Studies on aquaporin 4, a molecular determinant of brain water homeostasis

Sammanfattning: A well controlled brain water homeostasis is of utmost importance for an appropriate control of neuronal activity. Astrocytes express the water channel aqauporin 4 (AQP4) and play a key role for the maintenance of brain water homeostasis. Brain edema is a result of perturbed water homeostasis and involves astrocyte swelling. Emerging evidence supports that AQP4 is important in physiological potassium clearance, but also that AQP4 aggravates brain edema. The overall aim of this study has been to study the regulation of water permeability in astrocytes with particular emphasis on the role of AQP4. In study 1 we presented a new; third form of mouse AQP4 mRNA not previously known. It was found to be predominantly expressed in brain and to be developmentally regulated. The finding may be relevant for the understanding of regulation of water homeostasis in the immature brain. In study 11 and 111 we investigated the short term-regulation of astrocyte water permeability and the role of two substances associated with perturbed water homeostasis, the heavy metal lead and the neurotransmitter glutamate. Lead and glutamate were both found to induce a significant increase in astrocyte water permeability, an effect attributable to an effect on AQP4. The molecular target for the effects of lead and glutamate was identified as the AQP4 serine 111 residue, which is in a strategic position for control of water channel gating. The glutamate effect was mediated by activation of group 1 metabotropic glutamate receptors (mGluRs), intracellular calcium oscillations, activation of CaMKII and the NOS pathway. NOS can activate cGMP/PKG. We could show that PKG phosphorylates a peptide corresponding to 196-CI23 of WT AQP4 It is suggested that the glutamate-effect on AQP4 play a physiological role, but also that the lead- and glutamate-triggered stimulation of water transport in AQP4expressing astrocytes may contribute to adverse effects on brain water metabolism. Erythropoietin (EPO) is neuroprotective in several models of brain injury that may induce cytotoxic brain edema as a secondary event. In study IV we found that EPO and an EPOderivate significantly reduced the neurological symptoms in a rodent model of primary brain edema. We showed that EPO counteracts the effect of glutamate, mediated by mGluRs, on AQP4 water permeability in astrocytes. Our data indicate that EPO modifies the fast intracellular calcium oscillations caused by activation of mGluRs. Thus EPO may reduce the risk of astrocyte swelling in stroke and other brain insults. In conclusion, this thesis demonstrates that astrocyte water permeability can be dynamically regulated via changes in the opening state of AQP4. The data support that the phosphorylation state of a serine residue in the first intracellular loop of AQP4 determines the opening state of AQP4 and that the regulation involves a calcium signaling pathway including CaMKII, NOS and PKG. We propose that up-regulation of astrocyte AQP4 water permeability is involved in the development of cytotoxic brain edema, and that inhibition of astrocyte AQP4 activity may offer a new therapeutic strategy in situations of perturbed brain water homeostasis.