Epigenetic regulation of stress-induced glucocorticoid action in the dentate gyrus and its behavioural implications

Stress affects the lives of both humans and animals in our society. Psychological stress, like marital problems and bullying, is very debilitating for mental health and wellbeing in humans. Our farmed and companion animals can also suffer from psychological stress such as overcrowding, long-distance transportation and abuse. 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. Some events, certainly when uncontrollable and repeated, can be highly traumatic leading to psychosomatic and behavioural disturbances and psychiatric diseases (anxiety and depression). To help people to cope with stress in their lives, to develop directives to reduce stress and to improve wellbeing of our companion and farmed animals, we need to obtain better insight into how the brain responds to psychologically stressful events. 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. Work of the PI over the past 35+ years has contributed greatly to the concept that 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 resulting in translocation to the cell nucleus. The hormone-receptor complex can then bind to certain genes within the DNA and regulate the expression of those genes. These genes are important to change the function of nerve cells in order to respond and adapt properly to the challenge i.e. elicit an appropriate behavioural response. Presently, however, it is unclear which receptor-bound genes are important in the adaptive responses to stress. Furthermore, accessibility of the DNA for MRs and GRs may be regulated by so-called epigenetic mechanisms as these involve changes in the structure and function of proteins to which the DNA is attached. Such epigenetic mechanisms are however still unresolved.

We recently discovered a specific epigenetic 'mark' ('H3K9ac-S10p') that may play a role in controlling the binding of MR and GR to genes leading to changes in the expression of these genes. Interestingly, in previous research we found that this epigenetic mark is strongly linked with the learning of an adaptive behavioural response after a stressful challenge (forced swimming). Combined assessment of these observations has led us to the idea that the presence of the H3K9ac-S10p mark can distinguish MR- and GR-bound genes that are specifically involved in learning the adaptive behavioural response after stress. After identifying these genes in hippocampal neurons, we wish to select a few of the genes and test if suppression of their activity would impair the expression of behavioural adaptation to forced swim stress. This work is of fundamental importance to increase our understanding into how the brain adapts to stressful events and will help to develop novel approaches to alleviate the burden of stress-related disorders in humans and animals.

GC hormones play a critical role in the DG in the consolidation of adaptive behavioural responses after a stressful challenge like FS; the genes underpinning these responses are however unknown. We reported the confluence of FS stress-induced (non-genomic) GC- and NMDA-R-mediated signalling resulting in the enhanced formation of H3k9ac-S10p specifically within the genome of sparse DG neurons. This epigenetic mark is associated with the opening of condensed chromatin and is critically involved in the consolidation of the adaptive behavioural immobility response in DG neurons after FS. Recently, we conducted chromatin immuno-precipitation (ChIP) and whole genome sequencing (Seq) for the two GC-binding receptors (mineralocorticoid (MRs) and glucocorticoid receptors (GRs)) and H3K9ac-S10p on hippocampus of baseline and FS stressed rats. This study revealed a substantial overlap as well as a significant positive correlation between the level of H3K9ac-S10p formation and MR and GR binding after FS across the entire genome. In view of the selective localisation of H3K9ac-S10p in the DG, we hypothesise that this histone mark may play an important role in allowing access of GR and MR to their recognition sites within genes in this hippocampal subregion after FS. We also hypothesise that expression of these genes underlies the behavioural immobility response seen after repeated FS. To test our hypotheses, we will apply pharmacological as well as gene deletion models to investigate the link between signalling, H3K9ac-S10p formation, and MR/GR binding to GC target genes after FS in the DG. We will conduct H3K9ac-S10p/GR and H3K9ac-S10p/MR Tandem ChIP-Seq as well as RiboZero RNA-Seq on DG tissue collected under baseline and FS conditions. We will use bioinformatics analyses to identify genes of high interest which we will test for their role in FS-induced behavioural immobility using viral knock-down technology and behavioural assessment.

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. In the long run, 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').