Role of two-pore channels in the astroglial Mechanosensitivity controlling water balance

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In mammals, water balance is maintained by physiological processes such as appetite for salt or thirst, as well as by renal secretion or reabsorption of Na+ or water for which the neuropeptides vasopressin (VP) and oxytocin (OT) play crucial roles. The latter are centrally produced in paraventricular and supraoptic nuclei of the hypothalamus. These nuclei are essential in hydromineral balance regulation as they are able to sense osmotic changes in extracellular fluids on the one hand, and receive inputs from other fluid imbalance sensing areas of the lamina terminalis circumventricular organs devoid of blood brain barrier on the other hand. Neuro-glial interactions play a key role in brain functions, particularly in regions strongly connected to blood flow such as the hypothalamus. In this regard, astrocytes sense and integrate changes in blood composition as well as in neuronal activities. Accordingly, recent studies point at astrocytes as primary sensors of osmotic changes in the brain. In spite of this primary physiological importance, our knowledge on the osmoreceptors expressed in astrocytes remain limited, in particular with regard to dehydration/hyperosmotic stress. One line of research regards their ability to respond to activation of mechanosensitive channels by eliciting cytosolic Ca2+ elevations that trigger morphological changes leading to modulation of VP and OT neurons activity. In this regard, lysosomes are underexplored local source of Ca2+ that are involved in astroglial activation. Most interestingly, recent discoveries support that endolysosomes act as osmosensors as there are highly dynamic intracellular compartments that adapt their volume in response to cell swelling or shrinking induced by osmotic challenges. Recently, we have identified that lysosomal Ca2+ permeable two-pore channels (TPC) act as key components in the control of VP and OT secretions. Our preliminary data further show that TPCs are involved in hyperosmolarity-induced modulation of VP/OT neuronal activity via a mechanism that remains to be elucidated. Since our current data indicate that TPC partake in hypothalamic astrocyte Ca2+ elevations, we hypothesize they are key contributors to the mechanosensitive machinery allowing astroglial osmosensitivity by driving Ca2+ signals triggering regulatory neuro-glial pathways. To assess this posit we propose to:1. Characterize in vitro the distribution and mechanosensitive functions of TPCs in isolated astrocytes. To do so, we will perform high resolution cell imaging and evaluate the mechanosentivity of endolysosomal channels with an innovative electrophysiological technique. 2. Identify in vivo the implication of astroglial TPC in VP/OT-driven physiological control of water balance, water intake and the associated mechanisms. To this end, water intake will be evaluated together with the in vivo imaging of OT and VP release under conditions of hyperosmotic stress in mice deleted for astroglial TPCs.