The analysis of the skeletal muscle metabolism is crucial for designing optimal exercise paradigms in type 2 diabetes mellitus.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease that commonly results from a high-calorie diet and sedentary lifestyle, leading to insulin resistance and glucose homeostasis perturbation. Physical activity is recommended as one first-line treatment in T2DM, but it leads to contrasted results. We hypothesized that, instead of applying standard exercise protocols, the prescription of personalized exercise programs specifically designed to reverse the potential metabolic alterations in skeletal muscle could result in better results. METHODS: To test this hypothesis, we drew the metabolic signature of the fast-twitch quadriceps muscle, based on a combined unbiased NMR spectroscopy and RT-qPCR study, in several T2DM mouse models of different genetic background (129S1/SvImJ, C57Bl/6J), sex and aetiology (high-fat diet (HFD) or HFD/Streptozotocin (STZ) induction or transgenic MKR (FVB-Tg Ckm-IGF1R*K1003R)1Dlr/J) mice. Three selected mouse models with unique muscular metabolic signatures were submitted to three different swimming-based programs, designed to address each metabolic specificity. RESULTS: We found that depending on the genetic background, the sex, and the mode of T2DM induction, specific muscular adaptations occurred, including depressed glycolysis associated with elevated PDK4 expression, shift to ß-oxidation, or deregulation of amino-acid homeostasis. Interestingly, dedicated swimming-based exercises designed to restore specific metabolic alterations in muscle were found optimal in improving systemic T2DM hallmarks, including a significant reduction in insulin resistance, the improvement of glucose homeostasis, and a delay in sensorimotor function alterations. CONCLUSION: The muscle metabolism constitutes an important clue for the design of precision exercises with potential clinical implications for T2DM patients.

Activating ATF6 in spinal muscular atrophy promotes SMN expression and motor neuron survival through the IRE1α-XBP1 pathway.

AIM: Spinal muscular atrophy (SMA) is a neuromuscular disease caused by survival of motor neuron (SMN) deficiency that induces motor neuron (MN) degeneration and severe muscular atrophy. Gene therapies that increase SMN have proven their efficacy but not for all patients. Here, we explored the unfolded protein response (UPR) status in SMA pathology and explored whether UPR modulation could be beneficial for SMA patients. METHODS: We analysed the expression and activation of key UPR proteins by RT-qPCR and by western blots in SMA patient iPSC-derived MNs and one SMA cell line in which SMN expression was re-established (rescue). We complemented this approach by using myoblast and fibroblast SMA patient cells and SMA mouse models of varying severities. Finally, we tested in vitro and in vivo the effect of IRE1α/XBP1 pathway restoration on SMN expression and subsequent neuroprotection. RESULTS: We report that the IRE1α/XBP1 branch of the unfolded protein response is disrupted in SMA, with a depletion of XBP1s irrespective of IRE1α activation pattern. The overexpression of XBP1s in SMA fibroblasts proved to transcriptionally enhance SMN expression. Importantly, rebalancing XBP1s expression in severe SMA-like mice, induced SMN expression and spinal MN protection. CONCLUSIONS: We have identified XBP1s depletion as a contributing factor in SMA pathogenesis, and the modulation of this transcription factor proves to be a plausible therapeutic avenue in the context of pharmacological interventions for patients.

Involvement of Aryl hydrocarbon receptor in myelination and in human nerve sheath tumorigenesis.

Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in xenobiotic metabolism. Plexiform neurofibromas (PNFs) can transform into malignant peripheral nerve sheath tumors (MPNSTs) that are resistant to existing therapies. These tumors are primarily composed of Schwann cells. In addition to neurofibromatosis type 1 (NF1) gene inactivation, further genetic lesions are required for malignant transformation. We have quantified the mRNA expression levels of AHR and its associated genes in 38 human samples. We report that AHR and the biosynthetic enzymes of its endogenous ligand are overexpressed in human biopsies of PNFs and MPNSTs. We also detect a strong nuclear AHR staining in MPNSTs. The inhibition of AHR by siRNA or antagonists, CH-223191 and trimethoxyflavone, induces apoptosis in human MPNST cells. Since AHR dysregulation is observed in these tumors, we investigate AHR involvement in Schwann cell physiology. Hence, we studied the role of AHR in myelin structure and myelin gene regulation in Ahr-/- mice during myelin development. AHR ablation leads to locomotion defects and provokes thinner myelin sheaths around the axons. We observe a dysregulation of myelin gene expression and myelin developmental markers in Ahr-/- mice. Interestingly, AHR does not directly bind to myelin gene promoters. The inhibition of AHR in vitro and in vivo increased ß-catenin levels and stimulated the binding of ß-catenin on myelin gene promoters. Taken together, our findings reveal an endogenous role of AHR in peripheral myelination and in peripheral nerve sheath tumors. Finally, we suggest a potential therapeutic approach by targeting AHR in nerve tumors.

Lineage tracing of sclerotome cells in amphibian reveals that multipotent somitic cells originate from lateral somitic frontier.

The two somite compartments, dorso-lateral dermomyotome and medio-ventral sclerotome are major vertebrate novelties, but little is known about their evolutionary origin. We determined that sclerotome cells in Xenopus come from lateral somitic frontier (LSF) by lineage tracing, ablation experiments and histological analysis. We identified Twist1 as marker of migrating sclerotome progenitors in two amphibians, Xenopus and axolotl. From these results, three conclusions can be drawn. First, LSF is made up of multipotent somitic cells (MSCs) since LSF gives rise to sclerotome but also to dermomytome as already shown in Xenopus. Second, the basic scheme of somite compartmentalization is conserved from cephalochordates to anamniotes since in both cases, lateral cells envelop dorsally and ventrally the ancestral myotome, suggesting that lateral MSCs should already exist in cephalochordates. Third, the transition from anamniote to amniote vertebrates is characterized by extension of the MSCs domain to the entire somite at the expense of ancestral myotome since amniote somite is a naive tissue that subdivides into sclerotome and dermomyotome. Like neural crest pluripotent cells, MSCs are at the origin of major vertebrate novelties, namely hypaxial region of the somite, dermomyotome and sclerotome compartments. Hence, change in MSCs properties and location is involved in somite evolution.

Xenopus SOX5 enhances myogenic transcription indirectly through transrepression.

In anamniotes, somite compartimentalization in the lateral somitic domain leads simultaneously to myotome and dermomyotome formation. In the myotome, Xenopus Sox5 is co-expressed with Myod1 in the course of myogenic differentiation. Here, we studied the function of Sox5 using a Myod1-induced myogenic transcription assay in pluripotent cells of animal caps. We found that Sox5 enhances myogenic transcription of muscle markers Des, Actc1, Ckm and MyhE3. The use of chimeric transactivating or transrepressive Sox5 proteins indicates that Sox5 acts as a transrepressor and indirectly stimulates myogenic transcription except for the slow muscle-specific genes Myh7L, Myh7S, Myl2 and Tnnc1. We showed that this role is shared by Sox6, which is structurally similar to Sox5, both belonging to the SoxD subfamily of transcription factors. Moreover, Sox5 can antagonize the inhibitory function of Meox2 on myogenic differentiation. Meox2 which is a dermomyotome marker, represses myogenic transcription in Myod-induced myogenic transcription assay and in Nodal5-induced mesoderm from animal cap assay. The inhibitory function of Meox2 and the pro-myogenic function of Sox5 were confirmed during Xenopus normal development by the use of translation-blocking oligomorpholinos and dexamethasone inducible chimeric Sox5 and Meox2 proteins. We have therefore identified a new function for SoxD proteins in muscle cells, which can indirectly enhance myogenic transcription through transrepression, in addition to the previously identified function as a direct repressor of slow muscle-specific genes.

IGF-1R Reduction Triggers Neuroprotective Signaling Pathways in Spinal Muscular Atrophy Mice.

Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by the selective loss of spinal motor neurons due to the depletion of the survival of motor neuron (SMN) protein. No therapy is currently available for SMA, which represents the leading genetic cause of death in childhood. In the present study, we report that insulin-like growth factor-1 receptor (Igf-1r) gene expression is enhanced in the spinal cords of SMA-like mice. The reduction of expression, either at the physiological (through physical exercise) or genetic level, resulted in the following: (1) a significant improvement in lifespan and motor behavior, (2) a significant motor neuron protection, and (3) an increase in SMN expression in spinal cord and skeletal muscles through both transcriptional and posttranscriptional mechanisms. Furthermore, we have found that reducing IGF-1R expression is sufficient to restore intracellular signaling pathway activation profile lying downstream of IGF-1R, resulting in both the powerful activation of the neuroprotective AKT/CREB pathway and the inhibition of the ERK and JAK pathways. Therefore, reducing rather than enhancing the IGF-1 pathway could constitute a useful strategy to limit neurodegeneration in SMA. SIGNIFICANCE STATEMENT: Recent evidence of IGF-1 axis alteration in spinal muscular atrophy (SMA), a very severe neurodegenerative disease affecting specifically the motor neurons, have triggered a renewed interest in insulin-like growth factor-1 (IGF-1) pathway activation as a potential therapeutic approach for motor neuron diseases. The present study challenges this point of view and brings the alternative hypothesis that reducing rather than enhancing the IGF-1 signaling pathway exerts a neuroprotective effect in SMA. Furthermore, the present data substantiate a newly emerging concept that the modulation of IGF-1 receptor expression is a key event selectively determining the activation level of intracellular pathways that lie downstream of the receptor. This aspect should be considered when designing IGF-1-based treatments for neurodegenerative diseases.

NADPH oxidase 4 inhibition is a complementary therapeutic strategy for spinal muscular atrophy.

Introduction: Spinal muscular atrophy (SMA) is a fatal neurodegenerative disorder, characterized by motor neuron (MN) degeneration and severe muscular atrophy and caused by Survival of Motor Neuron (SMN) depletion. Therapies aimed at increasing SMN in patients have proven their efficiency in alleviating SMA symptoms but not for all patients. Thus, combinational therapies are warranted. Here, we investigated the involvement of NADPH oxidase 4 (NOX4) in SMA-induced spinal MN death and if the modulation of Nox4 activity could be beneficial for SMA patients. Methods: We analysed in the spinal cord of severe type SMA-like mice before and at the disease onset, the level of oxidative stress and Nox4 expression. Then, we tested the effect of Nox4 inhibition by GKT137831/Setanaxib, a drug presently in clinical development, by intrathecal injection on MN survival and motor behaviour. Finally, we tested if GKT137831/Setanaxib could act synergistically with FDA-validated SMN-upregulating treatment (nusinersen). Results: We show that NOX4 is overexpressed in SMA and its inhibition by GKT137831/Setanaxib protected spinal MN from SMA-induced degeneration. These improvements were associated with a significant increase in lifespan and motor behaviour of the mice. At the molecular level, GKT137831 activated the pro-survival AKT/CREB signaling pathway, leading to an increase in SMN expression in SMA MNs. Most importantly, we found that the per os administration of GKT137831 acted synergistically with a FDA-validated SMN-upregulating treatment. Conclusion: The pharmacological inhibition of NOX4 by GKT137831/Setanaxib is neuroprotector and could represent a complementary therapeutic strategy to fight against SMA.

A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA.

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5' untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIV(Mac) coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role.

Evolution of Somite Compartmentalization: A View From Xenopus.

Somites are transitory metameric structures at the basis of the axial organization of vertebrate musculoskeletal system. During evolution, somites appear in the chordate phylum and compartmentalize mainly into the dermomyotome, the myotome, and the sclerotome in vertebrates. In this review, we summarized the existing literature about somite compartmentalization in Xenopus and compared it with other anamniote and amniote vertebrates. We also present and discuss a model that describes the evolutionary history of somite compartmentalization from ancestral chordates to amniote vertebrates. We propose that the ancestral organization of chordate somite, subdivided into a lateral compartment of multipotent somitic cells (MSCs) and a medial primitive myotome, evolves through two major transitions. From ancestral chordates to vertebrates, the cell potency of MSCs may have evolved and gave rise to all new vertebrate compartments, i.e., the dermomyome, its hypaxial region, and the sclerotome. From anamniote to amniote vertebrates, the lateral MSC territory may expand to the whole somite at the expense of primitive myotome and may probably facilitate sclerotome formation. We propose that successive modifications of the cell potency of some type of embryonic progenitors could be one of major processes of the vertebrate evolution.

HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon.

Eukaryotic translation initiation begins with assembly of a 48S ribosomal complex at the 5' cap structure or at an internal ribosomal entry segment (IRES). In both cases, ribosomal positioning at the AUG codon requires a 5' untranslated region upstream from the initiation site. Here, we report that translation of the genomic RNA of human immunodeficiency virus type 2 takes place by attachment of the 48S ribosomal preinitiation complex to the coding region, with no need for an upstream 5' untranslated RNA sequence. This unusual mechanism is mediated by an RNA sequence that has features of an IRES with the unique ability to recruit ribosomes upstream from its core domain. A combination of translation assays and structural studies reveal that sequences located 50 nucleotides downstream of the AUG codon are crucial for IRES activity.

Author Correction: Musashi 1 regulates the timing and extent of meiotic mRNA translational activation by promoting the use of specific CPEs.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Low-Intensity Running and High-Intensity Swimming Exercises Differentially Improve Energy Metabolism in Mice With Mild Spinal Muscular Atrophy.

Spinal Muscular Atrophy (SMA), an autosomal recessive neurodegenerative disease characterized by the loss of spinal-cord motor-neurons, is caused by mutations on Survival-of-Motor Neuron (SMN)-1 gene. The expression of SMN2, a SMN1 gene copy, partially compensates for SMN1 disruption due to exon-7 excision in 90% of transcripts subsequently explaining the strong clinical heterogeneity. Several alterations in energy metabolism, like glucose intolerance and hyperlipidemia, have been reported in SMA at both systemic and cellular level, prompting questions about the potential role of energy homeostasis and/or production involvement in disease progression. In this context, we have recently reported the tolerance of mild SMA-like mice (SmnΔ7/Δ7; huSMN2 +/+) to 10 months of low-intensity running or high-intensity swimming exercise programs, respectively involving aerobic and a mix aerobic/anaerobic muscular metabolic pathways. Here, we investigated whether those exercise-induced benefits were associated with an improvement in metabolic status in mild SMA-like mice. We showed that untrained SMA-like mice exhibited a dysregulation of lipid metabolism with an enhancement of lipogenesis and adipocyte deposits when compared to control mice. Moreover, they displayed a high oxygen consumption and energy expenditure through ß-oxidation increase yet for the same levels of spontaneous activity. Interestingly, both exercises significantly improved lipid metabolism and glucose homeostasis in SMA-like mice, and enhanced oxygen consumption efficiency with the maintenance of a high oxygen consumption for higher levels of spontaneous activity. Surprisingly, more significant effects were obtained with the high-intensity swimming protocol with the maintenance of high lipid oxidation. Finally, when combining electron microscopy, respiratory chain complexes expression and enzymatic activity measurements in muscle mitochondria, we found that (1) a muscle-specific decreased in enzymatic activity of respiratory chain I, II, and IV complexes for equal amount of mitochondria and complexes expression and (2) a significant decline in mitochondrial maximal oxygen consumption, were reduced by both exercise programs. Most of the beneficial effects were obtained with the high-intensity swimming protocol. Taking together, our data support the hypothesis that active physical exercise, including high-intensity protocols, induces metabolic adaptations at both systemic and cellular levels, providing further evidence for its use in association with SMN-overexpressing therapies, in the long-term care of SMA patients.

Musashi 1 regulates the timing and extent of meiotic mRNA translational activation by promoting the use of specific CPEs.

The translational reactivation of maternal mRNAs encoding meiotic drivers in vertebrates is accomplished mainly by cytoplasmic polyadenylation. The cytoplasmic polyadenylation elements (CPEs) present in the 3' untranslated regions (3' UTRs) of these transcripts, together with their cognate CPE-binding proteins (CPEBs), define a combinatorial code that determines the timing and extent of translational activation upon meiosis resumption. In addition, the RNA-binding protein Musashi1 (Msi1) regulates polyadenylation of CPE-containing mRNAs by a yet undefined CPEB-dependent or CPEB-independent mechanism. Here we show that Msi1 alone does not support cytoplasmic polyadenylation, but its binding triggers the remodeling of RNA structure, thereby exposing adjacent CPEs and stimulating polyadenylation. In this way, Msi1 directs the preferential use of specific CPEs, which in turn affects the timing and extent of polyadenylation during meiotic progression. Genome-wide analysis of CPEB1- and Msi1-associated mRNAs identified 491 common targets, thus revealing a new layer of CPE-mediated translational control.

Specific Physical Exercise Improves Energetic Metabolism in the Skeletal Muscle of Amyotrophic-Lateral- Sclerosis Mice.

Amyotrophic Lateral Sclerosis is an adult-onset neurodegenerative disease characterized by the specific loss of motor neurons, leading to muscle paralysis and death. Although the cellular mechanisms underlying amyotrophic lateral sclerosis (ALS)-induced toxicity for motor neurons remain poorly understood, growing evidence suggest a defective energetic metabolism in skeletal muscles participating in ALS-induced motor neuron death ultimately destabilizing neuromuscular junctions. In the present study, we report that a specific exercise paradigm, based on a high intensity and amplitude swimming exercise, significantly improves glucose metabolism in ALS mice. Using physiological tests and a biophysics approach based on nuclear magnetic resonance (NMR), we unexpectedly found that SOD1(G93A) ALS mice suffered from severe glucose intolerance, which was counteracted by high intensity swimming but not moderate intensity running exercise. Furthermore, swimming exercise restored the highly ALS-sensitive tibialis muscle through an autophagy-linked mechanism involving the expression of key glucose transporters and metabolic enzymes, including GLUT4 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Importantly, GLUT4 and GAPDH expression defects were also found in muscles from ALS patients. Moreover, we report that swimming exercise induced a triglyceride accumulation in ALS tibialis, likely resulting from an increase in the expression levels of lipid transporters and biosynthesis enzymes, notably DGAT1 and related proteins. All these data provide the first molecular basis for the differential effects of specific exercise type and intensity in ALS, calling for the use of physical exercise as an appropriate intervention to alleviate symptoms in this debilitating disease.

Selection and evolution of NTP-specific aptamers.

ATP occupies a central position in biology, for it is both an elementary building block of RNA and the most widely used cofactor in all living organisms. For this reason, it has been a recurrent target for in vitro molecular evolution techniques. The exploration of ATP-binding motifs constitutes both an important step in investigating the plausibility of the 'RNA world' hypothesis and a central starting point for the development of new enzymes. To date, only two RNA motifs that bind ATP have been characterized. The first one is targeted to the adenosine moiety, while the second one recognizes the 'Hoogsteen' face of the base. To isolate aptamers that bind ATP in different orientations, we selected RNAs on an affinity resin that presents ATP in three different orientations. We obtained five new motifs that were characterized and subsequently submitted to a secondary selection protocol designed to isolate aptamers specific for cordycepin. Interestingly, all the ATP-binding motifs selected specifically recognize the sugar-phosphate backbone region of the nucleotides. Three of the aptamers show some selectivity for adenine derivatives, while the remainder recognize any of the four nucleotides with similar efficiency. The characteristics of these aptamers are discussed along with implications for in vitro molecular evolution.

Translational control by changes in poly(A) tail length: recycling mRNAs.

Beyond the well-known function of poly(A) tail length in mRNA stability, recent years have witnessed an explosion of information about how changes in tail length and the selection of alternative polyadenylation sites contribute to the translational regulation of a large portion of the genome. The mechanisms and factors mediating nuclear and cytoplasmic changes in poly(A) tail length have been studied in great detail, the targets of these mechanisms have been identified--in some cases by genome-wide screenings--and changes in poly(A) tail length are now implicated in a number of physiological and pathological processes. However, in very few cases have all three levels--mechanisms, targets and functions--been studied together.

Transcription factor YY1 binds to the murine beta interferon promoter and regulates its transcriptional capacity with a dual activator/repressor role.

The induction of the beta interferon (IFN-beta) gene constitutes one of the first responses of the cell to virus infection. Its regulation is achieved through an intricate combination of virus-induced binding of transcription factors and local chromatin remodeling. In this work, we demonstrate that transcription factor YY1, known to interact with histone deacetylases (HDAC) and histone acetyltransferases, has a dual activator/repressor role during the regulation of the IFN-beta promoter activity. We show that YY1 specifically binds in vitro and in vivo to the murine IFN-beta promoter at positions -90 and -122. Overexpression of YY1 strongly repressed the transcriptional capacity of a stably integrated IFN-beta promoter fused to a chloramphenicol acetyltransferase reporter gene as well as the endogenous IFN activity of murine L929 cells via an HDAC activity. Stably integrated IFN-beta promoters mutated at the -90 site were no longer repressed by YY1, could no longer be activated by trichostatin A, displayed a retarded postinduction turn off, and a reduced virus-induced activity. Introduction of a mutation at the -122 site did not affect YY1-induced repression, but promoters with this mutation displayed a reduced virus-induced activity. Stably integrated full-length promoters (from position -330 to +20) mutated at both YY1-binding sites displayed extremely reduced promoter activities. We conclude that YY1 has a dual activator/repressor role on IFN-beta promoter activity depending on its binding site and time after infection.