van den Hove, Lesch, Schmitt-Böhrer
The primary goal of A05 is to validate, characterise and rescue the functional impact of epigenetically regulated genes previously identified by genome-wide mRNA expression and DNA methylation profiling in our gene x environment (G x E) paradigms and to translate the findings to anxiety disorders investigated in project C02. These findings will directly address questions about the role of G x E interactions in the developmental programming of anxiety disorders. Via this systematic approach we will identify genes as either novel vulnerability or protective factors for anxiety disorders, which will serve as potential diagnostic and therapeutic targets.
In order to achieve this goal, we will pursue the following objectives:
- Validation of epigenetically regulated risk and protective genes identified in previously established G x E paradigms. We will validate candidate genes derived from the 5-Htt x prenatal stress (PS) paradigm targeting genes that foster resilience to anxiety disorders by techniques which quantify gene expression and differentiate CpG site-specific DNA methylation in a sex-dependent manner and focusing on various brain regions from which specific neuronal cells have been isolated.
- Investigation of epigenetically regulated risk and protective genes validated in Objective 1 in a novel Tph2-deficient mouse model of panic disorder. For this purpose, we will validate candidate genes derived from the 5-Htt x PS paradigm in the Tph2 x PS panic disorder model to elucidate the degree of developmental epigenetic programming of anxiety-related traits. The results are expected to identify pathways underlying inadequate adaptability versus protection.
- Causality assessment and intervention using an in vivo gene- and site-specific epigenetic editing strategy. Zinc finger (ZF) proteins and guide RNAs (CRISPR-Cas9 system) will be designed to target transcriptional activators or repressors to candidate mRNA loci, such as myelin basic protein (Mbp) identified in our 5-Htt x PS study. Making use of stereotactical injections of lentiviruses, specific genes encoding the epigenetic editors (e.g. DNMT3a, TET) will be delivered into the brain (e.g. hippocampus for Mbp) of adult stressed 5-Htt- or Tph2-deficient mice, in order to induce normalize the degree of methylation and expression of the gene of interest (e.g. Mbp) in local neurons. Fear- and anxiety-related behavioural consequences of this approach will be examined, while brain tissue will be assessed for e.g. morphological, neurogenic, epigenetic and transcriptomic changes.