Assignment of functional impact on genetic data in two mouse models of affective disorders Dissertation Faculty of Biology Ludwig Maximilian University, Munich Regina Widner-Andrä December 14th 2011 Erstgutachter Prof. Dr. Rainer Landgraf Zweitgutachter Prof. Dr. Gisela Gruppe Tag der Abgabe: 14. Dezember 2011 Tag der mündlichen Prüfung: 8. Mai 2012 “Nothing in life is to be feared. It is only to be understood.” Marie Curie This dissertation is dedicated to my father, Gottfried Widner († February 2001). Table of contents Table of contents Table of contents ............................................................................................................. 1 1 Abstract .................................................................................................................... 7 2 Introduction .............................................................................................................. 9 2.1 The current concept of depression scale disorders ............................................ 9 2.2 Comorbidity of depression ............................................................................... 10 2.3 Generalized anxiety disorder ........................................................................... 11 2.4 Stress and reactivity of the hypothalamic-pituitary-adrenal axis ....................... 11 2.5 Genetics of depression and associated diseases ............................................ 13 2.6 Genome-wide association studies ................................................................... 14 2.7 Pharmacological therapy options for depression and associated diseases ...... 15 2.8 Psychopharmacotherapy of the future – a step towards personalized medicine? ................................................................................... 16 2.9 The use of animal models to unravel neurobiological underpinnings of depression and associated diseases ........................................................... 17 2.9.1 The HAB/LAB mouse model reflecting extremes in trait anxiety .................. 18 2.9.2 The stress reactivity mouse model .............................................................. 20 2.10 Technical progress in methods applied for the generation of transcriptome- and sequence-based genomic data ......................................... 22 2.11 Aim and scope of this study ............................................................................. 22 3 Animals, materials, and methods ............................................................................ 25 3.1 Experimental animals ...................................................................................... 25 3.1.1 Inbred mouse models .................................................................................. 25 3.1.2 Heterozygous Cofilin-1 knockout mice ........................................................ 25 3.1.3 C57BL/6 wild-type mice............................................................................... 26 3.1.4 CD-1 mice ................................................................................................... 26 3.2 Behavioral experiments ................................................................................... 26 3.2.1 Assessment of anxiety-related behavior ...................................................... 26 3.2.1.1 Open field test .................................................................................... 26 3.2.1.2 Elevated plus-maze test ..................................................................... 27 3.2.1.3 Dark-light box test .............................................................................. 28 3.2.2 Assessment of depression-like behavior ..................................................... 29 3.2.2.1 Tail suspension test ........................................................................... 29 3.2.2.2 Forced swim test ................................................................................ 30 3.2.3 Assessment of stress reactivity ................................................................... 31 3.2.4 Behavioral test batteries performed in this study ......................................... 32 1 Table of contents 3.2.4.1 Behavioral test battery conducted for phenotypic characterization of Cfl-1+/- vs. WT mice .............................................. 32 3.2.4.2 Behavioral test battery conducted for phenotypic characterization of an outbred population of CD-1 mice ............................................... 33 3.3 Sacrifice of animals ......................................................................................... 33 3.4 Dissection of embryos ..................................................................................... 34 3.5 Collection of brain and hippocampal tissue from embryonic and adult mice .... 34 3.6 Nucleic acid based methods ............................................................................ 34 3.6.1 RNA based methods ................................................................................... 34 3.6.1.1 Extraction of total RNA from hippocampal tissue ............................... 34 3.6.1.2 Control measures of RNA quality and determination of RNA concentration ............................................................................. 35 3.6.1.3 Reverse transcription of RNA into cDNA ............................................ 35 3.6.1.4 RNA amplification for array analysis .................................................. 36 3.6.1.5 Serial analysis of gene expression ..................................................... 36 3.6.2 DNA based methods ................................................................................... 37 3.6.2.1 DNA extraction from mouse tissue ..................................................... 37 3.6.2.2 Polymerase-chain reactions and gel electrophoresis ......................... 38 3.6.2.3 Gel electrophoresis ............................................................................ 39 3.6.2.4 Precipitation of cDNA ......................................................................... 39 3.6.2.5 Quantitative real-time PCR ................................................................ 40 3.6.2.6 Table of oligonucleotides ................................................................... 41 3.7 Protein based methods.................................................................................... 43 3.7.1 Mitochondrial proteomics ............................................................................ 43 3.7.1.1 Preparation of mitochondrial protein lysates ...................................... 43 3.7.1.2 Bradford assay .................................................................................. 44 3.7.1.3 Two-dimensional gel electrophoresis ................................................. 44 3.7.2 Cytochrome c immunoassay ....................................................................... 46 3.7.3 Cytochrome c oxidase staining ................................................................... 47 3.7.4 Quantification of plasma corticosterone via radioimmunoassays................. 48 3.8 Statistical and bioinformatical methods ............................................................ 49 3.8.1 Statistical analyses of microarray experiment ............................................. 49 3.8.1.1 Univariate analysis ............................................................................. 49 3.8.1.2 Multivariate analysis .......................................................................... 49 3.8.2 Statistical analyses of the SAGE experiment .............................................. 50 3.8.3 Cluster analyses of differentially regulated genes ....................................... 51 3.8.3.1 Database for annotation, visualization, and integrated discovery ....... 51 2 Table of contents 3.8.3.2 Gene ontology analyses ..................................................................... 51 3.8.4 High-density screening for single-nucleotide polymorphisms and detection of segmental copy number variants by means of the Jax Mouse Diversity Genotyping array ........................................................ 51 3.8.4.1 Detection of segmental copy number variation based on data from Jax Mouse Diversity Genotyping array ....................................... 52 3.8.5 Principle component analyses of phenotypic features ................................. 52 3.8.6 Calculation of association via WG-Permer .................................................. 53 3.8.7 Generation of CD-1 strain-specific haplotype map ...................................... 53 3.8.8 Statistical data analyses, graphical illustration, and common bioinformatical processes ............................................................................ 53 4 Results ................................................................................................................... 55 4.1 Gene expressional profiling of the SR mouse model........................................ 55 4.1.1 Differentially expressed genes according to univariate analysis of the microarray experiment ....................................................................... 55 4.1.2 Genes detected as differentially expressed in the SAGE experiment .......... 55 4.1.3 Overlapping pool of genes detected as differentially expressed in both gene expression profiling approaches ................................................. 55 4.1.4 Univariate vs. multivariate analysis of microarray data ................................ 56 4.1.5 Cluster