Glutamate-system defects behind psychiatric manifestations in a familial hemiplegic migraine type 2 disease-mutation mouse model

Migraine is a complex brain disorder, and understanding the complexity of this prevalent disease could improve quality of life for millions of people. Familial Hemiplegic Migraine type 2 (FHM2) is a subtype of migraine with aura and co-morbidities like epilepsy/seizures, cognitive impairments and psychiatric manifestations, such as obsessive-compulsive disorder (OCD). FHM2 disease-mutations locate to the ATP1A2 gene encoding the astrocyte-located α(2)-isoform of the sodium-potassium pump (α(2)Na(+)/K(+)-ATPase). We show that knock-in mice heterozygous for the FHM2-associated G301R-mutation (α(2)(+/G301R)) phenocopy several FHM2-relevant disease traits e.g., by mimicking mood depression and OCD. In vitro studies showed impaired glutamate uptake in hippocampal mixed astrocyte-neuron cultures from α(2)(G301R/G301R) E17 embryonic mice, and moreover, induction of cortical spreading depression (CSD) resulted in reduced recovery in α(2)(+/G301R) male mice. Moreover, NMDA-type glutamate receptor antagonists or progestin-only treatment reverted specific α(2)(+/G301R) behavioral phenotypes. Our findings demonstrate that studies of an in vivo relevant FHM2 disease knock-in mouse model provide a link between the female sex hormone cycle and the glutamate system and a link to co-morbid psychiatric manifestations of FHM2

Potassium Dependent Regulation of Astrocyte Water Permeability Is Mediated by cAMP Signaling

Astrocytes express potassium and water channels to support dynamic regulation of potassium homeostasis. Potassium kinetics can be modulated by aquaporin-4 (AQP4), the essential water channel for astrocyte water permeability regulation. We investigated whether extracellular potassium ([K+]o) can regulate astrocyte water permeability and the mechanisms of such an effect. Studies were performed on rat primary astrocytes and a rat astrocyte cell line transfected with AQP4. We found that 10mM [K+]o caused an immediate, more than 40%, increase in astrocyte water permeability which was sustained in 5min. The water channel AQP4 was a target for this regulation. Potassium induced a significant increase in intracellular cAMP as measured with a FRET based method and with enzyme immunoassay. We found that protein kinase A (PKA) could phosphorylate AQP4 in vitro. Further elevation of [K+]o to 35mM induced a global intracellular calcium response and a transient water permeability increase that was abolished in 5min. When inwardly rectifying potassium (Kir)-channels were blocked, 10mM [K+]o also induced a calcium increase and the water permeability increase no longer persisted. In conclusion, we find that elevation of extracellular potassium regulates AQP4 and astrocyte water permeability via intracellular signaling involving cAMP. A prolonged increase of astrocyte water permeability is Kir-channel dependent and this response can be impeded by intracellular calcium signaling. Our results support the concept of coupling between AQP4 and potassium handling in astrocytes

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

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

Potassium elevation induces cAMP production and increases astrocyte water permeability via PKA-dependent phosphorylation of AQP4.

<p>(A) 10mM potassium significantly increased WT AQP4 water permeability (*p<0.01), N = 6, but did not increase S111A AQP4 water permeability (NS; not significant p = 0.51), N = 6. Dark grey bars: WT AQP4, light grey bars: S111A AQP4. (B) Upper panel shows plot of mean values of normalized FRET ratio (donor/acceptor) with SE bars at every acquired point. Lower panel (left): cAMP FRET ratio at different time points: 2.5mM (at 3min in upper panel); 10mM 1min (arrow at 4min in upper panel); 10mM 5min (arrow at 8min in upper panel). 10mM potassium significantly increased cAMP FRET ratio in astrocyte cell line (*p<0.001), n = 26. Lower panel (right): 35mM potassium increased cAMP FRET ratio (*p<0.01), n =  16. (C) By using cAMP EIA kit, 10mM potassium significantly increased cAMP levels in primary cultured astrocytes (left) (*p<0.001), N = 7. 35mM potassium also increased cAMP levels in primary cultured astrocytes (right) (*p<0.001), N = 5. (D) Activated PKA phosphorylated the WT AQP4 peptide containing serine 111 <i>in vitro</i>, but failed to phosphorylate the S111A AQP4 peptide where serine 111 was substituted to alanine. Numbers indicate positions of molecular weight markers. (E) Incubation with the PKA inhibitor KT5720 abolished water permeability increase caused by 10mM potassium (*p<0.001), n = 126–152. Values are means±SEM, n; number of cells, N; number of plates.</p

Functional relationship between Kir-channels and potassium effect on astrocyte water permeability.

<p>(A) Left panel shows representative recordings of intracellular calcium ([Ca²⁺]_i) in three individual primary astrocytes loaded with Fura-2 AM during perfusion with indicated concentrations of potassium (mM) and 100µM barium (Ba²⁺). With barium, 10mM potassium triggered a global [Ca²⁺]_i increase (∼150–420s). Right panel shows summarized calcium data normalized to the peak [Ca²⁺]_i induced by ATP, n = 147. (B) The astrocyte water permeability increase caused by 10mM potassium was abolished when cells were preincubated with 100µM barium (*p<0.001). There was no difference in water permeability in control cells exposed to basal potassium concentrations with or without preincubation with barium (2.5mM potassium -/+ 100µM barium, p = 0.24), n = 60–89.</p

Elevated potassium regulates astrocyte water permeability.

<p>(A) Representative recordings from two independent primary astrocytes showing changes in calcein fluorescence (2.5mM potassium, dashed line; 10mM potassium, solid line) in water permeability measurements. The grey bar depicts the part of the curve used for calculation of plasma membrane water permeability (s; seconds). (B) Fluorescence intensity change induced by isosmotic high potassium: bars show quantified fluorescence intensity at 2min before high potassium (-2min), at start (0min) and after 1min and 5min of indicated potassium concentrations, respectively. Values were normalized to the initial intensity at 2.5mM potassium. Right hand panels show fluorescence intensity curves for isosmotic high potassium (grey: 10mM; black: 35mM) within the time window (∼20s) of water permeability recordings after 1 or 5 minutes. Arrows indicate starting points. Values are means and error bars of each individual acquired time point, n = 48–65. (C) Water permeability in primary astrocytes exposed to 10mM potassium for 1min and 5min, respectively. 10mM potassium caused a significant water permeability increase at 1min and 5min (*p<0.001), n = 57–92. (D) 35mM potassium caused a transient astrocyte water permeability increase at 1min (*p<0.001). The effect was abolished after 5min (NS; not significant p = 0.52), n = 53–92. (E) AQP4 specific water permeability calculated from astrocyte cell line. 10mM potassium significantly increased AQP4 water permeability at 1min (*p<0.001) and 5min (*p<0.05), N = 5-7. (F) 35mM potassium did not increase AQP4 water permeability either at 1min (p = 0.62) or 5min (p = 0.89), N = 5–16. (G) Water permeability in AQP4-negative astrocyte cell line. 10mM potassium for 1min did not have any effect on water permeability in AQP4-negative cells. A small but significant water permeability was observed after 5min of 10mM potassium (P = 0.045), n = 43–130. (H) 35mM potassium did not have any effect on water permeability in AQP4-negative astrocyte cell line (1min, p = 0.62; 5min p = 0.89), n = 33–119. Values are means±SEM, n; number of cells, N; number of plates.</p

Highly elevated potassium prevents astrocyte water permeability increase via calcium signaling.

<p>(A) Upper panel shows representative 340/380nm fluorescence ratio images of astrocyte primary cultures loaded with Fura-2 AM at indicated time points, respectively. Lower left panel shows representative curves of intracellular calcium ([Ca²⁺]_i) recordings in three individual cells during perfusion with potassium (marked with matched colors in upper panel). Potassium concentrations (mM) are marked in the horizontal bar. Arbitrary units (a.u.) represent ratio values corresponding to [Ca²⁺]_i changes. 10mM potassium (∼200–500s) did not cause any change in [Ca²⁺]_i. 35mM potassium (∼680–1000s) induced a global [Ca²⁺]_i increase. The [Ca²⁺]_i increase disappeared when potassium was returned to baseline (2.5mM). One cell (blue) exhibits a spontaneous [Ca²⁺]_i peak at about 180s. Lower right panel shows summarized calcium data normalized to the peak [Ca²⁺]_i induced by ATP, n = 103. (B) After preincubation with the calcineurin inhibitor cypermethrin, 35mM potassium significantly increased astrocyte water permeability at 5min (*p<0.01, n = 73–89). Values are means±SEM, n; number of cells.</p

Role of Na,K-ATPase α1 and α2 isoforms in the support of astrocyte glutamate uptake.

Glutamate released during neuronal activity is cleared from the synaptic space via the astrocytic glutamate/Na(+) co-transporters. This transport is driven by the transmembrane Na(+) gradient mediated by Na,K-ATPase. Astrocytes express two isoforms of the catalytic Na,K-ATPase α subunits; the ubiquitously expressed α1 subunit and the α2 subunit that has a more specific expression profile. In the brain α2 is predominantly expressed in astrocytes. The isoforms differ with regard to Na+ affinity, which is lower for α2. The relative roles of the α1 and α2 isoforms in astrocytes are not well understood. Here we present evidence that the presence of the α2 isoform may contribute to a more efficient restoration of glutamate triggered increases in intracellular sodium concentration [Na(+)]i. Studies were performed on primary astrocytes derived from E17 rat striatum expressing Na,K-ATPase α1 and α2 and the glutamate/Na(+) co-transporter GLAST. Selective inhibition of α2 resulted in a modest increase of [Na(+)]i accompanied by a disproportionately large decrease in uptake of aspartate, an indicator of glutamate uptake. To compare the capacity of α1 and α2 to handle increases in [Na(+)]i triggered by glutamate, primary astrocytes overexpressing either α1 or α2 were used. Exposure to glutamate 200 µM caused a significantly larger increase in [Na(+)]i in α1 than in α2 overexpressing cells, and as a consequence restoration of [Na(+)]i, after glutamate exposure was discontinued, took longer time in α1 than in α2 overexpressing cells. Both α1 and α2 interacted with astrocyte glutamate/Na(+) co-transporters via the 1st intracellular loop

Transient changes in [Na⁺]_i following exposure to glutamate 200 µM in astrocytes expressing predominantly Na,K-ATPase α1 or α2 isoforms.

<p>A. Astrocyte [Na⁺]_i (mean) in Na,K-ATPase α1 and Na,K-ATPase α2 expressing cells exposed to glutamate 200 µM for 10 min (indicated by horizontal white bar). B. Maximum [Na⁺]_i after 10 min exposure to glutamate 200 µM in α1 (white) and α2 (grey) expressing astrocytes (One-way ANOVA, N = 18 cells, *P<0.05). C. Residual [Na⁺]_i measured at 10 min (indicated with an arrow in Fig. 3A) after discontinuation of glutamate treatment in α1 (white) and α2 (grey) expressing astrocytes (One-way ANOVA, N = 18 cells, *P<0.05).</p