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Abstract Aim: The aim of this report is to show that eccentric exercise under well-controlled conditions is an alternative model, to chemical and mechanical analyses, and analyse the process of degeneration/regeneration in mouse soleus. Methods: For this, mice were submitted to a single bout of eccentric exercise on a treadmill down a 14° decline for 150 min and the soleus muscle was analysed at different times following exercise by histology and in situ hybridization in comparison with cardiotoxin-injured muscles. Results: We analyse the regenerative process by detection of the accumulation of transcripts coding for the two myogenic regulatory factors, Myf-5 and MyoD, which are good markers of the activated satellite cells. From 24 h post-exercise (P-E), clusters of mononucleated Myf-5/MyoD-positive cells were detected. Their number increased up to 96 h P-E when young MyoD-positive myotubes with central nuclei began to appear. From 96 to 168 h P-E the number of myotubes increased, about 10-fold, the new myotubes representing 58% of the muscle cells (168 h P-E). Conclusion: These results show that this protocol of eccentric exercise is able to induce a drastic degeneration/regeneration process in the soleus muscle. This offers the opportunity to perform biochemical and molecular analyses of a process of regeneration without muscle environment defects. The advantages of this model are discussed in the context of fundamental and therapeutical perspectives. [ABSTRACT FROM AUTHOR]

The somites are embryonic structures that give rise to the axial musculoskeletal system. In amniotes vertebrates, somites are composed of multipotent somitic cells that quickly compartmentalize into a dorsal dermomyotome and a ventral sclerotome. In the somites, the dermomyotome gives rise to skeletal muscle cells (the myotome) and the dorsal dermis (the dermatome), while the sclerotome gives rise to vertebrae, ribs, and dorsal tendons (the syndetome). The compartmentalization pattern differs in anamniotes, with the establishment of a primitive myotome that begins before somite formation while the LSF (lateral somitic frontier) give rise to both the sclerotome and the dermomyotome in Xenopus. In this synthesis, we describe the contribution of the LSF in establishing somitic lineages in Xenopus and propose a model that traces the evolutionary history of somites back to ancestral precursors associated with striated skeletal muscle., (© 2023 médecine/sciences – Inserm.)

Collagen Q (ColQ) is a nonfibrillar collagen that plays a crucial role at the vertebrate neuromuscular junction (NMJ) by anchoring acetylcholinesterase to the synapse. ColQ also functions in signaling, as it regulates acetylcholine receptor clustering and synaptic gene expression, in a manner dependent on muscle-specific kinase (MuSK), a key protein in NMJ formation and maintenance. MuSK forms a complex with low-density lipoprotein receptor-related protein 4 (LRP4), its coreceptor for the proteoglycan agrin at the NMJ. Previous studies suggested that ColQ also interacts with MuSK. However, the molecular mechanisms underlying ColQ functions and ColQ-MuSK interaction have not been fully elucidated. Here, we investigated whether ColQ binds directly to MuSK and/or LRP4 and whether it modulates agrin-mediated MuSK-LRP4 activation. Using coimmunoprecipitation, pull-down, plate-binding assays, and surface plasmon resonance, we show that ColQ binds directly to LRP4 but not to MuSK and that ColQ interacts indirectly with MuSK through LRP4. In addition, we show that the LRP4 N-terminal region, which contains the agrin-binding sites, is also crucial for ColQ binding to LRP4. Moreover, ColQ-LRP4 interaction was reduced in the presence of agrin, suggesting that agrin and ColQ compete for binding to LRP4. Strikingly, we reveal ColQ has two opposing effects on agrin-induced MuSK-LRP4 signaling: it constitutively reduces MuSK phosphorylation levels in agrin-stimulated myotubes but concomitantly increases MuSK accumulation at the muscle cell surface. Our results identify LRP4 as a major receptor of ColQ and provide new insights into mechanisms of ColQ signaling and acetylcholinesterase anchoring at the NMJ., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. Neuropathology and Applied Neurobiology published by John Wiley & Sons Ltd on behalf of British Neuropathological Society.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Della Gaspera, Weill and Chanoine.)

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., (Copyright © 2019 Elsevier Inc. All rights reserved.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Key Points: The real impact of physical exercise parameters, i.e. intensity, type of contraction and solicited energetic metabolism, on neuroprotection in the specific context of neurodegeneration remains poorly explored. In this study behavioural, biochemical and cellular analyses were conducted to compare the effects of two different long-term exercise protocols, high intensity swimming and low intensity running, on motor units of a type 3 spinal muscular atrophy (SMA)-like mouse model. Our data revealed a preferential SMA-induced death of intermediate and fast motor neurons which was limited by the swimming protocol only, suggesting a close relationship between neuron-specific protection and their activation levels by specific exercise. The exercise-induced neuroprotection was independent of SMN protein expression and associated with specific metabolic and behavioural adaptations with notably a swimming-induced reduction of muscle fatigability. Our results provide new insight into the motor units' adaptations to different physical exercise parameters and will contribute to the design of new active physiotherapy protocols for patient care., Abstract: Spinal muscular atrophy (SMA) is a group of autosomal recessive neurodegenerative diseases differing in their clinical outcome, characterized by the specific loss of spinal motor neurons, caused by insufficient level of expression of the protein survival of motor neuron (SMN). No cure is at present available for SMA. While physical exercise might represent a promising approach for alleviating SMA symptoms, the lack of data dealing with the effects of different exercise types on diseased motor units still precludes the use of active physiotherapy in SMA patients. In the present study, we have evaluated the efficiency of two long-term physical exercise paradigms, based on either high intensity swimming or low intensity running, in alleviating SMA symptoms in a mild type 3 SMA-like mouse model. We found that 10 months' physical training induced significant benefits in terms of resistance to muscle damage, energetic metabolism, muscle fatigue and motor behaviour. Both exercise types significantly enhanced motor neuron survival, independently of SMN expression, leading to the maintenance of neuromuscular junctions and skeletal muscle phenotypes, particularly in the soleus, plantaris and tibialis of trained mice. Most importantly, both exercises significantly improved neuromuscular excitability properties. Further, all these training-induced benefits were quantitatively and qualitatively related to the specific characteristics of each exercise, suggesting that the related neuroprotection is strongly dependent on the specific activation of some motor neuron subpopulations. Taken together, the present data show significant long-term exercise benefits in type 3 SMA-like mice providing important clues for designing rehabilitation programmes in patients., (© 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.)

Fish may be extremely hypoxia resistant. We investigated how muscle fibre size and oxidative capacity in zebrafish (Danio rerio) adapt during severe chronic hypoxia. Zebrafish were kept for either 3 or 6 weeks under chronic constant hypoxia (CCH) (10% air/90%N2 saturated water). We analyzed cross-sectional area (CSA), succinate dehydrogenase (SDH) activity, capillarization, myonuclear density, myoglobin (Mb) concentration and Mb mRNA expression of high and low oxidative muscle fibres. After 3 weeks of CCH, CSA, SDH activity, Mb concentration, capillary and myonuclear density of both muscle fibre types were similar as under normoxia. In contrast, staining intensity for Mb mRNA of hypoxic high oxidative muscle fibres was 94% higher than that of normoxic controls (P<0.001). Between 3 and 6 weeks of CCH, CSA of high and low oxidative muscle fibres increased by 25 and 30%, respectively. This was similar to normoxic controls. Capillary and myonuclear density were not changed by CCH. However, in high oxidative muscle fibres of fish maintained under CCH, SDH activity, Mb concentration as well as Mb mRNA content were higher by 86%, 138% and 90%, respectively, than in muscle fibres of fish kept under normoxia (P<0.001). In low oxidative muscle fibres, SDH activity, Mb and Mb mRNA content were not significantly changed. Under normoxia, the calculated interstitial oxygen tension required to prevent anoxic cores in muscle fibres (PO2crit) of high oxidative muscle fibres was between 1.0 and 1.7 mmHg. These values were similar at 3 and 6 weeks CCH. We conclude that high oxidative skeletal muscle fibres of zebrafish continue to grow and increase oxidative capacity during CCH. Oxygen supply to mitochondria in these fibres may be facilitated by an increased Mb concentration, which is regulated by an increase in Mb mRNA content per myonucleus., (© 2014. Published by The Company of Biologists Ltd.)

The calcineurin/NFAT (nuclear factor of activated T-cells) signaling pathway is involved in the modulation of the adult muscle fiber type, but its role in the establishment of the muscle phenotype remains elusive. Here, we show that the NFAT member NFATc2 cooperates with the basic helix-loop-helix transcription factor MyoD to induce the expression of a specific myosin heavy chain (MHC) isoform, the neonatal one, during embryogenesis. We found this cooperation to be crucial, as Myod/Nfatc2 double-null mice die at birth, with a dramatic reduction of the major neonatal MHC isoform normally expressed at birth in skeletal muscles, such as limb and intercostal muscles, whereas its expression is unaffected in myofibers mutated for either factor alone. Using gel shift and chromatin immunoprecipitation assays, we identified NFATc2 bound to the neonatal Mhc gene, whereas NFATc1 and NFATc3 would preferentially bind the embryonic Mhc gene. We provide evidence that MyoD synergistically cooperates with NFATc2 at the neonatal Mhc promoter. Altogether, our findings demonstrate that the calcineurin/NFAT pathway plays a new role in establishing the early muscle fiber type in immature myofibers during embryogenesis.

Unlabelled: Although Xenopus is a key model organism in developmental biology, little is known about the myotome formation in this species. Here, we assessed the expression of myogenic regulatory factors of the Myod family (MRFs) during embryonic development and revealed distinct MRF programs., Results: The expression pattern of each MRF during embryonic development highlights three successive myogenic waves. We showed that a first median and lateral myogenesis initiates before dermomyotome formation: the median cell population expresses Myf5, Myod, and Mrf4, whereas the lateral one expresses Myod, moderate levels of Myogenin and Mrf4. The second wave of myoblasts arising from the dermomyotome is characterized by the full MRF program expression, with high levels of Myogenin. The third wave is revealed by Myf5 expression in the myotome and could contribute to the formation of plurinucleated fibers at larval stages. Furthermore, Myf5- or Myod-expressing anlagen are identified in craniofacial myogenesis., Conclusions: The first median and lateral myogenesis and their associated MRF programs have probably disappeared in mammals. However, some aspects of Xenopus myogenesis have been conserved such as the development of somitic muscles by successive myogenic waves and the existence of Myf5-dependent and -independent lineages., (Copyright © 2012 Wiley Periodicals, Inc.)

Xenopus myotome is formed by a first medial and lateral myogenesis directly arising from the presomitic mesoderm followed by a second myogenic wave emanating from the dermomyotome. Here, by a series of gain and loss of function experiments, we showed that Mef2d, a member of the Mef2 family of MADS-box transcription factors, appeared as an upstream regulator of lateral myogenesis, and as an inducer of dermomyotome formation at the beginning of neurulation. In the lateral presomitic cells, we showed that Mef2d transactivates Myod expression which is necessary for lateral myogenesis. In the most lateral cells of the presomitic mesoderm, we showed that Mef2d and Paraxis (Tcf15), a member of the Twist family of transcription factors, were co-localized and activate directly the expression of Meox2, which acts upstream of Pax3 expression during dermomyotome formation. Cell tracing experiments confirm that the most lateral Meox2 expressing cells of the presomitic mesoderm correspond to the dermomyotome progenitors since they give rise to the most dorsal cells of the somitic mesoderm. Thus, Xenopus Mef2d couples lateral myogenesis to dermomyotome formation before somite segmentation. These results together with our previous works reveal striking similarities between dermomyotome and tendon formation in Xenopus: both develop in association with myogenic cells and both involve a gene transactivation pathway where one member of the Mef2 family, Mef2d or Mef2c, cooperates with a bHLH protein of the Twist family, Paraxis or Scx (Scleraxis) respectively. We propose that these shared characteristics in Xenopus laevis reflect the existence of a vertebrate ancestral mechanism which has coupled the development of the myogenic cells to the formation of associated tissues during somite compartmentalization.

MEF2 transcription factors are well-established regulators of muscle development. In this report, we describe the cloning of multiple splicing isoforms of the XMEF2A and XMEF2C encoding genes, differentially expressed during Xenopus development. Using whole-mount in situ hybridization, we found that the accumulation of XMEF2C mRNA in the tadpole stages was restricted to intersomitic regions and to the peripheral edges of hypaxial and cranial muscle masses in contrast to XMEF2A and XMEF2D, characterized by a continuous muscle cell expression. The XMEF2C positive cells express the bHLH transcription factor, Xscleraxis, known as a specific marker for tendons. Gain of function experiments revealed that the use of a hormone-inducible XMEF2C construct is able to induce Xscleraxis expression. Furthermore, XMEF2C specifically cooperates with Xscleraxis to induce tenascin C and betaig-h3, two genes preferentially expressed in Xenopus larval tendons. These findings 1) highlight a previously unappreciated and specific role for XMEF2C in tendon development and 2) identify a novel gene transactivation pathway where MEF2C cooperates with the bHLH protein, Xscleraxis, to activate specific gene expression.

Spinal muscular atrophy (SMA) is an inborn neuromuscular disorder caused by low levels of survival motor neuron protein, and for which no efficient therapy exists. Here, we show that the slower rate of postnatal motor-unit maturation observed in type 2 SMA-like mice is correlated with the motor neuron death. Physical exercise delays motor neuron death and leads to an increase in the postnatal maturation rate of the motor-units. Furthermore, exercise is capable of specifically enhancing the expression of the gene encoding the major activating subunit of the NMDA receptor in motor neurons, namely the NR2A subunit, which is dramatically downregulated in the spinal cord of type 2 SMA-like mice. Accordingly, inhibiting NMDA-receptor activity abolishes the exercise-induced effects on muscle development, motor neuron protection and life span gain. Thus, restoring NMDA-receptor function could be a promising therapeutic approach to SMA treatment.

Alexander disease (AxD) is a rare neurodegenerative disorder characterized by large cytoplasmic aggregates in astrocytes and myelin abnormalities and caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP), the main intermediate filament protein in astrocytes. We tested the effects of three mutations (R236H, R76H and L232P) associated with AxD in cells transiently expressing mutated GFAP fused to green fluorescent protein (GFP). Mutated GFAP-GFP expressed in astrocytes formed networks or aggregates similar to those found in the brains of patients with the disease. Time-lapse recordings of living astrocytes showed that aggregates of mutated GFAP-GFP may either disappear, associated with cell survival, or coalesce in a huge juxtanuclear structure associated with cell death. Immunolabeling of fixed cells suggested that this gathering of aggregates forms an aggresome-like structure. Proteasome inhibition and immunoprecipitation assays revealed mutated GFAP-GFP ubiquitination, suggesting a role of the ubiquitin-proteasome system in the disaggregation process. In astrocytes from wild-type-, GFAP-, and vimentin-deficient mice, mutated GFAP-GFP aggregated or formed a network, depending on qualitative and quantitative interactions with normal intermediate filament partners. Particularly, vimentin displayed an anti-aggregation effect on mutated GFAP. Our data indicate a dynamic and reversible aggregation of mutated GFAP, suggesting that therapeutic approaches may be possible.

Sprouty (Spry) proteins were identified as negative regulators of fibroblast growth factor (FGF) signaling in vertebrates and invertebrates. Given the importance of the FGFs in myogenesis, we performed cardiotoxin injury-induced regeneration experiments on soleus muscles of both, adult control and FGF6 ( - / - ) mutant mice and analyzed the accumulation of Spry (1, 2 and 4) transcripts using semi-quantitative and real-time RT-PCR assays and in situ hybridization. We also analyzed the effects of muscle denervation on the accumulation of Spry transcripts. The three Spry genes begin to be expressed as early as the first stages of muscle regeneration and are characterized by distinct expression patterns. Moreover, Spry gene expression was highly and differentially up-regulated, precociously by the lack of FGF6, and belatedly by muscle denervation strongly suggesting that the transient rise of Spry mRNA accumulation was associated to muscle differentiation. Rescue experiments supported the idea of a specific relationship between FGF6 and Spry 2, both being known for their particular involvement in myogenesis.

Myelin/oligodendrocyte glycoprotein (MOG) is a minor integral membrane protein specific to CNS myelin, encoded by a gene located in the major histocompatibility complex. MOG is an highly encephalitogenic autoantigen and a target for autoaggressive immune responses in CNS inflammatory demyelinating diseases. We performed transcriptomic analyses for a gene expressed only in mammalian CNS, myelin oligodendrocyte glycoprotein (MOG). Complex splicing patterns were exclusively found in primates and not in mice, unlike patterns found for other myelin protein genes. In addition to those shared with rodents, these multiple MOG isoforms likely support functions unique to the primate order, in particular maintenance of myelin structure, intracellular signaling, and modulation of CNS autoimmunity via exposure of specific MOG determinants. Developmentally, in human brain the splice variants of MOG appear at a late stage compared to the major isoform, coincidental with myelination and myelin maturation, unlike other myelin proteins. These findings are discussed within the framework of a biological basis for phenotype diversity in recent mammalian evolution and for the notoriously variable clinical expression of diseases such as multiple sclerosis.

Myelin oligodendrocyte glycoprotein (MOG) is an integral membrane protein expressed in CNS oligodendrocytes and outermost myelin lamellae. Anti-MOG Abs cause myelin destruction (demyelination) in animal models of multiple sclerosis (MS); however, such pathogenic Abs have not yet been characterized in humans. Here, a method that specifically detects IgG binding to human MOG in its native, membrane-embedded conformation on MOG-transfected mammalian cells was used to evaluate the significance of these auto Abs. Compared with healthy controls, native MOG-specific IgGs were most frequently found in serum of clinically isolated syndromes (P < 0.001) and relapsing-remitting MS (P < 0.01), only marginally in secondary progressive MS (P < 0.05), and not at all in primary progressive MS. We demonstrate that epitopes exposed in this cell-based assay are different from those exposed on the refolded, extracellular domain of human recombinant MOG tested by solid-phase ELISA. In marmoset monkeys induced to develop MS-like CNS inflammatory demyelination, IgG reactivity against the native membrane-bound MOG is always detected before clinical onset of disease (P < 0.0001), unlike that against other myelin constituents. We conclude that (i) epitopes displayed on native, glycosylated MOG expressed in vivo are early targets for pathogenic Abs; (ii) these Abs, which are not detected in solid-phase assays, might be the ones to play a pathogenic role in early MS with predominant inflammatory activity; and (iii) the cell-based assay provides a practical serologic marker for early detection of CNS autoimmune demyelination including its preclinical stage at least in the primate MS model.

Whereas there have been extensive studies of the expression of XMyf5 and XMyoD during Xenopus embryogenesis, nothing is known about the spatio-temporal accumulation of XMRF4 transcripts and protein. In this report, we describe the cloning and characterization of two full-length MRF4 cDNAs and of their proximal promoters in Xenopus laevis. The comparison of the relative transcript levels of the XMRF4-a and -b genes in developing and adult muscles is highly suggestive of specific functions for the corresponding XMRF4 proteins. Whole-mount embryo in situ hybridization revealed the first XMRF4 transcripts in the more differentiated anterior myocytes of the embryo when the myosin heavy chain E3 mRNA begins to be detectable. XMRF4 mRNA accumulation later extended posteriorly but was never detected in the posterior unsegmented mesoderm, in contrast to XMyoD and XMyf-5. Whole-mount embryo immunohistochemistry revealed that XMRF4 protein accumulated in somite nuclei slightly after XMRF4 transcripts., (Copyright 2005 Wiley-Liss, Inc.)

Several studies indicate that physical exercise is likely to be neuroprotective, even in the case of neuromuscular disease. In the present work, we evaluated the efficiency of running-based training on type 2 spinal muscular atrophy (SMA)-like mice. The model used in this study is an SMN (survival motor neuron)-null mouse carrying one copy of a transgene of human SMN2. The running-induced benefits sustained the motor function and the life span of the type 2 SMA-like mice by 57.3%. We showed that the extent of neuronal death is reduced in the lumbar anterior horn of the spinal cord of running-trained mice in comparison with untrained animals. Notably, exercise enhanced motoneuron survival. We showed that the running-mediated neuroprotection is related to a change of the alternative splicing pattern of exon 7 in the SMN2 gene, leading to increased amounts of exon 7-containing transcripts in the spinal cord of trained mice. In addition, analysis at the level of two muscles from the calf, the slow-twitch soleus and the fast-twitch plantaris, showed an overall conserved muscle phenotype in running-trained animals. These data provide the first evidence for the beneficial effect of exercise in SMA and might lead to important therapeutic developments for human SMA patients.

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage, but its precise role in vivo remains mostly unclear. Here, using FGF6 (-/-) mice and rescue experiments by injection of recombinant FGF6, we dissected the functional role of FGF6 during in vivo myogenesis. We found that the appearance of myotubes was accelerated during regeneration of the soleus of FGF6 (-/-) mice versus wild type mice. This accelerated differentiation was correlated with increased expression of differentiation markers such as CdkIs and calcineurin, as well as structural markers such as MHCI and slow TnI. We showed that an elevated transcript level for calcineurin Aalpha subunit correlated with a positive regulation of calcineurin A activity in regenerating soleus of the FGF6 (-/-) mice. Cyclin D1 and calcineurin were up- and down-regulated, respectively in a dose-dependent manner upon injection of rhFGF6 in regenerating soleus of the mutant mice. We showed an increase of the number of slow oxidative (type I) myofibers, whereas fast oxidative (type IIa) myofibers were decreased in number in regenerating soleus of FGF6 (-/-) mice versus that of wild type mice. In adult soleus, the number of type I myofibers was also higher in FGF6 (-/-) mice than in wild type mice. Taken together these results evidenced a specific phenotype for soleus of the FGF6 (-/-) mice and led us to propose a model accounting for a specific dose-dependent effect of FGF6 in muscle regeneration. At high doses, FGF6 stimulates the proliferation of the myogenic stem cells, whereas at lower doses it regulates both muscle differentiation and muscle phenotype via a calcineurin-signaling pathway., ((c) 2004 Wiley-Liss, Inc.)

The discovery, in the late 1980s, of the MyoD gene family of muscle transcription factors has proved to be a milestone in understanding the molecular events controlling the specification and differentiation of the muscle lineage. From gene knock-out mice experiments progressively emerged the idea that each myogenic regulatory factor (MRF) has evolved a specialized as well as a redundant role in muscle differentiation. To date, MyoD serves as a paradigm for the MRF mode of function. The features of gene regulation by MyoD support a model in which subprograms of gene expression are achieved by the combination of promoter-specific regulation of MyoD binding and MyoD-mediated binding of various ancillary proteins. This binding likely includes site-specific chromatin reorganization by means of direct or indirect interaction with remodeling enzymes. In this cascade of molecular events leading to the proper and reproducible activation of muscle gene expression, the role and mode of function of other MRFs still remains largely unclear. Recent in vivo findings using the Xenopus embryo model strongly support the concept that a single MRF can specifically control a subset of muscle genes and, thus, can be substituted by other MRFs albeit with dramatically lower efficiency. The topic of this review is to summarize the molecular data accounting for a redundant and/or specific involvement of each member of the MyoD family in myogenesis in the light of recent studies on the Xenopus model., (Copyright (c) 2004 Wiley-Liss, Inc.)

Important functions in myogenesis have been proposed for FGF6, a member of the fibroblast growth factor family accumulating almost exclusively in the myogenic lineage. However, the use of FGF6(-/-) mutant mice gave contradictory results and the role of FGF6 during myogenesis remains largely unclear. Using FGF6(-/-) mice, we first analysed the morphology of the regenerated soleus following cardiotoxin injection and showed hypertrophied myofibres in soleus of the mutant mice as compared to wild-type mice. Secondly, to examine the function of the IGF family in the hypertrophy process, we used semiquantitative and real-time RT-PCR assays and Western blots to monitor the expression of the insulin-like growth factors (IGF-I and IGF-II), their receptors [type I IGF receptor (IGF1R) and IGF-II receptor (IGF2R)], and of a binding protein IGFBP-5 in regenerating soleus muscles of FGF6(-/-) knockout mice vs. wild-type mice. In the mutant, both IGF-II and IGF2R, but not IGF-I and IGF1R, were strongly up-regulated, whereas IGFBP5 was down-regulated, strongly suggesting that, in the absence of FGF6, the mechanisms leading to myofibre hypertrophy were mediated specifically by an IGF-II/IGF2R signalling pathway distinct from the classic mechanism involving IGF-I and IGF1R previously described for skeletal muscle hypertrophy. The potential regulating role of IGFBP5 on IGF-II expression is also discussed. This report shows for the first time a specific role for FGF6 in the regulation of myofibre size during a process of in vivo myogenesis.

FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (-/-) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.

We studied the immunological basis for the very potent encephalitogenicity of myelin/oligodendrocyte glycoprotein (MOG), a minor component of myelin in the CNS that is widely used to induce experimental autoimmune encephalomyelitis (EAE). For this purpose, we generated a mutant mouse lacking a functional mog gene. This MOG-deficient mouse presents no clinical or histological abnormalities, permitting us to directly assess the role of MOG as a target autoantigen in EAE. In contrast to WT mice, which developed severe EAE following immunization with whole myelin, MOG-deficient mice had a mild phenotype, demonstrating that the anti-MOG response is a major pathogenic component of the autoimmune response directed against myelin. Moreover, while MOG transcripts are expressed in lymphoid organs in minute amounts, both MOG-deficient and WT mice show similar T and B cell responses against the extracellular domain of MOG, including the immunodominant MOG 35-55 T cell epitope. Furthermore, no differences in the fine specificity of the T cell responses to overlapping peptides covering the complete mouse MOG sequence were observed between MOG+/+ and MOG-/- mice. In addition, upon adoptive transfer, MOG-specific T cells from WT mice and those from MOG-deficient mice are equally pathogenic. This total lack of immune tolerance to MOG in WT C57BL/6 mice may be responsible for the high pathogenicity of the anti-MOG immune response as well as the high susceptibility of most animal strains to MOG-induced EAE.

In Xenopus, previous studies showed that the transcripts of the myogenic regulatory factor (MRF) MRF4 accumulate during skeletal muscle differentiation, but nothing is known about the accumulation of XMRF4 protein during myogenesis. In this report, an affinity-purified polyclonal antibody against Xenopus MRF4 was developed and used to describe the pattern of expression of this myogenic factor in the adult and in regenerating muscles. From young forming myotubes, XMRF4 protein persistently accumulated in nuclei during the regeneration process and was strongly expressed in nuclei of adult muscles. No selective accumulation of XMRF4 protein was detectable at neuromuscular junctions, but XMRF4 immunoreactivity was observed in sole plate nuclei as well as in extrasynaptic myofiber nuclei. We also report that XMRF4 protein accumulated before the establishment of neuromuscular connections, showing that innervation is not necessary for the appearance of XMRF4 protein during muscle regeneration., (Copyright 2003 Wiley-Liss, Inc.)

Follistatin and myostatin are two secreted proteins involved in the control of muscle mass during development. These two proteins have opposite effects on muscle growth, as documented by genetic models. The aims of this work were to analyze in mouse, by using in situ hybridization, the spatial and temporal expression patterns of follistatin and myostatin mRNAs during soleus regeneration after cardiotoxin injury, and to investigate the influence of innervation on the accumulation of these two transcripts. Follistatin transcripts could be detected in activated satellite cells as early as the first stages of regeneration and were transiently expressed in forming myotubes. In contrast, myostatin mRNAs accumulated persistently throughout the regeneration process as well as in adult control soleus. Denervation significantly affected both follistatin and myostatin transcript accumulation, but in opposite ways. Muscle denervation persistently reduced the levels of myostatin transcripts as early as the young myotube stage, whereas the levels of follistatin mRNA were strongly increased in the small myotubes in the late stages of regeneration. These results are discussed with regard to the potential functions of both follistatin, as a positive regulator of muscle differentiation, and myostatin, as a negative regulator of skeletal muscle growth. We suggest that the belated up-regulation of the follistatin mRNA level in the small myotubes of the regenerating soleus as well as the down-regulation of the myostatin transcript level after denervation contribute to the differentiation process in denervated regenerating muscle., (Copyright 2003 Wiley-Liss, Inc.)

Annexin (Anx) 1, 2, 5, and 6 expressions were determined at the transcriptional and translational levels in the rat hepatocytes from gestational day 15 to postnatal day 17. Dramatic shifts were observed in Anx 1 and 2 levels, which peaked at day 1 and gestational day 20, respectively, and reached low levels thereafter. However, Anx 5 and 6 rates were more constant. Prenatal administration of dexamethasone (dex) resulted in a decrease of Anx 1 mRNA levels, and a strong increase in Anx 2 mRNA contents. In adult hepatocytes cultured in the presence of EGF or HGF, Anx 1 and 2 expressions resumed. By immunohistochemistry, Anx 1 was detected only in the cytoplasm of hepatocytes of 1- to 3-day-old rats, Anx 2 and 6 both exhibited a redistribution from the cytoplasm toward the plasma membrane, and Anx 5 was present in the nucleus, cytoplasm, and plasma membrane. Thus, Anx 1, 2, 5, and 6 have individual modes of expression and localization in the differentiating hepatocytes, where they might play unique roles at well defined phases of liver ontogeny., (Copyright 2001 Wiley-Liss, Inc.)

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the mammalian central nervous system, is located on the surface of the oligodendrocyte plasma membrane and the outermost lamellae of mature myelin; it is expressed during the latter steps of myelinogenesis. It has been shown that MOG may play a pathological role in autoimmune demyelinating diseases of the central nervous system, although its physiological function remains unknown. MOG is an integral membrane glycoprotein with an extracellular immunoglobulin-like domain and two hydrophobic segments which were predicted to be membrane-spanning on the basis of hydropathy analysis. As a first step in elucidation of MOG function, we have investigated its membrane topology, combining immunofluorescence studies on cultured oligodendrocytes and MOG-transfected Chinese hamster ovary cells with biochemical analyses, including in vitro translation, membrane insertion and protease-digestion assays. Our results indicate that the C-terminal tail of MOG is located into the cytoplasm, and that only the first hydrophobic region of MOG spans the membrane whereas the second hydrophobic region appears to be semi-embedded in the lipid bilayer, lying partially buried in the membrane with its N-terminal and C-terminal boundaries facing the cytoplasm.

Myelin/oligodendrocyte glycoprotein (MOG) is a major target antigen in experimental autoimmune encephalomyelitis and it has been suggested that it may as well play a key role in the demyelination process in multiple sclerosis (MS). As MOG variants could be pathogenic in autoimmune demyelinating diseases of the central nervous system, we analysed the coding sequence of MOG in MS patients and described a G-->A transition occurring in exon 3 of the human MOG gene. The mutation predicts that isoleucine substitutes for a valine at codon 145 (Val 145 Ile) in the transmembrane region of the protein. This is the first aminoacid substitution reported in human MOG. The polymorphism can be detected by restriction enzyme digestion of genomic DNA or reverse-transcribed PCR amplified products, making it a simple tool to detect a potential implication of MOG alleles in susceptibility to MS by association study. The analysis of 83 unrelated MS patients and 82 unrelated healthy controls showed that the polymorphism is found in similar proportions in MS patients (18%) and controls (14.6%). It is therefore unlikely that the MOG Val 145 Ile variant is responsible for genetic susceptibility to MS.

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the central nervous system localized on the outermost lamellae of mature myelin, is a member of the immunoglobulin superfamily. We report here the organization of the human MOG gene, which spans approximately 17 kb, and the characterization of six MOG mRNA splicing variants. The intron/exon structure of the human MOG gene confirmed the splicing pattern, supporting the hypothesis that mRNA isoforms could arise by alternative splicing of a single gene. In addition to the eight exons coding for the major. MOG isoform, the human MOG gene also contains, in the 3' region, a previously unknown alternatively spliced coding exon, VIA. Alternative utilization of two acceptor splicing sites for exon VIII could produce two different C-termini. The nucleotide sequences presented here may be a useful tool to study further possible involvement of the MOG gene in hereditary neurological disorders.

In this report, we investigated the expression of annexins I, II, V, and VI by Northern and Western blot analysis in four cell lines isolated from human fetal tracheae. Two cell lines were obtained from normal fetuses and the two others from fetuses with cystic fibrosis (CF). One CF fetus was heterozygous for the S549N and N1303K substitutions, whereas the other was homozygous for the delta F508 deletion. We found that the four annexins are always coexpressed. The expression of annexins I, II, and VI was the same in the four cell lines. In contrast, that of annexin V was significantly higher in CF than in normal cells. These observations demonstrate that annexins I, II, V, and VI are independently regulated in tracheal epithelial cell lines. Moreover, they suggest that the overexpressed annexin V, a Ca2+ channel, might profoundly modify Ca2+ transport across the membranes of CF cells.

Myelin/oligodendrocyte glycoprotein (MOG) is expressed specifically in the central nervous system (CNS) by myelinating glial cells, the oligodendrocytes. The external location of MOG on myelin sheaths and its late expression during myelinogenesis argue for a role of MOG in the completion of myelin and maintenance of its integrity. MOG is a target autoantigen in demyelinating diseases, such as experimental autoimmune encephalomyelitis (EAE) in animals and multiple sclerosis (MS) in humans. We previously located the gene encoding MOG to the major histocompatibility complex (MHC), both in human, by cytogenetics, and in mouse, by analysis of recombinants. To refine the position, we have now selected yeast artificial chromosome clones (YAC) which contain the MOG gene. Physical mapping of the human MOG and the mouse Mog genes by characterization of these YAC clones indicated that the gene is located at the distal end of the major histocompatibility complex (MHC) class Ib region in both species. The human MOG gene lies 60 kilobases (kb) telomeric to HLA-F in a head-to-head orientation; the mouse Mog gene lies 25 (kb) telomeric to H2-M5 in a tail-to-head orientation. These orthologous genes provide markers for comparative analysis of the evolution of the MHC in the two species. The physical mapping of MOG should facilitate analysis of its role in hereditary neurological diseases, and the YAC clones identified here will permit the identification of new genes in the region.

We report here the characterization of a full-length cDNA encoding the human myelin/oligodendrocyte glycoprotein (MOG). The sequence of the coding region of the human MOG cDNA is highly homologous to that of other previously cloned mouse, rat, and bovine MOG cDNAs, but the 3' untranslated region differs by an insertion of an Alu sequence between nucleotides 1,590 and 1,924. Accordingly, northern blot analyzes with cDNA probes corresponding to the coding region or the 3' untranslated Alu-containing sequence revealed a single band of 2 kb, rather than the 1.6 kb of bovine, rat, or mouse MOG cDNA(s). Immunocytochemical analysis of HeLa cells transfected with human MOG cDNA, which was performed using a specific antibody raised against whole MOG, clearly indicated that MOG is expressed at the cell surface as an intrinsic protein. These data are in accordance with the predicted amino acid sequence, which contains a signal peptide and two putative transmembrane domains. The knowledge of the human MOG sequence should facilitate further investigations on its potential as a target antigen in autoimmune demyelinating diseases like multiple sclerosis.