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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]

Glucagon plays a major role in the regulation of glucose homeostasis during fed and fasting states. However, the mechanisms responsible for the regulation of pancreatic [alpha] cell mass and function are not completely understood. In the current study, we identified mTOR complex 1 (mTORC1) as a major regulator of [alpha] cell mass and glucagon secretion. Using mice with tissuespecific deletion of the mTORC1 regulator Raptor in [alpha] cells ([[alpha]Raptor.sup.KO]), we showed that mTORC1 signaling is dispensable for [alpha] cell development, but essential for [alpha] cell maturation during the transition from a milk-based diet to a chow-based diet after weaning. Moreover, inhibition of mTORC1 signaling in [[alpha]Raptor.sup.KO] mice and in WT animals exposed to chronic rapamycin administration decreased glucagon content and glucagon secretion. In [[alpha]Raptor.sup.KO] mice, impaired glucagon secretion occurred in response to different secretagogues and was mediated by alterations in [K.sub.ATP] channel subunit expression and activity. Additionally, our data identify the mTORC1/FoxA2 axis as a link between mTORC1 and transcriptional regulation of key genes responsible for [alpha] cell function. Thus, our results reveal a potential function of mTORC1 in nutrient-dependent regulation of glucagon secretion and identify a role for mTORC1 in controlling [alpha] cell-mass maintenance., Introduction Type 1 (T1D) and type 2 diabetes (T2D) are characterized by uncontrolled hyperglycemia associated with the progressive decrease in insulin. Glucagon, insulin's counterregulatory hormone, plays a major role in [...]

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., Introduction Laminin-211 ([alpha]2[beta]i[gamma]1 subunits, abbreviated herein as Lm211) is the major laminin of the basement membrane (BM) of skeletal muscle and peripheral nerve Schwann cells. Mutations within the LAMA2 gene [...]

Myotonic dystrophy type I (DM1) is a disabling multisystemic disease that predominantly affects skeletal muscle. It is caused by expanded CTG repeats in the 3'-UTR of the dystrophia myotonica protein kinase (DMPK) gene. RNA hairpins formed by elongated DMPK transcripts sequester RNA-binding proteins, leading to mis-splicing of numerous pre-mRNAs. Here, we have investigated whether DM1-associated muscle pathology is related to deregulation of central metabolic pathways, which may identify potential therapeutic targets for the disease. In a well-characterized mouse model for DM1 ([HSA.sup.LR] mice), activation of AMPK signaling in muscle was impaired under starved conditions, while mTORC1 signaling remained active. In parallel, autophagic flux was perturbed in [HSA.sup.LR] muscle and in cultured human DM1 myotubes. Pharmacological approaches targeting AMPK/mTORC1 signaling greatly ameliorated muscle function in [HSA.sup.LR] mice. AICAR, an AMPK activator, led to a strong reduction of myotonia, which was accompanied by partial correction of misregulated alternative splicing. Rapamycin, an mTORC1 inhibitor, improved muscle relaxation and increased muscle force in [HSA.sup.LR] mice without affecting splicing. These findings highlight the involvement of AMPK/mTORC1 deregulation in DM1 muscle pathophysiology and may open potential avenues for the treatment of this disease., Introduction Myotonic dystrophy type I (DM1; OMIM #160900) is a multisystemic neuromuscular disorder, which represents the most common form of muscular dystrophy in adults (1). In particular, DM1 patients suffer [...]

Retinitis pigmentosa (RP) is an inherited photoreceptor degenerative disorder that results in blindness. The disease is often caused by mutations in genes that are specific to rod photoreceptors; however, blindness results from the secondary loss of cones by a still unknown mechanism. Here, we demonstrated that the mammalian target of rapamycin complex 1 (mTORCI) is required to slow the progression of cone death during disease and that constitutive activation of mTORC1 in cones is sufficient to maintain cone function and promote long-term cone survival. Activation of mTORC1 in cones enhanced glucose uptake, retention, and utilization, leading to increased levels of the key metabolite NADPH. Moreover, cone death was delayed in the absence of the NADPH-sensitive cell death protease caspase 2, supporting the contribution of reduced NADPH in promoting cone death. Constitutive activation of mTORC1 preserved cones in 2 mouse models of RP, suggesting that the secondary loss of cones is caused mainly by metabolic deficits and is independent of a specific rod-associated mutation. Together, the results of this study address a longstanding question in the field and suggest that activating mTORC1 in cones has therapeutic potential to prolong vision in RP., Introduction The interneuronal relationship between rod and cone photoreceptors in humans and mice is such that rod death always leads to cone death, while conversely, loss of cones has only [...]

Diabetic nephropathy (DN) is among the most lethal complications that occur in type 1 and type 2 diabetics. Podocyte dysfunction is postulated to be a critical event associated with proteinuria [...]

Chronic glomerular diseases, associated with renal failure and cardiovascular morbidity, represent a major health issue. However, they remain poorly understood. Here we have reported that tightly controlled mTOR activity was [...]

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth that associates with raptor and rictor to form the mTOR complex 1 (mTORC1) and mTORC2, respectively. Raptor is required for oxidative muscle integrity, whereas rictor is dispensable. In this study, we show that muscle-specific inactivation of mTOR leads to severe myopathy, resulting in premature death, mTOR-deficient muscles display metabolic changes similar to those observed in muscles lacking raptor, including impaired oxidative metabolism, altered mitochondrial regulation, and glycogen accumulation associated with protein kinase B/Akt hyperactivation. In addition, mTOR-deficient muscles exhibit increased basal glucose uptake, whereas whole body glucose homeostasis is essentially maintained. Importantly, loss of mTOR exacerbates the myopathic features in both slow oxidative and fast glycolytic muscles. Moreover, mTOR but not raptor and rictor deficiency leads to reduced muscle dystrophin content. We provide evidence that mTOR controls dystrophin transcription in a cell-autonomous, rapamycin-resistant, and kinase-independent manner. Collectively, our results demonstrate that mTOR acts mainly via mTORC1, whereas regulation of dystrophin is raptor and rictor independent. /doi/ 10.1083/jcb.200903131

Mammalian target of rapamycin (mTOR), a highly conserved protein kinase that controls cell growth and metabolism in response to nutrients and growth factors, is found in 2 structurally and functionally distinct multiprotein complexes termed mTOR complex 1 (mTORC1) and mTORC2, mTORC2, which consists of rictor, mSIN1, mLST8, and mTOR, is activated by insulin/IGF1 and phosphorylates Ser-473 in the hydrophobic motif of Akt/PKB. Though the role of mTOR in single cells is relatively well characterized, the role of mTOR signaling in specific tissues and how this may contribute to overall body growth is poorly understood. To examine the role of mTORC2 in an individual tissue, we generated adipose-specific rictor knockout mice ([rictor.sup.ad-/-]). [Rictor.sup.ad-/-] mice are increased in body size due to an increase in size of nonadipose organs, including heart, kidney, spleen, and bone. Furthermore, [rictor.sup.ad-/-] mice have a disproportionately enlarged pancreas and are hyperinsulinemic, but glucose tolerant, and display elevated levels of insulin-like growth factor 1 (IGF1) and IGF1 binding protein 3 (IGFBP3). These effects are observed in mice on either a high-fat or a normal diet, but are generally more pronounced in mice on a high-fat diet. Our findings suggest that adipose tissue, in particular mTORC2 in adipose tissue, plays an unexpectedly central role in controlling whole-body growth. genetics | mTORC2 | signal transduction | rapamycin

Alternative mRNA splicing is becoming increasingly recognized as an important mechanism for the generation of structural and functional diversity in proteins. Recent estimations predict that ~50% of all eukaryotic proteins can be alternatively spliced. Several lines of evidence suggest that alternative mRNA splicing results in small changes in protein structure and is likely to fine-tune the function and specificity of the affected protein. However, knowledge of how alternative splicing regulates cellular processes on the molecular level is still limited. It is only recently that structures of alternatively spliced proteins have been solved. These studies have shown that alternative splicing affects the structure not only in the vicinity of the splice site but also at long distance.

Author(s): Joachim Moll [1]; Patrizia Barzaghi [1]; Shuo Lin [1]; Gabriela Bezakova [1]; Hanns Lochmüller [2]; Eva Engvall [3]; Ulrich Müller [4]; Markus A. Ruegg (corresponding author) [1] Congenital muscular [...]

The synapse is a key structure that is involved in perception, learning and memory. Understanding the sequence of steps that is involved in establishing synapses during development might also help to understand mechanisms that cause changes in synapses during learning and memory. For practical reasons, most of our current knowledge of synapse development is derived from studies of the vertebrate neuromuscular junction (NMJ). Several lines of evidence strongly suggest that motor axons release the molecule agrin to induce the formation of the postsynaptic apparatus in muscle fibers. Recent advances implicate proteins such as dystroglycan, MuSK, and rapsyn in the transduction of agrin signals. Recently, additional functions of agrin have been discovered, including the upregulation of gene transcription in myonuclei and the control of presynaptic differentiation. Agrin therefore appears to play a unique role in controlling synaptic differentiation on both sides of the NMJ.

Expression of the [Epsilon]-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with [Epsilon]-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of [Epsilon]-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina.