Assessing the complex nature of behavior: Sequence-based and transcriptomic analyses in a mouse model of extremes in trait anxiety Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München vorgelegt von Ludwig Czibere München, 30. September 2008 Erstgutachter: Prof. Dr. Rainer Landgraf Zweitgutachter: Prof. Dr. Thomas Cremer Datum der mündlichen Prüfung: 06. Mai 2009 “Nothing in biology makes sense, except in the light of evolution.” Theodosius Grygorovych Dobzhansky, 1973 Meinen lieben Eltern für ihre stetige Unterstützung Drága szüleimnek állandó támogatásukért Abstract Abstract To unravel the molecular pylons of innate anxiety, a well established animal model has been characterized using transcriptome- and sequence-based analyses. The animal model – hyper (HAB) and hypo (LAB) anxious mice – has been created by selective inbreeding based on outbred CD1 mice using the extreme values the mice spent on the open arm of the elevated plus-maze, a test also used to screen drugs for anxiolytic or anxiogenic effects. These mice proved a robust phenotypic divergence, also for depression-like behavior and stress-axis reactivity. In a first assay, brain regions unambiguously involved in regulating anxiety-related behavior were screened for gene expression differences between HAB and LAB animals in a microarray experiment covering the whole genome. This led to the identification of thousands of differentially expressed transcripts. The highest significant results were further validated by quantitative PCR or other techniques focusing either on protein quantification or enzyme activity. Applying this strategy, differential regulation of 15 out of 28 transcripts could be validated: vasopressin, tachykinin 1, transmembrane protein 132D, RIKEN cDNA 2900019G14 gene, ectonucleotide pyrophosphatase/phosphodiesterase 5, cathepsin B, coronin 7, glyoxalase 1, pyruvate dehydrogenase beta, metallothionein 1, matrix metallopeptidase 15, zinc finger protein 672, syntaxin 3, solute carrier family 25 member 17 and ATP-binding cassette, sub-family A member 2. Additionally, analysis of cytochrome c oxidase activity resulted in the identification of differences in long-term activity between HAB and LAB mice in the amygdala and the hypothalamic paraventricular nucleus pointing to an important role of these brain regions in shaping the anxiety-related extremes in these mice. In a second genome-wide screening approach, 267 single nucleotide polymorphisms were identified to constantly differ between HAB and LAB animals (i.e. to carry the opposite homozygous genotype at these loci) and subsequently genotyped in 520 F2 mice, the offspring of reciprocally mated HABxLAB animals. These F2 mice have been previously phenotyped in a broad variety of behavioral tests and show – as descendants of intermediate heterozygotes for all polymorphic genomic loci between HAB and LAB mice – a free segregation of all alleles, thus allowing genotype-phenotype associations based on whole-genome analysis. Only focusing on the most significant findings, associations have been observed between anxiety-related behavior and loci on mouse chromosomes 5 and 11, between i Abstract depression-like behavior and chromosome 2 and between stress-axis reactivity and chromosome 3. The locus on chromosome 11 is marked by a polymorphism located in the 3’ untranslated region of zinc finger protein 672, a gene also markedly overexpressed in LAB mice and expressed at lower levels in HAB mice leading to a probable causal involvement in shaping the phenotype. Further associations on chromosome 5 include two functional polymorphisms in enolase phosphatase 1 that result in a different mobility of the enzyme in proteomic assays and with a polymorphism located in the transmembrane protein 132D gene. Furthermore, independently, an association of a polymorphism in this particular gene, together with the resulting gene expression differences has been observed in a group of panic disorder patients, highlighting this gene as a causal factor underlying anxiety-related behavior and disorders in both the HAB/LAB mouse model and human patients. The combination of expression profiling and confirmation by quantitative PCR, single nucleotide polymorphism analysis and F2 association studies, i.e. unbiased and hypothesis driven approaches were key to the identification and functional characterization of loci, genes and polymorphisms causally involved in shaping anxiety-related behavior. Thus, it provides an overview of some new promising targets for future pharmaceutic treatment and will contribute to a better understanding of the molecular processes that shape anxiety and thereby also animal and human behavior. ii Table of contents Abstract _________________________________________________ i Table of contents _________________________________________ iii Table of abbreviations and comments _______________________ vi 1. Introduction _____________________________________________ 1 1.1. The evolution of threat assessment systems _________________ 1 1.2. The evolution of neuroactive compounds ____________________ 2 1.3. Anxiety and depression disorders __________________________ 5 1.4. Assessing anxiety/depression in mice _______________________ 7 1.5. The hypothalamo-pituitary-adrenal (HPA) axis, gene expression and translation ___________________________________________ 9 1.6. The influence of genetic polymorphisms _____________________ 10 1.7. The HAB/LAB mouse model _______________________________ 11 1.7.1. Phenotypic characteristics of HAB/LAB mice _________________ 12 1.7.2. Molecular characteristics of HAB/LAB mice __________________ 13 1.7.3. The F2 panel ____________________________________________ 13 2. Animals, materials and methods ___________________________ 15 2.1. The HAB phenotype reversal by selective breeding ___________ 15 2.2. Identification of candidate genes ___________________________ 16 2.2.1. Animals and behavioral tests for the gene expression studies __ 16 2.2.2. Gene expression profiling on the MPI24K-platform ____________ 16 2.2.2.1. Tissue dissection _________________________________________ 16 2.2.2.2. Total RNA isolation and amplification _______________________ 18 2.2.2.3. Array hybridization and quantification _______________________ 19 2.2.2.4. Statistical analysis ________________________________________ 20 2.2.3. Gene expression profiling on the Illumina-platform ____________ 20 2.2.3.1. Tissue laser-microdissection _______________________________ 20 2.2.3.2. Total RNA isolation and amplification _______________________ 21 2.2.3.3. Array hybridization and quantification _______________________ 21 2.2.3.4. Statistical analysis _______________________________________ 22 2.2.4. Analysis of candidate genes by quantitative PCR _____________ 22 2.2.5. Assessment of candidate gene functions ____________________ 26 2.2.5.1. Glyoxalase I (GLO1) and metabolic stimulation _______________ 27 2.2.5.1.1. Metabolism and blood plasma parameters ___________________ 27 2.2.5.1.2. Behavioral manipulation via metabolism _____________________ 27 2.2.5.1.3. Western Blot analysis of glyoxalase 1 (GLO1) ________________ 28 2.2.5.2. Tachykinin 1 ( Tac1) -encoded peptide quantification ___________ 29 iii Table of contents 2.2.5.3. Cytochrome c oxidase (COX) activity ________________________ 32 2.3. Identification of polymorphisms _____________________________ 32 2.3.1. Animals and behavioral tests _______________________________ 33 2.3.2. DNA isolation ____________________________________________ 33 2.3.3. Prescreening ____________________________________________ 34 2.3.4. Screening of the F2 panel and CD1 mice ____________________ 35 2.3.5. Sequencing of candidate genes ____________________________ 43 2.3.5.1. Cloning of fragments _____________________________________ 43 2.3.5.2. Cycle sequencing ________________________________________ 44 2.3.5.3. Vasopressin ( Avp ) ________________________________________ 45 2.3.5.4. Corticotropin-releasing hormone ( Crh ) ______________________ 46 2.3.5.5. Tachykinin 1 ( Tac1 ) ______________________________________ 47 2.3.5.6. Cathepsin B ( Ctsb ) _______________________________________ 49 2.3.5.7. Metallothionein 1 ( Mt1 ) ____________________________________ 51 2.3.5.8. Transmembrane protein 132D ( Tmem132d ) __________________ 52 2.3.6. Assessing the effects of polymorphisms _____________________ 53 2.3.7. Allele-specific transcription of vasopressin (Avp ) _____________ 54 2.4. Association and linkage of SNPs with behavioral parameters ___ 54 2.5. Statistical data analysis ___________________________________ 54 3. Results _________________________________________________ 56 3.1. The HAB phenotype reversal by selective breeding ___________ 56 3.2. Identification of candidate genes ___________________________ 56 3.2.1. Animals and behavioral tests ______________________________ 56 3.2.2. Gene expression profiling on the MPI24K-platform ___________ 59 3.2.2.1. Tissue dissection _________________________________________ 59 3.2.2.2. RNA amplification ________________________________________ 59 3.2.2.3. Differential expression data ________________________________ 60 3.2.3. Gene expression profiling on the Illumina-platform ____________ 61 3.2.3.1. Tissue laser-microdissection _______________________________ 61 3.2.3.2. Total RNA isolation and amplification _______________________ 62 3.2.3.3. Differential expression data ________________________________ 64 3.2.4. Validation of results by quantitative PCR ____________________ 65 3.2.5. Effects