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Loss of protein homeostasis is one of the hallmarks of aging. As such, interventions that restore proteostasis should slow down the aging process and improve healthspan. Two of the most broadly used anti-aging interventions that are effective in organisms from yeast to mammals are calorie restriction (CR) and rapamycin (RM) treatment. To identify the regulatory mechanisms by which these interventions improve the protein homeostasis, we carried out ribosome footprinting in the muscle of mice aged under standard conditions, or under long-term treatment with CR or RM. We found that the treatments distinctly impact the non-canonical translation, RM primarily remodeling the translation of upstream open reading frames (uORFs), while CR restores stop codon readthrough and the translation of downstream ORFs. Proteomics analysis revealed the expression of numerous non-canonical ORFs at the protein level. The corresponding peptides may provide entry points for therapies aiming to maintain muscle function and extend health span. [ABSTRACT FROM AUTHOR]

Calorie restriction (CR) and treatment with rapamycin (RM), an inhibitor of the mTORC1 growth-promoting signaling pathway, are known to slow aging and promote health from worms to humans. At the transcriptome and proteome levels, long-term CR and RM treatments have partially overlapping effects, while their impact on protein phosphorylation within cellular signaling pathways have not been compared. Here we measured the phosphoproteomes of soleus, tibialis anterior, triceps brachii and gastrocnemius muscles from adult (10 months) and 30-month-old (aged) mice receiving either a control, a calorie restricted or an RM containing diet from 15 months of age. We reproducibly detected and extensively analyzed a total of 6960 phosphosites, 1415 of which are not represented in standard repositories. We reveal the effect of these interventions on known mTORC1 pathway substrates, with CR displaying greater between-muscle variation than RM. Overall, CR and RM have largely consistent, but quantitatively distinct long-term effects on the phosphoproteome, mitigating age-related changes to different degrees. Our data expands the catalog of protein phosphorylation sites in the mouse, providing important information regarding their tissue-specificity, and revealing the impact of long-term nutrient-sensing pathway inhibition on mouse skeletal muscle.Phosphoproteomics reveals the impact of longterm anti-aging treatments on mouse skeletal muscle and expands the catalog of protein phosphorylation sites, providing further details about their tissue-specificity. [ABSTRACT FROM AUTHOR]

Agrin is a heparan sulfate proteoglycan that is best known for its crucial involvement in the organization and maintenance of postsynaptic structures at the neuromuscular junction. Consistent with this role, mice deficient of agrin die at birth due to respiratory failure. Here we examined the early postnatal development of agrin-deficient mice in which perinatal death was prevented by transgenic expression of neural agrin in motor neurons. Such transgenic, agrin-deficient mice were born at Mendelian ratio but exhibited severe postnatal growth retardation. Growth plate morpholgy was markedly altered in these mice, with changes being most prominent in the hypertrophic zone. Compression of this zone was not caused by reduced viability of hypertrophic chondrocytes, as no differences in the apoptosis rates could be observed. Furthermore, deposition of the major cartilage matrix components collagen type II and aggrecan was slightly reduced in these mice. Consistent with a role for agrin in skeletal development, we show for the first time that agrin is highly expressed by chondrocytes and localizes to the growth plate in wild-type mice. Our data show that agrin is expressed in cartilage and that it plays a critical role in normal skeletal growth. [ABSTRACT FROM AUTHOR]

The heparan sulphate proteoglycan agrin is expressed as several isoforms in various tissues. Agrin is best known as a crucial organizer of postsynaptic differentiation at the neuromuscular junction, but it has recently also been implicated in the formation of the immunological synapse, the organization of the cytoskeleton and the amelioration of function in diseased muscle. So the activities of agrin might be of broader significance than previously anticipated. [ABSTRACT FROM AUTHOR]

Reports on the design of a minigene of agrin, a protein known for its role in the formation of the neuromuscular junction, to restore muscle function in a mouse model for congenital muscular dystrophy. Amendment of muscle pathology by a mechanism that includes agrin-mediated stabilization of alpha-dystroglycan and the laminin alpha5 chain; Provision of in vivo evidence on the use of a non-homologous protein in combination with rational protein design, to devise therapeutic tools that may restore muscle function in human muscular dystrophies.

Skeletal osteoblasts are important regulators of B-lymphopoiesis, serving as a rich source of factors such as CXCL12 and IL-7 which are crucial for B-cell development. Recent studies from our laboratory and others have shown that deletion of Rptor, a unique component of the mTORC1 nutrient-sensing complex, early in the osteoblast lineage development results in defective bone development in mice. In this study, we now demonstrate that mTORC1 signalling in pre-osteoblasts is required for normal B-lymphocyte development in mice. Targeted deletion of Rptor in osterix-expressing pre-osteoblasts (Rptorob−/−) leads to a significant reduction in the number of B-cells in the bone marrow, peripheral blood and spleen at 4 and 12 weeks of age. Rptorob−/− mice also exhibit a significant reduction in pre-B and immature B-cells in the BM, indicative of a block in B-cell development from the pro-B to pre-B cell stage. Circulating levels of IL-7 and CXCL12 are also significantly reduced in Rptorob−/− mice. Importantly, whilst Rptor-deficient osteoblasts are unable to support HSC differentiation to B-cells in co-culture, this can be rescued by the addition of exogenous IL-7 and CXCL12. Collectively, these findings demonstrate that mTORC1 plays an important role in extrinsic osteoblastic regulation of B-cell development. [ABSTRACT FROM AUTHOR]