An integrated epigenomic/transcriptomic approach to elucidate glucocorticoid-regulated gene networks in stress-related cognitive behaviour

Stress affects the lives of both humans and animals in our society. Successful coping with such stressful events involves adaptive and cognitive processes in the brain that make the individual more resilient to similar stressors in the future. Currently, however, we do not fully understand how the healthy brain generates physiological and behavioural responses to stressful events and adapts in the long-term to such events.

Stressful events result in the secretion of 'stress hormones' or glucocorticoid (GC) hormones from the adrenal glands into the blood stream. These hormones act in the brain to coordinate physiological and behavioural responses to stress through binding to two different GC hormone-binding 'receptors'. These receptors, called MRs and GRs, are protein molecules located in nerve cells. As a result of a stressful challenge, GC hormone is secreted and binds to these receptors. The hormone-receptor complex then binds to certain genes within the DNA at specific docking sequences (so-called GREs) and regulate the expression of those genes. These genes are thought to be important to change the function of nerve cells in order to respond and adapt properly to the challenge.

Presently, there is only very limited information about the genes whose activity is altered due to MR or GR binding. We aim to obtain insight into the genes that are regulated by MRs and/or GRs and play a critical role in learning to cope with an adverse, stressful situation. We will use a behavioural animal model called the Morris water maze (MWM). This is a circular pool (diameter 1.8 meter) containing water from which a rat can escape by finding a small platform hidden underneath the water surface. Using signs ('spatial cues') on the walls around the pool, the rat learns quickly to find the platform. When the rat is put in the pool, GCs are secreted because the situation is stressful for the animals. These hormones are however extremely important as they act via MRs and GRs in the hippocampus where they stimulate learning of the platform location. The hippocampus is a brain region critical for spatial learning. We aim to reveal the identity of the MR- and GR-regulated genes by combination of two methods: 1. With chromatin immuno-precipitation (ChIP) and next-generation sequencing we will determine in which genes in the hippocampus MRs and GRs are binding to GREs throughout the entire rat genome (>20,000 genes) at different stages of MWM training compared to the undisturbed 'baseline' condition. We include a so-called swim control (SC) group consisting of rats placed in the pool without a platform for the same time as the MWM-trained (i.e. pool with platform) animals. Thus, these (SC) rats will experience the stress of being in the pool but not learn to find a platform. Therefore, including the SC group will help to differentiate between genes bound by MRs and/or GRs involved in the effects of stress and those involved in spatial learning to find the platform. As it is presently still unclear whether binding of MRs and GRs within genes indeed changes (mRNA) expression of these genes, we will apply a second technique: 2. Using RNA sequencing we will assess changes in mRNA concentrations across the entire hippocampal genome of MWM-trained, SC and baseline rats. Computational comparison (bioinformatics) of the two data sets will allow us to determine the genes whose activity is altered as a result of MR and/or GR binding specifically as a result of spatial learning. Subsequently, experiments will be conducted in which MRs, GRs or specific genes will be inhibited and effects on MR/GR binding, gene expression and MWM performance will be studied to obtain insight into the specific roles of these receptors and selected high-interest genes in spatial learning.

These studies will increase our understanding about how GCs secreted after stress help to cope actively with such a challenge and to be better prepared if a similar event would reoccur.

We aim to identify the genes that are regulated by mineralocorticoid receptors (MRs) and/or glucocorticoid receptors (GRs) and play a critical role in learning to proactively cope with an adverse, stressful situation. We will use the Morris water maze (MWM) to elucidate the hippocampal glucocorticoid (GC)-target genes critically required for successful coping (i.e. finding the hidden platform) as compared with those involved in non-spatial learning coping processes (swim controls (SCs)). We hypothesise that learning to escape from the MWM involves GC action via MRs and GRs interacting with GREs in a subset of GC-target genes in the hippocampus. Accordingly, we postulate that blocking MRs and/or GRs results in abrogated expression of these genes and inhibition of expression of these genes will impair MWM learning. To test our hypotheses we will conduct MR and GR ChIP on hippocampal chromatin of MWM-trained rats, and SC and baseline (BL) controls followed by next-generation sequencing (seq) to elucidate MR and GR binding to GC-responsive elements (GREs) throughout the entire genome. This ChIP-seq study will be complemented by an RNA-seq study in which we interrogate the entire hippocampal transcriptome regarding differences in mRNA levels between MWM-trained rats, and SC and BL controls. In addition, we will use these high-throughput methods to examine changes in MR/GR binding and mRNA expression after moving the platform and rats having to re-learn to escape. We will apply bioinformatics analyses on these ChIP- and RNA-seq data to identify MWM (re-)learning-specific genes responding to MR and/or GR binding. Pharmacological blockade of MRs and/or GRs and shRNA-mediated mRNA knock-down will be used to confirm the identity of those GC-dependent genes specifically involved in MWM learning and re-learning. This research will provide unique insight into the GC-regulated gene network involved in healthy pro-active coping with stressful challenges.

Who will benefit from this research?

There are a number of beneficiaries for whom this research could be helpful in the longer term:

1. Owners of companion and farmed animals with stress-related behavioural disturbances
2. Patients suffering from stress-related disorders
3. Family and friends of such patients
4. The economy
5. The government and the National Health Service
6. Academia


How will they benefit from this research?

1. Owners of companion and farmed animals with stress-related behavioural disturbances. Stress is a common problem for companion and farmed animals. It can lead to behavioural (e.g. stereotypy, aggression), reproductive (e.g. infertility) and other disturbances. Our research will help to improve treatment of such animals which will benefit their health and wellbeing. Owners of companion animals will have a much more contented pet and farmers will have less economic loss. From a different perspective, our research will increase the awareness that stress has indeed a long-term impact on the animals' behaviour. Since (bad) experiences of the animals appear to become hardwired into their brain it may take substantial efforts to undo these changes and obtain a healthy animal again. This should motivate pet owners and farmers to treat their animals properly. Our research may also help to improve legislation.

2. Patients suffering from stress-related disorders. Clearly, at present there is no satisfactory treatment for stress-related disorders such as psychosomatic disturbances (e.g. low-back pain, gastro-intestinal complaints) and psychiatric diseases (anxiety and major depressive disorders). The main reason for this situation is that the underlying neurobiology of these disorders is still unknown. Our research has the potential to lead to the development of new drugs and other therapies for the treatment of stress-related disorders in the future. It should be noted that these disorders are extremely disruptive for the patient's life in personal terms (misery, suicide), social terms (divorce) and economic terms (job loss, poverty).

3. Family and friends of such patients. As mentioned, stress-related disorders can be very disruptive for someone's social life often leading to divorce and social isolation which is devastating for the partners, children and friends of the patient. Thus, the social environment of the patient would benefit greatly from a better treatment of the patient.

4. The economy. The economy in general would benefit because patients suffering from stress-related disorders are often unable to work. If skilled people drop out of the work force it can have a major negative impact on the management, development and productivity of companies. Research has shown that stress leads to the loss of over 15 million work days per year and many billions of pound sterling in economic damages. A better treatment would doubtless be beneficial for the economy. The pharmaceutical industry would also benefit as this research would spark new avenues in drug development.

5. The government and the National Health Service (NHS). It is logical that the social, economic and health problems of such patients are a great burden for the government and the NHS. Clearly, an improved treatment of these patients would alleviate this burden significantly. Better insight into the effects of stress on animals could be beneficial to improve legislation for a better handling and management of our companion and farmed animals.

6. Academia. International academia in the fields of molecular, cellular and behavioural neuroscience and preclinical and clinical psychiatry would greatly benefit from the scientific progress made by this research. This is underlined by the fact that our research output has been highly cited in the scientific literature (see CV Reul). Therefore, it is anticipated that our results will have a major impact on academia (See also 'Academic Beneficiaries').