Frédéric Charbonnier

Regular Exercise Prolongs Survival in a Type 2 Spinal Muscular Atrophy Model Mouse

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.

A novel function for the survival motoneuron protein as a translational regulator

SMN1, the causative gene for spinal muscular atrophy (SMA), plays a housekeeping role in the biogenesis of small nuclear RNA ribonucleoproteins. SMN is also present in granular foci along axonal projections of motoneurons, which are the predominant cell type affected in the pathology. These so-called RNA granules mediate the transport of specific mRNAs along neurites and regulate mRNA localization, stability, as well as local translation. Recent work has provided evidence suggesting that SMN may participate in the assembly of RNA granules, but beyond that, the precise nature of its role within these structures remains unclear. Here, we demonstrate that SMN associates with polyribosomes and can repress translation in an in vitro translation system. We further identify the arginine methyltransferase CARM1 as an mRNA that is regulated at the translational level by SMN and find that CARM1 is abnormally up-regulated in spinal cord tissue from SMA mice and in severe type I SMA patient cells. We have previously characterized a novel regulatory pathway in motoneurons involving the SMN-interacting RNA-binding protein HuD and CARM1. Thus, our results suggest the existence of a potential negative feedback loop in this pathway. Importantly, an SMA-causing mutation in the Tudor domain of SMN completely abolished translational repression, a strong indication for the functional significance of this novel SMN activity in the pathology.

Lithium enhances remyelination of peripheral nerves

Glycogen synthase kinase 3β (GSK3β) inhibitors, especially the mood stabilizer lithium chloride, are also used as neuroprotective or anti-inflammatory agents. We studied the influence of LiCl on the remyelination of peripheral nerves. We showed that the treatment of adult mice with LiCl after facial nerve crush injury stimulated the expression of myelin genes, restored the myelin structure, and accelerated the recovery of whisker movements. LiCl treatment also promoted remyelination of the sciatic nerve after crush. We also demonstrated that peripheral myelin gene MPZ and PMP22 promoter activities, transcripts, and protein levels are stimulated by GSK3β inhibitors (LiCl and SB216763) in Schwann cells as well as in sciatic and facial nerves. LiCl exerts its action in Schwann cells by increasing the amount of β-catenin and provoking its nuclear localization. We showed by ChIP experiments that LiCl treatment drives β-catenin to bind to T-cell factor/lymphoid-enhancer factor response elements identified in myelin genes. Taken together, our findings open perspectives in the treatment of nerve demyelination by administering GSK3β inhibitors such as lithium.

Motoneuron survival is promoted by specific exercise in a mouse model of amyotrophic lateral sclerosis

Several studies using transgenic mouse models of familial amyotrophic lateral sclerosis (ALS) have reported a life span increase in exercised animals, as long as animals are submitted to a moderate‐intensity training protocol. However, the neuroprotective potential of exercise is still questionable. To gain further insight into the cellular basis of the exercise‐induced effects in neuroprotection, we compared the efficiency of a swimming‐based training, a high‐frequency and ‐amplitude exercise that preferentially recruits the fast motor units, and of a moderate running‐based training, that preferentially triggers the slow motor units, in an ALS mouse model. Surprisingly, we found that the swimming‐induced benefits sustained the motor function and increased the ALS mouse life span by about 25 days. The magnitude of this beneficial effect is one of the highest among those induced by any therapeutic strategy in this disease. We have shown that, unlike running, swimming significantly delays spinal motoneuron death and, more specifically, the motoneurons of large soma area. Analysis of the muscular phenotype revealed a swimming‐induced relative maintenance of the fast phenotype in fast‐twitch muscles. Furthermore, the swimming programme preserved astrocyte and oligodendrocyte populations in ALS spinal cord. As a whole, these data are highly suggestive of a causal relationship not only linking motoneuron activation and protection, but also motoneuron protection and the maintenance of the motoneuron surrounding environment. Basically, exercise‐induced neuroprotective mechanisms provide an example of the molecular adaptation of activated motoneurons.

Expression and Neural Control of Myogenic Regulatory Factor Genes During Regeneration of Mouse Soleus

Given the importance of the myogenic regulatory factors (MRFs) for myoblast differentiation during development, the aims of this work were to clarify the spatial and temporal expression pattern of the four MRF mRNAs during soleus regeneration in mouse after cardiotoxin injury, using in situ hybridization, and to investigate the influence of innervation on the expression of each MRF during a complete degeneration/regeneration process. For this, we performed cardiotoxin injury-induced regeneration experiments on denervated soleus muscle. Myf-5, MyoD, and MRF4 mRNAs were detected in satellite cell-derived myoblasts in the first stages of muscle regeneration analyzed (2–3 days P-I). The Myf-5 transcript level dramatically decreased in young multinucleated myotubes, whereas MyoD and MRF4 transcripts were expressed persistently throughout the regeneration process. Myogenin mRNA was transiently expressed in forming myotubes. These results are discussed with regard to the potential relationships between MyoD and MRF4 in the satellite cell differentiation pathway. Muscle denervation precociously (at 8 days P-I) upregulated both the Myf-5 and the MRF4 mRNA levels, whereas the increase of both MyoD and myogenin mRNA levels was observed later, in the late stages of regeneration (30 days P-I). This significant accumulation of each differentially upregulated MRF during soleus regeneration after denervation suggests that each myogenic factor might have a distinct role in the regulatory control of muscle gene expression. This role is discussed in relation to the expression of the nerve-regulated genes, such as the nAChR subunit gene family.

Exercise-Induced Activation of NMDA Receptor Promotes Motor Unit Development and Survival in a Type 2 Spinal Muscular Atrophy Model Mouse

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.

In Vivo NMDA Receptor Activation Accelerates Motor Unit Maturation, Protects Spinal Motor Neurons, and Enhances SMN2 Gene Expression in Severe Spinal Muscular Atrophy Mice

Spinal muscular atrophy (SMA), a lethal neurodegenerative disease that occurs in childhood, is caused by the misexpression of the survival of motor neuron (SMN) protein in motor neurons. It is still unclear whether activating motor units in SMA corrects the delay in the postnatal maturation of the motor unit resulting in an enhanced neuroprotection. In the present work, we demonstrate that an adequate NMDA receptor activation in a type 2 SMA mouse model significantly accelerated motor unit postnatal maturation, counteracted apoptosis in the spinal cord, and induced a marked increase of SMN expression resulting from a modification of SMN2 gene transcription pattern. These beneficial effects were dependent on the level of NMDA receptor activation since a treatment with high doses of NMDA led to an acceleration of the motor unit maturation but favored the apoptotic process and decreased SMN expression. In addition, these results suggest that the NMDA-induced acceleration of motor unit postnatal maturation occurred independently of SMN. The NMDA receptor activating treatment strongly extended the life span in two different mouse models of severe SMA. The analysis of the intracellular signaling cascade that lay downstream the activated NMDA receptor revealed an unexpected reactivation of the CaMKII/AKT/CREB (cAMP response element-binding protein) pathway that induced an enhanced SMN expression. Therefore, pharmacological activation of spinal NMDA receptors could constitute a useful strategy for both increasing SMN expression and limiting motor neuron death in SMA spinal cord.

Expression and neural control of follistatin versus myostatin genes during regeneration of mouse soleus

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. Developmental Dynamics 227:256–265, 2003.

Liver X receptors alpha and beta promote myelination and remyelination in the cerebellum.

Significance Liver X receptors (LXRs) α and β are the two major receptors of oxysterols, oxygenated derivatives of cholesterol. They control the homeostasis of cholesterol, an important lipid constituent of myelin sheaths. In the central nervous system, these insulating structures are generated by oligodendrocytes and are stabilized by myelin proteins. Here, we provide evidence of a new role of LXRs in the myelin physiology of the cerebellum. Mice invalidated for both LXRs exhibit alteration in motor coordination and spatial learning linked with myelination deficits. We demonstrated that LXRs intervene both in oligodendroglial cell maturation and in the transcriptional control of myelin gene expression during (re)myelination processes. The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) α and β are nuclear receptors activated by oxysterols that originated from the oxidation of cholesterol. They are crucial for cholesterol homeostasis, a major lipid constituent of myelin sheaths that are formed by oligodendrocytes. However, the role of LXRs in myelin generation and maintenance is poorly understood. Here, we show that LXRs are involved in myelination and remyelination processes. LXRs and their ligands are present in oligodendrocytes. We found that mice invalidated for LXRs exhibit altered motor coordination and spatial learning, thinner myelin sheaths, and reduced myelin gene expression. Conversely, activation of LXRs by either 25-hydroxycholesterol or synthetic TO901317 stimulates myelin gene expression at the promoter, mRNA, and protein levels, directly implicating LXRα/β in the transcriptional control of myelin gene expression. Interestingly, activation of LXRs also promotes oligodendroglial cell maturation and remyelination after lysolecithin-induced demyelination of organotypic cerebellar slice cultures. Together, our findings represent a conceptual advance in the transcriptional control of myelin gene expression and strongly support a new role of LXRs as positive modulators in central (re)myelination processes.

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice

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.