Eusebio Perdiguero

Skeletal muscle atrophy and weakness resulting from loss of motor neuron innervation are shared hallmarks of amyotrophic lateral sclerosis (ALS) and age-associated sarcopenia. However, muscle wasting in ALS is rapid and severe, whereas sarcopenia progresses gradually over decades, peaking at an advanced age. The molecular mechanisms underlying muscle wasting in these conditions remain incompletely defined. Here, we used the SOD1 ^G93A ALS mouse model and wild-type mice across the lifespan to generate an integrated single-nucleus transcriptomic atlas of skeletal muscle, mapping changes associated with ALS and aging. This atlas is complemented by structural and functional analyses. Our results identify transcriptionally distinct condition-specific myonuclear states, including disease-responsive programs within synaptic myonuclei at the neuromuscular junction, that are shared or distinct between the two conditions. Notably, at early stages, muscle from ALS mice exhibits molecular hallmarks of aged muscle at the myofiber level, indicating premature aging and reinforcing aging as a risk factor in ALS. At advanced disease stages and in old age, myonuclear subpopulations follow similar degenerative pathways in fast glycolytic fibers, consistent with their loss in both conditions. However, a distinct ALS-enriched myonuclei subpopulation displays uniquely regulated genes not observed in aged muscle, potentially underlying the more rapid and severe pathology observed in ALS. This atlas maps shared and divergent mechanisms of muscle degeneration in ALS and sarcopenia and provides a resource for identifying therapeutic targets tailored to each condition.

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.

Tissue regeneration requires coordination between resident stem cells and local niche cells^1,2. 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 scarcity³was 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.

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.

Tissue regeneration declines with ageing but little is known about whether this arises from changes in stem-cell heterogeneity. Here, in homeostatic skeletal muscle, we identify two quiescent stem-cell states distinguished by relative CD34 expression: CD34^High, with stemness properties (genuine state), and CD34^Low, committed to myogenic differentiation (primed state). The genuine-quiescent state is unexpectedly preserved into later life, succumbing only in extreme old age due to the acquisition of primed-state traits. Niche-derived IGF1-dependent Akt activation debilitates the genuine stem-cell state by imposing primed-state features via FoxO inhibition. Interventions to neutralize Akt and promote FoxO activity drive a primed-to-genuine state conversion, whereas FoxO inactivation deteriorates the genuine state at a young age, causing regenerative failure of muscle, as occurs in geriatric mice. These findings reveal transcriptional determinants of stem-cell heterogeneity that resist ageing more than previously anticipated and are only lost in extreme old age, with implications for the repair of geriatric muscle.

A unique property of skeletal muscle is its ability to adapt its mass to changes in activity. Inactivity, as in disuse or aging, causes atrophy, the loss of muscle mass and strength, leading to physical incapacity and poor quality of life. Here, through a combination of transcriptomics and transgenesis, we identify sestrins, a family of stress-inducible metabolic regulators, as protective factors against muscle wasting. Sestrin expression decreases during inactivity and its genetic deficiency exacerbates muscle wasting; conversely, sestrin overexpression suffices to prevent atrophy. This protection occurs through mTORC1 inhibition, which upregulates autophagy, and AKT activation, which in turn inhibits FoxO-regulated ubiquitin–proteasome-mediated proteolysis. This study reveals sestrin as a central integrator of anabolic and degradative pathways preventing muscle wasting. Since sestrin also protected muscles against aging-induced atrophy, our findings have implications for sarcopenia.

The maintenance of adult stem cells in their normal quiescent state depends on intrinsic factors and extrinsic signals originating from their microenvironment (also known as the stem cell niche). In skeletal muscle, its stem cells (satellite cells) lose their regenerative potential with aging, and this has been attributed, at least in part, to both age-associated changes in the satellite cells as in the niche cells, which include resident fibro-adipogenic progenitors (FAPs), macrophages, and endothelial cells, among others. To understand the regenerative decline of skeletal muscle with aging, there is a need for methods to specifically isolate stem and niche cells from resting muscle. Here we describe a fluorescence-activated cell sorting (FACS) protocol to simultaneously isolate discrete populations of satellite cells and niche cells from skeletal muscle of aging mice.