Aged Stem Cells Reprogram Their Daily Rhythmic Functions to Adapt to Stress.

Normal homeostatic functions of adult stem cells have rhythmic daily oscillations that are believed to become arrhythmic during aging. Unexpectedly, we find that aged mice remain behaviorally circadian and that their epidermal and muscle stem cells retain a robustly rhythmic core circadian machinery. However, the oscillating transcriptome is extensively reprogrammed in aged stem cells, switching from genes involved in homeostasis to those involved in tissue-specific stresses, such as DNA damage or inefficient autophagy. Importantly, deletion of circadian clock components did not reproduce the hallmarks of this reprogramming, underscoring that rewiring, rather than arrhythmia, is associated with physiological aging. While age-associated rewiring of the oscillatory diurnal transcriptome is not recapitulated by a high-fat diet in young adult mice, it is significantly prevented by long-term caloric restriction in aged mice. Thus, stem cells rewire their diurnal timed functions to adapt to metabolic cues and to tissue-specific age-related traits.

Multimodal cell atlas of the ageing human skeletal muscle.

Muscle atrophy and functional decline (sarcopenia) are common manifestations of frailty and are critical contributors to morbidity and mortality in older people1. Deciphering the molecular mechanisms underlying sarcopenia has major implications for understanding human ageing2. Yet, progress has been slow, partly due to the difficulties of characterizing skeletal muscle niche heterogeneity (whereby myofibres are the most abundant) and obtaining well-characterized human samples3,4. Here we generate a single-cell/single-nucleus transcriptomic and chromatin accessibility map of human limb skeletal muscles encompassing over 387,000 cells/nuclei from individuals aged 15 to 99 years with distinct fitness and frailty levels. We describe how cell populations change during ageing, including the emergence of new populations in older people, and the cell-specific and multicellular network features (at the transcriptomic and epigenetic levels) associated with these changes. On the basis of cross-comparison with genetic data, we also identify key elements of chromatin architecture that mark susceptibility to sarcopenia. Our study provides a basis for identifying targets in the skeletal muscle that are amenable to medical, pharmacological and lifestyle interventions in late life.

Senescence atlas reveals an aged-like inflamed niche that blunts muscle regeneration.

Tissue regeneration requires coordination between resident stem cells and local niche cells1,2. Here we identify that senescent cells are integral components of the skeletal muscle regenerative niche that repress regeneration at all stages of life. The technical limitation of senescent-cell scarcity3 was overcome by combining single-cell transcriptomics and a senescent-cell enrichment sorting protocol. We identified and isolated different senescent cell types from damaged muscles of young and old mice. Deeper transcriptome, chromatin and pathway analyses revealed conservation of cell identity traits as well as two universal senescence hallmarks (inflammation and fibrosis) across cell type, regeneration time and ageing. Senescent cells create an aged-like inflamed niche that mirrors inflammation associated with ageing (inflammageing4) and arrests stem cell proliferation and regeneration. Reducing the burden of senescent cells, or reducing their inflammatory secretome through CD36 neutralization, accelerates regeneration in young and old mice. By contrast, transplantation of senescent cells delays regeneration. Our results provide a technique for isolating in vivo senescent cells, define a senescence blueprint for muscle, and uncover unproductive functional interactions between senescent cells and stem cells in regenerative niches that can be overcome. As senescent cells also accumulate in human muscles, our findings open potential paths for improving muscle repair throughout life.

Cripto shapes macrophage plasticity and restricts EndMT in injured and diseased skeletal muscle.

Macrophages are characterized by a high plasticity in response to changes in tissue microenvironment, which allows them to acquire different phenotypes and to exert essential functions in complex processes, such as tissue regeneration. Here, we report that the membrane protein Cripto plays a key role in shaping macrophage plasticity in skeletal muscle during regeneration and disease. Conditional deletion of Cripto in the myeloid lineage (CriptoMy-LOF ) perturbs MP plasticity in acutely injured muscle and in mouse models of Duchenne muscular dystrophy (mdx). Specifically, CriptoMy-LOF macrophages infiltrate the muscle, but fail to properly expand as anti-inflammatory CD206+ macrophages, which is due, at least in part, to aberrant activation of TGFß/Smad signaling. This reduction in macrophage plasticity disturbs vascular remodeling by increasing Endothelial-to-Mesenchymal Transition (EndMT), reduces muscle regenerative potential, and leads to an exacerbation of the dystrophic phenotype. Thus, in muscle-infiltrating macrophages, Cripto is required to promote the expansion of the CD206+ anti-inflammatory macrophage type and to restrict the EndMT process, providing a direct functional link between this macrophage population and endothelial cells.

Autophagy maintains stemness by preventing senescence.

During ageing, muscle stem-cell regenerative function declines. At advanced geriatric age, this decline is maximal owing to transition from a normal quiescence into an irreversible senescence state. How satellite cells maintain quiescence and avoid senescence until advanced age remains unknown. Here we report that basal autophagy is essential to maintain the stem-cell quiescent state in mice. Failure of autophagy in physiologically aged satellite cells or genetic impairment of autophagy in young cells causes entry into senescence by loss of proteostasis, increased mitochondrial dysfunction and oxidative stress, resulting in a decline in the function and number of satellite cells. Re-establishment of autophagy reverses senescence and restores regenerative functions in geriatric satellite cells. As autophagy also declines in human geriatric satellite cells, our findings reveal autophagy to be a decisive stem-cell-fate regulator, with implications for fostering muscle regeneration in sarcopenia.

Geriatric muscle stem cells switch reversible quiescence into senescence.

Regeneration of skeletal muscle depends on a population of adult stem cells (satellite cells) that remain quiescent throughout life. Satellite cell regenerative functions decline with ageing. Here we report that geriatric satellite cells are incapable of maintaining their normal quiescent state in muscle homeostatic conditions, and that this irreversibly affects their intrinsic regenerative and self-renewal capacities. In geriatric mice, resting satellite cells lose reversible quiescence by switching to an irreversible pre-senescence state, caused by derepression of p16(INK4a) (also called Cdkn2a). On injury, these cells fail to activate and expand, undergoing accelerated entry into a full senescence state (geroconversion), even in a youthful environment. p16(INK4a) silencing in geriatric satellite cells restores quiescence and muscle regenerative functions. Our results demonstrate that maintenance of quiescence in adult life depends on the active repression of senescence pathways. As p16(INK4a) is dysregulated in human geriatric satellite cells, these findings provide the basis for stem-cell rejuvenation in sarcopenic muscles.

Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q10 Deficiency.

Coenzyme Q10 (CoQ10 ) plays a crucial role in mitochondria as an electron carrier within the mitochondrial respiratory chain (MRC) and is an essential antioxidant. Mutations in genes responsible for CoQ10 biosynthesis (COQ genes) cause primary CoQ10 deficiency, a rare and heterogeneous mitochondrial disorder with no clear genotype-phenotype association, mainly affecting tissues with high-energy demand including brain and skeletal muscle (SkM). Here, we report a four-year-old girl diagnosed with minor mental retardation and lethal rhabdomyolysis harboring a heterozygous mutation (c.483G > C (E161D)) in COQ4. The patient's fibroblasts showed a decrease in [CoQ10 ], CoQ10 biosynthesis, MRC activity affecting complexes I/II + III, and respiration defects. Bona fide induced pluripotent stem cell (iPSCs) lines carrying the COQ4 mutation (CQ4-iPSCs) were generated, characterized and genetically edited using the CRISPR-Cas9 system (CQ4ed -iPSCs). Extensive differentiation and metabolic assays of control-iPSCs, CQ4-iPSCs and CQ4ed -iPSCs demonstrated a genotype association, reproducing the disease phenotype. The COQ4 mutation in iPSC was associated with CoQ10 deficiency, metabolic dysfunction, and respiration defects. iPSC differentiation into SkM was compromised, and the resulting SkM also displayed respiration defects. Remarkably, iPSC differentiation in dopaminergic or motor neurons was unaffected. This study offers an unprecedented iPSC model recapitulating CoQ10 deficiency-associated functional and metabolic phenotypes caused by COQ4 mutation. Stem Cells 2017;35:1687-1703.

p38alpha MAP kinase is essential in lung stem and progenitor cell proliferation and differentiation.

Stem cell function is central for the maintenance of normal tissue homeostasis. Here we show that deletion of p38alpha mitogen-activated protein (MAP) kinase in adult mice results in increased proliferation and defective differentiation of lung stem and progenitor cells both in vivo and in vitro. We found that p38alpha positively regulates factors such as CCAAT/enhancer-binding protein that are required for lung cell differentiation. In addition, p38alpha controls self-renewal of the lung stem and progenitor cell population by inhibiting proliferation-inducing signals, most notably epidermal growth factor receptor. As a consequence, the inactivation of p38alpha leads to an immature and hyperproliferative lung epithelium that is highly sensitized to K-Ras(G12V)-induced tumorigenesis. Our results indicate that by coordinating proliferation and differentiation signals in lung stem and progenitor cells, p38alpha has a key role in the regulation of lung cell renewal and tumorigenesis.

Essential role of p18Hamlet/SRCAP-mediated histone H2A.Z chromatin incorporation in muscle differentiation.

The chromatin-remodelling complex SNF2-related CBP activator protein (SRCAP) regulates chromatin structure in yeast by modulating the exchange of histone H2A for the H2A.Z variant. Here, we have investigated the contribution of H2A.Z-mediated chromatin remodelling to mammalian cell differentiation reprogramming. We show that the SRCAP subunit named ZNHIT1 or p18(Hamlet), which is a substrate of p38 MAPK, is recruited to the myogenin promoter at the onset of muscle differentiation, in a p38 MAPK-dependent manner. We also show that p18(Hamlet) is required for H2A.Z accumulation into this genomic region and for subsequent muscle gene transcriptional activation. Accordingly, downregulation of several subunits or the SRCAP complex impairs muscle gene expression. These results identify SRCAP/H2A.Z-mediated chromatin remodelling as a key early event in muscle differentiation-specific gene expression. We also propose a mechanism by which p38 MAPK-mediated signals are converted into chromatin structural changes, thereby facilitating transcriptional activation during mammalian cell differentiation.

Brain-muscle communication prevents muscle aging by maintaining daily physiology.

A molecular clock network is crucial for daily physiology and maintaining organismal health. We examined the interactions and importance of intratissue clock networks in muscle tissue maintenance. In arrhythmic mice showing premature aging, we created a basic clock module involving a central and a peripheral (muscle) clock. Reconstituting the brain-muscle clock network is sufficient to preserve fundamental daily homeostatic functions and prevent premature muscle aging. However, achieving whole muscle physiology requires contributions from other peripheral clocks. Mechanistically, the muscle peripheral clock acts as a gatekeeper, selectively suppressing detrimental signals from the central clock while integrating important muscle homeostatic functions. Our research reveals the interplay between the central and peripheral clocks in daily muscle function and underscores the impact of eating patterns on these interactions.

Interleukin-6 is an essential regulator of satellite cell-mediated skeletal muscle hypertrophy.

Skeletal muscles adapt to increasing workload by augmenting their fiber size, through mechanisms that are poorly understood. This study identifies the cytokine interleukin-6 (IL-6) as an essential regulator of satellite cell (muscle stem cell)-mediated hypertrophic muscle growth. IL-6 is locally and transiently produced by growing myofibers and associated satellite cells, and genetic loss of IL-6 blunted muscle hypertrophy in vivo. IL-6 deficiency abrogated satellite cell proliferation and myonuclear accretion in the preexisting myofiber by impairing STAT3 activation and expression of its target gene cyclin D1. The growth defect was indeed muscle cell intrinsic, since IL-6 loss also affected satellite cell behavior in vitro, in a STAT3-dependent manner. Myotube-produced IL-6 further stimulated cell proliferation in a paracrine fashion. These findings unveil a role for IL-6 in hypertrophic muscle growth and provide mechanistic evidence for the contribution of satellite cells to this process.

Context-dependent roles of cellular senescence in normal, aged, and disease states.

Cellular senescence is a state of irreversible cell cycle arrest that often emerges after tissue damage and in age-related diseases. Through the production of a multicomponent secretory phenotype (SASP), senescent cells can impact the regeneration and function of tissues. However, the effects of senescent cells and their SASP are very heterogeneous and depend on the tissue environment and type as well as the duration of injury, the degree of persistence of senescent cells and the organism's age. While the transient presence of senescent cells is widely believed to be beneficial, recent data suggest that it is detrimental for tissue regeneration after acute damage. Furthermore, although senescent cell persistence is typically associated with the progression of age-related chronic degenerative diseases, it now appears to be also necessary for correct tissue function in the elderly. Here, we discuss what is currently known about the roles of senescent cells and their SASP in tissue regeneration in ageing and age-related diseases, highlighting their (negative and/or positive) contributions. We provide insight for future research, including the possibility of senolytic-based therapies and cellular reprogramming, with aims ranging from enhancing tissue repair to extending a healthy lifespan.

Mitochondrial dynamics maintain muscle stem cell regenerative competence throughout adult life by regulating metabolism and mitophagy.

Skeletal muscle regeneration depends on the correct expansion of resident quiescent stem cells (satellite cells), a process that becomes less efficient with aging. Here, we show that mitochondrial dynamics are essential for the successful regenerative capacity of satellite cells. The loss of mitochondrial fission in satellite cells-due to aging or genetic impairment-deregulates the mitochondrial electron transport chain (ETC), leading to inefficient oxidative phosphorylation (OXPHOS) metabolism and mitophagy and increased oxidative stress. This state results in muscle regenerative failure, which is caused by the reduced proliferation and functional loss of satellite cells. Regenerative functions can be restored in fission-impaired or aged satellite cells by the re-establishment of mitochondrial dynamics (by activating fission or preventing fusion), OXPHOS, or mitophagy. Thus, mitochondrial shape and physical networking controls stem cell regenerative functions by regulating metabolism and proteostasis. As mitochondrial fission occurs less frequently in the satellite cells in older humans, our findings have implications for regeneration therapies in sarcopenia.

3-deazaadenosine (3DA) alleviates senescence to promote cellular fitness and cell therapy efficiency in mice.

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.

Vg1RBP phosphorylation by Erk2 MAP kinase correlates with the cortical release of Vg1 mRNA during meiotic maturation of Xenopus oocytes.

Xenopus Vg1RBP is a member of the highly conserved IMP family of four KH-domain RNA binding proteins, with roles in RNA localization, translational control, RNA stability, and cell motility. Vg1RBP has been implicated in localizing Vg1 mRNAs to the vegetal cortex during oogenesis, in a process mediated by microtubules and microfilaments, and in migration of neural crest cells in embryos. Using c-mos morpholino, kinase inhibitors, and constitutely active recombinant kinases we show that Vg1RBP undergoes regulated phosphorylation by Erk2 MAPK during meiotic maturation, on a single residue, S402, located between the KH2 and KH3 domains. Phosphorylation temporally correlates with the release of Vg1 mRNA from its tight cortical association, assayed in lysates in physiological salt buffers, but does not affect RNA binding, nor self-association of Vg1RBP. U0126, a MAP kinase inhibitor, prevents Vg1RBP cortical release and Vg1 mRNA solubilization in meiotically maturing eggs, while injection of MKK6-DD, a constitutively activated MAP kinase kinase, promotes the release of both Vg1RBP and Vg1 mRNA from insoluble cortical structures. We propose that Erk2 MAP kinase phosphorylation of Vg1RBP regulates the protein:protein-mediated association of Vg1 mRNP with the cytoskeleton and/or ER. Since the MAP kinase site in Vg1RBP is conserved in several IMP homologs, this modification also has important implications for the regulation of IMP proteins in somatic cells.

Regulation of skeletal muscle gene expression by p38 MAP kinases.

The formation of skeletal muscle is a multistep process orchestrated by the basic helix-loop-helix myogenic regulatory factors (MRFs). A wide array of proteins can interact with the MRFs, resulting in either induction or repression of their myogenic potential and subsequent MRF-mediated muscle-specific transcription. Findings published over the past few years have unambiguously established a key role for the p38 MAP kinase pathway in the control of muscle gene expression at different stages of the myogenic process. Here, we discuss the mechanisms by which p38 MAP kinase controls skeletal muscle differentiation by regulating the sequential activation of MRFs and their transcriptional coactivators, including chromatin remodeling enzymes.

Regulation of skeletal muscle stem cells through epigenetic mechanisms.

Quiescent adult skeletal muscle stem cells (satellite cells) are the main players of myogenesis assuring the possibility of growth and regeneration of the muscle tissue throughout adult life. The environmental stimuli that activate satellite cells induce their proliferation, leading on one hand to self-renewal and maintenance of the muscle stem cell reservoir, and on the other hand to the production of progenitor cells that further proliferate, differentiate, and fuse to form new muscle fibers. Hence, satellite cells constitute a perfect system to study the transitions involved in stem cell differentiation. The multistep process of myogenesis is orchestrated by specific regulatory proteins, such as Pax3/Pax7 or members of the MyoD family of transcription factors. However, findings published over the past few years indicate that epigenetic mechanisms, such as covalent modification of histones, DNA methylation, or regulation of gene expression by microRNAs, also critically repress, maintain, or induce the muscle-specific transcriptional program during myogenesis. These studies have increased our understanding of how the information encoding the muscle lineage is molecularly controlled.

CHD4 ensures stem cell lineage fidelity during skeletal muscle regeneration.

Regeneration of skeletal muscle requires resident stem cells called satellite cells. Here, we report that the chromatin remodeler CHD4, a member of the nucleosome remodeling and deacetylase (NuRD) repressive complex, is essential for the expansion and regenerative functions of satellite cells. We show that conditional deletion of the Chd4 gene in satellite cells results in failure to regenerate muscle after injury. This defect is principally associated with increased stem cell plasticity and lineage infidelity during the expansion of satellite cells, caused by de-repression of non-muscle-cell lineage genes in the absence of Chd4. Thus, CHD4 ensures that a transcriptional program that safeguards satellite cell identity during muscle regeneration is maintained. Given the therapeutic potential of muscle stem cells in diverse neuromuscular pathologies, CHD4 constitutes an attractive target for satellite cell-based therapies.

Translational control by DHX36 binding to 5'UTR G-quadruplex is essential for muscle stem-cell regenerative functions.

Skeletal muscle has a remarkable ability to regenerate owing to its resident stem cells (also called satellite cells, SCs). SCs are normally quiescent; when stimulated by damage, they activate and expand to form new fibers. The mechanisms underlying SC proliferative progression remain poorly understood. Here we show that DHX36, a helicase that unwinds RNA G-quadruplex (rG4) structures, is essential for muscle regeneration by regulating SC expansion. DHX36 (initially named RHAU) is barely expressed at quiescence but is highly induced during SC activation and proliferation. Inducible deletion of Dhx36 in adult SCs causes defective proliferation and muscle regeneration after damage. System-wide mapping in proliferating SCs reveals DHX36 binding predominantly to rG4 structures at various regions of mRNAs, while integrated polysome profiling shows that DHX36 promotes mRNA translation via 5'-untranslated region (UTR) rG4 binding. Furthermore, we demonstrate that DHX36 specifically regulates the translation of Gnai2 mRNA by unwinding its 5' UTR rG4 structures and identify GNAI2 as a downstream effector of DHX36 for SC expansion. Altogether, our findings uncover DHX36 as an indispensable post-transcriptional regulator of SC function and muscle regeneration acting through binding and unwinding rG4 structures at 5' UTR of target mRNAs.