Project Outline

Background

Aquaporins facilitate the passive, bidirectional flow of water in all cells and tissues. In the brain and spinal cord, aquaporin-4 is highly expressed and enriched at astrocyte endfeet, synapses and the glia limitans. It facilitates the exchange of water across the blood-spinal cord and blood-brain barriers, controlling cell volume, extracellular space volume and astrocyte migration. The perivascular enrichment of aquaporin-4 is consistent with its central role in glymphatic function, although the mechanism by which that role is exerted remains unknown. Recently, we have demonstrated that aquaporin-4 localization is dynamically regulated at the subcellular level, affecting membrane water permeability. In animal models of ageing, stroke, traumatic injury and sleep disruption, impairment of glymphatic function is associated with changes in perivascular aquaporin-4 localization. Each of these conditions represent established and emerging risk factors in developing neurodegeneration. Brain and spinal cord oedema are caused by the influx of water through aquaporin-4 in response to osmotic imbalances that occur following insults such as traumatic injury, stroke or tumour development. We have demonstrated that reducing dynamic subcellular relocalization of aquaporin-4 to the blood-spinal cord or blood-brain barriers reduces oedema and accelerates functional recovery in rodent injury models. Given the difficulties in developing pore-blocking aquaporin-4 inhibitors or activators and controversies in the field over the status of many proposed molecules, targeting dynamic aquaporin-4 subcellular relocalization provides a novel new approach to modulating aquaporin-4 function. This approach also opens up new treatment avenues for CNS oedema, neurovascular and neurodegenerative diseases, and provides a framework to address fundamental unanswered questions about water homeostasis in health and disease.

Objective

The aim of this PhD project is to understand how AQP4 localization and controls membrane water permeability and water homeostasis in health and disease using in vitro and/or in vivo methods depending upon the interests of the student

Method

The measurement of constantly-fluctuating water availability either side of a cell membrane under normal conditions is a major technical challenge. Every cell depolarization event reflects this process and the circadian rhythm is also thought to be a factor. We have identified the hypoxia caused by stroke as a pragmatic way of observing the functional effects of AQP regulation. We will therefore use cutting edge techniques to characterize the control of water homeostasis underpinned by a suite of biochemical and biophysical approaches.

The long-term impacts of this project are the identification of a mechanistic framework to understand the fundamental physiological process of water homeostasis and the use of this knowledge to prevent brain swelling. Current therapies can only manage the symptoms once they have developed, while we wish to prevent swelling from developing by acutely controlling membrane water permeability. Traumatic injury to the brain and spinal cord affect 60 million people every year. Stroke affects 15 million people (5 million die; 5 million are permanently disabled). This project will contribute to the urgent and unmet clinical need of these patients for effective medicines.

References

Salman MM, Kitchen P, Halsey A, Xun Wang M, Törnroth-Horsefield S, Conner AC, Badaut J, Iliff JJ, Bill RM: Emerging roles for dynamic aquaporin-4 subcellular relocalization in CNS water homeostasis, Brain, 2022, 145, 64-75 (Featured on the front cover).

Salman MM, Kitchen P, Yool AJ and Bill RM: Recent breakthroughs and future directions in drugging aquaporins, Trends Pharmacol Sci, 2022, 43,30-42 (Featured on the front cover).

Salman MM, Kitchen P, Iliff, JJ, Bill RM: Aquaporin-4 and glymphatic flow have a central role in brain fluid homeostasis, Nat Rev Neurosci. 2021, 22, 650–651.

Kitchen P, Salman MM, Halsey AM, Clarke-Bland C, MacDonald JA, Ishida H, Vogel HJ, Almutiri S, Logan A, Kreida S, Al-Jubair T, Winkel Missel J, Gourdon P, Törnroth-Horsefield S, Conner MT, Ahmed Z, Conner AC and Bill RM: Targeting aquaporin-4 subcellular localization as a novel approach to treat CNS edema, Cell, 2020, 181, 784-799

Techniques

Cell culture (2D and 3D); Water transport functional assays; Quantifying biomolecules (cell surface biotinylation, Western blot, qRT-PCR, ELISA); Protein-protein interaction studies (immunoprecipitation, proximity ligation); Structural modelling; in vivo models; behavioural studies (as appropriate to the interests of the student).