Contact: Group queries: d.murphy@bris.ac.uk, Website queries: bg14337@bristol.ac.uk

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

The precise regulation water balance is essential for survival and good health, and when threatened, osmotic stability is aggressively defended. However, these mechanisms go wrong in old age, and disorders of fluid balance are a frequent cause of morbidity and mortality in the elderly. The brain mechanisms responsible are located in hypothalamic magnocellular neurones, the axons of which terminate in the posterior pituitary gland. These neurones make the antidiuretic peptide hormone vasopressin (AVP), which acts at the level of the kidney to provoke water conservation. As a consequence of the depletion of pituitary stores that accompanies chronic osmotic stimulation, there is a need to synthesise 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. We documented the transcriptome of the rat hypothalamus, and described changes following dehydration or salt loading. One of the genes identified was RNA binding protein Caprin-2. We showed that Caprin-2 binds to the AVP mRNA, and lentiviral mediated snRNA knockdown of Caprin-2 in the osmotically stimulated hypothalamus shortens the AVP mRNA poly(A) tail and reduces transcript abundance. In an in vitro system, Caprin-2 over-expression enhanced the abundance and poly(A) tail length of the AVP mRNA. Importantly, Caprin-2 knock-down leads to dysfunction of the normal physiological response to salt loading, an osmotic challenge which in healthy rats leads to a gradual increase of urine output and fluid intake. Caprin-2 knockdown results in a significant decrease in urine output and fluid intake, and an increase in urine osmolality and plasma AVP levels. We have gone on to show that osmoregulatory dysfunction in aged rats is associated with both an increase in Caprin-2 expression and an increase in AVP mRNA poly(A) tail length. Mathematical analysis of our transcriptome data revealed that Caprin-2 is a central hub in a putative regulatory network. Importantly, knockdown or over-expression of three putative Caprin-2 targets - Pdyn, prodynorphin (Pdyn), Opsin 3 (Opn3) and b-haemoglobin (Hbb) - in vitro had opposite effects on target mRNA abundance. These data raise new questions regarding the molecular nature and physiological functions of the regulatory interactions in the Caprin-2 gene network. Importantly, it affords us the opportunity to deconstruct the network in vivo, and ask about the physiological consequences in both young and old animals. We will now ask about the molecular nature of the network interactions, how they impact on hormone elaboration and secretion, and ultimately how they control homeostasis. Comparing young and old animals, we are now using a multi-disciplinary combination of state-of-the-art transcriptomic (RNAseq and eCLIP) and proteomic (mass spectrometry) to address 10 interrelated questions, the answers to which will reveal the detailed mechanisms Caprin-2 network regulation and function within the context of the brain mechanisms that regulate water balance - *Does Caprin-2 bind to target gene transcripts? *Does Caprin-2 modulate the poly(A) tail length of target mRNAs? *Does Caprin-2 binding and/or poly(A) tail extension affect mRNA translation? *Is Caprin-2 part of an mRNP granule? *What is the entire hypothalamic Caprin-2 binding mRNA binding repertoire? *How does Caprin-2 affect the hypothalamic transcriptome? *How does Caprin-2 affect neuropeptide biosynthesis in the hypothalamus? *Can we construct in silco models of the entire Caprin-2 regulatory network? *What are the physiological consequences of in vivo network deconstruction? *Can transgenetic modulation of the Caprin-2 network restore osmoregulatory function in old age?