Identification and molecular mechanisms of the rapid tonicity-induced relocalization of the aquaporin 4 channel

The aquaporin family of integral membrane proteins is comprised of channels that mediate cellular water flow. Aquaporin 4 (AQP4) is highly expressed in the glial cells of the central nervous system and facilitates the osmotically-driven pathological brain swelling associated with stroke and traumatic brain injury. Here we show that AQP4 cell surface expression can be rapidly and reversibly regulated in response to changes of tonicity in primary cortical rat astrocytes and in transfected HEK293 cells. The translocation mechanism involves protein kinase A (PKA) activation, influx of extracellular calcium and activation of calmodulin. We identify five putative PKA phosphorylation sites and use site-directed mutagenesis to show that only phosphorylation at one of these sites, serine- 276, is necessary for the translocation response. We discuss our findings in the context of the identification of new therapeutic approaches to treating brain oedema

Phosphorylation of rat aquaporin-4 at Ser111 is not required for channel gating.

Aquaporin 4 (AQP4) is the predominant water channel in the mammalian brain and is mainly expressed in the perivascular glial endfeet at the brain-blood interface. AQP4 has been described as an important entry and exit site for water during formation of brain edema and regulation of AQP4 is therefore of therapeutic interest. Phosphorylation of some aquaporins has been proposed to regulate their water permeability via gating of the channel itself. Protein kinase (PK)-dependent phosphorylation of Ser111 has been reported to increase the water permeability of AQP4 expressed in an astrocytic cell line. This possibility was, however, questioned based on the crystal structure of the human AQP4. Our study aimed to resolve if Ser111 was indeed a site involved in phosphorylation-mediated gating of AQP4. The water permeability of AQP4-expressing Xenopus oocytes was not altered by a range of activators and inhibitors of PKG and PKA. Mutation of Ser111 to alanine or aspartate (to prevent or mimic phosphorylation) did not change the water permeability of AQP4. PKG activation had no effect on the water permeability of AQP4 in primary cultures of rat astrocytes. Molecular dynamics simulations of a phosphorylation of AQP4.Ser111 recorded no phosphorylation-induced change in water permeability. A phospho-specific antibody, exclusively recognizing AQP4 when phosphorylated on Ser111, failed to detect phosphorylation in cell lysate of rat brain stimulated by conditions proposed to induce phosphorylation of this residue. Thus, our data indicate a lack of phosphorylation of Ser111 and of phosphorylation-dependent gating of AQP4

His 95 acts as a pH gate in Aquaporin-4

Aquaporin 4 (AQP4) is a transmembrane protein from the aquaporin family and is the predominant water channel in the mammalian brain. The regulation of permeability of this protein could be of potential therapeutic use to treat various forms of damage to the nervous tissue. In this work, based on data obtained from insilico and invitro studies, a pH sensitivity that regulates the osmotic water permeability of AQP4 is demonstrated. The results indicate that AQP4 has increased water permeability at conditions of low pH in atomistic computer simulations and experiments carried out on Xenopus oocytes expressing AQP4. With molecular dynamics simulations, this effect was traced to a histidine residue (H95) located in the cytoplasmic lumen of AQP4. A mutant form of AQP4, in which H95 was replaced with an alanine (H95A), loses sensitivity to cytoplasmic pH changes in invitro osmotic water permeability, thereby substantiating the insilico work

Effects of erythropoietin on memory-relevant neurocircuitry activity and recall in mood disorders.

Objective Erythropoietin (EPO) improves verbal memory and reverses sub-field hippocampal volume loss across depression and bipolar disorder. This study aimed to investigate with functional magnetic resonance imaging (fMRI) whether these effects were accompanied by functional changes in memory-relevant neuro-circuits in this cohort. Method Eighty-four patients with treatment-resistant unipolar depression who were moderately depressed or bipolar disorder in remission were randomized to eight weekly erythropoietin (40,000 IU) or saline infusions in a double-blind, parallel-group design. Participants underwent whole-brain fMRI at 3T, mood ratings and blood tests at baseline and week 14. During fMRI, participants performed a picture encoding task followed by post-scan recall. Results Sixty-two patients had complete data (EPO: N=32, saline: N=30). EPO improved picture recall and increased encoding-related activity in dorsolateral prefrontal cortex (dlPFC) and temporo-parietal regions, but not in hippocampus. Recall correlated with activity in the identified dlPFC and temporo-parietal regions at baseline, and change in recall correlated with activity change in these regions from baseline to follow-up across the entire cohort. The effects of EPO were not correlated with change in mood, red blood cells, blood pressure or medication. Conclusion The findings highlight enhanced encoding-related dlPFC and temporo-parietal activity as key neuronal underpinnings of EPO-associated memory improvement. </p

Effects of erythropoietin on memory-relevant neurocircuitry activity and recall in mood disorders

Objective Erythropoietin (EPO) improves verbal memory and reverses sub-field hippocampal volume loss across depression and bipolar disorder. This study aimed to investigate with functional magnetic resonance imaging (fMRI) whether these effects were accompanied by functional changes in memory-relevant neuro-circuits in this cohort. Method Eighty-four patients with treatment-resistant unipolar depression who were moderately depressed or bipolar disorder in remission were randomized to eight weekly erythropoietin (40,000 IU) or saline infusions in a double-blind, parallel-group design. Participants underwent whole-brain fMRI at 3T, mood ratings and blood tests at baseline and week 14. During fMRI, participants performed a picture encoding task followed by post-scan recall. Results Sixty-two patients had complete data (EPO: N=32, saline: N=30). EPO improved picture recall and increased encoding-related activity in dorsolateral prefrontal cortex (dlPFC) and temporo-parietal regions, but not in hippocampus. Recall correlated with activity in the identified dlPFC and temporo-parietal regions at baseline, and change in recall correlated with activity change in these regions from baseline to follow-up across the entire cohort. The effects of EPO were not correlated with change in mood, red blood cells, blood pressure or medication. Conclusion The findings highlight enhanced encoding-related dlPFC and temporo-parietal activity as key neuronal underpinnings of EPO-associated memory improvement. </p