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With human median lifespan extending into the 80s in many developed countries, the societal burden of age-related muscle loss (sarcopenia) is increasing. mTORC1 promotes skeletal muscle hypertrophy, but also drives organismal aging. Here, we address the question of whether mTORC1 activation or suppression is beneficial for skeletal muscle aging. We demonstrate that chronic mTORC1 inhibition with rapamycin is overwhelmingly, but not entirely, positive for aging mouse skeletal muscle, while genetic, muscle fiber-specific activation of mTORC1 is sufficient to induce molecular signatures of sarcopenia. Through integration of comprehensive physiological and extensive gene expression profiling in young and old mice, and following genetic activation or pharmacological inhibition of mTORC1, we establish the phenotypically-backed, mTORC1-focused, multi-muscle gene expression atlas, SarcoAtlas (https://sarcoatlas.scicore.unibas.ch/), as a user-friendly gene discovery tool. We uncover inter-muscle divergence in the primary drivers of sarcopenia and identify the neuromuscular junction as a focal point of mTORC1-driven muscle aging. mTORC1 expression is increased during ageing of muscle, and on the other hand, its activation promotes muscle hypertrophy. Here, the authors assess whether mTORC1 has positive or negative effects on ageing, and show that its long-term inhibition preserves muscle mass and function and neuromuscular junction integrity, whereas muscle-specific activation is associated with sarcopenia.

The extracellular matrix protein laminin-α2 is essential for preserving the integrity of skeletal muscle fibers during contraction. Its importance is reflected by the severe, congenital LAMA2-related muscular dystrophy (LAMA2 MD) caused by loss-of-function mutations in the LAMA2 gene. While laminin-α2 has an established role in structurally supporting muscle fibers, it remains unclear whether it exerts additional functions that contribute to the maintenance of skeletal muscle integrity. Here, we report that in healthy muscle, activated muscle stem cells (MuSCs) express Lama2 and remodel their microenvironment with laminin-α2. By characterizing LAMA2 MD-afflicted MuSCs and generating MuSC-specific Lama2 knockouts, we show that MuSC-derived laminin-α2 is essential for rapid MuSC expansion and regeneration. In humans, we identify LAMA2 expression in MuSCs and demonstrate that loss-of-function mutations impair cell-cycle progression of myogenic precursors. In summary, we show that self-secreted laminin-α2 supports MuSC proliferation post-injury, thus implicating MuSC dysfunction in LAMA2 MD pathology. The extracellular matrix protein laminin-α2 provides muscle fibers with essential structural support. Here, the authors show that muscle stem cells also secrete laminin-α2 to support their own proliferation during muscle regeneration.

Hippocampal long-term potentiation (LTP) represents the cellular response of excitatory synapses to specific patterns of high neuronal activity and is required for learning and memory. Here we identify a mechanism that requires the calcium-binding protein Copine-6 to translate the initial calcium signals into changes in spine structure. We show that Copine-6 is recruited from the cytosol of dendrites to postsynaptic spine membranes by calcium transients that precede LTP. Cpne6 knockout mice are deficient in hippocampal LTP, learning and memory. Hippocampal neurons from Cpne6 knockouts lack spine structural plasticity as do wild-type neurons that express a Copine-6 calcium mutant. The function of Copine-6 is based on its binding, activating and recruiting the Rho GTPase Rac1 to cell membranes. Consistent with this function, the LTP deficit of Cpne6 knockout mice is rescued by the actin stabilizer jasplakinolide. These data show that Copine-6 links activity-triggered calcium signals to spine structural plasticity necessary for learning and memory. Learning and memory depend on the structural and functional plasticity of synapses. Reinhard et al . show that the calcium sensor Copine-6 is required for memory, controls spine structure by regulating Rac signalling, and through its modulation of actin, supports hippocampal LTP.

Mutations in laminin α2-subunit (Lmα2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lmα2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of αLNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of αLNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. αLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in αLNNd-transgenic dy2J mice. In cultured myotubes, we determined that αLNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lmα2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of αLNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.

Background The mammalian target of rapamycin complex 2 (mTORC2), containing the essential protein rictor, regulates cellular metabolism and cytoskeletal organization by phosphorylating protein kinases, such as PKB/Akt, PKC, and SGK. Inactivation of mTORC2 signaling in adult skeletal muscle affects its metabolism, but not muscle morphology and function. However, the role of mTORC2 in adult muscle stem cells (MuSCs) has not been investigated. Method Using histological, biochemical, and molecular biological methods, we characterized the muscle phenotype of mice depleted for rictor in the Myf5-lineage (RImyfKO) and of mice depleted for rictor in skeletal muscle fibers (RImKO). The proliferative and myogenic potential of MuSCs was analyzed upon cardiotoxin-induced injury in vivo and in isolated myofibers in vitro. Results Skeletal muscle of young and 14-month-old RImyfKO mice appeared normal in composition and function. MuSCs from young RImyfKO mice exhibited a similar capacity to proliferate, differentiate, and fuse as controls. In contrast, the number of MuSCs was lower in young RImyfKO mice than in controls after two consecutive rounds of cardiotoxin-induced muscle regeneration. Similarly, the number of MuSCs in RImyfKO mice decreased with age, which correlated with a decline in the regenerative capacity of mutant muscle. Interestingly, reduction in the number of MuSCs was also observed in 14-month-old RImKO muscle. Conclusions Our study shows that mTORC2 signaling is dispensable for myofiber formation, but contributes to the homeostasis of MuSCs. Loss of mTORC2 does not affect their myogenic function, but impairs the replenishment of MuSCs after repeated injuries and their maintenance during aging. These results point to an important role of mTORC2 signaling in MuSC for muscle homeostasis.

Abstract LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is a devastating congenital muscular dystrophy that is caused by mutations in the LAMA2 gene encoding laminin-α2, the long chain of several heterotrimeric laminins. Laminins are essential components of the extracellular matrix that interface with underlying cells. The pathology of LAMA2 MD patients is dominated by an early-onset, severe muscular dystrophy that ultimately leads to death by respiratory insufficiency. However, pathology in nonmuscle tissues has been described. Prior work in the dyW/dyW mouse model for LAMA2 MD has shown that two linker proteins, mini-agrin and αLNNd, when expressed in skeletal muscle fibers, greatly increase survival from a few months up to more than 2 years. However, the restoration of skeletal muscle function accentuates the pathology in nonmuscle tissue in dyW/dyW mice, first and foremost in the peripheral nerve resulting in paralysis of the hind limbs. We now show that the expression of the two linker proteins in all tissues ameliorates the muscular dystrophy and prevents the appearance of the hind limb paralysis. Importantly, the same ameliorating effect of the linker proteins was seen in dy3K/dy3K mice, which represent the most severe mouse model of LAMA2 MD. In summary, these data show that the two linker proteins can compensate the loss of laminin-α2 in muscle and peripheral nerve, which are the two organs most affected in LAMA2 MD. These results are of key importance for designing appropriate expression constructs for mini-agrin and αLNNd to develop a gene therapy for LAMA2 MD patients.

Mutations in laminin [alpha]2-subunit (Lm[alpha]2, encoded by LAMA2) are linked to approximately 30% of congenital muscular dystrophy cases. Mice with a homozygous mutation in Lama2 (dy2J mice) express a nonpolymerizing form of laminin-211 (Lm211) and are a model for ambulatory-type Lm[alpha]2-deficient muscular dystrophy. Here, we developed transgenic dy2J mice with muscle-specific expression of [alpha]LNNd, a laminin/nidogen chimeric protein that provides a missing polymerization domain. Muscle-specific expression of [alpha]LNNd in dy2J mice resulted in strong amelioration of the dystrophic phenotype, manifested by the prevention of fibrosis and restoration of forelimb grip strength. aLNNd also restored myofiber shape, size, and numbers to control levels in dy2J mice. Laminin immunostaining and quantitation of tissue extractions revealed increased Lm211 expression in [alpha]LNNd-transgenic dy2J mice. In cultured myotubes, we determined that [alpha]LNNd expression increased myotube surface accumulation of polymerization-deficient recombinant laminins, with retention of collagen IV, reiterating the basement membrane (BM) changes observed in vivo. Laminin LN domain mutations linked to several of the Lm[alpha]2-deficient muscular dystrophies are predicted to compromise polymerization. The data herein support the hypothesis that engineered expression of [alpha]LNNd can overcome polymerization deficits to increase laminin, stabilize BM structure, and substantially ameliorate muscular dystrophy.

LAMA2-related muscular dystrophy (LAMA2 MD or MDC1A) is caused by mutations in the LAMA2 gene encoding laminin-α2, the long chain of several heterotrimeric laminins. Laminins are essential components of the extracellular matrix that interface with underlying cells. The pathology of LAMA2 MD patients is dominated by the severe muscular dystrophy but also involves other tissues. In the dyW/dyW mouse model for LAMA2 MD, amelioration of muscle function by skeletal muscle-specific expression of the two linker proteins, mini-agrin and αLNNd, is sufficient to greatly increase survival. In such survivors, the phenotype is dominated by the hindlimb paralysis. We now show that ubiquitous expression of the two linker proteins in dyW/dyW mice improves muscle function and prevents hindlimb paralysis. The same ameliorating effect of the linker proteins was seen in dy3K/dy3K mice, which represent the most severe mouse model of LAMA2 MD. In summary, these data show that the two linker proteins can compensate the loss of laminin-α2 in many, if not all tissues affected in LAMA2 MD. Competing Interest Statement JRR and MAR are co-founders and shareholders of SEAL Therapeutics AG and inventors on a patent application filed by the University of Basel related to this work.

Adeno-associated virus (AAV)-mediated gene replacement holds promise for treating genetic diseases but faces challenges due to AAV’s limited packaging capacity and potential immune responses to transgene products, especially in patients lacking endogenous protein. LAMA2-related muscular dystrophy (LAMA2 MD), a severe congenital disorder caused by loss of laminin-α2, presents both hurdles: the LAMA2 gene exceeds AAV capacity, and severely affected patients do not produce the native protein. Here, we developed an AAV-based therapy using two engineered linker proteins derived from endogenously expressed components. These linker proteins restore laminin receptor binding and polymerization, enabling reassembly of a functional basement membrane. Dual AAV delivery of the linkers in a severe LAMA2 MD mouse model resulted in robust expression and significant improvements in muscle histology and function. Employing myotropic capsids enabled therapeutic efficacy at lower vector doses. However, muscle-specific targeting unmasked a LAMA2-related peripheral neuropathy. To address this, we expressed one linker under a muscle-specific promoter and the other under a ubiquitous promoter, delivered via AAV9 or AAV8. This approach achieved near-complete phenotypic restoration when administered neonatally and provided significant benefit when given at progressed disease stages. Our strategy offers a mutation-independent, size-compatible, and potentially immune-tolerable treatment for LAMA2 MD with broad clinical potential.

Muscle cell death in muscular dystrophies depends upon calcium ion (Ca++) leakage through sarcolemma and sarcoplasmic reticulum, triggered by muscle stretch during eccentric contraction. We show here that Ca++ spikes are detected in dystrophic myogenic cells in culture since early differentiation, before sarcomere assembly and contraction. Healthy and genetically corrected dystrophic myotubes do not display Ca++ spikes which are blocked by co-culturing DMD myogenic cells with embryonic mouse motoneurons or treating them with agrin proteoglycan. Same effect is elicited by a muscle spliced, COOH peptide of agrin (termed here mini-agrin) that interacts with dystroglycan, favouring its binding to the basal lamina. Lack of dystrophin in DMD myotubes results in decreased expression of CaV1.1 (CACNA1S), a Ca++ sensor component of the Dihydropyridine Receptor (DHPR) complex, known to regulate Ryanodine Receptor 1 (RyR1). These events explain the emergence of Ca++ spikes. Mini-agrin addition to medium, or lentivector-mediated mini-agrin expression in transplanted cells in vivo, stabilize the expression of CaV1.1 on the membrane. This leads to disappearance of Ca++ spikes and to reappearance of α-dystroglycan, α-sarcoglycan and n-NOS, indicating the reconstitution of the dystrophin complex in the absence of dystrophin. These findings unveil a novel regulatory mechanism and offer a new therapeutic opportunity for targeting calcium ion influx as a co-treatment strategy.