Long-term exercise-specific neuroprotection in spinal muscular atrophy-like mice.

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.

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.

Shift from extracellular signal-regulated kinase to AKT/cAMP response element-binding protein pathway increases survival-motor-neuron expression in spinal-muscular-atrophy-like mice and patient cells.

Spinal muscular atrophy (SMA), a recessive neurodegenerative disease, is characterized by the selective loss of spinal motor neurons. No available therapy exists for SMA, which represents one of the leading genetic causes of death in childhood. SMA is caused by a mutation of the survival-of-motor-neuron 1 (SMN1) gene, leading to a quantitative defect in the survival-motor-neuron (SMN) protein expression. All patients retain one or more copies of the SMN2 gene, which modulates the disease severity by producing a small amount of stable SMN protein. We reported recently that NMDA receptor activation, directly in the spinal cord, significantly enhanced the transcription rate of the SMN2 genes in a mouse model of very severe SMA (referred as type 1) by a mechanism that involved AKT/CREB pathway activation. Here, we provide the first compelling evidence for a competition between the MEK/ERK/Elk-1 and the phosphatidylinositol 3-kinase/AKT/CREB signaling pathways for SMN2 gene regulation in the spinal cord of type 1 SMA-like mice. The inhibition of the MEK/ERK/Elk-1 pathway promotes the AKT/CREB pathway activation, leading to (1) an enhanced SMN expression in the spinal cord of SMA-like mice and in human SMA myotubes and (2) a 2.8-fold lifespan extension in SMA-like mice. Furthermore, we identified a crosstalk between ERK and AKT signaling pathways that involves the calcium-dependent modulation of CaMKII activity. Together, all these data open new perspectives to the therapeutic strategy for SMA patients.

Physical exercise reduces cardiac defects in type 2 spinal muscular atrophy-like mice.

Spinal muscular atrophy (SMA), the leading genetic cause of death in infants worldwide, is due to the misexpression of the survival of motor neuron protein, causing death of motor neurons. Several clinical symptoms suggested that, in addition to motor neurons, the autonomic nervous systems could be implicated in the cardiac function alterations observed in patienst with SMA. These alterations were also found in a severe SMA mouse model, including bradycardia and a reduction of sympathetic innervation, both associated with autonomic imbalance. In the present study, we investigate the extent of autonomic dysfunction and the effects of a running-based exercise on the altered cardiorespiratory function in type 2 SMA-like mice. We observed that the SMA induced: (1) a dramatic alteration of intrinsic cardiac conduction associated with bradycardia; (2) a severe cardiomyopathy associated with extensive ventricular fibrosis; and (3) a delay in cardiac muscle maturation associated with contractile protein expression defects. Furthermore, our data indicate that the sympathetic system is not only functioning, but also likely contributes to alleviate the bradycardia and the arrhythmia in SMA-like mice. Moreover, physical exercise provides many benefits, including the reduction of cardiac protein expression defect, the reduction of fibrosis, the increase in cardiac electrical conduction velocity, and the drastic reduction in bradycardia and arrhythmias resulting in the partial restoration of the cardiac function in these mice. Thus, modulating the cardiorespiratory function in SMA could represent a new target for improving supportive care and for developing new pharmacological and non-pharmacological interventions that would most certainly include physical exercise.

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.

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.

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.

Exercise-induced modulation of calcineurin activity parallels the time course of myofibre transitions.

This study establishes a causal link between the limitation of myofibre transitions and modulation of calcineurin activity, during different exercise paradigms. We have designed a new swimming-based training protocol in order to draw a comparison between a high frequency and amplitude exercise (swimming) and low frequency and amplitude exercise (running). We initially analysed the time course of muscle adaptations to a 6- or 12-week swimming- or running-based training exercise program, on two muscles of the mouse calf, the slow-twitch soleus and the fast-twitch plantaris. The magnitude of exercise-induced muscle plasticity proved to be dependent on both the muscle type and the exercise paradigm. In contrast to the running-based training which generated a continuous increase of the slow phenotype throughout a 12-week training program, swimming induced transitions to a slower phenotype which ended after 6 weeks of training. We then compared the time course of the exercise-induced changes in calcineurin activity during muscle adaptation to training. Both exercises induced an initial activation followed by the inhibition of calcineurin. In the muscles of animals submitted to a 12-week swimming-based training, this inhibition was concomitant with the end of myofibre transition. Calcineurin inhibition was a consequence of the inhibition of its catalytic subunit gene expression on one hand, and of the expression increase of the modulatory calcineurin interacting proteins 1 gene (MCIP1), on the other. The present study provides the first experimental cues for an interpretation of muscle phenotypic variation control.

Sprouty gene expression is regulated by nerve and FGF6 during regeneration of mouse muscles.

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.

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.

FGF6 regulates muscle differentiation through a calcineurin-dependent pathway in regenerating soleus of adult mice.

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.

IGF-II is up-regulated and myofibres are hypertrophied in regenerating soleus of mice lacking FGF6.

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.

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.