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Title: The mechanisms of aquaporin expression and translocation in cerebral pathologies
Author: Salman, Mootaz
ISNI:       0000 0004 6500 5339
Awarding Body: Sheffield Hallam University
Current Institution: Sheffield Hallam University
Date of Award: 2017
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Aquaporins (AQPs) are membrane proteins that regulate the selective bidirectional water and solute transport across biological membranes and are essential for maintaining cellular integrity. In humans, the AQP family consists of 13 established members (AQP0-12). AQPs 1, 3, 4, 5, 8, and 9 are collectively known as "cerebral AQPs" as they are known to be present in the mammalian brain. AQPs are distributed throughout a wide range of tissues and therefore play a role in a diverse range of physiologies and pathologies including oedema, hypertension, diabetes insipidus and cancer. Consequently, AQPs have been highlighted as key drug targets. AQP4 is the most abundant AQP in the central nervous system (CNS), predominantly in CNS glial cells, particularly astrocytes and at the blood-brain barrier. Astrocytes are the most abundant cells in the CNS and are a major site of stroke or traumatic brain injury (TBI) induced cell swelling during osmotically-driven cytotoxic oedema formation. Current treatments for cerebral oedema use solutes at high concentrations or surgical intervention to relieve pressure. Neither works extremely well with having surgery having significant negative outcomes. AQP4-deficient animals were protected from early oedema formation but there are no clinically-available inhibitors of the AQP4 water channel. Modulating AQP4 surface localisation and expression rather than direct blocking of its pore could provide novel therapeutic benefits. The research in this thesis primarily elucidates the regulatory mechanisms involved in processes of hypotonicity and hypoxia mediated AQP4 translocation in primary astrocytes and examines current therapies for cerebral oedema including hypothermia. Using confocal microscopy, cell surface biotinylation, mutants and specific inhibitors, this study showed that AQP4 rapidly relocalised to astrocyte plasma membranes from intracellular vesicles in a calmodulin- and protein kinase A (PKA)-dependent manner. PKA is activated by a calmodulin-dependent adenylyl cyclase (AC) in response to osmotic stress, and calmodulin binds directly to AQP4 mediated by phosphorylation of AQP4 at S276. Additionally, the mechanisms of hypoxia induced cell swelling have been shown to be similar to hypotonicity-mediated cellular swelling. The potential of therapeutic hypothermia for the reduction of early stage (cytotoxic) oedema following TBI and stroke has been investigated with a focus on the effects on AQP regulatory mechanisms. Hypothermia increased AQP4 surface expression, but not total protein expression in primary human astrocyte adding novel insight to previously known hypothermia induced changes in membrane Na+ and K+ permeability and Na+-H+ antiporter activation. Human primary cortical astrocytes were cultured in hypoxic conditions, with and without therapeutic hypothermia intervention, at time points that model TBI and treatment. Hypoxia down-regulated AQP4, at both gene and protein levels, which then increased significantly with hypothermic intervention. However, hypoxia also caused a significant increase in AQP4 surface availability that was significantly reduced after hypothermic intervention. These results show that changes in AQP4 membrane localisation, rather than protein expression, may provide the protective effects of hypothermia against hypoxia induced cytotoxic oedema. This relocalisation of AQP4 was shown to involve mechanisms dependent on the transient receptor potential vanilloid 4 (TRPV4) channels, calcium and calmodulin activation. Investigation into the role of AQP expression and regulation in temporal lobe epilepsy (TLE) was conducted using transcriptomic (microarray, RT-qPCR) and proteome profiling of matched human sclerotic (TLE-HS) and non-sclerotic (TLE-NC) patient brain samples. AQP1 and AQP4 transcript expression was significantly increased, while AQP9 mRNA was significantly reduced in TLE-HS compared to TLE-NC. AQP4 protein expression was also increased while AQP1 protein remained unchanged and AQP9 was undetected. The differential expression of the MAPK pathway components p38 and JNK were revealed in patient samples and ELISA data showed that p38 and JNK inhibitors decreased AQP4 protein levels in cultured human primary cortical astrocytes. The components of the MAPK signalling pathway involved in the regulation of AQP4 expression may provide future TLE drug targets. The novel findings in this study further the understanding of the physiological mechanisms of AQP4 translocation, expression and regulation involved in astrocytic swelling during ischemic stroke or TBI induced cytotoxic oedema and also TLE. These mechanisms might help in improving the therapeutic effectiveness of hypothermia on cytotoxic oedema and/or treating other CNS condition such as TLE using pharmacological disruption of AQP expression and translocation mechanism.
Supervisor: Woodroofe, Nicola ; Conner, Matthew Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available