Regulatory and functional pathways mediating the control of central osmotic defences by hypothalamic transcription factor CREB3L1

The bodily fluids of the mammalian organism are in a constant state of flux. Even in the absence of challenges such as dehydration, haemorrhage or starvation, salt and water are constantly being lost as a consequence of normal, obligatory renal excretory functions, and by the processes of respiration and perspiration. The body has two mechanisms that function to control the consumption and the excretion of water and salt, in order to maintain the optimal bodily content required for good health. The first mechanism involves the production, by a part of the brain called the hypothalamus, of a hormone called "arginine vasopressin" (AVP) that tells the kidney to conserve water. The second mechanism is behavioural, and involves the instincts of thirst and salt appetite that emotionally drive the organism to correct its fluid balance. These mechanisms can go wrong resulting in ill-health. For example, disorders of fluid balance are evident in a substantial proportion of elderly patients admitted to hospital, and dehydration is a frequent cause of morbidity and mortality in old people.
AVP is a peptide hormone that is encoded by the corresponding AVP gene. When an animal becomes dehydrated, AVP is released from the hypothalamus and there is thus a need to make more. Twenty five years ago, we showed that the expression of the AVP gene is hence activated, and more messenger RNA (mRNA) is made from the gene, a process known as "transcription". It is the AVP mRNA that take the information coded by the gene to the cytoplasm of the cell. Here, the information in the mRNA is "translated" into protein. However, the exact molecular mechanisms involved have remained elusive, until now.
Using methods that allow us to simultaneously describe the expression of all genes, we identified another gene, called CREB3L1, as being increased in expression in the hypothalamus following dehydration. CREB3L1 is a specialised protein (a "transcription factor") that controls the expression of target genes. We showed that CREB3L1 is expressed in AVP neurones, binds to the promoter region of the AVP gene, and hence drives its expression.
Our aims are now to decipher the detailed molecular mechanisms by which CREB3L1 affects gene expression in the hypothalamus, and hence regulates the crucial hormonal processes that govern salt and water homeostasis. We will:
*work out which signals tell the hypothalamus to increase the expression of CREB3L1 following dehydration.
*find out what other proteins are bound to CREB3L1, which will tell us about upstream factors that regulate CREB3L1, and downstream effectors through which CREB3L1 might exerts its action.
*find out what other genes in the AVP cell are regulated by CREB3L1.
*determine how CREB3L1 globally affects the elaboration of neuropeptides in the hypothalamus.
*embark upon studies that will tell us about the physiological roles of CREB3L1, the proteins that it interacts with and the genes that it regulates. Importantly, these experiments will take place in the physiological integrity of the whole organism.
These unique studies will tell us about the mechanisms by which transcription factor protein affects gene expression in the brain, and hence mediates physiological stability.

The brain reflexes regulating water balance are centred on hypothalamic neurones that synthesise the antidiuretic hormone vasopressin (AVP). These neurones send axonal projections to the posterior pituitary, from where AVP is released into the blood. As a consequence of the depletion of pituitary stores that accompanies chronic osmotic stimulation, there is a need to synthesise more AVP. We showed 25 years ago that this starts with an increase in transcription, which leads to more abundant mature mRNAs, but the molecular mechanisms responsible remained elusive until we described transcriptome changes in the rat hypothalamus following osmotic stimulation. We identified basic leucine zipper transcription factor CREB3L1 as being robustly up-regulated in AVP neurones by hypersomostic cues, and showed binding to the AVP gene promoter and activation of AVP gene transcription. Further, we have now shown that CREB3L1 mRNA expression is up-regulated by cAMP pathways, and that shRNA knockdown of CREB3L1 blocks the cAMP-mediated activation of AVP expression. Interestingly, inhibition of protein synthesis blocks the cAMP-mediated up-regulation of CREB3L1 expression, suggesting the requirement for an intermediary "Mystery Transcription Factor". We will now we now address 6 questions, the answers to which will reveal the detailed mechanisms CREB3L1 regulation and function, within the context of the brain mechanisms that regulate water balance:
1. What are the molecular mechanisms that mediate the regulation of CREB3L1 by physiological cues? What is the identity of the "Mystery Transcription Factor".
2. What are the molecular interactions in the hypothalamus that facilitate CREB3L1 actions?
3. What genes does CREB3L1 regulate in the SON and PVN?
4. How does CREB3L1 affect neuropeptide biosynthesis in the HNS?
5. Can we construct and test in silico models of the integrated mechanisms of CREB3L1 function?
6. What are the physiological roles of CREB3L1 and interacting genes.

Academic. In the short-term, the proposed studies will generate important new basic scientific knowledge about the environment-genome interface, and the way that the brain regulates water and salt homeostasis. The new information gleaned from these studies will be published in high impact, peer reviewed international journals, and will also be presented at national and international conferences. The research will bolster in vivo systems physiology in the UK, which has been in serious decline of late, yet is crucial for the understanding of the function of the genome.

Economic. It is unlikely that our results will be clinically or commercially relevant, at least in the short term.

Social. It is estimated that by 2050, 40% of the population of the UK will be over 50 years of age, and 25% will be over 65. Whilst this dramatic increase in lifespan should be celebrated, the resultant demographic change represents a major challenge. This is because wellbeing and health in old age has not improved proportionally with longevity. This imbalance not only impacts on the individual, who would obviously wish to enjoy a happy and healthy old age and retirement, but it also imposes considerable economic and social burdens on families and society. However, the reasons why homeostatic systems deteriorate in the elderly is not well understood, and it remains a major challenge for the biomedical research community to address these issues, with a view to ultimately improving the quality of life of our senior citizens. It is possible that hypothalamic genes might be possible targets for the treatment or amelioration of disorders of water balance, which are prevalent in old age. Thus, in the long-term, it may well be that our findings will improve quality of life, health and wellbeing, and functionality in old age. This would reduce welfare and health costs whilst boosting economic productivity. This has clear benefits for the individual, for families and for society in general. We also envisage a contribution towards evidence based policy-making and influencing public policies and legislation at a local, regional, national and international level; these scientific advancements will have global impact, as an ageing population is a world-wide phenomenon. Policy makers and governments are keen to encourage us to consume a healthy diet, and that includes imploring us to take less salt with our food. Food companies, by signing up to the Public Health Responsibility Deal Companies, are committed to reducing the amount of salt in their products in order to meet Government targets for individual consumption. However, these endeavours have not been based on too much solid empirical evidence of the benefits, particularly in relation to combating chronic diseases. Our studies will contribute towards providing such evidence. As such we also expect an impact with nutritionists, dieticians and occupational therapists.