Düsseldorf, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Düsseldorf, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

Düsseldorf, Universiẗat, Diss., 2003.

Düsseldorf, Universiẗat, Diss., 2001.

Hohenheim, Univ., Diss., 2007.

Würzburg, Univ., Diss., 2009. / Zsfassung in dt. Sprache.

Charcot-Marie-Tooth 1B (CMT1B) is a progressive inherited demyelinating disease of human peripheral nervous system leading to sensory and/or motor function disability and is caused by mutations in the P0 gene. Mice heterozygously deficient for P0 (P0+/-) are an adequate model of this human disorder showing myelin degeneration, formation of onion bulbs, remyelination and a reduced motor conduction velocity of around 30m/s similar to patients. Previously, it had been shown that T-lymphocytes and macrophages play a crucial role during pathogenesis in peripheral nerves of P0+/- mice. Both, T-lymphocytes and macrophages increase in number in the endoneurium and deletion of T-lymphocytes or deletion of a macrophage-directed cytokine ameliorates the disease. In this study the monocyte chemoattractant protein-1 (MCP-1) was identified as an early regulated cytokine before onset of disease is visible at the age of six months. MCP-1 mRNA and protein expression could be detected in femoral quadriceps and sciatic nerves of P0+/- mice already at the age of one month but not in cutaneous saphenous nerves which are never affected by the disease. MCP-1 was shown to be expressed by Schwann cells and to mediate the immigration of immune cells into peripheral nerves. Deletion of MCP-1 in P0+/- mice accomplished by crossbreeding P0 and MCP-1 deficient mice revealed a substantial reduction of immune cells in peripheral nerves of P0+/-/MCP-1+/- and P0+/-/MCP-1-/- mice at the age of six months. In twelve months old mice reduction of immune cells in peripheral nerves is accompanied by amelioration of demyelinating disease in P0+/-/MCP-1+/- and aggravation of demyelinating disease in lumbar ventral roots of P0+/ /MCP-1-/- mice in comparison to P0+/ /MCP 1+/+ mice. Furthermore, activation of the MEK1/2-ERK1/2 signalling cascade could be demonstrated to take place in Schwann cells of affected peripheral nerves of P0+/- mice overlapping temporarily and spatially with MCP-1 expression. An animal experiment using a MEK1/2-inhibitor in vivo, CI-1040, revealed that upon reduction of ERK1/2 phosphorylation MCP-1 mRNA expression is diminished suggesting that the activation of the MEK1/2-ERK1/2 signalling cascade is necessary for MCP-1 expression. Additionally, peripheral nerves of P0+/- mice showing reduced ERK1/2 phosphorylation and MCP-1 mRNA expression also show reduced numbers of macrophages in the endoneurium. This study shows a molecular link between a Schwann cell based mutation and immune cell function. Inhibition of the identified signalling cascade might be a putative target for therapeutic approaches. / Die humane Erkrankung Charcot-Marie-Tooth 1B (CMT1B) ist eine erbliche, chronisch fortschreitende Erkrankung des peripheren Nervensystems die durch Mutation des P0-Gens verursacht wird und zu motorischen und/oder sensorischen Defiziten führt. Sehr ähnlich der humanen Erkrankung weist das Mausmodell, eine für das Myelinprotein P0 heterozygot-defiziente Maus (P0+/-), Degeneration peripheren Myelins, aufeinanderfolgende Zyklen von De- und Remyelinisierung als auch reduzierte Nervenleitgeschwindigkeiten auf. Wissenschaftliche Untersuchungen am Mausmodell ergaben eine Beteiligung von T-Lymphozyten und Makrophagen an der Pathogenese. In dieser Studie wurde das Chemokin „Monocyte Chemoattractant Protein-1“ (MCP-1) als pathogen-relevant in P0+/- Mäusen identifiziert. MCP-1 mRNA und Protein wurden sowohl im Alter von sechs und zwölf Monaten nachgewiesen, Stadien, in denen morphologische Veränderungen peripherer Nerven von P0+/- Mäusen zu erkennen sind, aber auch im Alter von einen und drei Monaten, ein Alter bei dem pathologischen Veränderungen nicht zu finden sind. Mit Hilfe von MCP-1 defizienten Mäusen (MCP-1-/-) und Verpaarung mit P0-defizienten Mäusen konnten weiterführende Untersuchungen zur Rolle von MCP-1 im peripheren Nerv der Maus durchgeführt werden. So zeigte es sich mittels Transplantation von GFP-positivem Knochenmark, dass MCP 1 die Infiltration von Makrophagen aus dem Blut in periphere Nerven vermittelt. Weiterhin konnte gezeigt werden, dass periphere Nerven von sechs Monate alten P0+/-/MCP-1+/- und P0+/-/MCP-1-/- Mäusen trotz signifikant niedrigerer Anzahl von Immunzellen keine Milderung der Demyelinisierung zeigen. Hingegen weisen periphere Nerven von zwölf Monate alten P0+/ /MCP-1+/- Mäusen sowohl weniger Makrophagen und T-Lymphozyten als auch wesentlich weniger pathologische Veränderungen auf. Periphere Nerven von P0+/-/MCP-1-/- Tieren dagegen zeigen nur eine nicht signifikante Reduktion von Immunzellen und sogar eine Verschlechterung des Phänotyps im Vergleich zu ventralen Spinalwurzeln von P0+/-/MCP-1+/+ Mäusen. Weiterführende Untersuchungen ergaben, dass eine Aktivierung der MEK1/2-ERK1/2 Signalkaskade sowohl in peripheren Nerven von drei und sechs Monate alten P0+/- Mäusen zu finden ist, allerdings, ähnlich der Expression von MCP-1, nur in peripheren Nerven, die von der Demyelinisierung betroffen sein können. Unter Verwendung eines Inhibitors der Kinasen MEK1 und 2 konnte in vivo gezeigt werden, dass Phosphorylierung von ERK1/2 für die erhöhte MCP-1 Expression in peripheren Nerven von P0+/- Mäusen notwendig ist. Darüber hinaus wurde durch Verminderung der ERK1/2-Phosphorylierung eine Reduktion von Makrophagen im Endoneurium von P0+/- Tieren erzielt.

Charcot-Marie-Tooth-1A (CMT1A) is the most prevalent inherited peripheral neuropathy and no curing treatment is available. Even though the genetic cause (a duplication of the myelin protein PMP22) is known since the 90th, the pathological mechanisms are poorly understood. Clinically, affected patients suffer from muscular atrophies, foot deformities and sensory deficits. Histologically, diseased peripheral nerves show primary myelin abnormalities during development followed by a slowly progressing demyelination and subsequent axonal loss in adulthood, which accounts for clinical symptoms. Most specifically, CMT1A nerves exhibit onion bulb formations, which are supernumerary Schwann cells, wrapping concentrically around a central axon-Schwann cell unit. Here, we show that Schwann cells of various demyelinating neuropathies express the paracrine growth factor Neuregulin-1 type-I, which is undetectable in healthy nerves but essential for regeneration after acute nerve injury. We could demonstrate a correlation of Nrg1-I expression in Schwann cells with clinical and histological disease hallmarks in various genetically modified mouse models. Induction of glial Nrg1-I expression on a wildtype background induced symptoms reminiscent of a demyelinating neuropathy, whereas ablation of Nrg1 in Schwann cells of a CMT1A mouse model ameliorated neuropathological hallmarks. Especially, formation of onion bulbs, the key hallmark of CMT1A, could be attributed to Nrg1-I expression in Schwann cells. Furthermore, we showed that this is facilitated via the ErbB2/MEK/ERK signaling cascade. This study proposes a model in which the beneficial, transient upregulation of glial Nrg1-I after acute nerve injury turns into a detrimental constantly active repair-response, due to functional loss of axonal contact in CMT1A and supposedly in other demyelinating neuropathies. This putative common pathway in peripheral neuropathies might open up promising new therapeutic strategies that interfere with the Nrg/ErbB2/MEK/ERK pathway, especially since ErbB2 receptor inhibitors are already applied in breast cancer therapies.:Abbreviations ........................................................................................................................................... i 1 Introduction ..................................................................................................................................... 1 1.1 The central and peripheral nervous system .............................................................................1 1.2 Schwann cells and Myelination ................................................................................................3 1.3 Neuregulin ................................................................................................................................6 1.4 Acute injury of the peripheral nervous system and Wallerian degeneration ....................... 11 1.5 Diseases of the peripheral nervous system: Neuropathies ................................................... 12 1.6 Charcot-Marie-Tooth disease ................................................................................................ 13 2 Aims ............................................................................................................................................... 16 3 Material and methods ................................................................................................................... 17 3.1 Animal models ....................................................................................................................... 17 3.2 Genotyping ............................................................................................................................ 18 3.3 Phenotyping tests .................................................................................................................. 18 3.4 Histology ................................................................................................................................ 19 3.5 Molecular biology .................................................................................................................. 22 3.6 Material ................................................................................................................................. 26 4 Results ........................................................................................................................................... 29 4.1 Ablation of glial Neuregulin-1 improves the clinical phenotype in a mouse model of CMT1A ………………………………………………………………………………………………………………………………………… 29 4.2 Overexpression of Neuregulin-1 type-I induces hallmarks of peripheral neuropathy ......... 34 4.3 Glial Neuregulin-1 type-I signaling promotes survival and attraction of Schwann cells ....... 36 4.4 Dedifferentiation promoting signaling pathways are induced by SC-Nrg1-I ........................ 40 5 Discussion ...................................................................................................................................... 45 6 Abstract ......................................................................................................................................... 50 7 Zusammenfassung ......................................................................................................................... 51 8 References ..................................................................................................................................... 52 9 Appendix .......................................................................................................................................... iii 9.1 List of figures ........................................................................................................................... iii 9.2 List of tables............................................................................................................................. iv 9.3 Erklärung über die eigenständige Abfassung der Arbeit .......................................................... v Content 9.5 Publications ........................................................................................................................... viii

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