Inhibition of the Neuregulin1 – ERBB2 – ERK signaling axis as a therapeutic approach for the Charcot-Marie-Tooth Disease 1A

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 DanksagungenThe 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 Danksagunge