Aquaporin Regulation: A New Translational Route to Cytotoxic CNS Oedema Therapies

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Aquaporins (AQP) control water flow in all tissues. We have discovered that AQP function is controlled by regulating the number of AQP4 water pores in the plasma membrane in response to changes in tonicity or hypoxia. Reversible vesicle-to-plasma-membrane relocalization is triggered by activation of mechanosensitive channels leading to increased intracellular Ca2+ levels and cAMP accumulation, resulting in calmodulin-dependent phosphorylation and cytoskeletal reorganization. To move the field forward, we now need to fully understand the mechanisms involved, which is the overall aim of this project. This new understanding will provide novel targets for diseases of water imbalance and a platform to guide the search for new inhibitors. We have developed an assay for endogenous AQP plasma membrane localization based on cell-surface biotinylation and ELISA detection of cell-surface AQP, currently in 24-well plate format. Scaling this to 96- and 384-well format will provide a high-throughput screening system for inhibitors of AQP relocalization using high content compound libraries.The objectives of this studentship are:Objective 1: Identify AQP inhibitors using high-content screening. Our 96-well assay measures cell swelling. Advances in high-content screening will be used to adapt this assay into a robust image-based 384-well transfluor format that quantifies cytoplasmic-to-membrane translocation and recycling of cell-surface molecules. Screening across a carefully-selected panel of approved, annotated and diverse chemical sets will identify new molecules that specifically modulate AQP4 sub-cellular localization. Confirmed hits will progress to our cell-surface biotinylation assay. Pharmacokinetically-appropriate hits will be selected for proof-of-concept testing in vivo. Prioritized hits will be profiled in-depth using a suite of cellular pharmacology tools to elucidate mechanism-of-action at phenotypic, proteomic and transcriptomic levels.Objective 2: Define the complete mechanism of AQP4 regulation. To obtain a complete mechanistic framework, we will examine in molecular detail how calcium influx triggers AQP4 relocalization. The currently-unknown molecular identity of vesicles and regulatory proteins transferring AQP4 to and from the plasma membrane in response to tonicity changes in astrocytes will be identified using molecular- and cell-biology tools, our inhibitor panel and proteomic/lipidomic analyses. The link between AQP4 homo-oligomerisation and trafficking will be investigated. Interactions between AQP4 and proteins that regulate its membrane abundance will be characterized. Structural characterization of full-length AQP4 and regulatory AQP4 complexes will provide a platform that can be directly exploited in the search for new inhibitors.