CHARBONNIER Frederic

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Charcot Marie Tooth 1A (CMT1A) is the most common hereditary neuropathy in the world. It is due to a duplication of the PMP22 gene. To date, most of the tested treatments are more palliative than curative. The objective of this project is to develop a new treatment for CMT1A based on the normalization of PMP22 gene overexpression by targeted siRNA therapy. For this purpose, eight PMP22 siRNAs were designed and tested in the MSC-80 cell line. In vitro, siPM22 #7 showed 50% inhibition of the PMP22 gene and corresponding protein expression without affecting cell viability. This siRNA was chosen for further studies. In order to protect the PMP22 siRNA from enzymatic degradation and to improve its cellular internalization, we coupled it to a cholesterol precursor, squalene (SQ), which has the particularity of forming nanoparticles (NP) in water. After bioconjugation, obtaining and characterization of PMP22-SQ siRNA NPs, we show that their lipophilic character remains stable over time and that they remain biologically active. In vitro, in MSC80 cells, the PMP22-SQ siRNA NPs inhibit, like the transfected PMP22 siRNA, the expression of the PMP22 gene at 50%. In vivo, i.v. injected PMP22-SQ siRNA NPs resituate motor activity as well as nerve conduction velocity and muscle action potential, in two CMT1A transgenic mouse models, carrying one extra copy (JP18 B6) or two extra copies (JP18/Y13 B6) of PMP22. After sacrifice of the mice, we show by confocal microscopy that the 2 transcription factors KROX20 and SOX10 as well as neurofilaments are increased in sciatic nerve sections treated with PMP22-SQ siRNA NPs showing myelination and axonal regeneration. In electron microscopy, we observe a normalization of the phenotype which translates into myelin compaction after internalization of PMP22-SQ siRNA NPs in Schwann cells and axons. In conclusion, we show that a targeted treatment with PMP22 siRNA NPs normalizing PMP22 expression could be a future therapy for CMT1A disease. This work is supported by a public funding supervised by the French National Research Agency (ANR) within the framework of the "investment of future" program: Labex NanoSaclay, reference: ANR-10-LABX-0035 and, protected by an international patent No.

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

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

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Infantile spinal muscular atrophy (SMA) is a rare autosomal recessive disease that occurs in childhood and for which no therapy has yet been found to be effective. SMAs are characterized by a specific degeneration of motor neurons leading to critical muscle weakness which, when it reaches the respiratory muscles, causes the death of the patients. The origin of these diseases is the mutation of the Survival of Motor Neuron 1 (Smn1) gene which induces a deficiency of the Survival of Motor Neuron (SMN) protein. All SMA patients have at least 2 copies of the Smn2 gene, which modulates the severity of the disease by producing a low amount of complete SMN protein. Indeed, due to alternative splicing of exon-7 which encodes a stability domain of the SMN protein, the transcripts produced by the expression of Smn2 are in most cases incomplete, and generate unstable, rapidly degraded SMN proteins. Thus, a major therapeutic goal in SMA is to increase SMN protein levels in patients. By deciphering the molecular mechanisms underlying the beneficial effects of exercise in a mouse model of severe SMA, the lab has shown that direct stimulation of NMDA receptors (NMDAR) results in a significant increase in exon 7 inclusion in SMN transcripts generated from Smn2 gene expression in SMA tissues. During my thesis, I was involved in the identification of a second exercise-induced mechanism related to the reduction of expression levels of the IGF-1 receptor (IGF-1R), which is overexpressed in the spinal cord of SMA mice. Reduction of IGF-1R is sufficient to promote SMN expression, both through transcriptional and post-transcriptional effects. Using a transcriptomic approach to identify genes whose expression is comparably altered by modulation of NMDARs or IGF-1R, we identified a set of genes that belong to the cholesterol biosynthetic pathway. RT-qPCR and western blot analyses in the spinal cord of control and SMA model mice revealed that i) cholesterol biosynthetic pathway enzymes are under-expressed in the spinal cord of SMA mice and in a culture of SMA patient fibroblasts and ii) NMDA treatment or IGF-1R reduction reactivates the expression of cholesterol biosynthetic pathway enzymes to levels comparable to controls. Importantly, we demonstrated that specific inhibition of DHCR24, a key enzyme in the terminal pathway of cholesterol biosynthesis, in control fibroblasts induces a significant reduction in SMN expression, both at the mRNA and protein level, and conversely, restoration of DHCR24 expression levels in human SMA fibroblasts induces a significant increase in SMN expression. Moreover, GC-MS quantification of cholesterol, its metabolites, and various oxysterols indicates that the whole cholesterol metabolism is altered in the SMA mouse spinal cord, and enhanced by NMDAR activation. Together, these results demonstrate for the first time that cholesterol homeostasis is disturbed in SMA and that modulating the molecular pathways dependent on cholesterol and its derivatives may constitute a promising therapeutic approach.

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

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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. 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.

The identification of new pathways governing myelination provides innovative avenues for remyelination. Liver X receptors (LXRs) a and beta 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 LXR alpha/beta 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.

Disease processes and trauma affecting nerve-evoked muscle activity, motor neurons, synapses and myofibers cause different levels of muscle weakness, i.e., reduced maximal force production in response to voluntary activation or nerve stimulation. However, the mechanisms of muscle weakness are not well known. Using murine models of amyotrophic lateral sclerosis (SOD1(G93) transgenic mice), congenital myasthenic syndrome (AChE knockout mice and Musk(V789m/-) mutant mice), Schwartz Jampel syndrome (Hspg2(C1532YNEO/C1532YNEO) mutant mice) and traumatic nerve injury (Neurotomized wild-type mice), we show that the reduced maximal activation capacity (the ability of the nerve to maximally activate the muscle) explains 52%, 58% and 100% of severe weakness in respectively SOD1(G93A), Neurotomized and Musk mice, whereas muscle atrophy only explains 37%, 27% and 0%. We also demonstrate that the impaired maximal activation capacity observed in SOD 1, Neurotomized, and Musk mice is not highly related to Hdac4 gene upregulation. Moreover, in SOD1 and Neurotomized mice our results suggest LC3, Fn14, Bcl3 and Gadd45a as candidate genes involved in the maintenance of the severe atrophic state. In conclusion, our study indicates that muscle weakness can result from the triggering of different signaling pathways. This knowledge may be helpful in designing therapeutic strategies and finding new drug targets for amyotrophic lateral sclerosis, congenital myasthenic syndrome, Schwartz Jampel syndrome and nerve injury.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular degenerative disease. It is accompanied by metabolic alterations, which manifest themselves early in mouse models. The objective of this thesis was to identify the molecular targets involved in these changes. To do so, we studied the glycolytic muscle, the first one affected by denervation during the disease in an ALS model, the SOD1G86R mouse. We showed an imbalance between carbohydrate and lipid metabolic pathways at a presymptomatic stage, with a preference for the lipid catabolic pathway. This alteration may explain our observation on the change in exercise capacity of presymptomatic SOD1G86R mice. In this context, we showed that pharmacological inhibition of PDK4, a major glycolysis inhibitor, is beneficial, delaying the onset of symptoms. By taking into account the metabolic specificities of the different components of the neuromuscular axis, this work opens new therapeutic avenues for the treatment of ALS.

Amyotrophic lateral sclerosis is a neurodegenerative disease associated with metabolic dysfunction. Alterations in lipid metabolism, described in ALS patients and animal models, could participate in the early stages of the disease. The objective of this thesis was to study the role of stearoyl-coenzyme A desaturase 1 (SCD1), a key enzyme in lipid metabolism, in ALS. By studying the peripheral fatty acid profile in an ALS mouse model, the SOD1m mice, we saw a decrease in SCD1 activity as early as the early (subclinical) stages of the disease. This decrease alone could explain the alterations in lipid metabolism characteristic of ALS. The impact of the loss of SCD1 activity on the motor axis has been studied. Gene deletion or pharmacological inhibition of SCD1 improves functional recovery after sciatic nerve injury in wild-type mice. We investigated whether the loss of SCD1 activity found in SOD1m mice is a protective mechanism against ALS progression. We treated SOD1m mice with an inhibitor of SCD1 activity. The treatment led to an increase in oxidative metabolism, preservation of neuromuscular integrity and improved motor neuron survival. We conclude that SCD1 inhibition represents a promising therapeutic target in ALS.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of adults characterized by selective loss of fast motor neurons (MN), muscle paralysis and lipid hypermetabolism. Physical exercise could be considered as a therapeutic treatment for ALS. In ALS mice, different running protocols have conflicting effects depending on their intensity. In this scientific context, we compared the neuromuscular adaptations of wild-type and ALS SOD1 G93A mice subjected to treadmill running training and swimming in a current pool that we developed. Under our experimental conditions, swimming preferentially recruits fast motor units and activates large surface MNs, in contrast to running, which preferentially recruits slow motor units (activation of reduced surface MNs and induction of fast-to-slow muscle fiber transitions). Only swimming delays the onset of symptoms and prolongs the life span of ALS mice. It limits MN death in the lumbar spinal cord and maintains skeletal muscle integrity. These effects of swimming are correlated with adaptation of energetic metabolism leading to increased glucose utilization and preservation of lipid stores. Our data highlight a relationship between activation and protection of ALS-sensitive fast motor units. Determining the neuroprotective mechanisms induced by swimming in ALS mice would allow us to propose new therapeutic drug pathways for ALS patients.

Infantile spinal muscular atrophy (ISA) is a neuromuscular disease of children, induced by a low level of SMN (Survival of Motor Neuron) protein in the body and for which no curative therapy is known to date. In this work, we show that the delayed maturation of motor units, observed in ASI type II mice, is correlated with motor neuron death. Physical exercise delays motor neuron death and induces an acceleration of motor unit maturation. Furthermore, exercise is able to specifically increase the expression of the gene coding for the activating subunit of the NMDA receptor and majority in motor neurons, named NR2A, which is under expressed in the spinal cord of ASI type II mice. Consequently, inhibiting NMDA receptor activity blocks the effects of exercise on muscle development, neuroprotection and lifespan. Thus, restoring NMDA receptor function may be a promising therapeutic approach for the treatment of ASI.

Infantile spinal muscular atrophy (ISA) and amyotrophic lateral sclerosis (ALS) are two very serious neurodegenerative diseases. We analyzed the effects of exercise in mouse models of these two diseases. Running is beneficial in the case of ASI. It increases life span, provides neuroprotection and decreases muscle atrophy due to the disease. A study comparing the effects of swimming and running in a mouse model of ALS shows that both types of exercise are neuroprotective. However, while running protects motor neurons in slow motor units, swimming protects those in fast motor units. Whether in ASI or ALS, exercise is very beneficial. These studies pave the way for the development of training programs for patients and will help identify these mechanisms and propose new therapeutic avenues.

Proteoglycans are macromolecules composed of a protein backbone on which are covalently bound particular polysaccharide chains, the glycosaminoglycans. Their role is associated with fundamental cellular processes: proliferation, adhesion and cell migration. In our laboratory, a peptide carrying chondroitin and heparan sulfate chains was isolated from human seminal plasma. The complementary dna generating this peptide was cloned, allowing the establishment of the complete deduced protein sequence of a new molecule, testis. A clone with a 112 bp insertion in the coding phase of the molecule and a shortened 3' non-coding region was characterized. The reality of this transcript was demonstrated by rt-pcr and the alternative exon coding for this insertion was characterized. The complete mapping of this gene and its chromosomal location have been established. The human testis is encoded by a single gene, located on chromosome 5q band 31 and is composed of twelve exons, for a size estimated at more than 100kb. Northern blot analysis on a representative set of human tissues revealed a majority messenger rna of 5.2kb, in the genital tract and particularly in the prostate. An antibody directed against the n-terminal region of the testis was performed. An immunohistochemical analysis allowed to localize the testis in the prostatic epithelial tissue, privileged target of neoplasia. This expression was confirmed by in situ hybridization, in healthy and cancerous tissues, for both types of transcripts. In a set of seven pathological tissues, a western analysis revealed, in only one case, a protein with unexpected electrophoretic mobility. An analysis of the gene of this patient, suffering from a severe form of hormone-independent prostate cancer, allowed to characterize, on one of the alleles, a 19 bp deletion in the alternatively spliced exon.