The role of hypothalamic RNA binding protein Caprin2 in osmoregulatory dysfunction in old age

By 2050, 25% of the population 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. The reasons why bodily systems deteriorate in the elderly is not well understood. However, there is growing evidence that brain dysfunction is involved in a wide range of chronic conditions of old age. 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. A specialised part of the brain called the hypothalamus controls the excretion of water through the production of a hormone called vasopressin (AVP) that tells the kidney to conserve water. AVP is a protein 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. At the same time, the AVP mRNA undergoes an unusual modification; the string of adenine (A) residues at the end of the mRNA - the so-called poly(A) tail - increases in length from around 200 to 400 residues. Importantly, we have recently shown that the AVP mRNA poly(A) tail length increases with age in rats.

Using methods that allow is to simultaneously describe the expression of all genes, we identified another gene, called Caprin2, an RNA binding protein, as being increased in expression in the hypothalamus following dehydration. There are over a thousand RNA binding proteins, but their physiological roles are not understood. We have shown that Caprin2 has a pivotal role in the brain mechanisms that control fluid balance. We showed that Caprin2 binds to the AVP mRNA and mediates the increase in poly(A) tail length. Importantly, when we knockdown expression of Caprin2 in the hypothalamus, we alter the response to a dehydrating stimulus - more AVP is released into the blood, suggesting that the normal function of Caprin2 is to inhibit this in some way. Importantly, we have shown that old age in rats results in an increase in Caprin2 expression, in parallel with the increase in AVP mRNA poly(A) tail length. We then used unbiased mathematical tools that use global gene expression data to predict gene interactions. We tested this gene network and validated regulatory interactions between Caprin2 and the genes encoding the neuropeptide dynorphin, haemoglobin-beta and the receptor Opsin-3.

Or aims are now to decipher the detailed molecular mechanisms by which Caprin2 affects a network of gene expression in the hypothalamus, and hence regulates the crucial hormonal and physiological processes that govern salt and water homeostasis. Further, we will test the hypothesis that changes in Caprin2 activity underlie, at least in part, the homeostatic dysfunction of old age. These unique studies will tell us about the mechanisms by which an RNA binding protein affect gene expression and hence mediate physiological stability, and how this goes wrong in old age.

The reflexes regulating water balance are centred on hypothalamic neurones that synthesise the antidiuretic peptide hormone vasopressin (AVP). As a consequence of the depletion of pituitary stores that accompanies osmotic stimulation, there is a need to make more AVP. This starts with an increase in transcription, and results in an increase in the abundance of mature mRNAs. In addition, the AVP mRNA is subject to post-transcriptional modification in the form of an increase in the length of the 3' poly(A) tail. These mechanisms go wrong in old age, with fatal consequences. Transcriptome analysis revealed that expression of RNA binding protein Caprin2 is increased in the osmotically stressed hypothalamus. Caprin2 binds to the AVP mRNA, and lentiviral mediated snRNA knockdown of Caprin2 in vivo shortens the AVP mRNA poly(A) tail and reduces transcript abundance. In an in vitro system, Caprin2 over-expression enhanced the abundance and poly(A) tail length of the AVP mRNA. Importantly, Caprin2 knockdown leads to perturbed osmoregulation. In old animals, Caprin2 expression increases in parallel with AVP mRNA poly(A) tail length. Mathematical analysis of transcriptome data revealed that Caprin2 is a central hub in a putative network. Knockdown or over-expression of Caprin2 in vitro had opposite effects on target mRNA abundance. We will now deconstruct the Caprin2 network in vivo, and ask about the physiological consequences. Focusing on three putative Caprin2 target genes (Prodynorphin, Opsin 3, Beta-haemoglobin), we will ask about the molecular nature of the network interactions, how they impact on hormone elaboration and secretion, and, ultimately, how they control homeostasis in vivo in both young and old animals. Further, we use transcriptomics (eCLIP) and proteomics (mass spectrometry) to reveal the global mechanisms Caprin2 network regulation and action, particularly in the context of their dysfunction in old animals.

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 balance in the context of the homeostatic deterioration that accompanies ageing. 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. The named Postdoc, Dr Michael Greenwood, will receive state-of-the art training in genomic bioinformatics with our collaborator, Dr Colin Campbell.

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

Social. Having defined molecular and neural mechanisms of salt and water intake in rat models, we will be in a position to explore their relevance to the human condition. There are a number of disease states related to our work, namely dehydration in old people, hyponatremia as a result of the "tea and toast" diet of many lonely, often bereaved elderly people, the syndrome of inappropriate antidiuretic hormone secretion (SIADH), which causes considerable morbidity in elderly and hospitalised patients, and sudden death in young people because of hyperacute hyponatremia (profuse sweating during strenuous exercise and rehydration with water only). Finally, in the longer term, we would like to explore the fascinating hypothesis that dehydration induced delirium is associated with cognitive decline in the elderly.
It is estimated that by 2050, 40% of the population of the UK will 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 advancement will have global impact, as the 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 and therapists.