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The AMPK/p27Kip1 Axis Regulates Autophagy/Apoptosis Decisions in Aged Skeletal Muscle Stem Cells

Summary Skeletal muscle stem cell (MuSC) function declines with age and contributes to impaired muscle regeneration in older individuals. Acting through AMPK/p27Kip1, we have identified a pathway regulating the balance between autophagy, apoptosis, and senescence in aged MuSCs. While p27Kip1 is implicated in MuSC aging, its precise role and molecular mechanism have not been elucidated. Age-related MuSC dysfunction was associated with reduced autophagy, increased apoptosis, and hypophosphorylation of AMPK and its downstream target p27Kip1. AMPK activation or ectopic expression of a phosphomimetic p27Kip1 mutant was sufficient to suppress in vitro apoptosis, increase proliferation, and improve in vivo transplantation efficiency of aged MuSCs. Moreover, activation of the AMPK/p27Kip1 pathway reduced markers of cell senescence in aged cells, which was, in part, dependent on p27Kip1 phosphorylation. Thus, the AMPK/p27Kip1 pathway likely regulates the autophagy/apoptosis balance in aged MuSCs and may be a potential target for improving muscle regeneration in older individuals.

Aged mammalian MuSCs have alterations in intracellular processes including autophagy and apoptosis (Garcia-Prat et al., 2016a;Jejurikar et al., 2006;Krajnak et al., 2006). Autophagy is initiated during periods of negative energy stress and generates substrates for energy production via degradation of cellular organelles and proteins (Mizushima et al., 2004). In the MuSC, autophagy provides initial ener-getics necessary for proliferation (Tang and Rando, 2014). Inhibition of the autophagy program results in perturbations in MuSC function including alterations in mitochondrial function, ATP production and induction of senescence (Garcia-Prat et al., 2016a;Tang and Rando, 2014). As a result, whole muscle regeneration is compromised when autophagy is genetically reduced in the MuSC (Garcia-Prat et al., 2016a). Aging MuSCs have an inherent reduction in autophagy (Garcia-Prat et al., 2016a), which contributes to the age-related dysfunction. Rescuing autophagy through genetic or pharmacological methods can recover aged MuSC function and rescue muscle regenerative capacity (Garcia-Prat et al., 2016a). Although the initial reports have established the importance of autophagy in MuSC function, upstream regulatory pathways have not been investigated, especially in the context of aging.
When autophagy is unable to meet the energy needs of activated cells, they become susceptible to apoptosis. During cell fate decisions, younger stem cells can initiate autophagy over apoptosis, while aged MuSCs are more likely to undergo apoptosis (Jejurikar et al., 2006). In an unchallenged state, in vitro activation of aged human MuSCs results in increased cell death (Fulle et al., 2013). Similarly, when mouse MuSCs are treated with pro-apoptotic factors such as tumor necrosis factor alpha and actinomycin D, apoptosis is more prevalent in aged MuSCs (Jejurikar et al., 2006). Thus, aged MuSCs have alterations in both apoptosis and autophagy processes critical for muscle regenerative capacity; yet, nodal signaling pathways responsible for such perturbations are currently unknown.
The AMPK signaling pathway has emerged as a potent regulator of autophagy, apoptosis, and proliferation (Liang et al., 2007;Sanli et al., 2014;Sun et al., 2014). In times of energy stress AMPK can promote autophagy directly through phosphorylation of ULK1 (Egan et al., 2011) or indirectly through inhibition of mammalian target of rapamycin (mTOR) complex 1 by phosphorylation of the tuberous sclerosis complex 2 (Garami et al., 2003;Inoki et al., 2003aInoki et al., , 2003bTee et al., 2003;Zhang et al., 2003) and/or through phosphorylation of raptor (Gwinn et al., 2008). Furthermore, AMPK has been shown to regulate apoptosis in part, through phosphorylation of p27 Kip1 (CDKN1B) (Liang et al., 2007). In the context of the MuSC, AMPK function is necessary for optimal muscle regeneration (Fu et al., 2015;Theret et al., 2017) however, functional effects of downstream p27 Kip1 signaling in the aged MuSC warrants further investigation.
Once thought to simply function as a cyclin inhibitor, p27 Kip1 is now recognized as a critical mediator of cell fate during metabolic stress conditions. p27 Kip1 is involved in both cell-cycle inhibition and pathways related to autophagy and apoptosis (Liang et al., 2007). In times of cell stress, p27 Kip1 can prevent apoptosis by directly inhibiting Cdk2 activation and downstream activity of the proapoptotic factor Bax (Gil-Gomez et al., 1998;Hiromura et al., 1999). The function of p27 Kip1 is regulated by transcription (Rathbone et al., 2008), phosphorylation (Liang et al., 2002(Liang et al., , 2007Motti et al., 2005), degradation Montagnoli et al., 1999;Pagano et al., 1995), and subcellular location (Liang et al., 2002(Liang et al., , 2007Motti et al., 2005). Liang et al. (2007) reported that AMPK-dependent phosphorylation of p27 Kip1 on Thr198 promotes p27 Kip1 protein stability, resulting in more autophagy and less apoptosis. In addition, the mTOR-raptor complex can also regulate p27 Kip1 phosphorylation and cellular localization through the serum and glucocorticoid-inducible kinase (SGK) (Hong et al., 2008). In aged MuSC, there is less mRNA expression of p27 Kip1 (Chakkalakal et al., 2012), yet protein expression is greater in the nuclei where it can serve as cyclin inhibitor (Machida and Booth, 2004) with minimal effect on cell survival. In addition, p27 Kip1 expression associates with maintenance of satellite cell populations that proliferate less frequently, but have long-term self-renewal capacity (Chakkalakal et al., 2014). It follows that the functional regulation of p27 Kip1 may serve as a key regulatory pathway of both autophagy and apoptosis in MuSCs.
Here, we describe a molecular mechanism controlling apoptosis/autophagy and cell fate decisions involving AMPK signaling to the cyclin inhibitor, p27 Kip1 in MuSCs. Furthermore, our results suggest that AMPK/p27 Kip1 signaling is a critical regulatory step contributing to the phenotype of aged MuSCs.

Aging Leads to a Reduction in MuSC Autophagy and Increased Apoptosis
We first determined whether aging affects MuSC autophagy and apoptosis in vitro during the initial days (first 48 hr) in culture. To determine the temporal pattern of autophagic flux in MuSCs isolated from young mice, we measured LC3B puncta at 12, 24, and 48 hr in culture. To accumulate and quantify LC3B puncta, 12 hr prior to each time point, we treated cells with chloroquine (10 mM), an inhibitor of autophagosome and lysosomal fusion ( Figure S1A). During the first 48 hr in culture, puncta increased steadily in young MuSCs: puncta were abundant after 24 hr in culture and further increased at 48 hr. We next determined MuSC autophagic flux across a time course of physiological aging after 48 hr in culture. LC3B puncta was determined in MuSCs from 3 to 4 months (young), 12 months (middle-aged), 24 months (old), and >28 months (geriatric) mice. Compared with young cells, there was no change in flux in the middle-aged group (Figure 1A). In contrast, puncta formation was less in old MuSCs and even less in geriatric cells. A parallel experiment measuring protein expression of LC3B I and II isoforms showed similar trends. We observed a reduction in both LC3B I and II isoforms in old cells and a further reduction in geriatric cells ( Figure 1B). Using the same time course of murine aging, we quantified markers of apoptosis, observing an inverse relationship between autophagy and apoptosis with aging. Protein expression of a marker of apoptosis, cleaved poly(ADP-ribose) polymerase (PARP), was minimal in young and middle-aged cells; however, expression was observed in old cells and was even greater in geriatric cells ( Figures 1C and S1C). We also examined apoptosis by analysis of TUNEL and Annexin V staining. Again, compared with young and middle-aged cells, old, and geriatric cells had progressively more TUNEL and Annexin (+) cells ( Figures 1D and S1D). In sum, MuSCs from aging mice exhibited progressively less autophagy and increased apoptosis across the lifespan; these trends were especially apparent in the geriatric mice, a state associated with reduced MuSC proliferative capacity ( Figure 1E).

Geriatric MuSCs Are Preferentially Susceptible to Cell Death during Autophagy Suppression
To determine whether there was a causal relationship between the reduction in autophagy and increase in MuSC apoptosis, we inhibited autophagy via lentivirus containing a short hairpin antisense RNA to Atg5, a key protein in formation of the autophagosome (Hara et al., 2006). The knockdown resulted in an 82% less Atg5 mRNA ( Figure S2A) and 87% less Atg5 protein expression (data not shown). The reduction in Atg5 expression after 24 hr of infection resulted in a marked suppression of MuSC autophagy; this was indicated by the lack of LC3B isoform II protein expression ( Figure S2B).
Suppression of autophagy did not induce apoptosis in young MuSCs (Figure 2A). In contrast, geriatric MuSCs were especially sensitive to autophagy suppression. Apoptosis increased by nearly 2-fold. Similar effects were observed with measurement of cell proliferation. The inhi-bition of autophagy by Atg5 knockdown delayed, but did not inhibit young MuSC proliferation throughout 72 hr in culture ( Figure 2B). Geriatric MuSCs failed to undergo proliferation under these conditions ( Figure 2B). To confirm the induction of apoptosis is associated with cell death, we quantified cell number for each treatment (Figure S2C). A total of 250 cells were plated for both young and geriatric groups with or without Atg5 knockdown. After 72 hr of autophagy inhibition, nearly half the geriatric cells were lost ( Figure S2C). Autophagy has been linked to both canonical and non-canonical apoptotic pathways in adult stem cells (Chung and Yu, 2013). To investigate if the observed induction of cell death was through the canonical apoptotic pathways, we replicated the Atg5 knockdown experiments in both young and geriatric cells, with or without a pan-caspase inhibitor (Figure 2C). In both young and geriatric cells, the caspase inhibitor rescued the induction of cell death caused by inhibition of autophagy. Further investigation of the canonical apoptotic pathway showed overexpressed of Bcl2 under conditions of autophagy suppression via shATG5 expression was able to attenuate the onset of apoptosis ( Figure S2D). Thus, apoptosis-induction via autophagy withdrawal appears to be dependent on the canonical apoptotic pathway. In morphologic analyses, 48 hr of Atg5 knockdown modestly reduced cell size in the young MuSC. However, the surviving geriatric Atg5 knockdown MuSCs failed to hypertrophy based on quantification of cell size ( Figure 2D).

Restoring AMPK Activity Prevents Cell Death in Aged MuSCs
We next sought a mechanism for the susceptibility of geriatric MuSCs to cell death under autophagy withdrawal. AMPK can regulate both autophagy and apoptosis through phosphorylation of the downstream target, p27 Kip1 (Liang et al., 2007). In young MuSCs, phosphorylation of AMPK (Thr172) and p27 Kip1 (Thr198) increased during the initial 48 hr in culture ( Figure 3A). Comparing across age groups, we observed reduced AMPK/p27 Kip1 phosphorylation in old mice; this was further reduced in geriatric mice at 48 hr in culture ( Figure 3B). To determine if rescue of AMPK activation would prevent cell death in geriatric MuSCs, we treated cells with the AMP analog, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). AICAR was effective at activating AMPK in geriatric MuSCs, as indicated by phosphorylation at Thr172 and increased AMPK-specific phosphorylation of p27 Kip1 ( Figure S3A). As signified by reductions in TUNEL staining ( Figure 3C) and expression of cleaved PARP ( Figure 3D), AICAR was sufficient to prevent cell death. To investigate the functional role of AMPK in the MuSC we genetically enhanced or suppressed AMPK activity before transplantation into injured muscle from young severe combined immunodeficiency (SCID) hosts ( Figure 3E). Transplantation efficiency was high in young control MuSCs, showing numerous GFP(+) cells 4 days after transplantation ( Figure 3F) and eventually incorporated into GFP(+) myofibers after 28 days (Figure 3F). Forced expression of a dominate negative AMPK prior to transplantation in young cells resulted in a roughly 70% reduction in GFP(+) donor cells/myofibers at each respective time point. Geriatric MuSCs had inherently poor transplantation efficiency, as represented by the presence of few GFP(+) cells at 4 days and GFP(+) myofibers at 28 days after transplant ( Figure 3G). Prior forced expression of constitutively active AMPK to geriatric cells increased the number of GFP(+) cells and myofibers at 4 and 28 days after transplantation, respectively ( Figure 3G). To show further physiological relevance to the AMPK/ p27 Kip1 pathway, we isolated MuSCs from old mice with  (legend continued on next page) or without short-term caloric restriction (CR) (40% CR), an established method to enhance MuSC function in old mice (Cerletti et al., 2012). After 48 hr in culture, phosphorylated and total forms of each protein were measured to determine if CR-mediated effects were associated with activation of the AMPK/p27 Kip1 pathway in old MuSCs. MuSCs from CR-treated old mice increased phosphorylation status of AMPK and p27 Kip1 compared with old mice fed ad libitum ( Figure S3B).
p27 Kip1 -Mediated Cell Survival Is Dependent on AMPK-Specific Thr198 Phosphorylation After establishing the importance of AMPK signaling for regulation of cell survival and proliferation in the aging MuSC, we investigated downstream regulation of p27 Kip1 function in the MuSC. Based on previously reported constructs (Liang et al., 2007), we generated lentiviral mutants of p27 Kip1 , identified as p27 Kip1(198A) and p27 Kip1(198D) . The p27 Kip1(198A) mutant cannot be phosphorylated at the AMPK-specific Thr 198, while p27 Kip1(198D) serves as a Thr 198 phosphomimetic. We confirmed the potent suppression of AMPK-specific phosphorylation in the p27 Kip1(198A) mutant after 24 hr of forced expression ( Figure S4A). Next, we showed the anti-apoptotic effects of AICAR were dependent on phosphorylation of p27 Kip1 . Overexpression of the p27 Kip1 mutant p27 Kip1(198A) , negated the effect of AICAR on MuSC cell death ( Figure 4A) and protein expression of cleaved PARP ( Figure 4B). To further validate AMPK/p27 Kip1 signaling as a cell survival mechanism, we suppressed autophagy in young and geriatric cells with concurrent overexpression of the p27 Kip1(198A) or p27 Kip1(198D) mutants. In the young MuSC, overexpression of the constitutively active p27 Kip1(198D) had minimal effect on cell death; however, overexpression of p27 Kip1(198A) resulted in increased cell death ( Figure 4C). In geriatric MuSCs, overexpression of p27 Kip1(198D) rescued the discernible induction of cell death during Atg5 knockdown, in contrast, the p27 Kip1(198A) mutant could not rescue the induction of cell death. Overexpression of the p27 Kip1(198D) mutant also prevented cleaved PARP expression in both untreated and ATG5 knockdown geriatric MuSCs ( Figure S4B).
Next, tested whether the phosphomimetic p27 Kip1(198D) mutant could prevent cell death without upstream AMPK activation. We treated young MuSCs with the AMPK inhibitor compound C (10 mM) for 48 hr with or without forced expression of the of p27 Kip1(198D) mutant. Compound C was effective in suppressing AMPK phosphorylation and phosphorylation of downstream AMPK target p27 Kip1 in young MuSCs 48 hr in culture ( Figure S4C). Compound C treatment nearly doubled the incidence of cell death in the young MuSC; this result was blocked by overexpression of p27 Kip1(198D) (Figure 4D), further establishing p27 Kip1 as a pro survival mechanism. We then tested the role of AMPK/p27 Kip1 signaling in transplanted geriatric MuSCs ( Figure 4E). Similar to genetic AMPK gain of function, AICAR treatment to geriatric MuSCs resulted in a high number of GFP(+) cells 4 days after transplantation ( Figures 4F  and 4G). The inability of AMPK to phosphorylate p27 Kip1 decreased AICAR-induced enhancement of transplantation efficiency as shown by a vast reduction in GFP(+) cells.

Cellular Location of p27 Kip1 Dictates Function in the Activating MuSC
The anti-apoptotic capacity of p27 Kip1 is dependent on its translocation from the nuclei to the cytoplasm (Liang et al., 2002(Liang et al., , 2007Wu et al., 2006). To investigate whether the same is true in activating MuSCs, we determined the localization pattern of p27 Kip1 in quiescent and activated MuSCs. Across all aging groups, p27 Kip1 was located in the nuclei in freshly isolated quiescent MuSCs ( Figure 5A). After 48 hr in culture, p27 Kip1 in young MuSCs was located purely in the cytoplasm; in contrast old MuSCs had roughly equal amount in the nuclei and cytoplasm; in geriatric MuSCs  (legend continued on next page) p27 Kip1 remaining primarily in nuclei ( Figure 5B). We made similar observations with p27 Kip1 staining in young and geriatric MuSCs after 48 hr in culture ( Figure S5A). Furthermore, we demonstrated the efficacy of AICAR treatment in geriatric MuSCs to translocate p27 Kip1 to the cytosol (Figure S5A). We also tested the cellular location of the p27 Kip1 mutants. Overexpression of the p27 Kip1(198A) mutant was located primarily in the nuclei; in contrast, the AMPK phosphomimetic p27 Kip1(198D) mutant was located in the cytoplasm ( Figure S5B). Next, we determined if the cytoplamic p27 Kip1 could induce autophagy as previously reported in non-muscle cell lines (Liang et al., 2007). Overexpression of the p27 Kip1(198D) in young activated MuSCs increased LC3B puncta by roughly 50% ( Figure 5C); and increased the ratio of LC3B II:I by 2-fold ( Figure 5D). As indicated by immunofluorescence or protein analysis, overexpression of the p27 Kip1(198A) mutant did not affect MuSC autophagy (Figures 5C and 5D). The ability to translocate p27 Kip1 out of the nuclei appeared critical for cell proliferation. Young MuSCs overexpressing p27 Kip1(198A) had a marked reduction in EdU labeling ( Figure 5E). In contrast, overexpression of the p27 Kip1(198D) mutant rescued proliferation in geriatric MuSCs. This effect was dependent on the induction of autophagy, as Atg5 knockdown negated the proliferative effects on p27 Kip1(198D) overexpressing geriatric MuSCs ( Figure 5F). These data suggest p27 Kip1 induces cell survival in an autophagy-independent fashion, while its proliferative effects are autophagy dependent.

AMPK Activation through p27 Kip1 Regulates Cellular Senescence
Loss of autophagy in satellite cells of young mice results in the induction of senescence markers (Garcia-Prat et al., 2016a). We sought to determine if restoring AMPK signaling activity would suppress senescence markers. Beta-galactosidase (b-gal), an established senescence marker (Dimri et al., 1995), was greater in old MuSCs and even greater in geriatric cells ( Figure S6). AICAR treatment in geriatric cells sup-pressed b-gal staining ( Figure 6A). Overexpression of the p27 Kip1(198A) mutant attenuated AICAR-induced suppression of b-gal staining ( Figure 6A). Similar effects were observed when quantifying protein expression of phosphorylated S6 Kinase ( Figure 6B). mRNA expression of senescence genes p16INK4a and p21CIP1 increased in geriatric MuSCs ( Figure 6C). AICAR treatment prevented p16INK4a and p21CIP1 expression while overexpression of p27 Kip1(198A) attenuated this effect. Thus, the AMPK/ p27 Kip1 pathway appears to regulate MuSC cellular senescence in addition to the autophagy and apoptotic processes.

DISCUSSION
Stem cell transition from quiescence to cell-cycle entry is a critical transformation. As cells proceed to the ''blast'' phenotype, in order to facilitate energy production and morphological changes, there is cytoplasmic hypertrophy (Rodgers et al., 2014). Cellular anabolism comes with high energy demands. As these processes require energy in excess of the minimal energy stores of the activating stem cell, this is a period of relative energy deficit and nutrient stress. Until energy demands are met or adequate survival measures are in place, the cell is susceptible to apoptosis. Our data support the role of AMPK/p27 Kip1 signaling in ensuring cell survival through prevention of apoptosis and enhancing the autophagy program during early activation ( Figure 6D). Defects in this pathway, through aging or genetic manipulation, lead stem cells to default to apoptosis or senescence (Garcia-Prat et al., 2016b).
Cell fate is determined by a delicate balance between energy availability and survival mechanisms. The interplay between autophagy and apoptosis is becoming increasing apparent across many cell types (Ravanan et al., 2017). In the MuSC, autophagy is critical for activation and proliferation, acting as a temporary energy reserve to fuel the initial stages of cell proliferation (Tang and Rando, 2014). Aging MuSCs have a reduction in autophagy while (B) Upper: representative blot of total and cleaved PARP; lower: quantification of protein expression normalized to untreated geriatric cells (N = 3 independent experiments). (C) Percentage of TUNEL-positive cells during Atg5 knockdown with concurrent overexpression of p27 Kip1 mutants p27 Kip1(198D) or p27 Kip1(198A) (N = 3 independent experiments). (D) Percentage of TUNEL-positive cells during treatment of compound C to young MuSCs with concurrent overexpression of p27 Kip1 mutant p27 Kip1(198D) (N = 3 independent experiments). (E) Geriatric MuSCs treated with or without AICAR and infected with respective p27 Kip1 mutant lentivirus. Cells were transplanted into injured young SCID muscle and quantified 4 or 28 days later.  (C) p27 Kip1 phosphorylation status-mediated cellular location and autophagy. Left: immunofluorescence staining of LC3B and p27 Kip1 expression of young MuSCs overexpressing GPF-labeled WT p27 Kip1 , p27 Kip1(198A) , or p27 Kip1(198D) . All cells were treated with chloroquine for (legend continued on next page) rescuing the aged-related defect has a rejuvenating effect, improving function and suppressing senescence (Garcia-Prat et al., 2016a). Aged MuSCs are more susceptibility to cell death when challenged with cellular stress (Jejurikar et al., 2006). We observed an age-dependent reduction in autophagy during the initial days of in vitro activation, associated with reduced proliferation and increased apoptosis. Furthermore, advanced age leaves the MuSC extremely susceptible to apoptosis under conditions of autophagy suppression. This is in contrast to what happens in young MuSCs during autophagy suppression (Tang and Rando, 2014), suggesting the possible presence of age-related perturbations in key survival mechanisms.
The AMPK pathway promotes cell survival during times of nutrient stress. Although AMPK targets numerus proteins and cellular pathways (Sanli et al., 2014), downstream signaling to the multifunctional p27 Kip1 can induce autophagy; increase proliferation; and increase cell survival (Liang et al., 2007;Sun et al., 2014). We hypothesized and subsequently observed this pathway to be relevant in activating MuSCs. In activated young MuSCs, there was greater phosphorylation of AMPK and its downstream target p27 Kip1 yet, in activated old and geriatric cells, there was less phosphorylation of these proteins. This observation is consistent with the idea that aging reduces AMPK sensitivity in muscle and likely throughout all organs and tissues (Salminen and Kaarniranta, 2012). Activation of the AMPK pathway by AICAR prevented apoptosis in aged cells; conversely, AMPK inhibition in young cells resulted in greater apoptosis. The lack of AMPK signaling could explain the differential age effects of apoptosis under autophagy inhibition. In the young MuSC, AMPK activation suppresses apoptosis and results in succeeding energy production, while the aged MuSC has a reduction in AMPK activity and the added stress of suppressed autophagy defaults the cell to apoptosis.
We observed the anti-apoptotic capacity of AMPK is dependent on phosphorylation and subsequent cytoplasmic translocation of p27 Kip1 . Machida and Booth (2004) reported aged MuSCs to have a greater expression of nuclear p27 Kip1 ; we confirmed these findings. However, our data suggest cellular location of p27 Kip1 is dynamic, shifting from the nucleus to the cytoplasm as the cell un-dergoes activation. Failure to translocate p27 Kip1 during activation is one mechanism of age-related cellular dysfunction. Maintaining p27 Kip1 in the nuclei not only inhibits cell-cycle progression, it also leaves the cell susceptible to apoptosis. In fact, inhibition of the cytoplasmic translocation of p27 Kip1 is a popular pharmacological target to encouraging cell death in various cancer cell line (Hiromura et al., 1999;Liang et al., 2002;Motti et al., 2005;Wu et al., 2006). Our current work shows the potency of this pathway and its dysfunction with age in the MuSC.
Our current work and that of others (Garcia-Prat et al., 2016a) show advanced age drives senescence in muscle MuSCs. Considering the potency of the AMPK/p27 Kip1 pathway in revitalizing cellular function in aged MuSCs, we strove to determine its regulatory role in senescence. AMPK activation was effective in blocking the induction of senescence markers. However, inability of AMPK to phosphorylate p27 Kip1 negated these effects. Autophagy is a negative regulator of MuSC senescence (Garcia-Prat et al., 2016a). The translocation of p27 Kip1 from the nuclei to the cytoplasm could play a role in preventing senescence by relieving the breaks on the cell cycle and promoting autophagy.
In sum, we observed a reduction in autophagy and more apoptosis in activating aged MuSCs when compared with their younger counterparts. This effect was due, in part by inactivation of the AMPK/p27 Kip1 pathway. Reactivation of AMPK and/or downstream p27 Kip1 translocation restored proliferation and cell survival in aged MuSCs. We observed functional differences in cytoplasmic and nuclear p27 Kip1 , the former enhancing cell survival and autophagy and the later promoting quiescence, apoptosis, and senescence. Thus, dysfunctional signaling of the AMPK/ p27 Kip1 pathway may be a central mechanism for ageinduced loss of MuSCs, subsequent decline in regenerative function and perhaps a mechanism of and a viable target for enhancing regeneration in aged muscle.

Lentivirus Infection
Lentivirus for GFP, sh-Atg5 and scrambled sh were purchased from Dharmacon (GE Lifesciences) with titer 10 8 transduction units (TU)/mL. Lentivirus for WT p27 Kip1 and p27 Kip1 mutant vectors, originally described in Liang et al. (2007) were cloned and amplified at the Duke Viral Vector Core with titer 10 9 TU/mL. For in vitro experiments lentivirus would be added shortly after plating and left overnight before replaced with fresh media.

Senescence-Associated b-Gal Activity
Senescence-associated (SA) b-gal activity was detected in MuSCs using the senescence b-Galactosidase Staining Kit (Cell Signaling (D) Summary figure illustrating role of AMPK/p27 Kip1 signaling on autophagy, proliferation, senescence, and apoptosis. * Signifies difference from untreated geriatric control cells; y signifies difference from AICAR-treated geriatric cells; # signifies difference from young cells;^signifies difference from untreated geriatric control cells (p < 0.05). Scale bars, 5 mm. Data are presented as means ± SE. Technology), according to manufacturer's instructions. SA b-gal + cells were quantified as percentage of the total number of cells analyzed.

In Vitro Immunofluorescence and Quantification
MuSCs were fixed with 4% paraformaldehyde for 15 min, washed with PBS and permeabilized with TBS-T (0.1% Triton X-[pH 8.0] TBS) for another 15 min. The cells were then blocked in 2% normal goat serum for 20 min. This was followed by incubation with respective antibodies diluted in blocking solution for 1 hr. An Alexa Fluor 488 secondary antibody (Invitrogen, cat. no. A-21121) was used for detection. The cells were counterstained with DAPI (Cell Signaling, cat. no. 4083) and Phalloidin (Cell Signaling Technology, cat. no. 8953) to visualize nuclei and actin, respectively. Images were taken using fluorescent microscopes (Zeiss). Total cell number and the percentage of cells expressing a particular fluorophore were quantified using the automated Cellomics ArrayScan. ImageJ/FIJI was used to measure densitometry for cytoplasmic/nuclear localization and quantification of cell size.

Muscle Injury
Skeletal muscle injury was performed by injection of barium chloride (BaCl 2 ) into the tibialis anterior (TA) muscle as described previously (White et al., 2009). Thirty microliters of 1.2% BaCl 2 (Sigma) was injected into the TA muscle.

Proliferation Assay
Quantification of cell proliferation was assayed with Click-iT EdU Alexa Fluor 594 Imaging Kit (Thermo Fisher Scientific) according to manufacturer's instructions. Quantification of EdU-positive cells was performed on the automated Cellomics ArrayScan.

Cell Apoptosis
Quantification of cellular apoptosis was assayed with TUNEL and Annexin V/PI staining. TUNEL Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor 488 dye according to manufacturer's instructions. Quantification of TUNEL-positive cells was performed on the automated Cellomics ArrayScan.

Satellite Cell Engraftment
Transplantation methods were performed as described previously (Garcia-Prat et al., 2016a;Sousa-Victor et al., 2014). In brief, FACS isolated satellite cells from young or geriatric C57BL/6 mice were plated and treated with AICAR and/or infected with the respective lentivirus overnight. The next day the media was briefly replaced and cells were collected, re-suspended in 20% fetal bovine serum F10 medium and injected into muscles previously injured with BaCl 2 the day before. Recipient mice were young (10-12 weeks) SCID mice. For each mouse, 10,000 cells were injected into the TA muscle. Transplantation experiments were analyzed 4 or 28 days post transplantation.

In Vivo Muscle Immunofluorescence
Immediately post dissection the whole muscle was incubated in 20% sucrose (w/v) for 12 hr then cut at the mid-belly and placed in OCT for later use. Sections (12 mm) were cut on a cryostat and dried at room temperature for 10 min. Sections were then blocked for 0.5 hr with 10% goat serum, followed by a 2-hr incubation with an anti-GFP antibody conjugated with Alex Flour 488 (Thermo Fisher Scientific, A-21311). After staining, slides were washed with PBS and mounted with mounting medium containing DAPI (Vector Laboratories, cat. no. H-1200). For quantification, six images (203) were taken per TA muscle and GFP(+) cells or myofibers were counted and totaled for each muscle.

Cell Culture Reagents
To quantify LC3B flux MuSCs were treated with 10 mM chloroquine (Sigma) for 12 hr. To inhibit AMPK activity MuSCs were treated with 10 mM compound C (Sigma) throughout time in culture. To induce AMPK activity MuSCs were treated with 250 mM AICAR (Sigma) for the duration of time in culture. For transplantation experiments, MuSCs were treated with AICAR overnight, typically during lentiviral infection and transplanted the following day.

Cell Fractionation
Cytosolic and nuclear fractionation was performed with NE-PER Nuclear and Cytoplasmic Extraction Reagent (Thermo Scientific, no. 78833) according to manufacturer's instructions.
Approximately 1 3 10 5 cells were plated for each sample to adequately quantify protein expression in nuclear and cytoplasmic compartments.

Statistical Analysis
Data were analyzed by one-way ANOVA using Tukey's post-hoc analysis or two-tailed Student's t test. All data are reported as means ± SEM. Statistical analysis was performed using GraphPad Prism 6 software.