Assessing Autophagy in Muscle Stem Cells.

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity's relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.Work in the authors’ laboratory has been supported by MINECO-Spain (RTI2018-096068), ERC-2016-AdG- 741966, LaCaixa-HEALTH-HR17-00040, MDA, Fundació LaMarató/TV3, MDA, UPGRADE-H2020-825825, AFM and DPP-Spain, as well as María-de-Maeztu-Program for Units of Excellence to UPF (MDM-2014-0370), Severo-Ochoa-Program for Centers of Excellence to CNIC (SEV-2015-0505). SC was supported by a Predoctoral Fellowship from Ayudas para la formación y contratación de personal investigador novel (FI) – AGAUR (Barcelona, Spain), IR-P was supported by a Predoctoral Fellowship from Programa de Formación de Personal Investigador (Spain), and XH was supported by a Severo-Ochoa Pre-doctoral Fellowship (Spain).S

Stem Cells and Aging:What's Next?

We asked 12 leaders in the stem cell and aging fields to share their personal perspectives on the future of the field and the unanswered questions that drive them to work in this exciting area

Attenuated epigenetic suppression of muscle stem cell necroptosis is required for efficient regeneration of dystrophic muscles

Somatic stem cells expand massively during tissue regeneration, which might require control of cell fitness, allowing elimination of non-competitive, potentially harmful cells. How or if such cells are removed to restore organ function is not fully understood. Here, we show that a substantial fraction of muscle stem cells (MuSCs) undergo necroptosis because of epigenetic rewiring during chronic skeletal muscle regeneration, which is required for efficient regeneration of dystrophic muscles. Inhibition of necroptosis strongly enhances suppression of MuSC expansion in a non-cell-autonomous manner. Prevention of necroptosis in MuSCs of healthy muscles is mediated by the chromatin remodeler CHD4, which directly represses the necroptotic effector Ripk3, while CHD4-dependent Ripk3 repression is dramatically attenuated in dystrophic muscles. Loss of Ripk3 repression by inactivation of Chd4 causes massive necroptosis of MuSCs, abolishing regeneration. Our study demonstrates how programmed cell death in MuSCs is tightly controlled to achieve optimal tissue regeneration

Brain-muscle communication prevents muscle aging by maintaining daily physiology

Muñoz-Cánoves, Pur

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.We thank M. Jardí, A. Navarro, J. M. Ballestero, K. Slobodnyuk, M. González, J. López and M. Raya for their technical contributions; A. Harada and K. Tanaka for assistance in ATAC-seq; all of the members of the P.M.-C. laboratory for discussions; J. Campisi for p16-3MR mice; J. A. Fernández-Blanco (PRBB Animal Facility); O. Fornas (UPF/CRG FACS Facility); E. Rebollo (IBMB Molecular Imaging Platform); V. A. Raker for manuscript editing; and the members of the Myoage network (A. Maier) for human material. We acknowledge funding from MINECO-Spain (RTI2018-096068, to P.M.-C. and E.P.); ERC-2016-AdG-741966, LaCaixa-HEALTHHR17-00040, MDA, UPGRADE-H2020-825825, AFM, DPP-Spain, Fundació La MaratóTV3-80/19- 202021 and MWRF to P.M.-C.; Fundació La MaratóTV3-137/38-202033 to A.L.S.; Maria-de-Maeztu ́ Program for Units of Excellence to UPF (MDM-2014-0370) and Severo-Ochoa Program for Centers of Excellence to CNIC (SEV-2015-0505). This work was also supported by JST-CREST JPMJCR16G1 and MEXT/JSPS JP20H00456/18H05527 to Y.O.; the Strategic Priority Research Program of the Chinese Academy of Sciences (XDA16030502) to M.A.E.; V.M. and A.C. were supported by FPI and Maria-de-Maeztu predoctoral fellowships, respectively, and V.S. by a Marie Skłodowska-Curie individual fellowship. Parts of the figures were drawn using pictures from Servier Medical Art. Servier Medical Art by Servier is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licences/by/3.0/).S

Marathons and myasthenia gravis: a case report.

The cardinal symptoms of auto-immune myasthenia gravis are fatigue and weakness. Endurance events such as marathon running would seem incompatible with this chronic disease. Many patients stop sport altogether. There is limited literature of patients with auto-immune myasthenia gravis undergoing regular endurance exercise. We report the case of a 36-year-old female who began long-distance running whilst experiencing initial symptoms of myasthenia gravis. She was diagnosed with auto-immune myasthenia gravis and whilst advised to stop all sport, her way of fighting and living with this chronic and unpredictable disease was to continue running to maintain a healthy body and mind. Despite suffering from ocular, bulbar and localized limb fatigability, she managed to complete multiple marathons and achieve disease stability with cholinesterase inhibitors. Marathon and half-marathon running lead to distinct changes in mediators of inflammation in an exercise-dose-dependent manner. Despite symptoms of weakness and fatigue in certain muscles in myasthenia gravis, physical exertion remains possible and may not worsen symptoms as demonstrated in this case and recent studies. The immunomodulatory role of exercise could be considered in this case however this hypothesis remains to be confirmed in future studies with quantitative data

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Autophagy: a decisive process for stemness

Mature skeletal muscle is a stable tissue imposing low homeostatic demand on its stem cells, which remain in a quiescent state in their niche over time. We have shown that these long-lived resting stem cells attenuate proteotoxicity and avoid senescence through basal autophagy. This protective "clean-up" system is lost during aging, resulting in stem cell regenerative decline. Thus, autophagy is required for muscle stem cell homeostasis maintenance.Work in the authors’ laboratories has been supported by: MINECO, Spain SAF2012-38547, SAF2015-67369-R, AFM, E-Rare/Eranet, Fundació Marató-TV3, MDA, EU-FP7 (Myoage, Optistem and Endostem) and DuchennePP-NL, to PM-C; and ISCIII, Spain (FISPS09/01267, FIS-PI13/02512, CP09/00184, PI14/01529) and CIBERNED to MM-V. L.G.-P. was supported by a Predoctoral Fellowship from Programa de Formación de Personal Investigador (Spain)