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Neuroscience Graduate Group, University of California, Davis, California 95618Center for Neuroscience, University of California, Davis, California 95618
Neuroscience Graduate Group, University of California, Davis, California 95618Center for Neuroscience, University of California, Davis, California 95618
Neuroscience Graduate Program, University of California, San Francisco, California 94158Department of Neurology, Kavli Institute for Fundamental Neuroscience, Weill Institute for Neurosciences, University of California, San Francisco, California 94158
Center for Neuroscience, University of California, Davis, California 95618Department of Physiology and Membrane Biology, UC Davis School of Medicine, Davis, CA 95616
Second generation antipsychotics (SGAs) are front-line treatments for serious mental illness. Often, individual patients benefit only from some SGAs and not others. The mechanisms underlying this unpredictability in treatment efficacy remain unclear. All SGAs bind the D3 dopamine receptor (D3R) and are traditionally considered antagonists for dopamine receptor signaling.
Methods
Here, we use a combination of two photon calcium imaging, in vitro signaling assays, and mouse behavior to assess signaling by SGAs at the D3R.
Results
We report that some clinically important SGAs function as arrestin-3-agonists at D3R, resulting in modulation of calcium channels localized to the site of action potential initiation in prefrontal cortex pyramidal neurons. We further show that chronic treatment with an arrestin-3-agonist-SGA, but not an antagonist-SGA, abolishes D3R function through post-endocytic receptor degradation by G-protein coupled receptor-associated sorting protein-1 (GASP1).
Conclusions
These results implicate D3R-arrestin-3 signaling as a source of SGA variability, highlighting the importance of including arrestin-3 signaling in characterizations of drug action. Furthermore, they suggest that post-endocytic receptor trafficking that occurs during chronic SGA treatment may contribute to treatment efficacy.
Second generation antipsychotics (SGAs) are important tools in the management of serious mental illness (SMI) including bipolar disorder, major depressive disorder, schizophrenia, and schizoaffective disorder (
). Each of these medications has unique dosing, pharmacokinetics, effect/side-effect profiles, and cost, and clinicians must often prescribe several drugs before finding a treatment regimen that suits individual patients (
). This approach primarily reflects an incomplete understanding of the molecular mechanisms underlying variable treatment efficacy in patients, even though these drugs have been in use for decades (
). All SGAs are antagonists or partial agonists for G-protein signaling at the D2 dopamine receptor (D2R), and their clinical efficacy is thought to rely on their ability to block dopamine signaling at D2R (
), and the positron emission tomography (PET) experiments in human patients that form the foundation of the D2R antagonism hypothesis of SGA efficacy did not distinguish between D2R and D3R occupancy (
Dopamine receptors, like most G-protein coupled receptors (GPCRs), not only engage G-proteins upon activation, but also recruit other effectors, including arrestin-3 (β-arrestin-2) (
). Arrestin-3 recruitment both “arrests” the canonical G-protein signal and also scaffolds non-canonical kinase activity, including Extracellular-Signal-Regulated-Kinase-1/2 (ERK) (
). We recently demonstrated that ERK signaling via arrestin-3 is required for D3R to modulate calcium channel CaV3.2 at the axon initial segment (AIS) after agonist activation (
). There has been no previous assessment of SGA ability to recruit arrestin-3 to D3R, despite the importance of D3R in SGA activity. Here, we report that some clinically-important SGAs are “arrestin-biased” agonists at D3R, promoting recruitment of arrestin-3 and ERK activation in the absence of G-protein activation. We further demonstrate that only the arrestin-biased SGAs modulate CaV3.2 activity in layer V neurons of the PFC. These findings provide a novel means by which SGAs can be divided into two functional classes based on their acute effects.
Importantly, the full therapeutic effect of SGAs often takes weeks of treatment. This suggests that mechanisms other than acute signaling contribute to drug effect. In addition to its acute signaling roles, arrestin-3 also facilitates endocytosis (internalization) of GPCRs through interaction with protein components of the clathrin-coated pit (
). Following endocytosis, both D2R and D3R are targeted for degradation in the lysosome via interaction with the GPCR-associated sorting protein-1 (GASP1) (
Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1.
). Therefore, agonists that drive endocytosis decrease receptor surface levels over time, while conversely, antagonists that block both G-protein and arrestin-3 recruitment prevent dopamine-mediated endocytosis and thereby maintain receptor levels over time (
). We hypothesized that chronic treatment with an arrestin-biased SGA would decrease the number of functional D3R and would do so with a time course more aligned with observed SGA treatment efficacy. We demonstrate here that chronic treatment with an arrestin-biased SGA eliminates the acute effects of D3R on AIS CaV3.2 channel function in wild type (WT) mice but not in mice with conditional deletion of GASP1. We further demonstrate that mice treated chronically with an arrestin-3-biased SGA develop tolerance to the locomotor-inhibitory effects of the drug and that this tolerance is abolished in mice lacking GASP1 in D3+ neurons. These findings provide a new mechanistic framework for understanding the therapeutic actions of SGA drugs.
METHODS AND MATERIALS
Briefly, acute and chronic signaling engaged at D3 receptors was assessed with molecular approaches in heterologous expression systems, then validated ex vivo using whole-cell electrophysiology and 2-photon imaging and in vivo with behavior. Please see Supplemental Methods for details.
RESULTS
Quinpirole modulates AIS calcium through D3R and arrestin-3 in PFC
The axon initial segment (AIS) is enriched with a number of ion channel classes, including CaV3.2 calcium channels that are the target of neuromodulation via D3R (
). D3R-dependent modulation hyperpolarizes steady-state voltage-dependent inactivation of CaV3.2 in a PKC- and ERK-dependent manner, leading to a reduction in high-frequency bursts of action potentials (APs) that depends in part on CaV3.2 activity (
). In cartwheel cells of the dorsal cochlear nucleus (DCN), we showed that this effect requires both D3R and non-canonical arrestin-3 signaling (Fig. 1A) (
). We also reported previously that quinpirole, a D2/3R agonist, modulates CaV3.2 at the AIS specifically in D3R-expressing (D3+) pyramidal cells in PFC (
). To examine whether this D3R-CaV3.2 modulation at the AIS in PFC was also mediated by arrestin-3, we performed whole-cell current-clamp recordings from D3+ PFC pyramidal neurons in wild type (WT), D3R knock-out (D3 KO), and arrestin-3 knock-out (arrestin-3 KO) mice. D3+ neurons were identified either by fluorescent labeling of D3+ neurons in D3-Cre::Ai14 mice or by targeting neurons with specific intrinsic electrophysiological properties that allow for unambiguous identification of D3+ pyramidal cells (
). Using this approach (Fig. 1B), we replicated our previous findings that AIS calcium was reduced by 30.4 ± 2.9% after 20-minutes of quinpirole (Fig. 1C-D) versus time-locked vehicle controls (Fig. 1D). There was no quinpirole effect on AIS calcium in D3 KO mice or arrestin 3-KO mice (Fig. 1D). Furthermore, inhibition of GPCR kinase 2/3 with CMPD101 (30 μM) blocked quinpirole-induced AIS calcium modulation (Fig. S1). Taken together these data indicate that, in PFC, quinpirole-mediated modulation of AIS calcium in D3+ neurons requires arrestin-3 (Fig. 1A).
Figure 1Quinpirole modulates AIS calcium through D3R and arrestin-3 in PFC. A. Schematic of quinpirole modulation of calcium at the AIS of a D3+ pyramidal cell in PFC. B. Left, 2PLSM z-stack of a D3+ pyramidal neuron visualized with Alexa Fluor 594. AIS denoted with bracket. Right, example linescan of AIS calcium averaged over 40 trials. APs were evoked with somatic current injection (3 at 50 Hz, 2 nA, 2 ms per stimulus). Linescan data displayed as mean ± SEM. C. Representative effect of quinpirole on AIS calcium in a D3+ neuron, averaged over 20 trials per condition. Baseline, black; quinpirole, purple. D. Peak AIS calcium transient amplitude normalized to baseline for vehicle and quinpirole in WT, D3 KO, and arrestin-3 KO mice. Vehicle controls include all 3 genotypes. For D3 KO, open circles denote previously published data (Clarkson, et al. 2017). Gray bar represents 95% confidence interval of control data. Vehicle: median normalized peak ΔG/Gsat = 94.8% of baseline, interquartile range (IQR) 89.9-104.0%, n = 8 cells from 6 mice; WT+quinpirole: 70.4% of baseline, IQR 64.2-77.2%, n = 9 cells from 7 mice, p <0.001; D3 KO+quinpirole: 100.3% of baseline, IQR 94.2-108.5%, n = 7 cells from 4 mice, p = 0.001; arrestin-3 KO+quinpirole: 92.2% of baseline, IQR 90.0-100.5%, n = 9 cells from 5 mice, p < 0.001. Kruskal-Wallis test with Mann-Whitney U test post hoc (Holm-Šídák correction).
Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1.
). We hypothesized that arrestin-3 recruitment would also depend on both PKC activation and agonist binding. Consistent with this hypothesis, D3R-dependent modulation of AIS CaV3.2 is both ligand- and PKC-dependent (
). We therefore examined whether PKC activity, concomitant with D3R agonist, is required for arrestin-3 recruitment to D3R. To test this, we visualized changes in the distribution of cytosolic GFP-tagged arrestin-3 relative to FLAG-tagged D3R on the plasma membrane in HEK293 cells under a series of treatment conditions (Fig. 2). In untreated cells, and cells treated with only quinpirole or only the PKC activator phorbol-12-myristate-13-acetate (PMA), arrestin-3 remained diffusely distributed and did not colocalize with D3R at the plasma membrane (Fig. 2B-C). By contrast, arrestin-3 redistributed close to surface D3R in cells treated with quinpirole plus PMA (Fig. 2B-C). Note that this timepoint preceded any significant receptor endocytosis. These results demonstrate that, in HEK293 cells, quinpirole is an agonist for arrestin-3 recruitment only with coincident PKC activation.
Figure 2Arrestin-3 recruitment to D3R is both ligand- and PKC-dependent. A. Schematic showing that D3R ligand and PKC activation are both necessary to recruit arrestin-3 to D3R in HEK293 cells. B. Sample images of HEK293 cells expressing FLAG-D3R and arrestin-3-GFP. Cells were treated with quinpirole (bottom row) and/or PMA (right column). Dotted lines in insets denote edge of cell membrane. C. Quantification of images in B, one region per condition, showing locations of peak FLAG-D3R and arrestin-3-GFP signal. FLAG-D3R in pink, arrestin-3-GFP in green.
There is mounting evidence that different GPCR drugs can stabilize distinct receptor conformations that preferentially engage either G-protein or arrestin-3 (
). Based on this, we hypothesized that some SGAs could promote arrestin-3 recruitment to D3R even in the absence of G-protein activation. To examine this hypothesis, we performed the arrestin-3-GFP redistribution experiments (Fig. 2), but with four commonly prescribed SGAs: roxindole, aripiprazole, quetiapine, and clozapine. Consistent with previous reports, among these ligands, only roxindole produced any G-protein stimulation from D3R (Fig. 4B). Despite this, we found that three of these SGA ligands—including roxindole, but also quetiapine and aripiprazole—recruited arrestin-3 (Fig. 3A, left; 3B-C). By contrast, clozapine did not produce a change in arrestin-3 localization (Fig. 3A, right; 3B-C). These results demonstrate that some SGAs promote arrestin-3 recruitment even in the absence of G-protein signaling (quetiapine, aripiprazole) while others engage neither G-protein nor arrestin-3 (clozapine) at D3R.
Figure 4Some SGAs promote ERK phosphorylation through D3R and arrestin-3 A. Schematic of experimental question: do SGAs promote ERK phosphorylation? B. Percent maximum cAMP inhibition, normalized to quinpirole Emax, as a readout of G-protein signaling for each drug. C. Top, representative immunoblot of phospho-ERK and total ERK after 5-minute drug treatment, with or without carbachol to activate PKC, in HEK293 cells stably expressing D3R. Bottom, quantification of phospho-ERK/total ERK across multiple experiments (n=3). Data displayed as mean ± SEM. *p <0.05 compared to drug alone, one-way ANOVA with Holm-Šídák multiple comparisons test.
Figure 3Some SGAs recruit arrestin-3 to D3R. A. Example HEK293 cells expressing FLAG-D3R and arrestin-3-GFP. Cells were either treated with drug alone (quetiapine or clozapine), top row, or with drug and PMA, bottom row. Yellow boxes denote regions of analysis. B. Quantification of peak arrestin-3 signal relative to D3R signal for each SGA without (top) or with (below) PMA to activate PKC. Each line profile is mean ± SEM. FLAG-D3R in pink, arrestin-3-GFP in green. C. Quantification of peak arrestin-3 signal relative to peak D3R signal (membrane association ratio), calculated at the gray bar in B. Black, drug alone; blue, drug plus PMA. Circles represent individual line profiles, 2 regions per cell, 5 cells per condition. *p <0.05, Mann-Whitney test. No change with vehicle or clozapine.
). ERK phosphorylation by some GPCRs has been shown to occur not only through G-protein activation, but also through arrestin/ERK scaffolding independent of G-protein (
). Therefore, we examined the ability of SGAs that did and did not recruit arrestin-3 to promote ERK phosphorylation in HEK293 cells expressing D3R (Fig. 4A). Given the PKC dependence shown in Fig. 2 and 3, all experiments were performed in either the absence or presence of PKC activation by carbachol, an agonist for the Gq-coupled muscarinic receptors that are expressed endogenously in HEK293 cells. This approach allowed us to titrate a dose of carbachol that stimulated little ERK phosphorylation on its own but synergized with quinpirole (Fig. 4C). ERK phosphorylation by carbachol+quinpirole was transient and returned to baseline within 30 minutes (Fig. S2). We then assessed whether SGAs could induce ERK phosphorylation in the presence of carbachol. Importantly, we found that the same SGAs that recruit arrestin-3 (Fig. 3) also promote ERK phosphorylation but only when PKC is activated (Fig. 4C). These data suggest that some SGAs (e.g., aripiprazole, quetiapine) can promote ERK phosphorylation even in the absence of any D3R-mediated G-protein signaling (Fig. 4B), classifying them as arrestin-biased agonists. Others (e.g., roxindole) are both G-protein (Fig. 4B) and arrestin-3-agonists, and others still (e.g., clozapine) are antagonists for both.
Arrestin-biased SGAs modulate AIS calcium in D3+ pyramidal cells in PFC
We established that arrestin-3 is necessary for quinpirole-activated D3R to modulate AIS calcium in PFC (Fig. 1). However, quinpirole is both a G-protein and arrestin-3-agonist at D3R, which precludes the ability to determine whether arrestin-3 recruitment alone is sufficient to modulate AIS calcium. The SGAs that promote arrestin-3 recruitment (Fig. 3) and ERK phosphorylation (Fig. 4C), even in the absence of G-protein activation (Fig. 4B), are ideal tools to address this question. Recordings were made from D3+ PFC neurons (Fig. 1B), and AIS calcium transients were imaged in the presence of vehicle, quinpirole (positive control), or SGA (Fig. 5A). These transients were stable for interleaved control cells treated with vehicle for 20 minutes, while 20 minutes of roxindole, aripiprazole, or quetiapine produced a ∼30% reduction in AIS calcium compared to baseline, a magnitude comparable to that observed with quinpirole (Fig. 5B). By contrast, clozapine did not modulate AIS calcium transients (Fig. 5B).
Figure 5Arrestin-biased SGAs modulate AIS calcium in D3+ pyramidal cells in PFC. A. Representative effects of time-locked vehicle control or drug after 20 minutes, averaged across 20 trials per condition. Linescan data displayed as mean ± SEM. Baseline, black; drugs, other colors. B. Data summarizing the effects of quinpirole and clinically relevant SGAs on AIS calcium. Gray bar represents 95% confidence interval of control data. Vehicle: 93.1% of baseline, IQR 86.9%-103.3%, n = 24 cells from 21 mice; quinpirole: 70.4% of baseline, IQR 64.2-77.2%, n = 9 cells from 7 mice, p <0.001 (same data as in Fig. 1); roxindole: 74.4% of baseline, IQR 71.9-81.7%, n = 9 cells from 6 mice, p <0.001; aripiprazole: 72.1% of baseline, IQR 66.7-82.0%, n = 8 cells from 6 mice, p = 0.002; quetiapine: 69.9% of baseline, IQR 59.9%-83.5%, n = 12 cells from 6 mice, p <0.001; clozapine: 90.0% of baseline, IQR 86.8%-105.6%, n = 7 cells from 6 mice, p = 0.944. Kruskal-Wallis test with Mann-Whitney U test post hoc (Holm-Šídák correction). Note: for clarity in the figure, we only indicated significant comparisons in relation to vehicle-treated cells. C. Data summarizing the lack of effect of quetiapine on AIS calcium in D3 KO and arrestin-3 KO mice. Vehicle data include D3 KO (open circles) and arrestin-3 KO (closed circles) mice. Gray bar represents 95% confidence interval of vehicle data from D3KO and arrestin-3 KO animals, while cyan bar represents 95% confidence interval quetiapine data in WT animals. n.s = not significant. Vehicle: 92.5% of baseline, IQR 91.4-94.6%, n = 9 cells from 7 mice; D3 KO: 94.2% of baseline, IQR 92.1-102.3%, n = 6 cells from 3 mice, p = 0.213; arrestin-3 KO: 93.7% of baseline, IQR 87.8-98.3%, n = 9 cells from 4 mice. p = 0.480, Kruskal-Wallis test with Mann-Whitney U test post hoc (Holm-Šídák correction). D. Left, coronal section of a D3-Cre::Ai14 mouse showing D3+ pyramidal and granule cells. Right, 2PSLM image showing D3+ pyramidal cells in dorsal CA1. E. Data summarizing the effects of quinpirole and quetiapine on AIS calcium in D3+ dorsal hippocampal CA1 pyramidal cells. Gray bar represents 95% confidence interval of data from vehicle-treated cells. Vehicle: 98.4% of baseline, IQR 94.5-107.8%, n = 12 cells from 11 mice; quinpirole: 77.0% of baseline, IQR 74.6-82.8%, n = 8 cells from 6 mice, p<0.001; quetiapine: 85.6%, IQR 85.1-96.4%, n = 6 cells from 5 mice, p = 0.002. Kruskal-Wallis test with Mann-Whitney U test post hoc (Holm-Šídák correction).
SGAs have high affinity for D3R but also bind to other GPCRs. To determine whether the AIS calcium modulation by SGAs occurs via D3R and to demonstrate that SGA modulation of CaV3.2 is arrestin-3 dependent, we assessed AIS calcium modulation by quetiapine in slices from both D3 KO and arrestin-3 KO mice. There was no change in AIS calcium with quetiapine treatment in either genotype (Fig. 5C).
To test whether these effects of some SGAs are unique to PFC, we also performed whole-cell recordings from D3+ dorsal hippocampal CA1 pyramidal cells (Fig. 5D). As in PFC, quinpirole and quetiapine both reduced AIS calcium (Fig. 5E). Taken together, these results suggest that some SGAs are arrestin-biased agonists at D3R and can modulate calcium at the AIS in PFC and hippocampus even in the absence of G-protein activation.
Mice treated chronically with quetiapine, but not clozapine, develop tolerance to the locomotor-inhibitory effects of drug
SGAs can cause locomotor effects, including dystonia and parkinsonism in humans (
Effects of dopamine D3 receptor antagonists on spontaneous and agonist-reduced motor activity in NMRI mice and Wistar rats: comparative study with nafadotride, U 99194A and SB 277011.
The role of dopamine D3 compared with D2 receptors in the control of locomotor activity: a combined behavioural and neurochemical analysis with novel, selective antagonists in rats.
Millan MJ, Gressier H, Brocco M (1997): The dopamine D3 receptor antagonist, ž q/ -S 14297, blocks the cataleptic properties of haloperidol in rats. 3.
). Individual SGAs have variable degrees of locomotor side-effects. We next tested the hypothesis that ability to act as an arrestin-3-agonist contributes to this variability by assessing the locomotor-inhibitory effects of an SGA that did (quetiapine) and did not (clozapine) recruit arrestin-3. Both quetiapine (15 mg/kg) and clozapine (4 mg/kg) inhibited locomotor activity (Fig. 6A), indicating that arrestin-3 recruitment to D3R is not necessary for locomotor inhibition by these SGAs.
Figure 6Mice treated chronically with quetiapine, but not clozapine, develop tolerance to locomotor-inhibitory effects of drug A. Top, mice were injected subcutaneously with drug or vehicle and immediately placed in the locomotor box, and distanced traveled was recorded for 60 minutes. Bottom, total distance traveled for each treatment condition. Gray bar represents 95% confidence interval of vehicle-treated mice. Vehicle: 59.1 m, IQR 51.6-74.3 m, n = 8 mice; quetiapine: 9.81 m, IQR 9.32-16.20 m, n = 8 mice, p <0.001; clozapine: 10.4 m, IQR 7.6-11.6 m, n = 8 mice, p <0.001. B. Top, same as A, except mice were pretreated with daily injections of drug or vehicle for 21 days. On day 21, they were placed in the locomotor box immediately following drug injection, and distance traveled was recorded for 60 minutes. Bottom, total distance traveled for each treatment condition. Open circles indicate clozapine vehicle control, while closed circles indicate quetiapine vehicle control. Gray bar represents 95% confidence interval of vehicle-treated mice. Vehicle: 49.0 m, IQR 29.0-60.0 m, n = 16 mice; quetiapine: 51.1 m, IQR 34.1-54.6 m, n = 8 mice, compared to vehicle p = 0.782; clozapine: 7.9 m, IQR 6.8-11.1 m, n = 8 mice, compared to vehicle and quetiapine p <0.001. Kruskal-Wallis test with Mann-Whitney U test post hoc (Holm-Šídák correction). C. Representative examples showing effect of quinpirole on AIS calcium transients in mice treated with quetiapine, left, blue, or clozapine, right, brown. Data shown averaged across 20 trials per condition. Linescan data displayed as mean ± SEM. Baseline, black; quinpirole, blue or brown. D. Summary data comparing vehicle or quinpirole effect on AIS calcium in animals treated for 21 days with either vehicle, quetiapine, or clozapine. Open circles indicate clozapine vehicle control, while closed circles indicate quetiapine vehicle control. Gray bar represents 95% confidence interval of control data. n.s. = not significant. 21-day vehicle treatment, vehicle slice treatment: 90.9% of baseline, IQR 88.3-99.8%, n = 17 cells from 12 mice; 21-day vehicle treatment, quinpirole slice treatment: 78.8% of baseline, IQR 73.7-86.5%, n = 23 cells from 13 mice, p <0.001; 21-day quetiapine treatment, vehicle slice treatment: 96.5% of baseline, IQR 94.7-97.7%, n = 8 cells from 6 mice; 21-day quetiapine treatment, quinpirole slice treatment: 92.5% of baseline, IQR 89.8-99.3%, n = 13 cells from 6 mice, p = 0.538; 21-day clozapine treatment, vehicle slice treatment: 103.5% of baseline, IQR 91.3-106.6%, n = 8 cells from 4 mice; 21-day clozapine treatment, quinpirole slice treatment: 78.5% of baseline, IQR 71.7-81.7%, n = 10 cells from 6 mice, p = 0.001. Kruskal-Wallis test with Mann-Whitney U test post hoc (Holm-Šídák correction). Note: for clarity, we only indicate significant comparisons within drug.
While both quetiapine and clozapine inhibited locomotion acutely, the optimal treatment efficacy of SGAs and an equilibrium of effect/side-effect profile requires weeks of treatment. Hence, we also examined the effects quetiapine and clozapine on locomotion following 21 days of drug treatment (15 mg/kg quetiapine or 4 mg/kg clozapine 1x per day). Remarkably, on day 21, quetiapine no longer reduced locomotion, while clozapine’s effect was indistinguishable from that observed on day 1 (Fig. 6B). These results demonstrate that mice treated chronically with quetiapine develop tolerance to the locomotor-inhibitory effects of drug, while animals treated with clozapine do not.
Chronic treatment with quetiapine, but not clozapine, eliminates D3R-CaV3.2 signaling at the AIS
We next examined whether we could observe drug tolerance on a single cell level. Mice were treated for 21 days with quetiapine or clozapine (as in Fig. 6). At the end of this regimen, AIS calcium transients were assessed in D3+ pyramidal cells in PFC. In mice treated with quetiapine for 21 days, quinpirole had no effect (Fig. 6C-D). In contrast, 21-day treatment with clozapine (or vehicle) did not interfere with quinpirole-mediated modulation of AIS CaV3.2 (Fig. 6C-D). These results suggest that chronic treatment with an arrestin-biased SGA, but not an SGA with no arrestin-3 engagement, causes a loss of D3R function at the AIS in PFC. Furthermore, these results, together with the locomotor tolerance phenotype (Fig. 6A-B), divide SGA function at D3R into two classes.
Quetiapine-induced loss of D3R-CaV3.2 modulation and development of locomotor tolerance are both mediated by post-endocytic sorting of D3R by GASP1
Recruitment of arrestin-3 to D3R not only arrests the G-protein signal and scaffolds ERK signaling but also promotes receptor endocytosis and sorting of D3R to the lysosome for degradation through interaction with the GPCR-associated sorting protein-1 (GASP1) (
Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1.
) (Fig. 7A). To examine whether this sorting mechanism contributed to tolerance, we generated mice with a selective disruption of the GASP1 gene only in cells expressing D3R by crossing D3-Cre driver mice to conditional floxed GASP1 mice (D3 GASP -cKO). We then treated these D3 GASP1 cKO mice for 21 days with quetiapine (15 mg/kg) (as in Fig. 6) and measured AIS calcium modulation by quinpirole in D3+ PFC cells. D3 GASP1 cKO mice treated with quetiapine for 21 days showed intact quinpirole-mediated modulation of AIS calcium (Fig. 7B-C) in stark contrast to WT mice (Fig. 6C-D), To test whether GASP1-mediated post-endocytic sorting of D3R was also necessary for tolerance to the locomotor-inhibitory effects of quetiapine, we treated D3 GASP1 cKO mice with quetiapine for 21 days (Fig. 7D-E). Unlike WT mice or floxed GASP1 mice not crossed to D3-Cre (21-day vehicle: median 33.9m, IQR 20.3-45.6m, n = 4 mice; 21 day quetiapine: median 24.8m, IQR 16.1-39.6m, n = 4 mice, p = 0.486, Mann-Whitney test), D3 GASP -cKO mice did not develop tolerance to the locomotor effects of quetiapine (Fig. 7D-E).
Figure 7Quetiapine-induced loss of D3R-CaV3.2 modulation and development of locomotor tolerance are both mediated by post-endocytic sorting of D3R by GASP1.A. Hypotheses for arrestin-dependent modulation of AIS Ca2+ following chronic SGA administration. Clozapine, which does not recruit arrestin-3, does not promote D3R endocytosis, preserving D3R-CaV3.2 modulation (case 1). Quetiapine, which does recruit arrestin-3, promotes D3R endocytosis and degradation, impairing subsequent D3R-CaV3.2 modulation (case 2). Quetiapine administration in GASP1 cKO eliminates lysosomal degradation and is predicted to preserve D3R-CaV3.2 modulation (case 3).B. Representative example showing effect of quinpirole on AIS calcium transients in a GASP1 cKO mouse treated with quetiapine. Data shown averaged across 20 trials per condition. Linescan data displayed as mean ± SEM. Baseline, black; quinpirole, blue.C. Summary data comparing the vehicle or quinpirole effect on AIS calcium in GASP1 cKO mice treated for 21 days with either vehicle or quetiapine. Gray bar represents 95% confidence interval of control data. 21-day quetiapine treatment, vehicle slice treatment: 92.8% of baseline, IQR 92.3-96.7%, n = 7 cells from 3 mice; 21-day quetiapine treatment, quinpirole slice treatment: 76.6% of baseline, IQR 65.0-82.8%, n = 9 cells from 3 mice. p = 0.005, Mann-Whitney test.D. D3 GASP1 cKO mice were treated with quetiapine for 21 days. On day 21, they were placed in the locomotor box immediately following drug injection, and distance traveled was recorded for 60 minutes. The following day, they were injected with vehicle and distance traveled was recorded for 60 minutes.E. Total distance traveled for D3 GASP1 cKO mice treated with vehicle or quetiapine. Gray bar represents 95% confidence interval of control data. Vehicle: 45.0 m, IQR 36.5-48.8 m, n = 6 mice; quetiapine: 15.9 m, IQR 11.6-18.8 m, n = 6 mice. p = 0.016, Wilcoxon signed-rank test.
Here, we demonstrate that some SGAs are agonists for arrestin-3 signaling at D3R in the absence of G-protein activity. In D3+ neurons of PFC, acute slice treatment with these “arrestin-biased” SGAs results in reduced AP-associated AIS calcium influx, demonstrating an acute mechanism of action of a subset of SGAs in addition to their role as D2R and D3R G-protein antagonists. We also establish that chronic treatment with an SGA that recruits arrestin-3 to D3R results in GASP1-mediated post-endocytic sorting, presumably to the lysosome as previously shown (
Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1.
), and subsequent reduction of both D3R function at the AIS in D3+ PFC pyramidal cells as well as SGA-induced locomotor inhibition. The receptor trafficking-mediated loss of D3R at sites of endogenous neuromodulation as a consequence of chronic SGA exposure may be an important aspect of treatment and could help explain why prolonged SGA treatment is necessary for full clinical efficacy.
Arrestin-3 signaling at D3R
Several studies have focused on arrestin-3 signaling at D2R (
), and, in the context of antipsychotic drugs, aimed to leverage G-protein or arrestin-3 signaling to maximize therapeutic benefits while minimizing side-effects (
). Here, we show that select SGAs can also engage arrestin-3 signaling at D3R. We found that in HEK293 cells, some SGAs, with coincident PKC activation, recruit arrestin-3 to D3R. Of note, aripiprazole recruits arrestin-3 to D3R (Fig. 3), but not to D2R (
), and promotes G-protein signaling at D2R but not D3R. Aripiprazole is thus a G-protein-biased agonist at D2R and an arrestin-biased agonist at D3R. This provides evidence that, even though D2R and D3R are highly homologous, SGAs do not engage the two receptors identically.
Importantly, arrestin-3 recruitment to D3R is sufficient to scaffold ERK phosphorylation even in the absence of G-protein signaling (Fig. 4). Specifically, aripiprazole and quetiapine promote increased P-ERK levels (Fig. 4C), even though they do not promote G-protein activation from D3R (Fig. 4B). We previously found that quinpirole modulates AIS-localized CaV3.2, hyperpolarizing the voltage-dependence of steady-state inactivation (
). We tested here whether “arrestin-biased” SGAs at D3Rs could activate this signaling pathway. We found that only the SGAs that recruit arrestin-3 in HEK293 cells reduce AIS calcium transients at timescales consistent with the timing of arrestin-3-dependent ERK phosphorylation (e.g., minutes rather than seconds following ligand application).
) found previously that arrestin-3 did not strongly translocate to D3R in heterologous cells, presumably because there was no concomitant activation of PKC. In HEK293 cells, we achieved PKC activation and ERK phosphorylation by either stimulating PKC directly or by activating a Gq-coupled GPCR. In neurons, PKC may be activated due to ongoing AP activity, as this can induce release of calcium from intracellular stores (
). In this way, AIS D3R signaling may serve as a coincidence detector for ligand binding and ongoing activity. If this were the case, receptors bound by an arrestin-recruiting dopaminergic ligand would promote signaling only if the neuron was recently firing APs at levels sufficient to activate PKC. Under endogenous conditions, this arrestin-recruiting ligand would be dopamine, but dopamine could be substituted with an arrestin-biased SGA during treatment.
Studies of some other GPCRs have indicated that arrestin-3 signaling cannot occur in the absence of G-protein activity (
). For D3R-arrestin-3-CaV3.2 modulation at the AIS, however, our results indicate that D3R can signal through arrestin-3 in a G-protein-independent manner, because SGAs that do not engage G-protein nevertheless produce channel modulation. Even in HEK293 cells, D3R ligands that do not promote G-protein activity recruit arrestin-3 and promote ERK phosphorylation, indicating that this signaling mechanism could be conserved across many cell types. While other groups have shown GPCR modulation of ion channels at the AIS (
), D3R’s inhibition of CaV3.2 is, to our knowledge, the first example of a GPCR directly inhibiting a channel solely through an arrestin-3 effector. This finding could have significant implications if it also occurs with other key GPCR drug targets. Future studies should examine whether drugs considered to be antagonists could likewise signal via arrestin-3 to channels or other downstream effectors.
Here, we show that acute application of an “arrestin-biased” SGA to an ex vivo slice of mouse PFC modulates CaV3.2 at the AIS via D3R, and that chronic treatment of mice with the same SGA results in loss of D3R and this modulatory effect. CaV3.2 channels play a critical role in the generation of high-frequency bursts of APs in many cell classes (
). Thus, arrestin-3-biased SGAs may acutely act to change bursting properties in D3+ neurons. Following chronic treatment and loss of membrane D3R, however, neurons may no longer alter their firing patterns based on the presence or absence of dopaminergic input. In PFC, this could be one potential mechanism of SGA action and one that differentiates SGAs into two distinct classes: those that do and do not recruit arrestin-3 and promote loss of D3R function at this site.
Because D3+ pyramidal cells are largely a distinct population of cells from those expressing D1R or D2R in PFC, we could isolate SGA-mediated arrestin-3 recruitment at D3R specifically (
). In future studies, it will be critical to understand fully how D3R regulates neuronal function via arrestin-dependent or more canonical signaling pathways as well as to understand more clearly how these signaling pathways are affected by SGAs (
While modulation of CaV3.2 is an acute SGA effect, antipsychotic drugs often take weeks to months to reach maximal therapeutic benefit. Here, we report two distinct effects of chronic treatment with an arrestin-biased SGA that are mediated by post-endocytic degradation of D3R by GASP1: 1) The ability of dopaminergic drugs, and by extension dopamine, to modulate calcium influx at the AIS is lost, and 2) mice become tolerant to the locomotor-inhibitory effects of drug. Specifically, we show that chronic treatment with the arrestin-3-agonist SGA quetiapine causes a loss of D3R function at the AIS (Fig. 6). This loss is prevented in mice with a disruption of the sorting protein GASP1 specifically in D3+ neurons (Fig. 7). Additionally, we show that chronic treatment with quetiapine results in loss of drug-mediated locomotor suppression (Fig. 6), an effect that was also eliminated in D3 GASP1cKO mice (Fig. 7). This result highlights the importance of D3R in the locomotor side-effects of SGAs and, by extension, the effect/side-effect profile of SGAs. Together these data directly implicate post-endocytic sorting as a key mechanism controlling the amount of functional D3R in multiple circuits, including cognitive and motor circuitry. Importantly, this trafficking mechanism regulates receptor function in a ligand-dependent manner, independent of mRNA expression, potentially reconciling why people living with SMI show altered receptor protein levels but small, if any, changes in dopamine receptor gene expression (
Purves-Tyson TD, Owens SJ, Rothmond DA, Halliday GM, Double KL, Stevens J, et al. (2017): Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain. Transl Psychiatry 7: e1003–e1003.
Comparative efficacy and tolerability of 32 oral antipsychotics for the acute treatment of adults with multi-episode schizophrenia: a systematic review and network meta-analysis.
). Patients living with SMI often rotate through a number of drug protocols before finding an effective strategy through a protracted and stochastic process. A more complete understanding of the molecular mechanisms underlying the variable acute and chronic actions of these drugs could help inform a more streamlined approach for an individual patient.
Here, we show that SGAs can be separated into two classes based on their D3R-mediated arrestin-3 signaling. SGAs that recruit arrestin-3 affect D3+ neurons by modulating calcium at the AIS acutely and by reducing D3R levels at the AIS over time. Historically, GPCR-targeting drugs have been characterized solely for their ability to alter G-protein activity. More recently, the role of arrestin-3-mediated signaling in drug response has come to light. Importantly, arrestin-3 not only arrests G-protein signaling and scaffolds signal transduction to other effectors, but also promotes receptor endocytosis. Therefore, when considering the implications of arrestin-3 engagement, it is important to account for not only acute signaling but also how engagement on a longer timescale can change receptor distribution and surface expression through endocytosis and post-endocytic degradation. This may be particularly relevant for GPCR drugs, such as SGAs, that have rapid pharmacokinetics, reaching the brain within minutes, but that take days or weeks to reach maximal efficacy.
SMI diagnoses describe constellations of symptoms that vary from patient to patient, and the genetic and environmental factors underlying SMI are complex (
). Therefore, two individuals with a similar SMI diagnosis may have different underlying disease etiologies. Moving forward, it would be helpful to track patient diagnosis and symptoms and map their effect/side-effect profiles onto whether an arrestin-3-biased SGA was or was not therapeutically beneficial. We posit that, for some patients, treatment with an arrestin-biased ligand to reduce D3R levels would be most efficacious. For others, treatment with an SGA that blocks dopamine-mediated arrestin-3 recruitment, receptor endocytosis, and post-endocytic degradation to increase membrane D3R levels may be more effective. In conclusion, the findings here contribute to a mechanistic understanding of how D3R signaling can vary across different effectors and brain regions and could inform a more personalized approach to treatment with SGAs. D3R has also been suggested as a potential therapeutic target for Parkinson’s disease and substance use disorder (
Newman AH, Xi Z-X, Heidbreder C (n.d.): Current Perspectives on Selective Dopamine D3 Receptor Antagonists/Partial Agonists as Pharmacotherapeutics for Opioid and Psychostimulant Use Disorders. Berlin, Heidelberg: Springer, pp 1–45.
). Hence, our findings may inform the development of D3R-selective ligands, either biased or not, for multiple indications.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Michael Roberts for help with image analysis, Dr. Matthew McGregor and Anna Lipkin for assistance with data analysis, Anirudh Gaur for guidance on in vitro experiments, Dr. Robert Lefkowitz for the arrestin-3 KO mice, Chenyu Wang and Henry Kyoung for assistance with IP injections, and members of the Bender and Whistler labs for critically assessing this work. This work was supported by the National Institute of Mental Health (R01MH112729 to JLW and KJB).
Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1.
Effects of dopamine D3 receptor antagonists on spontaneous and agonist-reduced motor activity in NMRI mice and Wistar rats: comparative study with nafadotride, U 99194A and SB 277011.
The role of dopamine D3 compared with D2 receptors in the control of locomotor activity: a combined behavioural and neurochemical analysis with novel, selective antagonists in rats.
Millan MJ, Gressier H, Brocco M (1997): The dopamine D3 receptor antagonist, ž q/ -S 14297, blocks the cataleptic properties of haloperidol in rats. 3.
Purves-Tyson TD, Owens SJ, Rothmond DA, Halliday GM, Double KL, Stevens J, et al. (2017): Putative presynaptic dopamine dysregulation in schizophrenia is supported by molecular evidence from post-mortem human midbrain. Transl Psychiatry 7: e1003–e1003.
Comparative efficacy and tolerability of 32 oral antipsychotics for the acute treatment of adults with multi-episode schizophrenia: a systematic review and network meta-analysis.
Newman AH, Xi Z-X, Heidbreder C (n.d.): Current Perspectives on Selective Dopamine D3 Receptor Antagonists/Partial Agonists as Pharmacotherapeutics for Opioid and Psychostimulant Use Disorders. Berlin, Heidelberg: Springer, pp 1–45.
S.S., E.L., A.H., K.J.B., and J.L.W. were responsible for conceptualization; S.S., E.L., C.M.K., A.H., K.J.B., and J.L.W. were responsible for methodology; S.S., E.L., C.M.K., K.J.B., and J.L.W. were responsible for formal analysis; S.S., E.L., C.M.K., A.H., K.J.B., and J.L.W. were responsible for investigation; S.S. was responsible for writing-original draft; S.S., E.L., A.H., K.J.B., and J.L.W. were responsible for writing-review and editing; K.J.B. and J.L.W. were responsible for supervision, project administration, and funding acquisition.
DISCLOSURES
KJB receives research funding from BioMarin Pharmaceuticals. The other authors report no biomedical financial interests or potential conflicts of interest.
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