Role of corticosteroid receptor DNA binding in stress-induced hippocampal gene transcription in relation to glucocorticoid and behavioural responses

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.

For many years it is known that 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 has been instrumental to the development of 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 thought to be important to change the function of nerve cells in order to respond and adapt properly to the challenge. Presently, however, it is unclear how these receptors bind to the genes. It has been assumed for decades that MR and GR binding to genes is proportional to the receptor's occupancy level by hormone; our pre-work indicate this is indeed the case for GRs but surprisingly not for MRs. Under baseline (Bs; non-stress) conditions GR occupancy by GCs is low as well as GR binding to genes whereas after stress GR occupancy is high and its gene binding is too. In contrast, despite a high level of MR occupancy by GCs under Bs conditions its DNA binding is low and only increases after exposure to a stressful challenge. This is an entirely new finding which could mean that the existing assumptions about the role of MRs and GRs in the brain need to be revised.

We aim to investigate under which conditions (different types of stress, specific hormone stimulation) MRs and GRs bind to genes in the hippocampus (a part of the brain that is particularly involved in the regulation of GC secretion and behavioural responses after stress) and the consequences of this binding for the expression of these genes and for glucocorticoid and behavioural responses to stress. We have also planned to study whether for the fine-tuning of MR and GR binding additional proteins ('steroid receptor co-regulators') is required. In addition to acute stress models (e.g. forced swim stress, restraint stress), we will use a model of repeated variable stress as well. In this model it has been shown that hippocampal MRs and GRs have declined and GC secretion and anxiety and learning behaviour are disturbed. This model will increase our insight into how changes in MR and GR binding to genes contribute to the changes in expression of these genes as well as hormonal and behavioural changes. This work is of fundamental importance to increase our understanding into how stress affects brain function and will help to develop new ways to reduce the burden of stress-related disorders in humans and animals.

Glucocorticoid (GC) hormones coordinate adaptive responses to stressful events to maintain health and wellbeing. Acute rises in GC secretion after stress are regarded as beneficial to establish adaptive responses, whereas sustained increases such as after repeated stress can be damaging. In the hippocampus GCs bind to two types of corticosteroid receptors, mineralocorticoid receptors (MRs) and GC receptors (GRs), with different affinity and thus have distinct occupancy levels under baseline (Bs) and stress conditions. MRs and GRs are ligand-dependent transcription factors that bind to the same GC-responsive elements (GREs) within GC-target genes (e.g. Fkbp5, Sgk1). Our recent work has shown that, after acute stress, GR binding to GREs increases in parallel to stress-induced GC secretion and GR occupancy as expected. In contrast, MRs, which are highly (>80%) occupied by GC hormone under any conditions (Bs or stress), showed a surprisingly low GRE-binding at Bs but increasing substantially after stress. This result was unexpected and clearly will have implications for the decennia-reigning concept of occupancy-dependent MR and GR action in the brain. We hypothesise that hippocampal MRs and GRs have different GRE-binding profiles within GC-target genes in response to acute and repeated stress which are associated with distinct transcriptional responses of these genes. We further postulate the involvement of steroid receptor co-regulator proteins as well as marked implications for HPA axis regulation and behavioural responses. To test our hypotheses we will use acute and repeated variable stress models and pharmacological approaches. We will apply state-of-the-art chromatin immuno-precipitation (ChIP) and Tandem ChIP in combination with qPCR and next-generation DNA sequencing, and RNA sequencing analysis to elucidate MR and GR GRE-binding profiles and m/hnRNA responses in relation to HPA hormone responses and changes in anxiety-like behaviour.

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').