The present address of Henrik Hansen, Senior Scientist, PhD, Gubra aps, Agern Allé 1, DK-2970 Hørsholm, Denmark.
Kv7 (KCNQ) channel openers normalize central 2-deoxyglucose uptake in a mouse model of mania and increase prefrontal cortical and hippocampal serine-9 phosphorylation levels of GSK3β
Article first published online: 14 MAR 2012
© 2012 Neurosearch A/S. Journal of Neurochemistry © 2012 International Society for Neurochemistry
Journal of Neurochemistry
Volume 121, Issue 3, pages 373–382, May 2012
How to Cite
Kristensen, L. V., Sandager-Nielsen, K. and Hansen, H. H. (2012), Kv7 (KCNQ) channel openers normalize central 2-deoxyglucose uptake in a mouse model of mania and increase prefrontal cortical and hippocampal serine-9 phosphorylation levels of GSK3β. Journal of Neurochemistry, 121: 373–382. doi: 10.1111/j.1471-4159.2012.07704.x
- Issue published online: 10 APR 2012
- Article first published online: 14 MAR 2012
- Accepted manuscript online: 22 FEB 2012 02:39PM EST
- Received January 6, 2012; revised manuscript received January 25, 2012; accepted February 15, 2012.
- bipolar disease;
- KCNQ channel;
J. Neurochem. (2012) 121, 373–382.
Several metabolic neuroimaging studies have indicated that bipolar patients with mania exhibit alterations in metabolic activity, suggesting that perturbations in corticolimbic function contribute to the functional deficits associated with the disease. Because pharmacological stimulation of Kv7 channel function has shown anti-manic like efficacy in the D-amphetamine and chlordiazepoxide (AMPH+CDP) induced hyperactivity mouse model of mania, we addressed whether this effect of Kv7 channels could be associated with changes in cerebral [14C]2-deoxyglucose (2-DG) uptake, a surrogate marker of brain metabolic activity. Acute administration of the Kv7 channel modulators, retigabine (pan Kv7.2-Kv7.5 channel opener) and ICA-27243 (Kv7.2/Kv7.3 channel-preferring opener) reduced 2-DG uptake in several mouse forebrain structures with a brain regional signature similar to the mood stabilizers, lithium and valproate. Combined administration of AMPH+CDP enhanced 2-DG uptake in the striatum, cortex and thalamus, and both retigabine and ICA-27243 fully prevented this stimulatory effect of AMPH+CDP. In addition, both Kv7 channel openers dose-dependently increased phospho-serine-9 levels of GSK3β in the prefrontal cortex and hippocampus, a common molecular mechanism shared by anti-manic drugs. In combination, these data emphasize the potential of Kv7 channel openers in the treatment of bipolar disorder, and suggest that heteromeric Kv7.2/Kv7.3 channels may present a novel anti-manic therapeutic target.
ventrolateral orbitofrontal cortex
Bipolar disorder (BD) is a common, chronic and recurring medical disorder characterized by episodes of mania, that is, extremely elevated mood, increases in motor activity, racing thought patterns, impaired judgment, decreased sleep and sometime psychosis, and depression. The prognosis for patients with BD is poor as the patients frequently experience relapse, lingering residual symptoms, cognitive impairments and psychosocial disability (Belmaker 2004). The prevalence of full-blown BD is thought to be around 1%, but may be up to 5% if less severe bipolar spectrum disease states are also accounted for (Merikangas et al. 2011). Despite advances in its diagnosis and treatment, the underlying neuropathobiology of BD remains largely unknown. A growing body of data, however, supports the view that BD arises from structural and functional impairments in various brain regions with several clinical metabolic neuroimaging studies utilizing [18F]2-deoxyglucose and cerebral blood flow techniques have reported differences in metabolic activity in corticolimbic structures in BD patients linked with disrupted emotional and executive functioning (al-Mousawi et al. 1996; Blumberg et al. 2000; Drevets 2000; Brooks et al. 2010).
Kv7 channels, the molecular correlate of the M-current, are voltage-dependent potassium channels composed of homo- and heteromeric complexes of five different Kv7 subunits (Kv7.1-5, encoded by the kcnq1-5 genes). Unlike Kv7.1, all Kv7.2-5 subunits are expressed in the CNS (Jentsch 2000), and they generally serve to dampen neuronal excitability (Gribkoff 2008; Hansen et al. 2008). A susceptibility locus for BD has been identified on chromosome 8q24, a region which contains the kcnq3 gene (Avramopoulos et al. 2004), and a genetic association between dominant-negative Kv7.2 splice variants in BD patients has been reported (Borsotto et al. 2007). Also, Kv7.2 channel function is inhibited by interaction with GSK3βin vitro, which can be rescued by lithium (Borsotto et al. 2007), a standard mood stabilizer known to increase inhibitory serine-9 phosphorylation [pSer9] which occludes GSK3β catalytic activity (Klein and Melton 1996; Stambolic et al. 1996; De Sarno et al. 2002). In pre-clinical settings, various Kv7 channel openers have been characterized in the D-amphetamine (AMPH) + chlordiazepoxide (CDP) behavioral hyperactivity model of mania. In the AMPH+CDP model, the inhibitory effect of retigabine and the Kv7.2/Kv7.3 channel-preferring opener, ICA-27243, was equivalent to lithium (Dencker et al. 2008; Redrobe and Nielsen 2009), thus supporting the theoretical rationale for the involvement of Kv7 channel function in mania.
We therefore tested the hypothesis that the potential anti-manic profile of Kv7 channel openers may be associated with changes in brain regional metabolic activity by mapping the influence of Kv7 channel openers on brain regional [14C]2-DG uptake under baseline conditions as well as in the mouse AMPH+CDP model of mania. In addition, we sought to determine whether Kv7 channel openers would also affect cerebral [pSer9]GSK3β levels in vivo.
Materials and methods
All animals were allowed at least 7 days of acclimatisation to the laboratories before use. C57Bl/6 male (20–25 g, Harlan, the Netherlands) mice were housed in groups of eight in Macrolon III cages (20 cm × 40 cm × 18 cm) contained in Scantainers (Scanbur A/S, Ejby, Denmark) under a 12-h light/dark cycle (lights on at 06:00 hours) with free access to standard chow and tap water. Experiments were conducted between 7:00 hours and 13:00 hours in temperature and humidity regulated rooms (22–24°C; relative humidity 60–70%). All procedures were carried out in accordance with internationally accepted principles for the care and use of laboratory animals and were approved by the Danish Committee for Animal Research.
Retigabine, ICA-27243 and XE-991 were synthesized at NeuroSearch A/S. Flumazenil, chlordiazepoxide hydrochloride, D-amphetamine sulfate, memantine hydrochloride and sodium valproate were obtained from Sigma (St Louis, MO, USA). Lithium chloride was obtained from Merck (Darmstadt, Germany). All compounds were administered intraperitoneally (i.p.) in an injection volume of 10 mL/kg. Doses were calculated using salt weights, where salts were used. Retigabine, ICA-27243, (S)-BMS-204352, XE-991, (S)-1, lithium chloride and sodium valproate were dissolved in 10% Tween-80 in saline. D-amphetamine sulphate (AMPH) and chlordiazepoxide (CDP) hydrochloride were dissolved in saline and administered as a cocktail (AMPH+CDP). [14C]2-deoxyglucose (2-DG, 2-[1-14C]-deoxy-d-glucose, 45–60 mCi/mmol) was purchased from Perkin Elmer (Skovlunde, Denmark) and diluted to standard injection solution with 0.9% NaCl.
The methodology was performed according to Dedeurwaerdere et al. (2011) with a minor modification, as animals were fasted prior to administration of 2-DG. A pilot study indicated that endogenous plasma glucose levels were lowered after a minimum of 17 h of fasting, as compared with non-fasted mice (data not shown). From 17 to 21 h of food deprivation endogenous plasma glucose levels were stabilized to approximately 55% of the non-fasted level, whereas further extension of the fasting period caused less reductions in plasma glucose levels, presumably due to compensatory endogenous glucose release from peripheral tissue deposits. In addition, fasting also facilitated more efficacious cerebral 2-DG uptake (data not shown). Importantly, the fasting procedure also mimics general human PET scan settings where subjects are fasted prior to the scanning procedures to increase the [18F]2-deoxyglucose signal-to-noise ratio.
Animals were single-housed the day before the experiment and subsequently fasted for 17–20 h before administration of 2-DG (0.16 μCi/g body weight). For initial assessment of time–response effects of retigabine on brain regional 2-DG levels, the mice were simultaneously administered retigabine and 2-DG, then returned to their individual home cages, whereupon they were sacrificed 30 or 60 min after drug administration. For single time-point analyses, all drugs were administered 15 min prior to 2-DG and the animals were killed 45 min after 2-DG administration, that is, total test drug exposure time was 60 min. To assess Kv7 channel specific effects of retigabine on 2-DG uptake, the Kv7 channel blocker XE-991 (0.3 or 1.0 mg/kg, i.p.) was administered 15 min prior to retigabine treatment. Plasma glucose levels were unaffected by any drug used in the 2-DG experiments (data not shown).
For time–response assessment of AMPH (4.0 mg/kg) + CDP (2.5 mg/kg) effects on 2-DG uptake, the AMPH+CDP cocktail and 2-DG, respectively, were administered simultaneously, whereafter the animals were killed at 15, 30, or 60 min after the 2-DG injection. For determination of retigabine–AMPH+CDP interactions, animals were pre-treated with either vehicle (10% Tween-80 in 0.9% NaCl) or retigabine (3.0 or 10 mg/kg) 30 min before simultaneous administration of the AMPH+CDP challenge dose and 2-DG, respectively, whereupon the experiment was terminated 60 min after the last drug injection.
For analysis of brain regional 2-DG uptake, the brain was instantly removed from the skull, and immersed in powderized dry ice. The brains were kept at −80°C until further processing. The brains were sectioned on a cryostat CM3050S (Leica, Ballerup, Denmark) in coronal sections of 20 μm thickness and serial sections (at least six sections per slide) were sampled on superfrost plus glass slides (Thermo Scientific, Braunschweig, Germany) at the following coronal levels: prefrontal cortex (bregma +1.98 mm), striatum (bregma +1.10 mm) and hippocampus (bregma −1.82 mm) according to Paxinos and Franklin (2004). The sections were dried on a hotplate (60°C) for approximately 20 min and mounted on Biomax MR films (35 × 43 cm; Sigma-Aldrich, Broendby, Denmark) together with a calibration [14C]-standard (GE Healthcare Life Sciences, Buckinghamshire, UK). The autoradiographic films were exposed for 2 days before development. The autoradiographic films were scanned on a Bio-Rad GS-800 Calibrated Imaging Densitometer scanner (Bio-Rad, Hercules, CA, USA) with highest resolution (36.3 × 36.3 μm) and densitometry was performed using QuantityOne (Bio-Rad). The optical density of individual regions of interest was calculated by extrapolating from the [14C]-standard calibration curve. Regions of interest were manually outlined with reference to Paxinos and Franklin (2004). The analysed brain regions were: medial prefrontal cortex (PFC), ventrolateral orbitofrontal cortex (VLO), striatum, hippocampal dentate gyrus (i.e. stratum lacunosum moleculare), thalamus, and retrosplenial cortex. Three representative coronal brain sections on every slide were analysed bilaterally, averaged and the corresponding glass slide background level was subtracted.
Analysis of GSK3β phosphorylation
The effect of reference Kv7 channel openers on [pSer9]GSK3β levels were assessed in the mouse prefrontal cortex and hippocampus. The mice were housed in groups of eight, and had free access to food and water during the experiment. Animals were treated with test compounds and returned to their home cage until termination of the experiment. To assess Kv7 channel-specific effects of retigabine on [pSer9]GSK3β levels, the Kv7 channel blocker XE-991 (1.0 mg/kg, i.p.) was administered 15 min prior to retigabine treatment. The animal was decapitated, the left and right hippocampus and prefrontal cortex were quickly removed from the brain, samples were placed individually in Eppendorf tubes, and snap-frozen in liquid nitrogen. They were kept at −80°C until further use. Total tissue protein was extracted with Milliplex Map Lysis buffer (Millipore, Copenhagen, Denmark) added a protease inhibitor cocktail (Complete, Roche, Denmark) and phosphatase inhibitor cocktail (Sigma-Aldrich). The tissue was homogenized in an Eppendorf tube on ice with a Pellet Pestle Motor for approximately 30 s, and incubated for 15 min on ice. The samples were then centrifuged at 16 000 g (4°C) for 10 min. The cleared supernatant was collected in aliquots and stored at −80°C until further use. The total protein content of the samples was determined using a DC protein assay, according to the manufacturer’s instructions (Bio-Rad). All samples were determined in triplicate in a 96-well microplate, and absorbance was measured at 655 nm (Bio-Rad MicroPlate Reader 680, Copenhagen, Denmark). Determination of [pSer9]GSK-3β levels was subsequently performed with a Luminex 200 instrument in 96-well plates with application of 20 μg total tissue protein per sample, 100 μL sample buffer and 50 μL mouse [pSer9]GSK3β antibody-coated microsphere beads (Invitrogen, Camarillo, CA, USA), according to the manufacturer’s instructions. At least 100 beads were counted per sample.
Data analysis, graphics and statistics were performed with GraphPad Prism 4 (Graphpad Software, San Diego, CA, USA). Statistical analyses were performed using unpaired t-test or one-way anova with Tukey’s post hoc test where appropriate. Data are presented as mean ± SEM. Difference between means were considered statistically significant when p < 0.05.
Drug effects on baseline 2-DG uptake
Following intraperitoneal administration of 2-DG to male C57 mice, autoradiographic analysis of the CNS uptake showed a brain regional signature with the thalamus, striatum and VLO having higher baseline levels relative to the cortex, septum and dentate gyrus, see Fig. 1a and b. This general pattern is in agreement with previous reports on rodent imaging studies using acute intraperitoneal (Miyamoto et al. 2000; Dedeurwaerdere et al. 2011) or intravenous routes of 2-DG administration (Sokoloff et al. 1977; Duncan et al. 1998).
Acute administration of retigabine or ICA-27243 resulted in a reduction of baseline 2-DG uptake in several brain regions in the mouse with both drugs significantly suppressing baseline 2-DG uptake in the PFC, VLO, striatum, and thalamus (Fig. 1a and b). Accordingly, acute administration of retigabine (10 mg/kg) significantly reduced basal 2-DG uptake in several brain regions in the mouse, including the PFC (−26%, p = 0.015), VLO (−17%, p = 0.032), striatum (−19%, p = 0.003), dentate gyrus (−14%, p = 0.018) and thalamus (−17%, p = 0.006). The effect of retigabine was comparable to ICA-27243 (5.0 mg/kg) in the VLO (−27%, p = 0.008), striatum (−22%, p = 0.007), and thalamus (−22%, p = 0.010). ICA-27243 also affected the retrosplenial cortex (−23%, p = 0.018), and showed a statistical trend in affecting the PFC (p = 0.06) and dentate gyrus (p = 0.07).
To compare with reference mood stabilizers, LiCl and valproate were also characterized for potential modulatory effects on basal 2-DG uptake. LiCl (120 mg/kg) significantly reduced baseline 2-DG uptake in the PFC (−21%, p = 0.012), VLO (−21%, p = 0.016), striatum (−23%, p = 0.003) and dentate gyrus (−18%, p = 0.029), whereas the thalamus (p = 0.117) and retrosplenial cortex (p = 0.208) were unaffected. Valproate (400 mg/kg) was observed to inhibit basal 2-DG uptake in the PFC (−17%, p = 0.01), VLO (−23%, p = 0.004), striatum (−20%, p = 0.001), thalamus (−29%, p = 0.0001) and retrosplenial cortex (−28%, p = 0.001), but only showed a statistical trend (p = 0.06) in reducing 2-DG uptake in the dentate gyrus.
To address the relevance of selective Kv7 channel activation by retigabine, the pan-Kv7 channel blocker XE-991 was administered prior to retigabine (10 mg/kg). XE-991 (0.3 or 1.0 mg/kg) had no significant effect on baseline 2-DG uptake in the PFC, VLO, dentate gyrus, and thalamus, whereas XE-991 (1.0 mg/kg) significantly stimulated 2-DG uptake in the striatum (+22%, p < 0.05) and thalamus (+53%, p < 0.001), see Fig. 2a and b. Both doses were further characterized in combination with retigabine. While pre-treatment with XE-991 (0.3 mg/kg) fully reversed the inhibitory effect of retigabine only in the striatum (p < 0.05), administration of a higher dose of XE-991 (1.0 mg/kg) prevented retigabine-induced 2-DG reductions in all brain regions examined, that is, the PFC (p < 0.001), striatum (p < 0.001), dentate gyrus (p < 0.05), and thalamus (p < 0.001). For the striatal and thalamic regions, it should be noted that the combination of XE-991 (1.0 mg/kg) and retigabine resulted in an augmented 2-DG uptake as compared with baseline levels, which reflects the stimulatory 2-DG uptake effect of XE-991 (1.0 mg/kg) per se (Fig 2a and b).
Determination of 2-DG uptake effects in the mouse AMPH+CDP model of mania
The time–response study revealed that acute administration of an AMPH+CDP cocktail stimulated 2-DG uptake at 1 h post-treatment, whereas shorter administration periods had no significant effects on 2-DG uptake in the CNS (Figs 3 and 4b). At 1 h post-treatment, AMPH+CDP significantly increased 2-DG uptake in the striatum (+17%, p = 0.033), thalamus (+22%, p = 0.009) and retrosplenial cortex (+23%, p = 0.042). Hence, the effect of acute retigabine pre-treatment on 2-DG uptake in the AMPH+CDP model was investigated after 1 h of AMPH+CDP exposure. Both doses of retigabine (3 and 10 mg/kg) normalized 2-DG uptake when administered prior to the AMPH+CDP cocktail (Fig. 4). The inhibitory effect of retigabine pre-administration was apparent in all brain regions where 2-DG uptake was affected by AMPH+CDP, that is, striatum, thalamus and retrosplenial cortex.
Effects of Kv7 channel openers on GSK3β phosphorylation
Figure 5 shows the regulation of [pSer9]GSK3β levels in the prefrontal cortex and hippocampus of mice acutely treated with retigabine or ICA-27243. Retigabine dose-dependently stimulated hippocampal [pSer9]GSK3β levels by +17% (10 mg/kg, p < 0.05) and +48% (20 mg/kg, p < 0.001 C), respectively (Fig. 5c). Although to a lesser extent, retigabine (20 mg/kg) also significantly increased [pSer9]GSK3β phosphorylation in the PFC (+9%, p < 0.05, Fig. 5a). ICA-27243 (10 mg/kg) showed a similar stimulatory effect on [pSer9]GSK3β levels with a greater stimulation in the hippocampus (+28%, p < 0.01, Fig. 5d) than in the PFC (+21%, p < 0.01, Fig. 5b).
The pan Kv7 channel blocker, XE-991 (1.0 mg/kg), had no effect on baseline [pSer9]GSK3β levels, however, when administered prior to retigabine, XE-991 fully prevented the stimulatory effect of retigabine (20 mg/kg) on hippocampal [pSer9]GSK3β levels (Fig. 5e).
In this report, we describe the impact of pharmacological Kv7 channel modulation in the mouse CNS on two important biochemical markers associated with BD, that is, cerebral glucose metabolism and [pS9]GSK3β.
The pan-Kv7 opener, retigabine, suppressed baseline 2-DG uptake in several brain regions in the mouse, including the PFC, VLO, striatum, dentate gyrus, and thalamus, which was blocked by pre-administration of the pan-Kv7 channel blocker, XE-991, thus being indicative of a Kv7 channel-specific effect of retigabine. The brain regional distribution as well as the magnitude of effect by ICA-27243, a Kv7.2/Kv7.3-preferring opener (Wickenden et al. 2008), was generally similar to retigabine which points to the possibility that activation of heteromeric Kv7.2/Kv7.3 channels in the mouse forebrain was the major Kv7 channel isoform responsible for the dampening of basal 2-DG uptake. Accordingly, Kv7.2 and Kv7.3 channels are the most widely distributed Kv7 channel isoforms in the murine CNS (Cooper et al. 2001; Geiger et al. 2006; Weber et al. 2006). It should be noted that the rather low doses of retigabine (3–10 mg/kg) and ICA-27243 (3–5 mg/kg) used were well within ranges known not to affect locomotor activity or body temperature regulation in mice (Redrobe and Nielsen 2009; Kristensen et al. 2011), thereby rendering it unlikely that the Kv7 channel-induced dampening effect on 2-DG uptake was caused by generalized inhibitory effects on motor function or thermogenesis.
Because Kv7 channels are strongly activated by depolarization events causing Kv7 channel opening and repolarization of the cell, hence potently regulating neuronal excitability, this likely indicates that suppression of cerebral 2-DG uptake by stimulation of Kv7 channel function was caused by inhibition of neuronal excitability. This is consistent with numerous studies showing that retigabine inhibits action potential generation and, conversely, pharmacological blockade of Kv7 channel activity leads to neuronal excitation in several brain regions, including the striatum, hippocampus, thalamus and prefrontal cortex (Yue and Yaari 2004; Shen et al. 2005; Hu et al. 2007; Kasten et al. 2007; Smith et al. 2007; Santini and Porter 2010).
The mapping of mouse brain regions affected by enhanced Kv7 channel activity indicates that Kv7 channel openers reduce basal 2-DG uptake and, by inference, consequently dampens cerebral metabolic activity. Notably, acute administration of lithium or valproate at doses typically used in animal models of BD (Einat 2007; O’Donnell and Gould 2007) also suppressed baseline 2-DG uptake with many similarities to the brain regional signature evoked by Kv7 channel openers. Hence, lithium reduced baseline 2-DG uptake levels in the PFC, VLO, striatum and dentate gyrus, and with the exception of the dentate gyrus, valproate also affected 2-DG uptake in all brain regions examined. Lithium and valproate both reduce cerebral glucose metabolic rate and blood flow at therapeutically relevant doses in humans, however, typically only assessed after long-term treatment (Leiderman et al. 1991; Gaillard et al. 1996; Bell et al. 2005), thus hampering direct comparison to the present pre-clinical acute settings.
Various animal models have been proposed as tools to characterize potential anti-manic properties of novel compounds. The AMPH+CDP hyperactivity paradigm is established as a rapid drug screening model, with the repetitive hyperlocomotor behavioral features thought to reflect core mania-like excessive psychomotor activity (Aylmer et al. 1987). In contrast to administration of AMPH alone, the AMPH+CDP model is more sensitive to standard mood stabilizers, e.g. lithium, valproate and carbamazepine, at doses not affecting basal locomotor activity (Poitou et al. 1975; Okada et al. 1990; Arban et al. 2005), thus showing predictive validity. Using the AMPH+CDP model, we observed a forebrain regional response where the striatum, thalamus and retrosplenial cortex exhibited significant increases in 2-DG uptake. Notably, retigabine normalized 2-DG uptake in these motor regions when administered prior to the AMPH+CDP cocktail, suggesting that this brain regional modulatory effect underlies the previously reported anti-manic like behavioural effects of retigabine and other Kv7 channel openers in this model (Dencker et al. 2008; Redrobe and Nielsen 2009).
The present regional pattern of AMPH+CDP-induced 2-DG uptake in mice is similar to that reported on acute administration of AMPH (Miyamoto et al. 2000). This argues for AMPH being the essential denominator for the forebrain regional 2-DG signature of the AMPH+CDP combination. Interestingly, AMPH administration to normal human volunteers precipitates a mania-like syndrome concomitantly with a significant increase in 2-DG uptake in the cortex, caudate-putamen and thalamus being sensitive to lithium and valproate treatment (Vollenweider et al. 1998; Bell et al. 2005). While several neuroimaging studies on BD patients with mania also indicate hypermetabolism in corticolimbic regions which may be linked to disrupted emotional and executive functioning (Blumberg et al. 2000; Rubinsztein et al. 2001; Altshuler et al. 2005), others report a mixed profile of hypo- and hypermetabolic brain regional activity in manic patients (Gonul et al. 2009; Brooks et al. 2010). Hence, no clear consensus exists as to whether manic patients display a uniform pattern of cerebral metabolic changes and we may thus conclude that the 2-DG signature in the AMPH+CDP mania model to a greater degree resembles the generalized hypermetabolic state observed in a subset of neuroimaging studies.
While blocking the effect of retigabine in all brain regions examined, the higher dose (1.0 mg/kg) of XE-991 also triggered hypermetabolic 2-DG activity in the striatum per se, a key brain region involved in dopaminergic neurotransmission. The anti-dopaminergic effects of retigabine have been addressed extensively by our laboratory and others, leading to the notion that Kv7 channel activation strongly suppresses dopaminergic activity by targeting both intrastriatal and extrastriatal Kv7 channels (Hansen et al. 2008). This aspect of Kv7 channel pharmacology may be applied to the BD pathophysiology, as altered function of the dopaminergic system is associated with mania (Cousins et al. 2009; Gonul et al. 2009).
It is noteworthy that both retigabine and ICA-27243 increased [pSer9]GSK3β levels in the hippocampus and prefrontal cortex, regions highly implicated in the emotional and cognitive aspects of BD (Cousins et al. 2009), hence lending further support to the hypothesis that positive modulation of Kv7 channel function affects a key signalling pathway in mania similar to standard anti-manic drugs. A genetic association between dominant-negative Kv7.2 splice variants in BD patients has recently been reported, and it is proposed that splice isoforms may heteromerize with normal Kv7.2 subunits to impair inhibitory Kv7.2 channel influence on excitatory neuronal activity (Borsotto et al. 2007). Interestingly, the authors also showed that the GSK3β occludes Kv7.2 channel activity by catalyzing inhibitory phosphorylation of the Kv7.2 channel, and lithium rescues Kv7.2 channel function in vitro. The association of GSK3β with Kv7 channel function is relevant, as genome-wide association BD studies have identified polymorphisms implicating GSK3β signalling cascades (Baum et al. 2008), and a subset of GSK3β gene polymorphisms are also linked to BD (Luykx et al. 2010). In agreement, GSK3β-over-expressing mice show a mania-like phenotype of hyperactivity and increased hedonic drive and defective [pSer9]GSK3β function in mice results in impaired memory (Prickaerts et al. 2006; Dewachter et al. 2009). In reverse analogy, heterozygous loss of GSK3β expression mimics the behavioral and molecular responses to lithium treatment (O’Brien et al. 2004) and several anti-manic agents, including lithium, valproate and antipsychotics, are known to inhibit GSK3β function by increasing inhibitory serine-9 phosphorylation of GSK3β or directly interfering with its catalytic site (Klein and Melton 1996; Stambolic et al. 1996; De Sarno et al. 2002). In addition, [pSer9]GSK3β levels are found inversely correlated with the severity of manic-like symptoms and lithium treatment increase [pSer9]GSK3β levels in BD patients (Li et al. 2007; Polter et al. 2010).
In this report, we demonstrate that Kv7 channel openers reduce baseline cerebral glucose metabolic activity similar to standard mood stabilizing agents. The Kv7 channel openers also prevented CNS hypermetabolic activity in the acute AMPH+CDP mouse model of mania. In addition, Kv7 channel openers increase prefrontal cortical and hippocampal [pSer9]GSK3β levels, a common feature also shared by mood stabilizers. These pre-clinical data emphasizes the potential utility of Kv7 channel openers in the treatment of bipolar disorders, and further suggests that Kv7.2/Kv7.3 channels present a novel potential molecular target in the management of mania. Interestingly, retigabine has recently been approved for adjunctive therapy in partial-onset seizures (French et al. 2011) and because bipolar patients often experience therapeutic benefit from treatment with anticonvulsant drugs (Altamura et al. 2011), this implies that Kv7 channel openers may also prove to have clinical utility in the treatment of BD.
The authors would like to acknowledge Charlotte Holtoft, Pernille O. Hulgaard, Tine Østergaard, Bettina Lyngsø Bengtsen and Hanne Nord Søndergaard for excellent technical assistance. The authors have no conflict of interest to declare.
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