mTORC1 activation during repeated regeneration impairs somatic stem cell maintenance

Introduction

Regenerative processes in somatic tissues require coordinated regulation of stem cell proliferation and daughter cell differentiation to ensure long-term tissue homeostasis (Chandel et al., 2016; Jones and Rando, 2011). Studies in a wide range of model systems indicate that the loss of this coordination contributes to regenerative dysfunction in aging tissues. Understanding the causes and consequences of age-related dysregulation of these processes is likely to identify intervention strategies to maintain stem cell function and improve regenerative capacity in aging tissues.

Barrier epithelia are exposed to frequent environmental challenges, and are thus under repeated regenerative pressure during the lifespan of an organism. Accordingly, age-related stem cell dysfunction is particularly evident in barrier epithelia of aging organisms, resulting in dysplasias, degenerative diseases, and cancers (Li and Jasper, 2016; Wansleeben et al., 2014). The Drosophila posterior midgut epithelium has emerged as an excellent model system to study the causes and consequences of age-related regenerative dysfunction of barrier epithelia (Ayyaz and Jasper, 2013). Excessive proliferation and mis-differentiation of intestinal stem cells (ISCs) is a common phenotype in aging flies, resulting in epithelial dysplasia and the breakdown of the epithelial barrier function. These phenotypes contribute to mortality in old flies, and interventions that limit and delay their progression frequently result in lifespan extension (Guo et al., 2014; Li et al., 2016; Wang et al., 2015).

In young animals, ISCs divide infrequently under homeostatic conditions, but are rapidly and transiently activated in response to damage to the intestinal epithelium (Ayyaz et al., 2015; Biteau et al., 2008; Jiang et al., 2009). During such regenerative episodes, ISCs divide to self-renew and produce enteroblasts (EB), which undergo differentiation to become either enterocytes (ECs) or enteroendocrine cells (EEs) (Ayyaz and Jasper, 2013; Li et al., 2016). To adjust proliferative responses to changing local, systemic, and environmental conditions, ISCs integrate a wide range of growth factor, inflammatory, and stress signals by modulating intracellular calcium levels (Ayyaz and Jasper, 2013; Biteau et al., 2011; Deng et al., 2015a; Li et al., 2016). Differentiation in the ISC lineage is controlled by Delta/Notch (Dl/N) signaling (Ayyaz and Jasper, 2013; Li et al., 2016). Dl is expressed in ISCs and triggers N activation in EBs. In these cells, N coordinates cell specification with cell growth and proliferation by activating the TOR signaling pathway (Kapuria et al., 2012).

As a constituent of the mTORC1 complex, TOR kinase is part of an evolutionarily conserved nutrient sensing pathway that coordinates cellular responses to nutrients by promoting anabolic functions, including translation, and by inhibiting catabolic processes like autophagy (Laplante and Sabatini, 2012). Accordingly, it has a major impact on cell growth, and is among the best understood regulators of tissue and organ size in metazoans (Laplante and Sabatini, 2012). Its repression extends lifespan in different organisms, including flies and mice (Kennedy and Lamming, 2016). mTORC1 can be activated by multiple mechanisms, including by growth factors through Akt-mediated phosphorylation of Tuberous Sclerosis Complex 2 (TSC2; encoded by the gene gigas in Drosophila) and subsequent inhibition of the TSC1/2 complex (Laplante and Sabatini, 2012). TSC1 promotes the stability of TSC2, a GTPase activating protein for the small GTPase Rheb, inhibiting Rheb-mediated mTORC1 activation. Active mTORC1 phosphorylates translational regulators (including ribosomal protein S6 Kinase, S6K, and eIF4E Binding Protein, 4EBP), autophagy regulators (including ATG1/ULK1), and TFEB transcription factors, resulting in a net increase of protein production, and a decrease in autophagy and lysosome biogenesis (Laplante and Sabatini, 2012, 2013).

Results from (1) fly germline and intestinal stem cell lineages, (2) mouse embryonic, hematopoietic, follicle, muscle and neuronal SCs, (3) induced pluripotent stem cells, and (4) human embryonic SCs suggest that TSC/mTORC1 signaling has an important regulatory role in stem cell maintenance and differentiation (Chen et al., 2011; Magri et al., 2011). In mouse hematopoietic SCs (HSCs), the expression of constitutively active AKT leads to mTORC1-dependent SC loss (Kharas et al., 2010). Loss of Pten in HSCs or myogenic progenitors leads to constitutively active AKT and mTORC1 signaling and SC activation that is associated with long-term SC loss (Yilmaz et al., 2006; Yue et al., 2016; Zhang et al., 2006). Sustained activation of mTORC1 in hair follicle SCs (through the activation of Wnt signaling) leads to SC exhaustion (Castilho et al., 2009). In human embryonic stem cells, activation of S6K by mTORC1 has been reported to induce differentiation (Easley et al., 2010), and reduction of mTORC1 activity in Paneth cells (ISC support cells) under dietary restriction promotes ISC maintenance and proliferation through a non-autonomous mechanism (Yilmaz et al., 2012). mTORC1 also appears to play a critical role in the activation of SC proliferation during regenerative episodes in various systems: short term induction of AKT promotes HSC pool expansion (Kharas et al., 2010), while in a mouse model of muscle injury, mTORC1 activation is necessary to allow rapid proliferation of muscle SCs (MuSCs) by promoting a switch from the G₀ to a mTORC1-dependent ‘G_alert’ state (Rodgers et al., 2014). During hair regeneration, mTORC1 signaling promotes hair follicle SC activation by repressing BMP signaling (Deng et al., 2015b).

In flies, TSC/TOR signaling regulates proliferation and maintenance of SCs according to nutritional conditions both in the germline and the intestine (Amcheslavsky et al., 2011; Hsu and Drummond-Barbosa, 2009; Jasper and Jones, 2010; Kapuria et al., 2012; LaFever and Drummond-Barbosa, 2005; LaFever et al., 2010; McLeod et al., 2010; O’Brien et al., 2011; Quan et al., 2013; Sun et al., 2010). In quiescent ISCs, TOR is repressed by high levels of TSC2. The activation of N in EBs results in the repression of TSC2 expression and subsequent activation of TOR, which is sufficient and required for growth and differentiation of these cells into ECs (Kapuria et al., 2012). TSC/TOR signaling thus serves as a critical determinant of cell identity in the ISC lineage, and repression of TOR by TSC1/2 is required to prevent ectopic ISC differentiation (Amcheslavsky et al., 2011; Kapuria et al., 2012; Quan et al., 2013).

The conflicting requirement for mTORC1 activation to trigger proliferative activity in a wide variety of SCs in flies and mice, and for mTORC1 repression in the long-term maintenance of SCs in these animals, suggests that precise mTORC1 regulation is essential if long-term tissue homeostasis and regenerative capacity is to be preserved. Here, we have asked how the repeated activation of mTORC1 during regenerative episodes impacts long-term maintenance of SCs in the fly intestine and in the mouse tracheal epithelium, two barrier epithelia with similar morphology and regenerative processes. The mouse airway epithelia contain a SC population, the basal cells (BC), which differentiate into either ciliated or secretory cells (Rock et al., 2009). BCs are characterized by expression of Keratin 5 (Krt5) and the transcription factor p63 (Trp63). After injury, BCs rapidly segregate into two populations, expressing either the intracellular domain of Notch2 (N2ICD) or c-myb that commit these cells to the secretory (N2ICD expressing BCs) or ciliated (c-myb expressing BCs) cell lineages (Pardo-Saganta et al., 2015). Activated BCs express both Krt5 and the differentiation marker Keratin 8 (Krt8), transitioning through a so-called Basal Luminal Progenitor (BLP) state, before becoming Early Precursor cells (EPs) which express only Krt8, but no markers of fully differentiated cells (Scgb1a1, Muc5ac, Foxj1, CCSP, AcTub). Secretory cells still have the ability to self-renew and produce ciliated cells (Rock et al., 2011; Watson et al., 2015) and can also dedifferentiate into BCs (Tata et al., 2013). The regulation of BC proliferation closely resembles that of Drosophila ISCs (Biteau et al., 2011; Rock et al., 2011). Similar to ISCs, BC differentiation is controlled by Notch activity (Biteau et al., 2011; Rock et al., 2011).

Our study shows that in both systems, mTORC1 is transiently activated in SCs after a regenerative stimulus. The induction of repeated regenerative episodes results in mTORC1-dependent loss of ISCs and BCs, while chronic activation of mTORC1 (by loss of TSC1) causes loss of ISCs or BCs, respectively. Critically, mTORC1 activation is a likely cause of BC loss in aging mice, as long-term treatment with the mTORC1 inhibitor Rapamycin is sufficient to prevent BC loss and restore BC numbers in aging mice. Our data indicate that this loss may be caused by mTORC1-dependent differentiation of BCs. These effects are conserved in the muscle, as activation of mTORC1 promotes reprogramming of the MuSC transcriptome towards a differentiation signature, and the age-dependent loss of MuSCs can be prevented by limiting mTORC1 activity genetically in these cells. Our findings highlight the evolutionarily conserved role of mTORC1 in coordinating SC proliferation and differentiation, and demonstrate the usefulness of mTORC1 suppressing interventions in promoting regenerative homeostasis of various organ systems.

Results

In homeostatic conditions, TOR signaling is specifically active in EBs of the Drosophila intestine, and has to be suppressed in ISCs to prevent their differentiation (Fig. 1A, (Kapuria et al., 2012). At the same time, we observed previously that TOR is essential for normal proliferative activity, as ISCs homozygous for TOR loss of function alleles fail to generate clonal lineages that grow as fast as lineages from wild-type ISCs (Kapuria et al., 2012). A possible explanation for this paradox is that TOR becomes transiently activated in active ISCs. To test this idea, we performed infection studies using Erwinia Carotovora Carotovora (Ecc15), a mild enteropathogen that induces a synchronized and transient proliferative response in the epithelium (as measured by the increase in mitotic, phospho-histone H3 expressing ISCs; Fig. 1B). Using phosphorylated 4EBP as a readout for TOR activity, we confirmed that TOR is inactive in ISCs under homeostatic conditions (Fig. 1C & C′), but also found that TOR activity increases within 4 hours after Ecc15 treatment. The p-4EBP antibody reliably detects changes in TOR signaling activity (Kapuria et al., 2012; LaFever et al., 2010; Penney et al., 2012; Cheng et al., 2011; Vachias et al., 2014). ISCs were labeled by YFP expressed under the control of the ISC/EB driver esg::Gal4, combined with the heat-sensitive Gal4 inhibitor Gal80^ts and with Gal80 expressed under the control of the Su(H)Gbe promoter to inhibit UAS-linked transgene expression in EBs (McGuire et al., 2003; Micchelli and Perrimon, 2006; Ohlstein and Spradling, 2006; Wang et al., 2014).

The increase in p-4EBP in ISCs depends on TSC/TOR signaling, as over-expression of TSC1 and 2 was sufficient to prevent 4EBP phosphorylation in ISCs, and as knocking down TSC1 results in significantly elevated p-4EBP levels (Fig. 1C′). Consistently, ISC size increases transiently during the regenerative response, and this increase depends on TSC/TOR signaling (Fig. 1D). The ISC phenotypes observed under homeostatic conditions when TSC1 is knocked down suggest that ISCs may also receive signals that activate TOR transiently during homeostatic renewal of the epithelium.

These observations support the notion that TOR activity is induced in ISCs that are under regenerative pressure. To test whether this activation of TOR would be required for normal regeneration in the gut, we knocked-down the TORC1 component dRaptor in ISCs and quantified the number of mitotic ISCs at two timepoints after the challenge. Strikingly, ISC proliferation was significantly lower in raptor-deficient animals in the early phase of the regenerative response, but was higher than in wild-type animals in the recovery phase, suggesting that the induction of ISC proliferation is not inhibited, but delayed in TOR loss-of-function conditions (Fig. 1B). Together, these results indicate that TOR activation in ISCs is required for the rapid activation of ISC proliferation in response to a challenge. This observation is reminiscent of the role of mTORC1 activation in the regenerative response of mouse satellite cells during muscle regeneration (Rodgers et al., 2014). We cannot exclude, however, that the function of TOR in fly ISCs changes from pro-proliferative in the early phase of the response to anti-proliferative in the late response.

Since activation of TOR in ISCs also promotes their differentiation into ECs (Kapuria et al., 2012), we hypothesized that its repeated activation as part of multiple regenerative episodes could contribute to stem cell exhaustion by triggering spontaneous differentiation. We tested this hypothesis by performing multiple successive infections with Ecc15 and determining the consequence of these repeated proliferative response for ISC numbers in the epithelium of the posterior midgut (Fig. 1 E–G). We found a significant, progressive decline of ISC numbers in animal exposed to 2 or more successive infections (Fig. 1F). This loss of ISCs was prevented when TOR activation was limited by over-expression of TSC1 and 2, and exacerbated when TOR was activated in the ISC lineage by knocking down TSC1 (Fig. 1G). When TOR activity was genetically induced in a repeated fashion by inducing the expression of a dsRNA against TSC1 3 times, ISC loss was also observed (Fig. 1H). Interestingly, the number of mitotic figures during the activated phase of the regenerative response was maintained even after 3 Ecc15 exposures, indicating that under these conditions the remaining pool of ISCs was still large enough to fuel a vigorous regenerative response (Fig. S1A). However, there was a mild, but significant change in cell composition of the intestinal epithelium, with an elevated percentage of enteroendocrine cells at the expense of enterocytes (Fig. 1I).

At older ages, flies develop epithelial dysplasia in the intestine, characterized by chronic activation of ISCs (Biteau et al., 2010; Guo et al., 2014; Li et al., 2016; Wang et al., 2015). While this leads to a significant increase in mitotic figures even in unchallenged conditions, exposure to Ecc15 still elevated mitotic figures in old flies (Fig. S1B). To test whether chronic TOR activity may contribute to ISC dysfunction in older animals, we asked whether marked lineages derived from individual ISCs (using the MARCM system, (Lee and Luo, 2001)) would be lost with age, and whether Rapamycin exposure was sufficient to rescue this phenotype (Fig. S1C). Indeed, we found that clone numbers declined in aging animals, and that this loss of functional ISCs was significantly slower when animals were exposed to Rapamycin (Fig. S1C).

The requirement for TOR activation to achieve a rapid regenerative response in the midgut thus also contributes to a progressive decline in ISC numbers and function. This observation has critical implications for our understanding of the loss of tissue homeostasis not only in flies, but also in aging vertebrates, and points to possible intervention strategies for the preservation of regenerative capacity in barrier epithelia. We thus sought to test whether a similar transient role for mTORC1 activity in regeneration, coupled to a loss of stem cells, is observed in a vertebrate barrier epithelium.

We chose the tracheal epithelium of the mouse as a system with high similarity to the fly intestinal epithelium. Basal Cells (BCs) in this epithelium are triggered to proliferate in response to epithelial injury by SO2 exposure, and differentiation of early precursor cells is induced by BC-derived Delta-like signals (Fig. 2A). SO2 exposure results in the elimination of all differentiated cells of the epithelium, resulting in a monolayer of activated BCs at 1 day after exposure, which proliferate to restore epithelial cells (Fig. 2B and (Tata et al., 2013)). Further highlighting the similarity of epithelial regeneration in the fly intestine and the mouse trachea, we observed a transient induction of S6 phosphorylation (which we chose as a readout for mTORC1 pathway activity, as an antibody against p-4EBP is not reliable in paraffin sections) in basally located cells around 1 day after the SO2 exposure (Fig. 2B, D, S2A). This activation correlated with proliferative activation (as determined by Ki67 staining) of BCs. pS6/Ki67+ cells (the only remaining cells after the SO₂-induced ablation of differentiated tracheal epithelial cells) also expressed both the BC marker Keratin 5 (Krt5) and the differentiation marker Keratin 8 (Krt8), indicating that they constitute transient Basal Luminal Progenitor (BLP) cells (Fig. 2B, C, S2A–C). Between 3 and 8 days after the challenge, the structure of the epithelium was largely re-established, and S6 phosphorylation and Ki67 staining returned to basal levels (Fig. 2B, D). Furthermore, Krt5+ and Krt8+ cells segregated into Krt5+ BCs and Krt8+ differentiated cells (Fig. 2B, C, S2B). The transient induction of S6 phosphorylation was also observed in western blots of tracheal extracts (Fig. 2D, S2D), and correlated with a transient increase in size of Krt5+ cells (Fig. 2E).

Tracheal BC can be isolated and cultured in 3D cultures to generate ‘Tracheospheres’, organoids that consist of an epithelium with Trp63+/Krt5+ BCs, as well as ciliated and secretory differentiated cells. In growing spheres, sporadic activation of mTORC1 signaling (based on p-4EBP staining) can be observed in cells emerging from the basal layer, indicating that transient mTORC1 activity is part of the normal differentiation pathway in these organoids (Fig. 2F). Accordingly, inhibition of mTORC1 by Rapamycin treatment results in a decrease in Tracheosphere growth, thinning of the Tracheosphere epithelium, and accumulation of Trp63+ BCs (Fig. 2 G–I). We also observed a significant reduction in mitotic (pH3+) cell numbers, but not in apoptotic cells (detected using anti-cleaved Caspase 3 antibody) in Rapamycin-treated Tracheospheres (Fig. S2E, F). These observations suggest that mTORC1 signaling is critical for the establishment of a functional epithelium with differentiated cells, as well as for proliferation of BCs in the tracheal epithelium.

To ask whether, similar to the fly intestine, chronic or repeated activation of mTORC1 may negatively impact BC maintenance in mice, we performed repeated ablation of the epithelium by SO2 exposure, allowing the epithelium to recover for 2 weeks in between exposures (Fig. 3A). This regimen resulted in a significant loss of Trp63+ BCs in the epithelium, and this loss was dependent on mTORC1 signaling, as concurrent dietary supplementation with microencapsulated Rapamycin (42 ppm) (Flynn et al., 2013; Harrison et al., 2009; Miller et al., 2011; Wilkinson et al., 2012) resulted in a significant rescue of this phenotype (Fig. 3A). Repeated SO2 exposure further resulted in a mTORC1-dependent increase in secretory cells at the expense of BCs (Fig. S3A).

To further confirm the role of mTORC1 signaling in the loss of BCs, we genetically activated mTORC1 signaling in these cells. We used a Krt5::Cre^ERT2 Tamoxifen-inducible driver to knock out TSC1 in BCs of young mice in combination with the Rosa26R-LacZ transgene (Rock et al., 2009)(Fig. 3B). This resulted in a sustained increase of phospho-S6 in BCs (Fig. 3B, C, S3B), along with an increase in their size and activation of BC proliferation (Fig. 3C, S3B). Similar to fly ISCs, BCs of the tracheal epithelium may receive signals that activate mTORC1 in the absence of TSC1 during homeostatic renewal. While loss of TSC1 did not cause a significant change in the overall proportion of differentiated ciliated vs secretory cells under basal conditions (Fig 3C), epithelia with TSC1-deficient BCs accumulated Krt5+/Krt8+ cells after SO2 exposure, and these cells did not segregate into distinct Krt5+ and Krt8+ cell populations after the SO₂ treatment at the same rate as wild-type cells (Fig. 3D). Interestingly, loss of TSC1 in BCs caused epithelia to reacquire differentiated cells (both secretory and ciliated cells) at a faster rate than wild-type epithelia after SO2 exposure, suggesting that elevated mTORC1 activity may promote differentiation of BC-derived progenitors (Fig. S3B). We did not observe elevated levels of apoptosis in tracheal epithelia in which TSC1 was deleted from BCs (Fig. S3C).

These results indicate that, while mTORC1 activity in BCs contributes to activation of BCs, downregulation of mTORC1 activity is required for cells to return from an activated BLP state into a quiescent BC state.

TSC1-deficient BCs were observed at roughly the same rates as wild-type BCs in the tracheal epithelium up to 10 days after deleting TSC1, yet their numbers decline later on, resulting in a significant reduction of Krt5+ BCs at 28 days after Cre induction (Fig. 3E, F, S3D, E). To characterize the consequences of TSC1 loss in individual BC lineages, we used a Rosa26R::eYFP lineage tracer and reduced the numbers of Tmx injections from 4 to 3 (generating more isolated eYFP-marked BC lineages). 40 days after the Tamoxifen treatment, only 70% of TSC1-deficient clones contained a BC (based on Krt5 expression), while the rest contained only cells in a more differentiated state (Krt8+ cells). 100% of wild-type eYFP clones, instead, contained a stem cell (Fig. 3G, S3E). The decline in BC numbers in these lineages was primarily due to the increased differentiation of secretory cells (Fig. 3G, S3E). We observed similar phenotypes in tracheosphere cultures, where the number of Trp63+ BCs declined within 48 hours after induction of Cre (Fig. 3H, S3F).

We further tested whether other stem cell types may be subject to differentiation when mTORC1 is activated, and found that the muscle stem cell (MuSC) transcriptome acquires hallmarks of myogenic differentiation when TSC1 is knocked out, while differentiation markers are depleted when the mTORC1 component Raptor is knocked out (Fig. S3G, H). Accordingly, mTORC1 promotes the rapid expression of the myogenic differentiation marker, MyoG, in MuSCs following isolation. (Fig. S3I).

These results support the notion that sustained mTORC1 activation promotes the differentiation of mammalian tracheal and muscle stem cells, although further studies are needed to fully characterize the cellular response of stem cells to long-term mTORC1 activity.

Aging is accompanied by chronic activation of mTORC1 signaling in many tissues, and inhibition of TOR is sufficient to extend lifespan in a wide range of organisms (Bjedov et al., 2010; Garcia-Prat et al., 2016; Harrison et al., 2009; Markofski et al., 2015; Miller et al., 2011; Wilkinson et al., 2012). Tracheal BC numbers decline with age (Wansleeben et al., 2014) through an unknown mechanism. Our results indicated that elevated or frequent mTORC1 activity, either during repeated regenerative episodes, or due to chronic activation of mTORC1, could cause this loss of BCs. To test this hypothesis, we first analyzed regeneration in the trachea of young and old mice, and found a sustained increase of BLP-like Krt5+/Krt8+ cells, accompanied by a mild (not statistically significant) reduction in proliferating cells (Ki67+) in response to SO2 exposure (Fig. 4, S4). These observations are consistent with mTORC1 gain-of-function phenotypes, and support the idea that elevated mTORC1 signaling impairs tracheal regeneration in old mice by increasing the probability of BCs entering or remaining in the BLP pre-differentiated state. Accordingly, we found elevated p-S6 levels in old BCs compared to BCs of young animals, both under homeostatic conditions and during regeneration (Fig. S4B, S5C). Furthermore, exposure to dietary Rapamycin was sufficient to promote return of Krt5+/Krt8+ BLPs into Krt5+ only BCs (Fig. 4A).

To test this hypothesis further, we asked whether inhibiting mTORC1 signaling chronically would be sufficient to maintain BC numbers in aging mice. Using dietary supplementation with microencapsulated rapamycin (14ppm) (Flynn et al., 2013; Harrison et al., 2009; Miller et al., 2011; Wilkinson et al., 2012), we tested the effects of long-term, chronic rapamycin treatment on BC maintenance and function. Female mice were treated either for 3 months (starting the rapamycin regimen at 9 or 15 months of age and sacrificed at 12 or 18 months, respectively), or for 12 months (starting at 12 months, sacrificed at 24 months; Fig. 5A). We observed increased proliferating cell numbers in basal conditions (Fig. S5A), restoration of the balance between secretory, ciliated and basal cells (Fig. S5B), reduced chronic mTORC1 activity in the epithelium (Fig. S5C), recovery of epithelial thickness in the trachea (Fig. 5B, S5D), as well as a significant increase in BC numbers in response to Rapamycin exposure in each of these conditions (Fig. 5C, D, S5B). Interestingly, tracheospheres derived from 18 months old animals exposed to Rapamycin for 3 months also showed a significant reduction in the accumulation of BLP-like Trp63+/Krt8+ cells. As observed in the tracheal epithelium of old mice, BCs from old mice generate tracheospheres with higher numbers of these BLPs than BCs from young mice. The fact that this phenotype was reduced in tracheospheres derived from Rapamycin-fed mice suggests that the beneficial effects of chronic mTORC1 inhibition is maintained in BCs ex vivo (Fig. 5E).

To test whether mTORC1 inhibition is sufficient to allay stem cell loss also in other tissues, we analyzed the consequences of mTORC1 signaling perturbation in aging muscle stem cells (MuSCs). Using a Pax7::Cre^ER driver associated with a Rosa26::EYFP lineage tracer, we observed a loss of MuSCs in aged mice. This loss of MuSC can either be amplified by knocking out TSC1, or rescued by knocking out Raptor in MuSCs (Fig. 5F, S5E).

Discussion

Our results suggest that the age-related loss of stem cells in various tissues is a consequence of the repeated activation of mTORC1 signaling during regenerative episodes (Fig. 6). The mechanism(s) of this activation remain to be established, but recent studies in fly imaginal discs point to a possible activation of mTORC1 downstream of CycD/cdk4 and/or E2F1 activation in the cell cycle (Kim et al., 2017; Romero-Pozuelo et al., 2017). In the fly intestine, the mouse trachea, and the mouse muscle, the need for mTORC1 activation to induce stem cell activity contrasts with the requirement for mTORC1 repression to maintain stem cell identity, and as a consequence, repeated episodes of regenerative pressure increase the probability of SC differentiation, resulting in the progressive loss of SCs. Critically, our findings also indicate that dietary supplementation with Rapamycin is sufficient to promote tracheal SC maintenance, and thus preserve regenerative capacity of airway epithelia. This is consistent with the fact that suppression of mTORC1 activity, through Rapamycin or genetic means, increases lifespan and slows the progression of other age-related phenotypes (Kennedy and Lamming, 2016).

Our results, and the observation that mTORC1 activation is required for the exit from quiescence of muscle stem cells, while sustained mTORC1 activation (e.g. through the loss of PTEN) also results in a decline of SC numbers in the hematopoietic system, the muscle, and the hair follicle, suggests that the tradeoff between mTORC1-mediated induction of SC proliferation and differentiation-induced loss is widely conserved (Castilho et al., 2009; Chen et al., 2011; Deng et al., 2015b; Easley et al., 2010; Kharas et al., 2010; Magri et al., 2011; Rodgers et al., 2014; Yilmaz et al., 2012; Yilmaz et al., 2006; Yue et al., 2016; Zhang et al., 2006). Based on our findings, it can thus be anticipated that long-term Rapamycin feeding will also promote SC maintenance in tissues other than the tracheal epithelium.

Our findings in fly ISCs, and the loss of TSC – deficient BCs and MuSCs, indicate that the preservation of BC numbers in the aging tracheal epithelium by Rapamycin feeding is a consequence of cell-autonomous repression of mTORC1 activity in these cells. It is possible, however, that Rapamycin elicits other local or systemic effects that promote stem cell maintenance. Non-autonomous effects of mTORC1 signaling on SC maintenance have been described for intestinal crypts of the mouse for example (Yilmaz et al., 2012), and additional studies will reveal whether such non-autonomous effects also apply to the mouse trachea or muscle.

Our data show that repeated transient activation of mTORC1 during regenerative episodes is sufficient to cause SC loss in both the fly intestine and the mouse tracheal epithelium. We propose that such repeated regenerative events along the lifespan of the animal results in increased probability of SC differentiation, thus causing SC loss over time. However, mTORC1 signaling activity may also be chronically elevated in SCs, as has been proposed for MuSCs (Garcia-Prat et al., 2016). Such chronic activation could contribute to the loss of SCs even in the absence of regenerative pressure. Whether conditions in which regenerative pressure is specifically reduced would result in improved SC maintenance in aging mice remains unclear. Identifying and studying such conditions would allow distinguishing whether repeated mTORC1 activation or chronic age-related elevation of mTORC1 activation are the primary drivers of SC loss in aging animals.

Our studies in the fly intestine have revealed that TOR activation is sufficient to promote ISC differentiation (Kapuria et al., 2012). In the mouse trachea, the transient formation of Krt5+/Krt8+ BLPs during regeneration, the correlation of their formation with mTORC1 activation, and their increased formation with elevated mTORC1 activity and in old animals, are consistent with a similar differentiation-promoting role of mTORC1 in mammals. Accordingly, we find that TSC1-deficient BCs populate the SO2 depleted tracheal epithelium faster with differentiated ciliated and secretory cells than wild-type BCs. However, based on work in the hair follicle, it has also been proposed that mTORC1 may promote senescence of SCs (Castilho et al., 2009). We can thus not exclude that the effects of long-term Rapamycin exposure on BC maintenance are a consequence of reduced senescence or apoptosis rather than reduced differentiation. Studies that will explore the downstream mechanisms of mTORC1-mediated BC loss will likely resolve this question. In particular, the involvement of repressed autophagy or increased translation in mTORC1-mediated BC loss is going to provide critical insight. It is striking that the restoration of BC numbers in the old tracheal epithelium can be achieved even when Rapamycin is supplemented late in life (as late as 15 months of age). This may be explained by an increase in symmetric BC division when mTORC1 is inhibited and is consistent with a role for mTORC1 in promoting differentiation, as well as with the observation that mTORC1-deficient fly ISCs undergo symmetric divisions that result in small clusters of undifferentiated ISCs (Kapuria et al., 2012). However, these results may also indicate that de-differentiation of tracheal epithelial cells, which has been reported previously (Tata et al., 2013), may be increased in conditions of reduced mTORC1 activity. Importantly, this finding has important implications for the use of Rapamycin or Rapamycin analogues in clinical settings. Restoration of regenerative capacity of barrier epithelia by late-life Rapamycin administration emerges as a promising strategy for functional restoration in aging humans.

STAR METHODS

QUANTIFICATION AND STATISTICAL ANALYSIS

All quantifications were done manually using FIJI (NIH ImageJ) software.

Supplementary Material