Clozapine acts as an agonist at serotonin 2A receptors to counter MK-801-induced behaviors through a βarrestin2-independent activation of Akt

Abstract

The G protein-coupled serotonin 2A receptor (5-HT2AR) is a prominent target for atypical antipsychotic drugs, such as clozapine. Although clozapine is known to inhibit 5-HT2AR signaling through G protein-dependent mechanisms, it differs from classic GPCR antagonists, in that it also induces 5-HT2AR internalization and activates Akt signaling via a 5-HT2AR-mediated event. In this regard, clozapine may also be considered a functionally selective agonist. The cognate neurotransmitter at the 5-HT2AR, serotonin, also induces 5-HT2AR internalization and Akt phosphorylation. Serotonin promotes interactions with the scaffolding and regulatory protein, βarrestin2, which results in the recruitment and activation of Akt. These interactions prove to be critical for serotonin-induced, 5-HT2AR-mediated behavioral responses in mice. Herein, we sought to determine whether clozapine also utilizes βarrestin2-mediated mechanisms to induce 5-HT2AR signaling, and whether this interaction contributes to its behavioral effects in mice. We demonstrate that unlike serotonin, clozapine-mediated 5-HT2AR internalization and Akt phosphorylation is independent of receptor interactions with βarrestin2. Moreover, clozapine-mediated suppression of MK-801 and phencyclidine (PCP)-induced hyperlocomotion is βarrestin2 independent, although it is dependent upon Akt. These results demonstrate that pharmacologically oppositional ligands, serotonin and clozapine, utilize differential mechanisms to achieve the same 5-HT2AR-meadiated downstream events: Akt phosphorylation and receptor internalization. Although βarrestin2 has no effect on clozapine's actions in vivo, Akt phosphorylation is required for clozapine's efficacy in blocking MK-801- and PCP-induced models of schizophrenic behaviors in mice.

Figure 1

Clozapine antagonizes serotonin-induced 5-HT2AR:βarrestin2 interactions. (a and b) The DiscoveRx PathHunter enzyme complementation assay was utilized to quantify βarrestin2 interactions with the 5-HT2AR in U2OS cells. Mean±SEM are provided. (a) Serotonin (5-HT), but neither clozapine nor M100907, induces robust interactions between βarrestin2 and the 5-HT2AR (two-way analysis of variance (ANOVA) for interaction of drug × concentration: 5-HT vs clozapine, F_(9,60)=354.64, p<0.0001; 5-HT vs M100907, F_(9,50)=397.81, p<0.0001; n=3–4 independent experiments performed in triplicate). (b) 5-HT2AR U2OS DiscoveRx cells were pretreated with either clozapine or M100907 for 60 min before stimulation with 500 nM serotonin. Both M100907 and clozapine fully block serotonin-induced βarrestin2 interactions with the 5-HT2AR, although M100907 is more potent than clozapine (two-way ANOVA for interaction of drug × concentration: M100907 vs clozapine, F_(11,156)=78.27, p<0.0001; n=6–9 independent experiments performed in triplicate). (c) βarrestin2 translocation to the 5-HT2AR was visualized by confocal microscopy of HEK-293 cells transiently transfected with HA-5-HT2AR and βarr2-GFP. βArr2-GFP is localized throughout the cytosol in vehicle (20 μM ascorbate, 0.1% DMSO)-treated cells. Serotonin induces robust recruitment of βarr2-GFP to the plasma membrane (arrow). Clozapine on its own does not induce detectable βarr2-GFP translocation. Pretreatment with clozapine for 10 min before the addition of serotonin abrogates βarr2-GFP recruitment to the membrane. Representative images from three independent transfections are provided (1 μM, 10 min). (d) Confocal microscopy was utilized to visualize HA-5-HT2AR internalization in WT and βarr1/2-KO MEFs. Cells were serum starved for 2 h before live-cell staining of the receptor and drug treatment (10 μM for 30 min). The receptor is localized to the plasma membrane of both WT and βarr1/2-KO MEFs treated with vehicle (20 μM ascorbate, 0.1% DMSO). Serotonin induces HA-5-HT2AR internalization in WT MEFs (arrow), but not MEFs lacking βarrestins. Clozapine induces noticeable internalization in both WT and βarr1/2-KO MEFs (arrows). Representative images from three independent transfections are provided. (e) The percentage of the receptor internalized in MEFs is quantified. Serotonin induces significant internalization in WT MEFs alone, whereas clozapine induces significant internalization in both genotypes (Student's t-test: vehicle vs drug within each genotype, ***p<0.001; n=9–10 cells per condition from at least three independent transfections).

Figure 2

Clozapine induces 5-HT2AR-mediated Akt phosphorylation in a βarrestin2- and Src-independent manner. Primary neurons cultured from frontal cortices of WT and βarr2-KO neonates were serum starved for 1 h before treatment with agonist (1 μM, in 20 μM ascorbate) for 10 min. Lysates were prepared and Akt phosphorylation was determined by western blot analysis. Fold over vehicle was determined by normalizing phosphorylated Akt (p-Akt) to total Akt levels (t-Akt) and then normalizing to the average vehicle (Veh) levels within each experiment. Densitometric analysis (mean±SEM) and representative western blots are provided. (a) Serotonin (5-HT) and clozapine induce Akt phosphorylation over vehicle levels in cortical neurons cultured from WT neonates, but only clozapine activates Akt in βarr2-KO neurons. M100907 does not activate Akt in either genotype (Student's t test: vehicle vs drug within each genotype, **p<0.01, ***p<0.001; n=12–18 WT, 4–10 βarr2-KO, from at least three independent preparations of each genotype). (b and c) Pretreatment with the 5-HT2AR antagonist M100907 (M100, 100 nM) for 15 min blocks serotonin- and clozapine (Cloz)-induced Akt phosphorylation in WT (b) and βarr2-KO (c) neurons (Student's t-test: vehicle vs drug within the same pretreatment group, *p<0.05, **p<0.01; n=6–9, from at least three independent preparations of each genotype). (d) Pretreatment with the Src inhibitor PP2 (1 μM, during the 1 h serum-starvation period) blocks serotonin-induced Akt phosphorylation, but does not block clozapine-induced Akt phosphorylation in WT neurons (Student's t-test: vehicle vs drug within the same pretreatment group, *p<0.05, **p<0.01, ***p<0.001; n=5–8, from four independent preparations).

Figure 3

Clozapine-induced locomotor suppression occurs in the absence of βarrestin2 and with inhibition of Akt. The locomotor activity of WT and βarr2-KO mice was monitored during a 30-min habituation period and for 1 h following treatment with vehicle (0.9% saline) or clozapine (i.p.) (a and b). Both the 3 and 5 mg/kg doses of clozapine significantly inhibit both horizontal activity (a), sum beam breaks, and stereotypy (b), sum stereotypy counts, below vehicle levels (Student's t-test: vehicle vs dose within the same genotype, *p<0.05, **p<0.01, WT vs βarr2-KO of the same dose, ^#p<0.05; n: vehicle=7–9, 1 mg/kg=4–5, 3 mg/kg=5–8, 5 mg/kg=4–8 of each genotype). (c and d) Mice were pretreated with either vehicle (1% DMSO in dH₂O) or 100 ng Akti (i.c.v.) for 30 min before clozapine administration. (c) Clozapine (vehicle+clozapine) decreases total beam breaks (c) and total stereotypy counts (d) observed in the 60 min following treatment compared with vehicle pretreated (vehicle+vehicle) animals. Akti by itself (Akti+vehicle) does not affect locomotion, nor does it modulate clozapine's ability (Akti+clozapine) to decrease horizontal activity (Student's t-test, vehicle vs drug within the same pretreatment group: *p<0.05, **p<0.01; n=5–6 mice per treatment group).

Figure 4

Clozapine suppresses NMDA receptor antagonist-induced hyperlocomotion in both WT and βarr2-KO mice. Locomotor activity was monitored for WT and βarr2-KO mice during a 30-min habituation period, in some cases, a 30-min pretreatment with 1 mg/kg clozapine (i.p.) or vehicle (0.9% saline) and for 1 h following treatment (i.p.) with either MK-801 (a–h) or PCP (i–p). Mean±SEM are shown. (a) WT and βarr2-KO mice do not differ in the sum of horizontal beam breaks observed following administration of three different doses of MK-801 (Student's t-test, p>0.05; n: 0.1 mg/kg=4, 0.3 mg/kg=12, 0.5 mg/kg=6 of each genotype). (b and c) Clozapine pretreatment decreases MK-801-induced (0.3 mg/kg) horizontal beam breaks in WT (b) and βarr2-KO (c) mice (two-way analysis of variance (ANOVA) for WT: for pretreatment, F_(1,480)=43.28, p<0.0001, for time, F_(11,480)=34.67, p<0.0001; for βarr2-KO: for pretreatment, F_(1,612)=70.34, p<0.0001, for time, F_(11,612)=51.19, p<0.0001; n=19–23 WT, 23–30 βarr2-KO per treatment group). (d) Clozapine inhibits the level of horizontal activity observed in the 1-h period following administration of MK-801 (0.3 mg/kg) in both WT and βarr2-KO mice to a similar extent. Total activity observed following clozapine treatment was normalized the average activity in mice that received vehicle and are shown as a percent (Student's t-test: p>0.05). (e) There is also no difference between the genotypes in the sum of the stereotypy counts observed following administration of three different doses of MK-801 (Student's t-test, p>0.05). (f and g) Clozapine pretreatment decreases MK-801-induced (0.3 mg/kg) stereotypy counts in both WT (f) and βarr2-KO (g) mice (two-way ANOVA for WT: for pretreatment F_(1,480)=29.83, p<0.0001, for time, F_(11,480)=23.00, p<0.0001; for βarr2-KO: for pretreatment F_(1,564)=42.06, p<0.0001, for time, F_(11,564)=27.85, p<0.0001). (h) Clozapine inhibits the sum stereotypy counts observed in the 1-h period following administration of MK-801 (0.3 mg/kg) in both WT and βarr2-KO mice to a similar extent (Student's t-test: p>0.05). (i) WT and βarr2-KO mice do not differ in the sum of horizontal beam breaks observed following administration of three different doses of PCP (Student's t-test, p>0.05; n: 3 mg/kg=4; 6 mg/kg=10, 10 mg/kg=10 of each genotype). (j and k) Clozapine pretreatment decreases PCP-induced (10 mg/kg) horizontal beam breaks in WT (j) and βarr2-KO (k) mice (two-way ANOVA for WT: for pretreatment, F_(1,144)=59.02, p<0.0001, for time, F_(11,144)=2.17, p=0.0191; for βarr2-KO: for pretreatment, F_(1,132)=224.12, p<0.0001, for time, F_(11,132)=3.09, p=0.0010). (l) Clozapine inhibits the total level of horizontal activity observed following administration of PCP (10 mg/kg) in both WT and βarr2-KO mice to a similar extent (Student's t-test: p>0.05). (m) WT and βarr2-KO mice do not differ in the total stereotypy counts observed following administration of three different doses of PCP (Student's t-test: p>0.05). (n and o) Clozapine pretreatment decreases PCP-induced (10 mg/kg) stereotypy counts in both WT (n) and βarr2-KO (o) mice (two-way ANOVA for WT: for pretreatment F_(1,144)=65.43, p<0.0001, for time, F_(11,144)=1.80, p=0.0594; for βarr2-KO: for pretreatment F_(1,132)=101.08, p<0.0001, for time, F_(11,132)=2.05, p=0.0288). (p) Clozapine inhibits the total levels of stereotypy counts observed following administration of PCP (10 mg/kg) in both WT and βarr2-KO mice to a similar extent (Student's t-test: p>0.05).

Figure 5

Akti-1/2 inhibits clozapine's ability to suppress MK-801-induced hyperlocomotion in WT mice. (a–d) WT mice were habituated for 30 min, pretreated with either Akti-1/2 (Akti, 100 ng, i.c.v.) or vehicle (0.1% DMSO in dH₂O) for 30 min, treated with either clozapine (1 mg/kg, i.p.) or vehicle (0.9% saline) and then challenged with MK-801 (0.3 mg/kg, i.p.). Locomotor activity was monitored and mean±SEM are shown (n=14 vehicle+vehicle, 8 vehicle+clozapine, 9 Akti+vehicle, 8 Akti+clozapine). (a) Clozapine (vehicle+clozapine) decreases MK-801-induced horizontal beam breaks compared with vehicle pretreated mice (vehicle+vehicle; two-way analysis of variance (ANOVA) for pretreatment: F_(1,240)=65.64, p<0.0001; for time: F_(11,240)=9.30, p<0.0001). Akti on its own (Akti+vehicle) does not decrease MK-801-induced hyperactivity (two-way ANOVA for pretreatment: F_(1,252)=2.22, p<0.1375; for time: F_(11,252)=17.06, p<0.0001). Akti pretreatment (Akti+clozapine) blocks clozapine's ability to decrease MK-801-induced hyperactivity (vehicle+clozapine; two-way ANOVA for pretreatment: F_(1,168)=49.10, p<0.0001; for time: F_(11,168)=12.72, p<0.0001). (b) Analysis of the sum beam breaks observed within each treatment group compared with mice, which received MK-801 alone (vehicle+vehicle) shows that Akti attenuates clozapine's ability to inhibit hyperlocomotion (Student's t-test, vehicle+vehicle vs vehicle+clozapine, **p<0.01; vehicle+clozapine vs Akti+clozapine, ^#p<0.05). (c) Analysis of the counts observed demonstrates that Akti inhibits clozapine's ability to block MK-801-induced stereotypy (two-way ANOVA: vehicle+clozapine vs vehicle+vehicle, for pretreatment: F_(1,240)=61.97, p<0.0001, for time: F_(11,240)=7.39, p<0.0001; Akti+vehicle vs vehicle+vehicle, for pretreatment: F_(1,252)=3.71, p=0.0551, for time: F_(11,252)=13.30, p<0.0001; Akti+clozapine vs vehicle+clozapine, for pretreatment: F_(1,168)=46.98, p<0.0001, for time: F_(11,168)=9.64, p<0.0001). (d) Analysis of the total stereotypy counts observed within each treatment group, compared with mice, which received MK-801 alone (vehicle+vehicle), shows that Akti attenuates clozapine's ability to inhibit hyperlocomotion (Student's t-test, vehicle+vehicle vs vehicle+clozapine, *p<0.05; vehicle+clozapine vs Akti+clozapine, ^#p<0.05). (e) WT mice were pretreated with Akti (100 ng, i.c.v.) or vehicle (01.% DMSO in dH₂O) for 30 min and then treated with either clozapine (1 mg/kg, i.p.) or vehicle (0.9% saline). Forty-five minutes later frontal cortex was isolated, lysates were prepared, and Akt phosphorylation levels were determined by western blot analysis. Mean±SEM are shown. Pretreatment with Akti significantly decreases clozapine-mediated Akt phosphorylation in the mouse frontal cortex (Student's t-test: vehicle+vehicle vs drug challenge, *p<0.05, **p<0.01; vehicle+clozapine vs Akti+clozapine, ^#p<0.05; n=8–11 for each treatment group).