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Functional Role of the Overexpression of the Myelin and Lymphocyte

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Functional Role of the Overexpression of the Myelin and Lymphocyte Functional role of the overexpression of the myelin and lymphocyte protein MAL in Schwann cells Inauguraldissertation Zur Erlangung der Würde eines Doktors der Philosophie Vorgelegt der Philosophisch‐Naturwissenschaftlichen Fakultät der Universität Basel von Daniela Schmid aus Ramsen (SH) Basel, 2013 Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von: Prof. M.A. Rüegg (Fakultätsverantwortlicher) Prof. N. Schaeren-Wiemers (Dissertationsleiterin) Prof. J. Kapfhammer (Korreferent) Basel, den 18. Juni 2013 Prof. J. Schibler (Dekan) To Michael 1. Acknowledgments 1. ACKNOWLEDGMENTS ................................................................................................................................ 6 2. ABBREVIATIONS ......................................................................................................................................... 7 3. SUMMARY ............................................................................................................................................... 10 4. INTRODUCTION ........................................................................................................................................ 11 4.1. THE NERVOUS SYSTEM AND MYELIN SHEATH COMPOSITION ..................................................................................... 11 4.2. SCHWANN CELL ORIGIN AND LINEAGE ................................................................................................................. 12 4.3. THE FUNCTIONAL ROLE OF THE BASAL LAMINA ...................................................................................................... 14 4.3.1. Cytoskeleton in Schwann cells........................................................................................................... 15 4.4. SCHWANN CELL SIGNALING ............................................................................................................................... 16 4.4.1. The Neuregulin 1/ ErbB system ......................................................................................................... 17 4.4.2. Neurotrophins – TrkA, B, C / p75NTR system ...................................................................................... 18 4.4.3. The cAMP signaling pathway ............................................................................................................ 19 4.4.4. The PI3‐kinase/ Akt signaling pathway ............................................................................................. 21 4.4.5. The MAP‐kinase signal transduction pathway .................................................................................. 21 4.4.5.1. The ERK1/2 signaling cascade ........................................................................................................................ 22 4.4.5.2. The c‐Jun N‐terminal kinases (JNK) cascade .................................................................................................. 22 4.5. THE MYELIN AND LYMPHOCYTE PROTEIN MAL ...................................................................................................... 23 4.5.1. Characterization of MAL and its biochemical properties .................................................................. 23 4.5.2. Expression pattern of MAL in the nervous system ............................................................................ 23 4.5.3. Putative functional role of MAL ........................................................................................................ 24 4.5.4. Phenotype of MAL overexpression in the peripheral nervous system ............................................... 24 4.5.5. Septin 6 is an interaction partner of MAL ......................................................................................... 25 5. AIM OF THE WORK ................................................................................................................................... 26 6. MATERIAL AND METHODS ........................................................................................................................ 27 6.1. CELL CULTURE ................................................................................................................................................ 27 6.1.1. Primary Schwann cell cultures .......................................................................................................... 27 6.1.1.1. Primary mouse Schwann cell cultures ........................................................................................................... 27 6.1.1.2. Stimulation assays of mouse Schwann cell cultures ...................................................................................... 27 6.1.1.3. Primary rat Schwann cell cultures ................................................................................................................. 27 6.1.2. Standard cell cultures ........................................................................................................................ 29 6.2. IMMUNOHISTOCHEMISTRY ............................................................................................................................... 30 6.2.1. Immunohistochemistry on cell cultures ............................................................................................ 30 6.2.2. Immunohistochemistry on fresh frozen mouse tissue ....................................................................... 30 6.2.3. Antibodies ......................................................................................................................................... 30 6.3. EXPRESSION ANALYSIS...................................................................................................................................... 31 6.3.1. RNA isolation of Schwann cells ......................................................................................................... 31 6.3.2. RNA isolation of sciatic nerves .......................................................................................................... 31 6.3.3. Reverse transcription reaction .......................................................................................................... 31 6.3.4. Quantitative RT‐PCR analysis ............................................................................................................ 32 6.3.4.1. Quantitative RT‐PCR analysis ......................................................................................................................... 32 6.3.4.2. Primer pairs for quantitative RT‐PCR ............................................................................................................. 32 6.3.5. Whole genome expression profiling .................................................................................................. 33 6.4. GENERATION OF MCHERRY TAGGED MAL AND PMP22 CONSTRUCTS ...................................................................... 33 ‐3‐ Table of Content 6.4.1. Eliminating STOP sequence of the pMX‐mCherrySTOP vector ............................................................. 34 6.4.2. Cloning MAL into the pMX‐mCherry vector ...................................................................................... 34 6.4.3. Cloning PMP22 into the pMX‐mCherry vector .................................................................................. 34 6.4.4. Primer pairs for cloning the retroviral constructs ............................................................................. 36 6.4.5. Production of retroviral stocks in PhoenixEco packaging cell line ...................................................... 36 6.4.6. Retroviral infection ........................................................................................................................... 37 6.5. IN SITU HYBRIDIZATION ................................................................................................................................... 37 6.5.1. Cloning of the riboprobes .................................................................................................................. 37 6.5.2. DIG RNA labeling by in vitro transcription ........................................................................................ 39 6.5.3. In situ hybridization........................................................................................................................... 39 6.5.4. Buffer composition for in situ hybridization ...................................................................................... 40 7. RESULTS ................................................................................................................................................... 41 7.1. MAL‐DEPENDENT GENE REGULATION IN SCHWANN CELLS ...................................................................................... 41 7.1.1. Differential expression analysis on developing sciatic nerves ........................................................... 41 7.1.2. Establishment of primary mouse Schwann cell cultures ................................................................... 44 7.1.3. Investigation of Schwann cell differentiation in vitro ....................................................................... 44 7.1.3.1. Stimulation with forskolin led to Schwann cell differentiation ..................................................................... 45 7.1.3.2. MAL‐overexpressing Schwann cells manifest reduced P0 and p75NTR expression ........................................ 47 7.1.3.3. Most transcription
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