Philippe Lopes

In situ localisation and quantification of surfactins in a Bacillus subtilis swarming community by imaging mass spectrometry

Surfactins are a family of heptacyclopeptides in which the C‐terminal carbonyl is linked with the β‐hydroxy group of a fatty acid acylating the N‐terminal function of a glutamic acid residue. The fatty acyl chain is 12–16 carbon atoms long. These compounds, which are secreted by the Gram‐positive bacterium Bacillus subtilis in stationary phase in liquid cultures, play an important role in swarming communities on the surface of agar media in the formation of dendritic patterns. TOF secondary ion MS (TOF‐SIMS) imaging was used to map surfactins within 16–17 h swarming patterns, with a 2 μm spatial resolution. Surfactins were mainly located in the central mother colony (the site of initial inoculation), in a ‘ring’ surrounding the pattern and along the edges of the dendrites. In the mother colony and the interior of the dendrites, surfactins with shorter chain lengths are present, whereas in the ring surrounding the swarm community and between dendrites, surfactins with longer fatty acyl chain lengths were found. A quantitative analysis by MALDI‐TOF MS showed a concentration gradient of surfactin from the mother colony to the periphery. The concentration of surfactin was ∼400 pmol/mL in the mother colony and ∼10 pmol/mL at the base of the dendrites, decreasing to 2 pmol/mL at their tips.

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.

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.

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.

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. J. Cell. Physiol. 214:126–135, 2008.

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.

Isolation and Characterization of α,β-Unsaturated γ-Lactono-Hydrazides from Streptomyces sp.

Two novel α,β-unsaturated γ-lactono-hydrazides, geralcin A (2) and geralcin B (3), were isolated from Streptomyces sp. LMA-545. This unusual scaffold consists of the condensation of alkyl-hydrazide with an α,β-unsaturated γ-lactone, 3-(5-oxo-2H-furan-4-yl)propanoic acid (1), which was isolated from the same broth culture. Amberlite XAD-16 solid-phase extraction was used during the cultivation step, and the trapped compounds (1-3) were eluted from the resin with methanol. The structures were elucidated using (1)H, (13)C, and (15)N NMR spectroscopic analysis and high-resolution mass spectrometry. Geralcin B (3) was cytotoxic against MDA231 breast cancer cells with an IC(50) of 5 μM.

Resistance Strength Training Exercise in Children with Spinal Muscular Atrophy

Introduction: Preliminary evidence in adults with spinal muscular atrophy (SMA) and in SMA animal models suggests exercise has potential benefits in improving or stabilizing muscle strength and motor function. Methods: We evaluated feasibility, safety, and effects on strength and motor function of a home‐based, supervised progressive resistance strength training exercise program in children with SMA types II and III. Up to 14 bilateral proximal muscles were exercised 3 times weekly for 12 weeks. Results: Nine children with SMA, aged 10.4 ± 3.8 years, completed the resistance training exercise program. Ninety percent of visits occurred per protocol. Training sessions were pain‐free (99.8%), and no study‐related adverse events occurred. Trends in improved strength and motor function were observed. Conclusions: A 12‐week supervised, home‐based, 3‐day/week progressive resistance training exercise program is feasible, safe, and well tolerated in children with SMA. These findings can inform future studies of exercise in SMA. Muscle Nerve 52: 559–567, 2015

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

•  The present study provides evidence that the cardiomyopathy observed in spinal muscular atrophy (SMA) model mice is mainly due to intrinsic cardiac alteration but not to autonomic impairment. •  We demonstrated a non‐pathological sympathetic activity on the heart of type 2 SMA‐like mice, which likely counteracts the dramatic alteration of the cardiac function, such as arrhythmia and reduced heart rate. •  We demonstrated for the first time that physical exercise partially restores cardiac conduction efficiency, prevents fibrosis, attenuates defects in protein expression, bradycardia and arrhythmias leading to the partial recovery of heart and respiratory rates in exercised SMA‐like mice. •  This study indicates a profound involvement of cellular, structural and vascular cardiac dysfunction in the pathogenesis of SMA, widely opening alternative pharmacological and non‐pharmacological therapeutic strategies that would most certainly include physical exercise.