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Examining New Interactors of Notch/Rbpjκ Signalling in Adult

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Examining New Interactors of Notch/Rbpjκ Signalling in Adult TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Entwicklungsgenetik Examining new interactors of Notch/RBPJ signalling in adult neural stem cells Dissertation von Tobias Johannes Schwarz Helmholtz Zentrum München Institut für Entwicklungsgenetik TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl Entwicklungsgenetik Examining new interactors of Notch/RBPJ signalling in adult neural stem cells Tobias Johannes Schwarz Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzende: Univ.-Prof. A. Schnieke, Ph.D. Prüfer der Dissertation: 1. Univ.-Prof. Dr. W. Wurst 2. Univ.-Prof. Dr. C. Lie, (Friedrich-Alexander Universität Erlangen-Nürnberg) Die Dissertation wurde am 20. 03. 2012 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 25. 07. 2012 angenommen. Table of contents 1 Zusammenfassung......................................................................................... 4 2 Summary .......................................................................................................... 6 3 Introduction ...................................................................................................... 7 3.1 Adult neurogenesis and neural stem cells ..................................................... 7 3.2 Signal integration by adult neural stem cells ............................................... 10 3.3 Notch signalling pathway............................................................................. 12 3.4 Forkhead box transcription factors O........................................................... 17 3.5 TDP-43 ........................................................................................................ 20 3.6 Nuclear Factor 1 transcription factors.......................................................... 22 3.7 Objectives of this study................................................................................ 25 4 Results ............................................................................................................ 26 4.1 Notch signalling pathway and FOXO........................................................... 26 4.1.1 RBPJ interacts with FOXO proteins .................................................... 26 4.1.2 FOXO transcription factors modulate expression of the Notch target gene Sox2............................................................................................. 28 4.2 New regulators of adult neural stem cell maintenance ................................ 32 4.2.1 Cross-linking of RBPJ antibodies to Protein G Sepharose beads....... 32 4.2.2 Identification of proteins co-purified from RBPJ immunoprecipitations in adult neural stem- and progenitor cells ............................................. 33 4.2.3 Validation of proteins co-purified with RBPJ and identified by MS/MS 52 4.2.4 Confirmation of RBPJ interacting proteins .......................................... 54 4.3 Notch signalling pathway and TDP-43......................................................... 56 4.3.1 RBPJ interacts with TDP-43................................................................ 56 4.3.2 TDP-43 enhances Notch signalling mediated Hes1 promoter activity... 59 4.3.3 A TDP-43 (A315T) mutant associated with ALS and FTLD-U induces lower activation of Hes1 promoter......................................................... 65 4.3.4 TDP-43 promotes expression of endogenous Hes1 ............................. 66 4.4 Notch signalling pathway and NFIA............................................................. 68 4.4.1 RBPJ interacts with NFIA.................................................................... 68 4.4.2 NFIA inhibits Notch signalling-mediated Hes1 and Hes5 promoter activation............................................................................................... 71 1 4.4.3 RBPJ conditional knockout mice display increased NFIA expression in the dentate gyrus .............................................................................. 74 5 Discussion ...................................................................................................... 77 5.1 Interaction of the Notch signalling pathway with FOXOs ............................. 77 5.2 Core transcriptional network in stem cell maintenance................................ 80 5.3 Interaction of the Notch signalling pathway with TDP-43............................. 83 5.4 Interaction of the Notch signalling pathway with an ALS and FTLD-U associated TDP-43 mutant .......................................................................... 84 5.5 Interaction of the Notch signalling pathway and NFIA ................................. 85 5.6 Cross-talk of Notch signalling pathway with other signalling pathways ....... 88 5.7 Is Notch signalling impaired in neurodegenerative disease?....................... 90 6 Material and Methods .................................................................................. 92 6.1 Material........................................................................................................ 92 6.1.1 Histological solutions ............................................................................ 92 6.1.2 Cell culture media and solutions ........................................................... 93 6.1.3 Molecular biology solutions................................................................... 94 6.1.4 Protein isolation solutions ..................................................................... 95 6.1.5 Western blot analysis solutions............................................................. 96 6.1.6 Mass spectrometry analysis solutions................................................... 97 6.1.7 Commercial kits .................................................................................... 98 6.1.8 Primary antibodies ................................................................................ 98 6.1.9 Secondary antibodies ........................................................................... 99 6.1.10 Plasmids ............................................................................................... 99 6.1.11 Primers ................................................................................................100 6.1.12 Software...............................................................................................100 6.2 Methods......................................................................................................101 6.2.1 Immunohistochemical methods............................................................101 6.2.1.1 Animals..........................................................................................101 6.2.1.2 Tissue processing .........................................................................101 6.2.1.3 Immunohistochemistry...................................................................101 6.2.2 Cell Culture Methods ...........................................................................102 6.2.2.1 HEK293T.......................................................................................102 6.2.2.2 Mouse adult neural stem cells (neurospheres)..............................102 6.2.2.3 Embryonic stem cells ....................................................................103 2 6.2.2.4 Calcium chloride mediated transfection.........................................103 6.2.2.5 Luciferase reporter assay..............................................................104 6.2.3 Molecular Biological Methods ..............................................................104 6.2.3.1 Prediction of putative transcription factor binding sites..................104 6.2.3.2 Statistical analysis .........................................................................104 6.2.3.3 Transformation ..............................................................................104 6.2.3.4 Isolation and purification of DNA ...................................................104 6.2.3.5 Isolation of RNA ............................................................................105 6.2.3.6 cDNA synthesis and DNA sequencing ..........................................105 6.2.3.7 Mutagenesis of transcription factor binding sites on DNA .............105 6.2.3.8 Cloning procedures .......................................................................105 6.2.4 Protein Biochemical Methods...............................................................106 6.2.4.1 Protein isolation from free floating neurospheres ..........................106 6.2.4.2 Protein isolation from monolayer cell cultures ...............................106 6.2.4.3 Immunoprecipitation ......................................................................106 6.2.4.4 SDS-polyacrylamid gel electrophoresis (SDS PAGE) ...................107 6.2.4.5 Silver staining of protein gels.........................................................108 6.2.4.6 Coomassie staining of protein gels................................................108 6.2.4.7 Western blot ..................................................................................108 6.2.4.8 Stripping of antibodies from PVDF membrane ..............................109 6.2.4.9 DMP Cross-linking of antibodies to Protein G Sepharose .............109 6.2.4.10 Tandem Mass spectrometry..........................................................110
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