Dissecting nucleolar functions in dopaminergic neurons and Parkinson's disease by targeted inactivation of the transcription-initiation factor IA (TIF-IA) [Elektronische Ressource] / presented by Claus Rieker

Claus Rieker

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2008
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation





submitted to the

Combined Faculties for the Natural Sciences
and for Mathematics
Ruperto-Carola University of Heidelberg, Germany


for the degree of
Doctor of Natural Sciences






presented by
Diplom-Biologist Claus Rieker

born in Geislingen / Steige





oral examination:





Dissecting nucleolar functions in dopaminergic
neurons and Parkinson’s disease
by targeted inactivation of the
transcription-initiation factor IA (TIF-IA)



























Referees:

Prof. Dr. Günther Schütz
Prof. Dr. Hilmar Bading


Table of contents



Table of contents




1. SUMMARY...............................................................................1
1.1 Zusammenfassung ............................................................................2



2. INTRODUCTION......................................................................3
2.1 Neural development...........................................................................3
2.2 Dopaminergic neurons.......................................................................3
2.3 Dopamine and associated cellular pathologies..................................5
2.4 Parkinson’s disease7
2.5 Molecular mechanism of PD..............................................................8
2.6 The nucleolus: site of ribosomal RNA synthesis..............................10
2.7 RNA polymerase I............................................................................10
2.8 Transcription initiation factor I A (TIF-IA) .........................................11
2.9 Aim of this thesis .............................................................................12









I Table of contents


3. RESULTS: .............................................................................14
3.1 MPTP affects nucleolar integrity and function..................................14
3.2 Progressive loss of dopaminergic neurons by genetic ablation of ......
TIF-IA...............................................................................................16
DATCre3.3 Loss of striatal dopamine in TIF-IA mutants............................19
DATCre3.4 Rescue of TIF-IA mutant mice by L-DOPA treatment. ............21
3.5 Reduced mitochondrial activity upon nucleolar impairment.............22
3.6 Generation of a mouse line expressing an inducible Cre-
recombinase exclusively in dopaminergic neurons..........................25
T23.7 Faithfull expression of the CreER fusion protein in dopaminergic
neurons............................................................................................26
3.8 Targeted tamoxifen-induced recombination in the adult SN/VTA ....28
DATCreERT23.9 Generation of the inducible TIF-IA mice............................30
3.10 Progressive loss of dopaminergic neurons is occasionally
accompanied with Lewy body formation..........................................34
3.11 Linking nucleolar activity and mitochondrial integrity .......................36
3.12 Increased nucleolar disruption in dopaminergic neurons of PD
patients............................................................................................39


4. DISCUSSION.........................................................................42
4.1 The nucleolus acts as a stress sensor in dopaminergic neurons.....42
4.2 Generation of mice with artificial impairment of the nucleolus .........43
II Table of contents
4.3 Treatment with L-DOPA restores normal locomotor performance in
transgenic mice ...............................................................................45
4.4 Impairment of nucleolar function affects mitochondrial activity and
induces oxidative stress...................................................................45
4.5 Generation of mice expressing an inducible Cre recombinase
specifically in dopaminergic neurons ...............................................46
4.6 Inducible ablation of TIF-IA leads to nucleolar disruption ................47
4.7 Increased expression of Lewy body components ............................48
4.8 Linking nucleolar activity and mitochondrial integrity .......................48
4.9 Increased nucleolar disruption in dopaminergic neurons of PD
patients............................................................................................52


5. MATERIALS AND METHODS...............................................53
5.1 Materials..........................................................................................53
5.1.1 Chemicals and enzymes ............................................................................53
5.1.2 Standard solutions......................................................................................54
5.1.3 Plasmids.....................................................................................................55

5.2 Plasmid constructs and probes........................................................55
5.3 Standard techniques in molecular biology .......................................55
5.3.1 Cloning into plasmid vectors and sequencing ............................................55
5.3.2 Isolation of DNA .........................................................................................56
5.3.2.1 Miniprep of plasmid from bacteria DNA from bacteria................................56
5.3.2.2 Miniprep of BAC DNA.................................................................................56
5.2.2.3 Midiprep of BAC DNA57
III Table of contents
5.4 Generation of transgenic mice.........................................................57
5.4.1 Modification of a BAC by homologous recombination in bacteria ..............57
5.4.2 Preparation of competent bacteria for transformation with the BAC ..........58
5.4.3 Re-transformation of the BAC ....................................................................58
5.4.4 homologous recombination............59
5.4.5 ET recombination and removal of the ampicillin resistance cassette.........59
5.4.6 Preparative and analytical pulse-field gel electrophoresis..........................60
5.4.7 DNA microinjection in mouse oocytes........................................................61

5.5 Genotyping ......................................................................................63
5.5.1 Isolation of genomic mouse DNA by NID lysis buffer.................................63
5.5.2 Polymerase Chain Reaction (PCR)............................................................63
5.5.3 Agarose gel electrophoresis.......................................................................65

5.6 RNA analysis – in situ hybridization.................................................66
5.6.1 Synthesis of digoxigenin (DIG) -labeled RNA-probes ................................66
5.6.2 Synthesis of riboprobes by using PCR products as template.....................66
5.6.3 Denaturating RNA gel ................................................................................67
5.6.4 RNA dot blot ...............................................................................................68
5.6.5 In situ hybridization on paraffin sections.....................................................68

5.7 Protein analysis ...............................................................................71
5.7.1 Preparation of vibratome sections..............................................................71
5.7.2 Preparation of embryos for paraffin sections..............................................71
5.7.3 Preparation of tissue for cryosections ........................................................72

5.8 Immunohistochemistry.....................................................................72
5.8.1 Immunohistochemistry using paraffin sections...........................................72
IV Table of contents
5.8.2 Immunohistochemistry using vibratome sections.......................................73
5.8.3 Immunoflorescence using paraffin sections ...............................................74
5.8.4 COX staining using cryosections................................................................75
5.8.5 Hematoxylen/eosin staining of paraffin sections ........................................75
5.8.6 ß-galactosidase staining.............................................................................76
5.8.7 HPLC-Electrochemical Detection ...............................................................76
5.8.8 Quantitative analysis of dopaminergic neurons..........................................77

5.9 Mouse work .....................................................................................78
5.9.1 C57/Bl6.......................................................................................................78
5.9.2 Tamoxifen treat