The role of aquaporin-4 in limiting acute oedema after spinal cord injury

Impairment of the blood–spinal cord barrier (BSCB) leading to spinal cord oedema is the second most common insult after ischemia reperfusion (IR) injury and it is associated with poor prognosis, such as paralysis or even death. Several studies have indicated that disruption of BSCB causes pathological changes including swollen perivascular glial end feets (cytotoxic edema), breakdown of endothelial tight junctions and vascular basal lamina (vasogenic edema), associated with deregulated expression of water-channel protein aquaporins (AQPs). AQPs are small membrane proteins of epithelial and/or glial cell origin and permit passive water diffusion in the development of cytotoxic as well as vasogenic edema during various pathophysiological injury such as neuroinflammation, ischemia and trauma and AQP4 is recognized as the major pathway for water homeostasis in the CNS. AQP4 is expressed in white and gray matter spinal cord astrocytes and normally functions in astrocytes to enable fast water influx or efflux, driven by osmotic or hydrostatic pressures.

AQP4 function has been implicated in spinal cord trauma, spinal cord ischemia; neuromyelitis optica and multiple sclerosis. In the spinal cord, however, simply removing AQP4 expression, as in AQP4-/- mice, is not the answer since these mice exhibit significantly impaired locomotor function and prolonged bladder dysfunction compared to wild-type littermates after SCI. AQP4-/- mice also showed increased spinal cord water content and blood vessels near the SCI site had incomplete barrier function due to sparse tight junctions. These results suggest that AQP4 has different functions in the acute phase compared to the later phase. The identification of molecules that inhibit water transport through specific AQP isoforms is not a simple proposition for drug discovery on account of the high level of structural conservation within the AQP family. The small diameter of all AQP pores together with the chemical properties of the pore-lining amino acid side-chains mean that hydrophilic compounds are unlikely to enter and block them. The lack of reliable in vitro phenotypic assays suitable for screening and validating the pharmacological regulation of AQP function, means that pharmaceutical companies have been unable to meet the challenge of developing small molecules that block the AQP pore. However, it has recently been shown that endogenous AQP4 regulation occurs through vesicle-to-plasma-membrane relocalisation in astrocytes, providing a new translation route to effective therapies; namely the pharmacological reduction of the number of AQP4 water channels in the plasma membrane. Our preliminary data (Fig. 1A), building on the recent discoveries by others in cultured astrocytes, shows that CNS oedema is suppressed after mouse SCI by inhibition of cAMP-dependent protein kinase (PKA) or calmodulin (CaM); these inhibitors were selected to demonstrate proof-of-concept by targeting two different points in the likely AQP4 relocalisation mechanism (Fig. 1B). One of these inhibitors is a licensed drug, trifluoperazine (TFP) which inhibits CaM. The drugs therefore represent a step in the right direction for SCI patients and fill and unmet clinical need that targets the early events after injury and may potential reduce damaging long-term effects. This project will aim to translate our studies on AQP4 relocalisation into a treatment that reduces cytotoxic CNS oedema formation for SCI patient benefit.

In preliminary experiments, we have described a partial mechanism of AQP regulation which provides the rationale for this project. We have identified 2 inhibitors (one experimental and one an FDA-approved drug) that significantly reduce odema after spinal cord injury (SCI). We will therefore evaluate these two drugs in an already validated model of swelling after SCI, by answering the following questions:
1. How effective are known AQP4 relocalization inhibitors identified from our in vitro model at reducing oedema in a rat model?
2. What functional consequences occur after inhibiting AQP4 relocalisation?

By joining this exciting project you can help to test these hypotheses in experiments involving in vitro and in vivo models of CNS trauma. Your project will take place within Birmingham’s Neuroscience and Ophthalmology Group where you will use cutting edge research facilities covering molecular, cellular and imaging techniques to explore the role of aquaporins in spinal cord injury. You will also benefit from the vibrant interdisciplinary research environment provided by the wider Institute of Inflammation and Ageing groupings, which include scientists focussed on neuroprotection and neuroregeneration after CNS trauma and disease.