Deciphering the physiological roles of the Rasd1 interactome in the central control of hydromineral homeostasis.

The brain mechanisms responsible for osmotic stability are located in the hypothalamo-neurohypophyseal system (HNS), which consists of magnocellular neurons located in the supraoptic nucleus (SON) and paraventicular nucleus (PVN), the axons of which terminate in the posterior pituitary gland. These neurones make the antidiuretic hormone vasopressin (AVP), which acts on the kidney to provoke water conservation. As a consequence of dehydration, the release of AVP from the hypothalamus is associated with many plastic changes that all work together to optimise hormone release and replenishment. These mechanisms go wrong in old age, and diminished thirst and salt-appetite can result in disorders of fluid balance that are a frequent cause of morbidity and mortality in the elderly. We used microarrays to document the transcriptome of the rat hypothalamus, and described changes following the osmotic challenges of dehydration or salt loading. One of the genes identified as being robustly up-regulated in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus following chronic dehydration for 3 days, and following consumption of 2% (w/v) NaCl (salt-loading, SL) was small G-protein Rasd1, a multifunctional intracellular signaling molecule with a role in numerous pathways governed by an extensive interactome that includes not only other signaling proteins, but also transcription factors. We have demonstrated that Rasd1 is a small G protein with a big role in the function related plasticity exhibited by the osmotically challenged hypothalamus. We have shown that in vivo knockdown of Rasd1 by viral-mediated delivery of shRNAs has no effect on water intake in euhydrated rats, but induced insatiable consumption of salt during SL as well as increased levels of circulating AVP. These data suggest that the role of Rasd1 is normally to inhibit these processes. However, as Rasd1 is a multifunctional protein with an extensive interactome, the exact pathways involved still remain to be deciphered. We thus hypothesise that Rasd1 has a unique interactome in the HNS, and that Rasd1 binding proteins and Rasd regulated genes mediate diverse vital physiological functions that cooperatively contribute to osmotic defense mechanisms. We will thus explore the interactions between Rasd1 and partner proteins, and Rasd1 and the genome, within the context of HNS osmoregulatory plasticity. Our specific objectives are as follows: *In the different compartments of the HNS, we will determine whether Rasd1 forms complexes with proteins previously described as being part of the Rasd1 interactome. *Two unbiased methodologies will define the entirety of the Rasd1 interactome in different compartments of the HNS, thus confirming previously described interactions, as well as identifying new ones. This information will place Rasd1 into functional pathways that can be tested physiologically in vivo. *We will identify genes regulated by transcription factors that complex with Rasd1. *A major bottleneck of the “Omic” era is the sheer scale and complexity of the datasets, and the resulting daunting problem of identifying suitable targets for often expensive and time consuming physiological studies. Rather than “cherry-pick” our data, we will use unbiased mathematical criteria to select genes worthy of functional study. *It is important to confirm expression of interacting protein and Rasd1-regulated genes in Rasd1- and AVP-expressing cells. *We will dissect the role of interacting genes and proteins in overall physiological role of Rasd1. These studies are a necessary prelude to future molecular and cellular mechanistic studies, particularly in regard to the ageing process.