Les nerfs périphériques sont sujets à de nombreuses pathologies et l’étiologie des neuropathies périphériques (NP) est vaste (troubles métaboliques, infections, toxines, blessures physiques et mutations génétiques, ect.). Par exemple, les NP d’origine traumatique sont courantes et sont caractérisées par une dégénérescence dite Wallérienne des fibres nerveuses. Autre exemple, la maladie de Charcot-Marie-Tooth 1A (CMT1A) qui est la NP génétique héréditaire la plus fréquente. Elle est caractérisée par une surexpression de la protéine PMP22 impliquée dans le maintien de la gaine de myéline. Actuellement, il n’existe pas de traitement pharmacologique de ces deux affections des nerfs. Récemment, l’intérêt pour le rôle des antioxydants alimentaires, tels que la curcumine, a suscité de nombreuses recherches. Cette molécule est depuis longtemps utilisée en médecine asiatique pour ces propriétés thérapeutiques. Cependant, elle possède une très faible biodisponibilité et nécessite donc l’emploi de doses très élevées pour obtenir des effets bénéfiques. Dans une première étude, nos résultats ont montré que des faibles doses de curcumine administrées localement et en continu, améliorent la récupération fonctionnelle, les paramètres électrophysiologiques et histologiques, et l’expression des principales protéines de la myéline dans un modèle d’écrasement du nerf sciatique chez le rat. Ces effets bénéfiques ont été attribués aux propriétés antioxydantes de la curcumine. Dans une seconde étude, nos résultats ont montré qu’une faible dose de nanocristaux de curcumine (Nano-Cur), injectée en IP, améliorent le phénotype, les paramètres électrophysiologiques et histologiques dans un modèle transgénique de rat CMT1A. Dans cette étude, les effets positifs ont été attribués aux propriétés antioxydantes des Nano-Cur, couplés à l’activation de la voie de dégradation associée au réticulum endoplasmique, permettant la réduction de la surexpression nocive de PMP22 chez les rats CMT1A. L'ensemble de ces résultats démontrent que, l’administration de faibles doses de curcumine constitue un traitement prometteur dans la réparation des nerfs périphériques. / Peripheral nerves are subject to many pathologies and the etiology of peripheral neuropathies (PN) is vast (metabolic disorders, infections, toxins, physical injuries and genetic mutations, etc.). For example, PN of traumatic origin are common and are characterized by a called Wallerian degeneration of nerve fibres. Another example is Charcot-Marie-Tooth disease 1A (CMT1A), which is the most common hereditary genetic PN. It is characterized by an overexpression of the PMP22 protein involved in maintaining the myelin sheath. Currently, there is no pharmacological treatment for these two nerve disorders. Recently, interest in the role of dietary antioxidants, such as curcumin, has led to much research. This molecule has long been used in Asian medicine for its therapeutic properties. However, it has a very low bioavailability and therefore requires the use of very high doses to obtain beneficial effects. In a first study, our results showed that low doses of curcumin administered locally and continuously improve functional recovery, electrophysiological and histological parameters, and expression of major myelin proteins in a rat sciatic nerve crush model. These beneficial effects have been attributed to the antioxidant properties of curcumin. In a second study, our results showed that a low dose of curcumin nanocrystals (Nano-Cur), injected in IP, improves phenotype, electrophysiological and histological parameters in a transgenic model of CMT1A rats. In this study, the positive effects were attributed to the antioxidant properties of the Nano-cur, coupled with the activation of the endoplasmic reticulum associated degradation pathway, allowing the reduction of harmful overexpression of PMP22 in CMT1A rats. All these results show that the administration of low doses of curcumin is a promising treatment for peripheral nerve repair

L’existence de douleurs neuropathiques et/ou de perte de la sensibilité douloureuse sont souvent le reflet d’une neuropathie sensitive affectant plus particulièrement les fibres nerveuses sensitives amyélinique Aδ et C, dites neuropathie des petites fibres (NPF). Ces fibres innervent, notamment, le derme et l’épiderme de la peau. Elles communiquent la sensibilité thermique et algique au système nerveux central et contribuent à l’homéostasie cutanée, entre autres, par la libération de neuropeptides en périphérie. De nombreuses pathologies sont associées à une altération de ces petites fibres dans la peau. Deux pathologies impliquant une NPF ont été étudiées au cours de ce travail : les escarres et la maladie de Charcot-Marie-Tooth type 1A. Un travail expérimental a été réalisé chez la souris pour répondre à la question suivante ; est-ce qu’une seule atteinte des fibres nociceptives, responsables de la perte de sensibilité peut entraîner un déséquilibre de l’homéostasie cutanée, responsable de l’apparition des escarres ? La mise en place d’un modèle de neuropathie sensitive fonctionnelle réversible a permis de mettre en en évidence l’implication des neuropeptides, substance P (SP) et « calcitonin gene-related peptide » (CGRP), libérés par les fibres nerveuses cutanées, dans la formation d’ulcères de pression. Un traitement préventif à la rhEPO (Recombinant Human Erythropoietin) dans ce modèle associant une neuropathie et des plaies de pression, protège la peau contre une pression ischémiante induisant une escarre par son effet neuroprotecteur sur les petites fibres cutanées. L’association CMT1A et NPF a été étudiée à partir de biopsies cutanées humaines. La quantification des fibres intraépidermiques révèle que 48% des patients CMT1A sont atteints d’une NPF. L’analyse des biopsies cutanées révèle également une altération du nombre et de la morphologie de cellules de Langerhans dans la maladie de CMT1A. L'ensemble de ces résultats confirme l'intérêt de l'étude des petites fibres dans des pathologies variées et confirme le potentiel thérapeutique neuroprotecteur de l'EPO / The neuropathic pain and/or hypoalgesia often reflect a sensory neuropathy that affects particularly sensory, Aδ (thinly myelinated) and C (unmyelinated) nerve fibers. This kind of neuropathy is named "small fiber neuropathy" (SFN). These small fibers innervate the dermis and epidermis. C and Aδ free nerve endings respond to a variable range of stimuli including mechanical, thermal and pain stimuli. They conduct nociceptive signals to central nervous system and contribute to skin homeostasis, among others, by the release of neuropeptides in the periphery. Many diseases are associated with an alteration of these cutaneous small fibers. Two pathologies involving SFN were studied in this work: pressure ulcers and Charcot-Marie-Tooth disease Type 1A (CMT1A). Experimental studies on mice were performed to determine if impairment of nociceptive fibers could lead to an imbalance of skin homeostasis and could be involved in development of pressure ulcers, apart from its role in pain signal transduction. A functional reversible sensory neuropathy mouse model was set up and helped to demonstrate the involvement of the neuropeptides, substance P (SP) and "calcitonin gene-related peptide" (CGRP), released by cutaneous nerve fibers in the formation of pressure ulcers. By its neuroprotective effect on small nerve fibers, a preventive rhEPO (Recombinant Human Erythropoietin) treatment in this model protects the skin against an ischemic pressure-induced Stage 2 ulcer. The CMT1A and SFN association has been studied from human skin biopsies. Quantification of intraepidermal nerve fibers reveals that 48% of CMT1A patients have a SFN. The analysis of skin biopsies also revealed an alteration in the number and morphology of Langerhans cells in CMT1A disease. All these results confirm the interest of the study of small fibers in various pathologies and confirm the neuroprotective therapeutic potential of EPO.

Die Charcot-Marie-Tooth-Krankheit 1A (CMT1A) gehört zur Familie der erblichen Neuropathien des peripheren Nervensystems (PNS) und ist mit einer Prävalenz von 1 zu 2500 die häufigste Subform. Die Patienten leiden an einer distal ausgeprägten Muskelatrophie und sensorischen Beeinträchtigungen. Die Histopathologie zeigt sich im PNS durch Myelinisierungsdefekte, die Bildung von Zwiebelschalenformationen, eine langsam fortschreitende Demyelinisierung und einen axonalen Verlust in späteren Krankheitsstadien, die für die klinische Symptomatik verantwortlich sind. Bis heute gibt es keine Behandlungsmöglichkeiten. Die genetische Duplikation des Gens, das für das periphere Myelinprotein von 22kDa (PMP22) kodiert, ist Hauptursache der Erkrankung und betrifft speziell die myelinisierenden Gliazellen des PNS, die Schwannzellen. Erkrankte Schwannzellen induzieren eine de novo Expression des glialen Wachstumsfaktors Neuregulin 1-Typ I (NRG1-I). Die chronische NRG1-I Überexpression wurde als ein maßgeblicher Faktor der Krankheitspathogenese der CMT1A identifiziert. Aberrantes NRG1-I überstimuliert den ERBB2 Rezeptor, was zu einer Hyperaktivierung des MEK-ERK Signalwegs führt. Die genetische Ablation von NRG1 spezifisch in Schwannzellen verbesserte die klinische Präsentation und Histopathologie in CMT1A Mäusen. Damit einhergehend waren die ERBB2 und ERK Hyperaktivität in Schwannzellen normalisiert. Die Inhibition der NRG1-I-vermittelten pathologischen Signalkaskade stellt damit einen vielversprechenden therapeutischen Ansatz für die CMT1A dar. Die langfristige pharmakologische Inhibition entweder der ERBB2 oder der ERK Aktivierung verschlechterte jedoch die klinische Präsentation von adulten CMT1A Ratten. Die Verringerung der ERBB2 Aktivität verschlimmerte die axonale Funktion und bewirkte eine Zunahme von Zwiebelschalenformationen. Die Reduktion der ERK Hyperaktivität führte zu einem frühen behandlungsinduzierten Gewichtsverlust und einer Zunahme an demyelinisierten Axonen. Das Auftreten sekundär dysregulierter Signalkaskaden könnte zu dem Scheitern der Studien beigetragen haben. Das Scheitern beider Studien weist jedoch auch darauf hin, dass die Komplexität der CMT1A Pathobiologie besonders während der späten chronischen Phase noch immer unzureichend verstanden ist und weiterer Erforschung bedarf. Zusammenfassend ist die Interferenz mit der ERBB2-ERK Signalkaskade für die Behandlung der CMT1A ungeeignet. Dies weist auf die Notwendigkeit hin, ein tieferes Verständnis der Komplexität der späten chronischen CMT1A Pathobiologie und vor allem der NRG1-vermittelten Pathomechanismen zu erlangen. Beides schafft die Grundlage für die Identifizierung neuer therapeutischer Behandlungsansätze:Abbreviations 1. Introduction 1.1 The peripheral nervous system 1.1.1 Structure and functions of the nervous system 1.1.2 Neurons and glial cells 1.1.3 Signal transduction and myelination 1.1.4 Development and functions of Schwann cells 1.2 Peripheral neuropathies 1.2.1 Acquired and inherited peripheral neuropathies 1.2.2 Clinical presentation and classification of Charcot-Marie-Tooth diseases 1.2.3 The peripheral myelin protein of 22 kDa (PMP22) 1.2.4 Animal models of PMP22 – associated CMTs 1.2.5 Pathobiology and treatment options of CMT1A 1.2.6 Ablation of Schwann cell Neuregulin 1 ameliorates the phenotype of a CMT1A mouse model 1.3 The Neuregulin 1 – ERBB2 – ERK signaling axis in the Charcot-Marie-Tooth 1A disease 1.3.1 Functions of Neuregulin 1 in the peripheral nervous system 1.3.2 ERBB receptor tyrosine kinase signaling in Schwann cells 1.3.3 NRG1-I – ERBB mediated pathophysiological effects in CMT1A 1.3.4 Options for pharmacological interference with NRG1-I – mediated ERBB2 and ERK activation 1.4 Aim of the dissertation 2. Materials and Methods 2.1 Materials 2.1.1 Chemicals and Reagents 2.1.2 Consumable Supplies 2.1.3 Technical Equipment 2.1.4 Kits 2.1.5 Buffers and Solutions 2.1.6 Antibodies 2.1.7 Genotyping Primer 2.1.8 qPCR Primer 2.1.9 Enzymes 2.1.10 Software 2.2 Animal caretaking and treatment 2.2.1 Animal strains and breeding 2.2.2 Animal breeding and caretaking 2.2.3 Identification of animals 2.2.4 Herceptin® and Selumetinib treatment of CMT1A rats 2.2.5 Mechanical phenotyping 2.2.6 Electrophysiological examination 2.2.7 Euthanasia 2.2.8 Preparation and handling of peripheral nerve tissues 2.3 Histological methods 2.3.1 Embedding of isolated peripheral nerves in Agar 100 resin 2.3.2 Generation, imaging, and analysis of semi-thin sections 2.3.3 Coating of support copper grids for ultrastructure analysis 2.3.4 Generation, contrasting, imaging, and analysis of ultra-thin cross-sections 2.4 Protein Biochemistry 2.4.1 Isolation and quantification of (phospho-) proteins from frozen peripheral nerves 2.4.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) 2.4.3 Western blot and Fast Green staining 2.5 Molecular Biology Methods 2.5.1 Isolation of genomic DNA from tissues 2.5.2 Polymerase chain reaction of genomic DNA 2.5.3 Agarose gel electrophoresis of PCR products 2.5.4 RNA Isolation 2.5.5 Quality and quantity assessment of isolated RNA 2.5.6 cDNA Synthesis of total RNA 2.5.7 Quantitative real-time PCR analysis 2.6 Bioinformatics 2.6.1 Power analysis 2.6.2 Statistical methods 3. Results 3.1 NRG1 – ERBB2/ERBB3 – ERK signaling is increased in an adult CMT1A-resembling rat model 3.2 Late long-term blockage of ERBB2 signaling deteriorates the clinical and histopathological outcome of CMT1A rats 3.2.1 The clinical phenotype and histopathological hallmarks worsen after long-term Herceptin treatment of CMT1A rats 3.2.2 Herceptin application inhibits NRG1-I – mediated ERBB2/ERBB3 activation and concomitant downstream signaling 3.2.3 Inhibition of ERBB2 – mediated downstream signaling does not affect CMT1A Schwann cell dedifferentiation 3.3 Late long-term application of Selumetinib deteriorates the clinical presentation of CMT1A rats 3.3.1 Late short-term Selumetinib application ameliorates NRG1-I – ERBB2/3 – mediated downstream signaling 3.3.2 Long-term Selumetinib treatment deteriorates the clinical phenotype and promotes demyelination in CMT1A rats 3.3.3 Selumetinib treatment improved the dysbalance of the ERK and AKT pathways in CMT1A 4. Discussion 4.1 NRG1-I-mediated malsignaling may not have been sufficiently inhibited to impact the clinical outcome of CMT1A rats after ERBB2 suppression 4.2 Herceptin-binding of ERBB2 may interfere with the regeneration ability of Schwann cells in CMT1A 4.3 Dysregulation of ERK activity in late CMT1A promotes demyelination 5. Abstract 6. Zusammenfassung 7. References 8. Appendix 8.1 List of figures 8.2 List of tables 8.3 Eigenständigkeitserklärung 8.4 Curriculum vitae 8.5 Publikationen 8.6 Danksagungen / The Charcot-Marie-Tooth disease 1A (CMT1A) belongs to the family of inherited neuropathies of the peripheral nervous system (PNS) and is the most frequent subform with a prevalence of 1 in 2500. Patients present with a distally pronounced muscle atrophy and sensory impairments. Histologically, peripheral nerves display developmental myelination abnormalities, onion bulb formations, slowly progressive demyelination, and axonal loss during later disease stages, which account for clinical symptomatology. No treatment options are available upon today. Genetic testing revealed a genomic duplication of the gene encoding for the peripheral myelin protein of 22kDa (PMP22) as the primary cause of disease that affects specifically the myelinating glia of the PNS, the Schwann cells. Pmp22 overexpression has been shown to dysregulate physiological axon-glia interactions. Diseased Schwann cells respond with a de novo expression of the glial growth factor, Neuregulin 1-type I (NRG1-I). While not present under physiological conditions, chronic NRG1-I signaling was identified to be a major driver of disease pathogenesis in CMT1A. On the mechanistic level, aberrant NRG1-I has been demonstrated to overstimulate the ERBB2 receptor resulting in hyperactivation of the MEK-ERK pathway. Genetic disruption of NRG1 specifically in Schwann cells strongly improved the clinical presentation and histopathological manifestation of disease hallmarks in CMT1A-resembling rodents. This beneficial outcome was accompanied by a downregulation of ERBB2 and ERK hyperactivity in Schwann cells. Therefore, interfering with NRG1-I-mediated pathological signaling was proposed to constitute a promising therapeutic approach for CMT1A. However, long-term pharmacological obstruction of either ERBB2 or ERK activation deteriorated the clinical presentation of adult CMT1A rats. ERBB2 inhibition worsened axonal function and induced an increase in onion bulb formations. Reduction of ERK hyperactivity resulted in an early treatment-induced weight loss and an increase in demyelinated axons. The development maladaptive signaling mechanisms may have caused the observed outcome of the trials. However, failing of both trials instead also demonstrates that the complexity of late chronic CMT1A pathobiology is still poorly understood and has to be further investigated. In conclusion, interfering with ERBB2-ERK signaling, downstream of NRG1-I, turned out to be not a viable approach for CMT1A treatment. This points to the necessity to gain a deeper understanding of the late chronic CMT1A intricacy and especially of NRG1-mediated pathomechanisms in neuropathy. Both may build the foundation for the identification of novel therapeutic approaches and drug targets.:Abbreviations 1. Introduction 1.1 The peripheral nervous system 1.1.1 Structure and functions of the nervous system 1.1.2 Neurons and glial cells 1.1.3 Signal transduction and myelination 1.1.4 Development and functions of Schwann cells 1.2 Peripheral neuropathies 1.2.1 Acquired and inherited peripheral neuropathies 1.2.2 Clinical presentation and classification of Charcot-Marie-Tooth diseases 1.2.3 The peripheral myelin protein of 22 kDa (PMP22) 1.2.4 Animal models of PMP22 – associated CMTs 1.2.5 Pathobiology and treatment options of CMT1A 1.2.6 Ablation of Schwann cell Neuregulin 1 ameliorates the phenotype of a CMT1A mouse model 1.3 The Neuregulin 1 – ERBB2 – ERK signaling axis in the Charcot-Marie-Tooth 1A disease 1.3.1 Functions of Neuregulin 1 in the peripheral nervous system 1.3.2 ERBB receptor tyrosine kinase signaling in Schwann cells 1.3.3 NRG1-I – ERBB mediated pathophysiological effects in CMT1A 1.3.4 Options for pharmacological interference with NRG1-I – mediated ERBB2 and ERK activation 1.4 Aim of the dissertation 2. Materials and Methods 2.1 Materials 2.1.1 Chemicals and Reagents 2.1.2 Consumable Supplies 2.1.3 Technical Equipment 2.1.4 Kits 2.1.5 Buffers and Solutions 2.1.6 Antibodies 2.1.7 Genotyping Primer 2.1.8 qPCR Primer 2.1.9 Enzymes 2.1.10 Software 2.2 Animal caretaking and treatment 2.2.1 Animal strains and breeding 2.2.2 Animal breeding and caretaking 2.2.3 Identification of animals 2.2.4 Herceptin® and Selumetinib treatment of CMT1A rats 2.2.5 Mechanical phenotyping 2.2.6 Electrophysiological examination 2.2.7 Euthanasia 2.2.8 Preparation and handling of peripheral nerve tissues 2.3 Histological methods 2.3.1 Embedding of isolated peripheral nerves in Agar 100 resin 2.3.2 Generation, imaging, and analysis of semi-thin sections 2.3.3 Coating of support copper grids for ultrastructure analysis 2.3.4 Generation, contrasting, imaging, and analysis of ultra-thin cross-sections 2.4 Protein Biochemistry 2.4.1 Isolation and quantification of (phospho-) proteins from frozen peripheral nerves 2.4.2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) 2.4.3 Western blot and Fast Green staining 2.5 Molecular Biology Methods 2.5.1 Isolation of genomic DNA from tissues 2.5.2 Polymerase chain reaction of genomic DNA 2.5.3 Agarose gel electrophoresis of PCR products 2.5.4 RNA Isolation 2.5.5 Quality and quantity assessment of isolated RNA 2.5.6 cDNA Synthesis of total RNA 2.5.7 Quantitative real-time PCR analysis 2.6 Bioinformatics 2.6.1 Power analysis 2.6.2 Statistical methods 3. Results 3.1 NRG1 – ERBB2/ERBB3 – ERK signaling is increased in an adult CMT1A-resembling rat model 3.2 Late long-term blockage of ERBB2 signaling deteriorates the clinical and histopathological outcome of CMT1A rats 3.2.1 The clinical phenotype and histopathological hallmarks worsen after long-term Herceptin treatment of CMT1A rats 3.2.2 Herceptin application inhibits NRG1-I – mediated ERBB2/ERBB3 activation and concomitant downstream signaling 3.2.3 Inhibition of ERBB2 – mediated downstream signaling does not affect CMT1A Schwann cell dedifferentiation 3.3 Late long-term application of Selumetinib deteriorates the clinical presentation of CMT1A rats 3.3.1 Late short-term Selumetinib application ameliorates NRG1-I – ERBB2/3 – mediated downstream signaling 3.3.2 Long-term Selumetinib treatment deteriorates the clinical phenotype and promotes demyelination in CMT1A rats 3.3.3 Selumetinib treatment improved the dysbalance of the ERK and AKT pathways in CMT1A 4. Discussion 4.1 NRG1-I-mediated malsignaling may not have been sufficiently inhibited to impact the clinical outcome of CMT1A rats after ERBB2 suppression 4.2 Herceptin-binding of ERBB2 may interfere with the regeneration ability of Schwann cells in CMT1A 4.3 Dysregulation of ERK activity in late CMT1A promotes demyelination 5. Abstract 6. Zusammenfassung 7. References 8. Appendix 8.1 List of figures 8.2 List of tables 8.3 Eigenständigkeitserklärung 8.4 Curriculum vitae 8.5 Publikationen 8.6 Danksagungen