with certain chemotherapeutic drugs, such as tamoxifen, inhibit the mTOR signaling pathway, 33 which has a central role in inducing autophagy and increasing the expression of Beclin1, 33 an upstream regulator of autophagy. 41 Simultaneously, Cer promotes the dissociation of Beclin1 from the Beclin1/Bcl-2 complex. 34,42,43 A short-chain Cer analog has also been associated with the induction of autophagyassociated cell death by increasing the transcription of the BH3-only protein, a mitochondria-associated protein that induces nonapoptotic cell death. 35 A recent study showed that Cer directly interacts with microtubule-associated protein light chain 3 (LC3) on mitochondrial membranes to induce deadly autophagy via an increase in intracellular mitophagy. 44 S1P has emerged as a cell-proliferative lipid messenger. S1P has been found to induce survival-mediated or protective autophagy under nutrient starvation, distinct from Cerassociated autophagy-associated cell death. 45,46 In contrast to Cer-related autophagy, S1P-mediated autophagy has not been shown to be related to the accumulation of Beclin1 protein or to the suppression of class I PI3K or Akt. 45

Figure 2
Model of SL-related autophagy and its consequences on cell fate. CerSs, ceramide synthases; DES, dihydroceramide desaturase; So, sphingosine; SPPs, sphingosine-1-phosphate phosphatases However, mTOR was still inhibited by increased S1P levels, [45][46][47] which suggests that S1P induces autophagy via inhibiting mTOR and is independent of the class I PI3K signal. dhCer has been used as a negative control for Cer treatments to induce autophagy 33,34,48 because dhCer has long been thought to be biologically inactive. However, dhCer was recently identified as a novel SL-based mediator of autophagy. 3,49,50 dhCer induces both protective autophagy and autophagy-associated cell death. Endogenously added dhCer induced a transient, early increase in dhCer levels via inhibition of dhCer desaturase, such as XM462, which successively promoted autophagy and reduced etoposide toxicity in gastric and colon cancer cells. 49,51 However, anticancer agents such as fenretinide (4-hydroxy (phenyl) retinamide or 4-HPR) 50 and gamma tocotrienol (a lipophilic antioxidant of vitamin E) 52 induced cancer cell death through the elevation of intracellular dhCer levels. Interestingly, none of the reported Cer-mediated autophagic pathways is induced by both Cer and dhCer. 33 Intriguingly, the accumulation of dhCer may serve as an additional 'switch' to regulate cell fate; however, the biological activity of dhCer remains controversial and unclear.
In addition to the major SLs that induce autophagy, gangliosides and several rare SLs are also involved in autophagy-related cell fate decisions. Gangliosides induce autophagy-associated cell death in both isolated astrocytes and astrocytoma cells. 53 An increase in the amount of LC3-II and an accumulation of autophagic vacuoles were observed in cells that underwent ganglioside treatment. 53,54 Cer methylaminoethyl phosphonate and sphingadienes are two rare SLs that promote autophagy-associated cell death through the downregulation of PI3K/Akt and the activation of Beclin1, similar to Cer. 55,56 Overall, although Cer, dhCer, and S1P are all able to induce autophagy, the effects of S1P-mediated autophagy are markedly mild compared with the effects elicited by Cer. dhCer-related autophagic effects are moderate, and their intensity level lies between the levels caused by Cer and S1P. Treatment with chemotherapeutic agents often promotes the biosynthesis of intracellular Cer and leads to autophagyassociated cell death; however, elevation in S1P levels is typically a cell response to nutrition starvation and mediates cytoprotective autophagy. Furthermore, cells maintain a dynamic equilibrium between the levels and the effects of Cer, dhCer, and S1P. The conversion of Cer to S1P simultaneously accumulates the survival effects and removes the death signals. This observation has led to the concept of a so-called 'SL rheostat' or 'SL biostat,' based on the relative amounts and reciprocal roles of these antagonistic metabolites, which are critical in guiding the destiny of cells. 12,57,58 Possible SL Involvement in the Cross Talk Between Autophagy and Apoptosis Apoptosis and autophagy have been recognized as two principal forms of programmed cell death. Apoptosis generally initiates cell death, whereas autophagy is primarily a protective process for the cell that may also contribute to cell death. 59 There is a sophisticated and not yet fully understood association between autophagy-related cell death and survival that depends on various biological situations. 1 In certain cellular contexts, autophagy functions as a stress response to suppress apoptosis and promote cell survival. 16,60 However, in other cases, autophagy may serve as a mechanism for caspase-dependent or -independent cell death. 61,62 Certain SLs are now thought to elicit autophagy; however, they have also been known to induce apoptosis. Cer is a wellestablished inducer of apoptosis via activation of the mitochondrial pathway. In a number of different cell types, increased Cer levels arrest cell growth and promote cell apoptosis. [63][64][65][66] In contrast to Cer, S1P facilitates cell survival and suppresses cell apoptosis. [67][68][69] dhCer has also been suggested to exhibit antiapoptotic effects because the lipid inhibits Cer channel assembly in isolated mitochondria. 70 In addition, other SL metabolites, such as sphingosine and ganglioside, are involved in the modulation of apoptosis and autophagy. 71 SL-associated apoptosis and autophagy are interlinked. The conversion of Cer to S1P switches the cell fate from apoptosis to autophagy-induced survival. 57,58 Several exogenous stimuli, and particularly the activation of the enzymes that interconvert Cer and S1P, such as sphingosine kinases, lead to an increase in S1P levels, a corresponding reduction in Cer levels, and the antagonistic effects of Cer. 68 In addition, dhCer has been proposed to exhibit antiapoptotic effects to promote cell survival during hypoxia through the induction of autophagy, while also serving as a lipid reserve for the rapid production of Cer to address cellular damage on reperfusion. 72 dhCer serves as a unique regulator of cell fate, controlling the 'switch' between cytoprotective autophagy and Cer-mediated apoptosis in response to stress. Intriguingly, Cer promotes both apoptosis and autophagy through the inhibition of class I PI3K and Akt signaling or the mTOR pathway. 33,40,73,74 Furthermore, suppressors of apoptosis, such as the Bcl-2 family, also directly bind and control Beclin1. 75 Disruption of the Beclin1-Bcl-2 complex has also emerged as a common mechanism in Cer-associated autophagy. 71 In addition to altering the balance between Beclin1 and Bcl-2 protein levels, endogenous Cer liberates Beclin1 for autophagy induction through the JNK-mediated phosphorylation of Bcl-2. 34 Notably, Bcl-2 has emerged as a critical regulator of the Cer-mediated cross talk between autophagy and apoptosis.

SL-Associated Autophagy and Diseases
Although the primary function of autophagy is to aid in adaptation to cellular stress under adverse conditions by enabling cells to degrade cytosolic proteins and organelles to generate a supply of essential nutrients, autophagy has also emerged in the pathological process of many disorders, including neurodegeneration, tumors, immunity responses, and heart diseases. 16 SL metabolism-associated autophagy also contributes to a range of diseases. 31 Sphingolipidoses are a collection of more than 40 genetically distinct disorders caused by inherited deficiencies of lysosomal hydrolytic activities or lipid transport. These deficiencies result in intracellular accumulations of cholesterol and lipids in the endosomal/lysosomal network, usually leading to signs of neurodegeneration. 76,77 The biochemical Orchestration of autophagy by sphingolipids Y Li et al abnormalities in sphingolipidoses are complex and lead to the accumulation of SL metabolites by diverse pathways (Table 1). 78,79 Niemann-Pick disease type C (NPC) is a complex neurodegenerative sphingolipidosis characterized by the accumulation of unesterified cholesterol, SLs, and complex gangliosides in late endosomes and lysosomes. 80 The disease is caused by mutations in either the NPC1 or the NPC2 gene, 81,82 which disturbs not only the regular transport of endocytosed lipids but also the autophagic flux, leading to an accumulation of autophagic vacuoles in cells. 83,84 NPC1 and NPC2 81,82 are genes whose protein products mediate proper intracellular lipid transport through pathways that are incompletely understood. The brains of afflicted humans and NPC-deficient animal models are marked by a loss of neurons. 85,86 Histological studies and cell culture experiments have shown that defective lysosomal degradation of autophagosomes may contribute to the abnormal autophagic flux. 84,87 The deficiency present in NPC promotes both the generation of autophagosomes and the impairment of autophagic flux. An imbalance between induction and flux through the autophagic pathway contributes to cell stress and neuronal loss in NPC. 77 Gaucher disease (GD), a kind of sphingolipidosis, is characterized by the accumulation of glucosylceramide or glucosylsphingosine in the lysosomes of the cells of the monocyte/macrophage system. 88,89 In GD, the point mutations within the GBA (glucosidase, beta, acid) gene lead to the production of acid b-glucosidase with functional, kinetic, trafficking, and/or stability defects and a resultant decrease in lysosomal function and increase in the accumulation of glucosylceramide and glucosylsphingosine. 76 GD can be classed into three subsets, based on the age of onset and the presence of central nervous system abnormalities. Type 1 is known as the non-neuronopathic form, and types 2 and 3 are differentiated from type 1 by neurodegeneration of the central nervous system with either rapid or chronic progression. Vaccaro et al. 89 reported that mutation of saposin (Sap) C, and not a direct GBA gene mutation, was associated with the type 3 or type 1 phenotype in cases of GD. The decrease in/absence of Sap C affected GCase intracellular localization, resulting in lysosomal lipid accumulation and enhanced autophagy. This study suggested that Sap C might have a role in intralysosomal SL transport, as indicated by the accumulation of glucosylceramide and Cer in the lysosomes of the Sap C-mutant cells. In addition, deficient degradation of autophagic substrates in cells in GD can lead to an increased risk of Parkinson disease. 90 For NPC, GD, and other sphingolipidoses, 76,91 the relevance of the impairment of autophagic flux to disease pathogenesis remains poorly defined. In addition, the extent to which the accumulation of autophagic substrates contributes to neuron dysfunction needs to be determined in future studies.
Dysregulated SL metabolism occurs in numerous cancers and has been shown to contribute to cancer progression and chemoresistance. 92,93 Several species of SLs have been shown to have aberrant expression or metabolism in cancer cells. 94 The tumor suppressor Cer and the tumor promoter S1P are generally recognized to trigger autophagy; however, Cer and S1P have different outcomes regarding cell death and survival. 29 Nevertheless, recent studies have suggested that de novo-generated Cer-associated autophagy can be lethal as well as protective for cells. 92 For example, Beljansky et al. 95 determined that enhancement of Cer levels through treatment with a SphK 2-selective inhibitor suppresses tumor growth by leading to autophagy-associated cell death. Conversely, Park et al. 96 suggested that Cer-CD95-PERK signaling promotes cell death via the cascade activation of caspase proteins; however, gene silencing of Atg5 further strengthened the cell death process, indicating that the autophagy was protective. The contradictory activities may correspond to variations among Cers in their carbon chain lengths, double bond numbers, subcellular distributions, and versatile targets. In addition, Cer is metabolized into S1P by SphK 1 or SphK 2, which are associated with protective autophagy and other biological behaviors, including survival, infiltration, angiogenesis, metastasis, and resistance to anticancer drugs in cancer cells. 92 Intact and impaired SL-related autophagy has significant implications for many other diseases and pathological processes. For example, CerS 5-mediated autophagy has recently been implicated in lipotoxic cardiomyopathy and hypertrophy. 97 In addition, Kdo 2-lipid A induced substantial alterations in SL metabolism and composition in RAW264.7 cells, a mouse macrophage-like cell line. These changes apparently promote the de novo SL biosynthesis that is required for autophagosome generation, which is suggested to have an essential role in the processes of the innate immune response. 24 Cer also mediates the augmentation of interleukin-1(IL-1) levels and the release of tumor necrosis factor (TNF) alpha that are induced by toll-like receptor 4, and this process may contribute to the enhanced inflammatory response in metabolic diseases, including obesity and diabetes, which are characterized by dyslipidemia. 98 Overall, the aberrations in the bioactive SLs that mediate autophagy have been associated with diverse pathological conditions, including neurodegeneration, carcinogenesis, metabolic diseases, and inflammatory responses. Understanding the specific mechanism connecting SL-related autophagy and these diseases has substantial implications for revealing these diseases' biochemical characteristics and for designing and developing new therapeutic strategies.

Compounds and Drugs that Might Trigger Autophagy-Associated Cell Death Through Regulation of SL Metabolism
Many human disease states may be caused by the aberrant regulation of SL metabolism-associated autophagy; therefore, drugs and compounds that target SL metabolism, including chemotherapeutic drugs, sphingomimetics, and enzyme inhibitors, are attractive candidates for therapeutic development (Table 2). Many chemotherapeutic drugs elicit autophagy and exert their anticancer properties by increasing the intracellular levels of SLs. A large number of malignant cells often acquire genetic deletions or mutations that render them resistant to the classic apoptotic cell death that is induced by many anticancer therapies, which ultimately results in poor prognosis. 99,100 In these cases, the use of autophagy-inducing drugs offers an alternative way to induce cell death in apoptosis-resistant tumors. 99,100 As reviewed in the present paper, one of the major results of the chemotherapy-mediated increase in the levels of intracellular SLs, such as Cer and dhCer, is cell death induction by autophagy in various human cancer cells. Autophagy is initiated by tamoxifen, an antagonist of the estrogen receptor, leading to an increase in endogenous Cer levels that acts against breast cancer cells. 33 Arsenic trioxide, a potent antineoplastic agent, has been used clinically for the treatment of certain types of leukemia. Arsenic trioxide was shown to induce the biosynthesis of Cer in both acute promyelocytic leukemia and adult T-cell leukemia cells, and the cytotoxic effects were attributed to the induction of both apoptosis and autophagy. 101,102 D 9 -Tetrahydrocannabinol, the major active component of marijuana, was reported to induce the activation of autophagy-mediated apoptotic cell death by increasing de novo Cer biosynthesis. 103 Moreover, SL metabolism may be involved in the regulation of autophagy by other chemotherapeutic drugs, including, but not limited to, etoposide, 61,104 doxorubicin, 105 and daunorubicin. 106,107 Resveratrol is a natural polyphenolic phytoalexin produced by grapes and other berries that exerts its cancer-preventive properties in several animal models and exhibits its potent anticancer activities in leukemia and many solid tumors. The capacity to trigger dhCer biosynthesis contributes to resveratrol-induced autophagy-associated cell death. Puissant et al. 108 found that resveratrol initiates autophagy in chronic myelogenous leukemia cells through the AMP-activated protein kinase (AMPK)-dependent induction of autophagy and that p62 is also required for resveratrol-mediated autophagy. Whether other polyphenolic compounds, such as curcumin, which has two phenolic hydroxyl groups: genistein, which has three phenolic hydroxyl groups; and quercetin, which has four phenolic hydroxyl groups, are implicated in SL metabolism and SL-mediated autophagy remains to be determined.
In clinical trials, 4-HPR has been encouraging as a chemopreventive and therapeutic agent for breast cancer. 109,110 The promise of 4-HPR as a therapeutic agent is enhanced by its unique ability to activate the autophagyassociated cell death pathway when the apoptotic pathway is deregulated, which is a characteristic that is not shared by other chemotherapeutic agents, including cisplatin and etoposide. 109 Furthermore, the accumulation of dhCer following treatment with 4-HPR was found to induce cell cycle arrest in neuroblastoma cells. 111,112 SL metabolism is an exploitable target for the discovery of novel chemotherapeutics, and this target has been validated through the development of several sphingomimetics. 8,113,114 Safingol, a synthetic sphinganine, triggers autophagy in several tumor cell types by the suppression of class I PI3K and Akt signaling. 115 Safingol has been assessed in phase I clinical trials alone or in combination with cisplatin for adults with advanced solid tumors and for children with neuroblastoma. Safingol treatment was shown to trigger a dosedependent decrease in S1P levels, and safingol is a promising representative drug whose primary mechanism for promoting tumor cell death is autophagy. 116 Targeting enzymes in SL metabolism is another attractive avenue to provide therapeutic benefits in SL-related human diseases. 117,118 Acid SMases (aSMases) and acid ceramidases (aCDases) are crucial enzymes that modulate the synthesis and degradation of Cer in SL metabolism. 2,6 aCDase overexpression has been observed in cancer cell lines and primary tumors and contributes to resistance to chemotherapy and radiation. [119][120][121] The consequence of aCDase overexpression is the ability to convert Cer, which is often produced as a proapoptotic response to stress, to sphingosine, which can then be converted to S1P. 122 In addition to the ability to metabolize the Cer produced in response to stress, Turner et al. 119 found that prostate cancer cell lines overexpressing aCDase also have an increased lysosomal density and increased levels of autophagy. Morad et al. 120 identified an analog of cyclosporin A, PSC 833 (Valspodar, Novartis Pharma AG, Basel, Switzerland), a second-generation, non-immunosuppressive P-glycoprotein antagonist that inhibits aCDase and generates autophagyassociated cytotoxicity in pancreatic cancer cells. B13 was found to be a potent CDase inhibitor that induced colon cancer cell death 123 and the inhibition of colon tumor growth in a xenograft model. 124 Furthermore, LCL385, a B13 analog, enhanced the sensitivity of prostate cancer cells to radiation and inhibited tumor growth in a nude mouse model. 125 Another novel B13 analog, LCL204, has been shown to overcome the resistance of head and neck squamous carcinoma cells to Fas-induced cell death in both in vitro and in vivo experiments. 126 Most aCDase inhibitors are lysosomotropic agents and cause lysosomal destabilization and a change in SL metabolism enzymes in the lysosomal compartment. 118 aSMase hydrolyzes sphingomyelin into Cer and phosphorylcholine. For the most part, aSMase seems to reside in classic lysosomes, where it mediates the catabolism of sphingomyelin. 117 aSMase activity is essential for lysosomal stability and the survival of cancer cells, as well as for the multidrug-resistant phenotype. Because of their ability to inhibit autophagic flux, certain aSMase inhibitors, such as chloroquine, siramesine, and clomipramine, are currently being tested as anticancer agents in several clinical trials and laboratory studies. 127 aSMase was also found to be a target for the treatment of Niemann-Pick disease type A (NPA), which is caused by loss-of-function mutations in the aSMase gene and is a lysosomal storage disorder leading to neurodegeneration. Fibroblasts from NPA patients and aSMase-knockout mouse brains show similar autophagolysosome accumulation and impaired autophagy. 128 A recent study has suggested that the control of lysosome trafficking and fusion by aSMase is essential to normal autophagic flux in coronary arterial smooth muscle cells and has a protective role in atherosclerosis. 129 Other chemotherapeutic treatments, such as several histone deacetylase inhibitors, were also demonstrated to lead to leukemic cell death by enhancing Cer production via the degradation of sphingomyelin. 130 One of these histone deacetylase inhibitors is suberoylanilide hydroxamic acid, which was demonstrated to trigger autophagy-associated cell death in apoptosis-deficient tumor cells, such as chondrosarcoma cells. 131

Conclusion
Numerous well-developed and convenient experimental methods and techniques that can be used to detect autophagy in different species systems exist. 132,133 To establish that an autophagic response is occurring in SL-related autophagy, multiple methods have been used, including cellular ultrastructure studies by transmission electron microscopy, 132,133 an LC3-puncta formation assay combined with immunoblots for autophagic cargoes (for example, p62 and NBR1), and the detection of autophagy-related molecules. 132,134 In addition, performing a large-scale and comprehensive sphingolipidomic analysis has been challenging in the study of SLassociated autophagy. 135,136 Both nets and hooks are indispensable tools to allow efficient 'fishing' for multiple intermediates and products directly from the sophisticated process of SL metabolism.
Because there is an obvious overlap between the subcellular localization of enzymes needed for SL biosynthesis and autophagosome formation, SLs have been increasingly implicated in the composition of the phagophore and the autophagosomal membrane.
Autophagy-associated cell death and cell survival represent the so-called 'Yin-Yang' regulatory mechanism. The balance Orchestration of autophagy by sphingolipids Y Li et al has been exemplified by interconvertible SLs that produce opposite autophagic effects on cells that were termed as 'SL rheostat' or 'SL biostat'. In addition, the increase in intracellular Cer levels usually results from chemotherapy and often stimulates autophagy-associated cell death, although this increase has also been found to be cytoprotective. In contrast, starvation treatment triggers protective autophagy via increasing S1P levels. However, the underlying mechanisms by which chemotherapy and nutrient deprivation mediate different regulation are still largely unknown. In addition to autophagy, SLs have long been identified to induce apoptosis. SLs mediate the cross talk between apoptosis and autophagy because certain SLs share several common kinase signaling pathways that regulate cell fate that affect both apoptosis and autophagy or execute a seesaw type of regulation of key molecules.
Increasing new knowledge on the biochemistry and cell biology of SL-mediated autophagy is beneficial for deepening our understanding of SL-associated diseases, including neurodegeneration, cancer pathogenesis, and inflammatory responses. An imbalance between induction of and flux through the autophagic pathway contributes to cell stress and neuronal loss in NPC. Intact and impaired SL-related autophagy has significant implications for the development of malignant tumors.
In addition, the current understanding of the effects of multiple SLs on the modulation of various autophagy-related cell fates has primarily arisen from exogenous SLs or pharmacological perturbations of SL metabolism. The identification of SL-mediated cytoprotective autophagy that can be shifted to cell death may provide a novel strategy for cancer therapy, including chemotherapy, sphingomimetics, SL metabolic enzyme inhibitors, and histone deacetylase inhibitors.

Conflict of Interest
The authors declare no conflict of interest.