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Regulation of Gene Expression in Specific Mouse Brain Cells During Neurodegenerative Prion Disease

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Regulation of Gene Expression in Specific Mouse Brain Cells During Neurodegenerative Prion Disease Regulation of gene expression in specific mouse brain cells during neurodegenerative prion disease Dissertation zur Erlangung des Doktorgrades (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn vorgelegt von Melvin Schleif aus Dormagen Bonn, September 2017 Angefertigt mit Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Rheinischen Friedrich-Wilhelms-Universität Bonn 1. Gutachter Prof. Dr. Ina Vorberg Gruppenleiterin Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn 2. Gutachter Prof. Dr. Walter Witke Institutsdirektor und Gruppenleiter Institut für Genetik, Universität Bonn Tag der Promotion: 24.04.2018 Erscheinungsjahr: 2018 Table of contents I. Table of contents I. Table of contents .................................................................................................... 1 II. List of abbreviations .............................................................................................. 4 II.A Used abbreviations .......................................................................................... 4 II.B Primary nucleobases ....................................................................................... 8 II.C Proteinogenic amino acids .............................................................................. 8 1. Introduction ............................................................................................................ 9 1.1 Neurodegenerative diseases ........................................................................... 9 1.2 Selective vulnerability .................................................................................... 10 1.3 Transmissible spongiform encephalopathies ................................................. 12 1.3.1 Prions ...................................................................................................... 13 1.3.2 Prion protein gene ................................................................................... 14 1.3.3 Prion protein ............................................................................................ 16 1.3.4 Prion replication ...................................................................................... 18 1.3.5 Prion strains, prion infectivity, neurotoxicity and species barrier ............. 20 1.3.6 Prion protein function .............................................................................. 23 1.3.7 Prion disease therapeutics ...................................................................... 23 1.3.8 Scrapie .................................................................................................... 24 1.4 RiboTag ......................................................................................................... 25 1.5 Next generation sequencing .......................................................................... 27 2. Aim of study ......................................................................................................... 29 3. Material and methods .......................................................................................... 31 3.1 Biosafety and animal experimentation ........................................................... 31 3.2 Mouse lines, holding and breeding ................................................................ 31 3.3 Genotyping .................................................................................................... 32 3.4 Video-based behavior-recognition ................................................................. 34 3.5 Intracranial injections and tissue dissection ................................................... 34 3.6 Electroencephalogram ................................................................................... 35 3.7 Immunoprecipitation ...................................................................................... 37 3.8 RNA-sequencing library preparation and RNA-sequencing ........................... 38 3.9 RNA-sequencing data analysis ...................................................................... 39 3.10 Pathway and gene ontology analysis .......................................................... 41 3.11 Immunohistochemistry ................................................................................. 41 3.12 Polyacrylamide gel electrophoresis and western blot .................................. 43 1 Table of contents 3.13 Dot blot ........................................................................................................ 45 3.14 Reverse transcription and real-time quantitative PCR ................................. 46 3.15 Chemicals and consumable goods .............................................................. 48 3.16 Software and web applications .................................................................... 48 4. Results ................................................................................................................ 50 4.1 Selection of mouse genetic background ........................................................ 50 4.2 Selection of disease time points .................................................................... 51 4.3 Experimental setup ........................................................................................ 53 4.3.1 Improvement of RiboTag immunoprecipitation ........................................ 53 4.3.2 RNA-sequencing sample generation....................................................... 55 4.3.3 RNA-sequencing quality control .............................................................. 62 4.4 Neuropathological changes ........................................................................... 67 4.5 RNA-sequencing data analysis methods ....................................................... 69 4.6 RNA-sequencing analysis based on unique exon reads ............................... 70 4.6.1 Cell type specificity .................................................................................. 70 4.6.2 Comparison of cell-type-specific gene expression regulation ................. 72 4.6.3 Gene expression regulation in glutamatergic neurons ............................ 80 4.6.4 Gene expression regulation in GABAergic neurons and subtypes ......... 83 4.6.5 Gene expression regulation in astrocytes ............................................... 83 4.6.6 Summary of unique exon read data analysis .......................................... 88 4.7 RNA-sequencing analysis based on total gene reads ................................... 89 4.7.1 Cell type specificity .................................................................................. 89 4.7.2 Comparison of cell-type-specific gene expression regulation ................. 91 4.7.3 Gene expression regulation in glutamatergic neurons ............................ 92 4.7.4 Gene expression regulation in GABAergic neurons and subtypes ......... 94 4.7.5 Gene expression regulation in astrocytes ............................................... 95 4.7.6 Summary of total gene read data analysis .............................................. 96 5. Discussion ........................................................................................................... 97 5.1 Selection of a disease model ......................................................................... 97 5.1.1 RML as a model of neurodegenerative prion disease ............................. 97 5.1.2 Selection of S4 genetic background ........................................................ 98 5.1.3 Selection of 10 and 18 wpi disease time points ...................................... 98 5.2 Neuropathological changes ........................................................................... 99 5.3 Quality of RNA samples and RNA-sequencing run ..................................... 100 5.4 Comparison of the two RNA-sequencing data analyses .............................. 101 5.5 Cell-type specificity ...................................................................................... 103 2 Table of contents 5.6 Gene expression regulation ......................................................................... 104 5.6.1 Gene expression regulation in glutamatergic neurons .......................... 106 5.6.2 Gene expression regulation in GABAergic neurons and subtypes ....... 108 5.6.3 Gene expression regulation in astrocytes ............................................. 109 5.7 Summary findings of RML infection ............................................................. 110 5.8 Outlook ........................................................................................................ 111 6. Summary ........................................................................................................... 113 7. References ........................................................................................................ 115 III. Acknowledgment / Danksagung ....................................................................... 130 3 List of abbreviations II. List of abbreviations II.A Used abbreviations °C Degree Celsius µ Micro 2logFC Log2 FoldChange 3’ Three prime end of nucleic acid 5’ Five prime end of nucleic acid A Ampere ADP Adenosine diphosphate AG Research group AP Anterior – posterior Astro Astrocytes ATP Adenosine triphosphate B6 C57Bl/6N inbred mouse strain bp Base pair BP Biological process BSE Bovine spongiform encephalopathy, “mad cow” disease CC Cellular compartment cDNA Complementary DNA CHO Carbohydrate (N-linked glycosylation site) CJD Creutzfeldt-Jakob
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