PACAP–PAC1 Signaling Regulates Serotonin 2A Receptor Internalization

Mice lacking pituitary adenylate cyclase-activating polypeptide (PACAP) display psychomotor abnormalities, most of which are ameliorated by atypical antipsychotics with serotonin (5-HT) 2A receptor (5-HT2A) antagonism. Heterozygous Pacap mutant mice show a significantly higher hallucinogenic response than wild-type mice to a 5-HT2A agonist. Endogenous PACAP may, therefore, affect 5-HT2A signaling; however, the underlying neurobiological mechanism for this remains unclear. Here, we examined whether PACAP modulates 5-HT2A signaling by addressing cellular protein localization. PACAP induced an increase in internalization of 5-HT2A but not 5-HT1A, 5-HT2C, dopamine D2 receptors or metabotropic glutamate receptor 2 in HEK293T cells. This PACAP action was inhibited by protein kinase C inhibitors, β-arrestin2 silencing, the PACAP receptor PAC1 antagonist PACAP6-38, and PAC1 silencing. In addition, the levels of endogenous 5-HT2A were decreased on the cell surface of primary cultured cortical neurons after PACAP stimulation and were increased in frontal cortex cell membranes of Pacap−/− mice. Finally, intracerebroventricular PACAP administration suppressed 5-HT2A agonist-induced head twitch responses in mice. These results suggest that PACAP–PAC1 signaling increases 5-HT2A internalization resulting in attenuation of 5-HT2A-mediated signaling, although further study is necessary to determine the relationship between behavioral abnormalities in Pacap−/− mice and PACAP-induced 5-HT2A internalization.

5-HT 2A has been implicated in many psychiatric disorders, such as schizophrenia and affective disorders (13). Clinical studies have indicated that impaired 5-HT 2A signaling plays a major role in schizophrenic episodes (14). Almost all currently available atypical antipsychotic drugs possess antagonistic effects against D2 and 5-HT 2A (15). Cellular internalization is known to play a critical role in the regulation of 5-HT 2A functions (16,17). 5-HT, dopamine, DOI and clozapine induce 5-HT 2A internalization and recycling, and the signaling processes through which each ligand induces its effect are differentially regulated (17). In addition, different classes of Gprotein-coupled receptors (GPCRs) can form heteromeric complexes that potentially contribute to the regulation of receptor internalization or alteration of pharmacological signaling properties (18, 19). 5-HT 2A /metabotropic glutamate receptor 2 (mGlu2) and 5-HT 2A /D2 form heteromeric complexes that induce unique hallucinogen-specific signaling (20)(21)(22)(23). Thus, the signaling pathways involved in 5-HT 2A function are complicated, and the precise signaling pathways responsible for hallucinogenic and therapeutic effects remain unclear.
Our previous studies indicated that there are no significant differences in 5-HT content in the cortex and striatum or in 5-HT 2A protein levels in the somatosensory cortex between PACAP mutant and wild-type mice (5,11,24). Therefore, here, we examined the effect of PACAP signaling on 5-HT 2A internalization and revealed that the PACAP-PAC1 signaling pathway regulates 5-HT 2A internalization in a protein kinase C (PKC)-and b-arrestin2-dependent manner. These results further suggest the existence of functional crosstalk between PACAP and 5-HT 2A -mediated signaling pathways in the brain.

Animals
ICR mice were purchased from Japan SLC (Shizuoka, Japan). Generation of Pacap −/− mice by gene targeting was reported previously (5). Pacap −/− mice and wild-type littermates on the ICR background were obtained by crossing Pacap +/− heterozygous mice.
All animal care and handling procedures were performed in accordance with protocols approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences, Osaka University. All efforts were made to minimize the number of animals used.

Vector Construction
The vector, pFN21A (HaloTag technology, Promega, Madison, WI, USA), encoding the secretory IL-6 signal peptide fused to the N-terminus of Halo-tag was a gift from Dr. Nagase (Kazusa DNA Research Institute). To generate the Halo-PAC1 construct, the hop1 splicing variant of a human PAC1 cDNA was subcloned into the pFN21A vector at SgfI and PmeI restriction sites as described previously (4). Human 5-HT 1A , 5-HT 2A , D2 and mGlu2 cDNAs were obtained from the Kazusa Collection of Flexi ORF Clones (Kazusa DNA Research Institute, Chiba, Japan). These clones were also subcloned into the pFN21A vector at SgfI and PmeI restriction sites.

Receptor Internalization in HEK293T Cells
Receptor internalization was quantitatively assessed using HaloTag technology (Promega) as described previously (4). HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM, 5919, Nissui, Tokyo, Japan) supplemented with 10% fetal bovine serum. The cells were transfected with Halo-expressing vector and labeled with the cell-impermeable Alexa Fluor 488 ligand (Promega) in Opti-MEM for 15 min at 37°C. Each inhibitor or antagonist pretreatment was for 30 min. The cells were then treated with 1 µM PACAP, 5-HT or saline, washed with phosphate-buffered saline and fixed in 4% paraformaldehyde. Cells were imaged using an FV1000D confocal microscope (Olympus, Tokyo, Japan) in sequential mode and membrane protein internalization was quantified using ImageJ software (NIH, MD, USA). To assess the internalization ratio, we defined the shape of a whole-cell (region of interest, ROI, A) and its cytoplasmic region (ROI B) by reducing the size by 5-10 pixels and then determining the fluorescence in both ROIs. The internalization ratio (%) was defined by dividing the amount of luminescence in ROI B by that in ROI A.
b-Arrestin Silencing siRNA-mediated silencing of b-arrestins was performed exactly as described in our previous study (4). b-arrestin1 (6218S; Cell Signaling Technology, Danvers, MA, USA), b-arrestin2 (sc-29743; Santa Cruz Biotechnology, Dallas, TX) or control siRNA (6568S; Cell Signaling Technology), each at 25 mM, were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer's protocol. We confirmed that the b-arrestin1 and b-arrestin2 siRNAs effectively decreased the respective b-arrestin levels to less than 35% in HEK293T cells in our previous study (4).

Surface Biotinylation Assay and Membrane Protein Isolation
A receptor biotinylation assay was performed using the Pierce cell surface protein isolation kit (Thermo Fisher Scientific, Waltham, MA, USA) as described previously (25). Primary cultures of cortical neurons were prepared as described previously (4). The surface proteins of mouse primary cultured cortical neurons at 14 days in vitro were biotinylated with EZ-Link Sulfo-NHS-SS-biotin for 30 min at 4°C. To collect the surface proteins, cells were lysed with lysis buffer and biotinylated proteins were precipitated with NeutrAvidin agarose. The collected surface proteins were analyzed by western blotting.
Membrane protein isolation was performed using a plasma membrane protein isolation kit (Invent Biotechnologies, Plymouth, MN, USA) according to the manufacturer's instructions. The collected membrane proteins were analyzed by western blotting.

Head Twitch Response and Intracerebroventricular Injections
Intracerebroventricular injections were performed as described previously (26). Head twitch responses were assessed as described previously (10). ICR mice were anesthetized and placed in a stereotaxic instrument (Narishige, Tokyo, Japan). A G-4 cannula (Eicom, Kyoto, Japan) was implanted, −0.4 mm posterior, 1.0 mm lateral, and 2.3 mm ventral from the bregma. After cannula implantation, each mouse was given 1 mg/kg buprenorphine (Sigma-Aldrich) to relieve pain and housed individually for at least 10 days before performing head-twitch experiments. Thirty minutes before DOI (Sigma-Aldrich) treatment, PACAP (10 pmol) was diluted in Ringer's solution (1:100, Fuso Pharmaceutical Industries, Osaka, Japan) and a 3 ml volume was injected at an infusion rate of 1 ml/min using a microinjection pump (KD Scientific, MA, USA). For the pretreatment of the PAC1 antagonist, PACAP 6-38 (100 pmol) were diluted and injected in the same way 30 min before PACAP treatment. The mice were individually placed in observation cages (19 × 10 × 11 cm) for a 30 min habituation period. They were then intraperitoneally injected with either saline or DOI, which were prepared just before use, and recordings were made for a duration of 60 min. Scoring began immediately after injection by trained observers who were blind to the treatment. The head twitch response is a distinctive paroxysmal head-twitching behavior that is easily distinguished from head-bobbing, lateral movements of the head and grooming. The intracerebroventricular injection was judged successful if the third ventricle was stained by Evans blue.

Statistical Analysis
Experimental data were analyzed using Student's t-test, or oneway, two-way or two-way repeated measures analysis of variance (ANOVA). The Tukey-Kramer post hoc test was also performed after significant main effects for interaction were observed. The criterion for statistical significance was p < 0.05. Statistical analyses were performed using StatView software (version 5.0; SAS Institute, Cary, NC, USA). All experiments were performed in a blinded manner. The observers were blinded to the group of samples during the analyses by random numbering.

PACAP-Induced Internalization of 5-HT 2A in HEK293T Cells
To examine whether PACAP signaling modulates the internalization of 5-HT 2A and related GPCRs in HEK293T cells, we constructed membrane-specific Halo-tagged receptors for PAC1, 5-HT 2A , 5-HT 1A , 5-HT 2c , D2 and mGlu2. As a first step, we examined whether PAC1, VPAC1, VPAC2, and 5-HT 2A mRNAs were expressed in HEK293T cells using reverse transcription (RT)-PCR analysis. In our HEK293T cell cultures, we detected the mRNA expression of PAC1 and VPAC1; however, the expression of VPAC2 and 5-HT 2A was below the detection limit of our RT-PCR analysis (Supplementary Figure 1A). Quantitative RT-PCR analysis showed that PC12 cells and SH-SY5Y cells expressed relatively higher levels of PAC1 mRNA as expected from the previous reports (27)(28)(29), and both our HEK293T cell cultures and the HEK293T cells provided by RIKEN BRC Cell Bank (RCB2202; the National Bio-Resource Project of the MEXT/AMED, Japan) moderately expressed PAC1 mRNA at similar levels. In Hela cells, PAC1 expression was below the detection limit of our quantitative RT-PCR analysis (Supplementary Figure 1B). The nucleotide sequence of the cDNA fragment amplified from our HEK293T cell cultures was identical to that of the cDNA encoding the human PAC1 hop1 splice variant (NCBI Reference Sequence: NM_001199635.2).
We then examined whether PACAP, maxadilan, a potent and specific PAC1 agonist (30), and VIP increase intracellular cyclic adenosine monophosphate (cAMP) levels in our HEK293T cell cultures and confirmed that PACAP and maxadilan, both at ≥ 0.01 nM, significantly increased intracellular cAMP levels, while VIP at higher concentrations (≥ 1 nM) increased intracellular cAMP levels (Supplementary Figure 1C).

Intracerebroventricular PACAP Administration Ameliorates the Hallucinogenic Head Twitch Response
We then addressed PACAP signaling involvement in 5-HT 2Adependent behavioral responses by examining the head twitch response, which is a characteristic head-shaking movement induced by a hallucinogenic drug through the stimulation of 5-HT 2 receptors (33). DOI (1.0 mg/kg)-induced head twitch responses were significantly fewer in mice administered PACAP (10 pmol) compared with vehicle control mice in the first, third and fourth 10 min-bins of a 60-min observation period ( Figure 5C). The numbers of head twitch responses induced by 0.3 and 1.0 mg/kg DOI during 60 min were significantly lower in mice administered PACAP compared with vehicle control mice (two-way ANOVA, PACAP effect, F (1, 12) = 39.80, p < 0.001; dose effect, F (2, 12) = 50.90, p < 0.001; interaction, F (2, 12) = 11.03, p = 0.0019) ( Figure 5D). In addition, we examined whether the inhibitory effect of PACAP on DOIinduced head twitch response is mediated by PAC1 by using the PAC1 antagonist PACAP  . Intracerebroventricular preadministration of PACAP 6-38 (100 pmol) significantly blocked the inhibitory effect of PACAP on DOI-induced head  twitch response (one-way ANOVA, F (3, 12) = 47.77, p < 0.001) ( Figure 5E).

DISCUSSION
In the present study, we investigated the mechanisms underlying the relationship between PACAP and 5-HT 2A signaling pathways. We found that PACAP time-and dose-dependently increased the internalization of 5-HT 2A , but not 5-HT 1A , 5-HT 2c , D2 or mGlu2, in HEK293T cells and that the effect of PACAP was mediated by PAC1, PKC and b-arrestin2. In addition, we showed that PACAP decreased the cell surface levels of endogenously expressed 5-HT 2A in mouse primary cultured cortical neurons and that 5-HT 2A levels in the membrane fraction of the frontal cortex were increased in Pacap -/mice compared with wild-type mice. Finally, we observed that intracerebroventricular administration of PACAP suppressed DOI-induced head twitch responses in mice. These results suggest that PACAP-PAC1 signaling increases 5-HT 2A internalization, resulting in attenuation of 5-HT 2A -meadiated signaling.
In the present study, it is still uncertain whether PACAPinduced 5-HT 2A internalization can be a mechanism for behavioral abnormalities including hyperactivity, PPI deficits, depressive-like behavior and memory impairment, reversal of the depressive-like behavior by the 5-HT 2A  Values are the mean ± SEM (n = 4 per group). **p < 0.01 vs. vehicle, one-way ANOVA followed by the Tukey-Kramer test.
potential colocalization of 5-HT 2A and PAC1 in the frontal cortex. In addition, 5-HT 2A expressed in the frontal cortex plays an important role in the pathophysiology and therapeutic effects of schizophrenia (12,14). However, further analyses in other brain regions are needed, which will be investigated in our future work. 5-HT 2A internalization is involved in diverse signaling pathways depending on different ligands. Recent studies indicate that 5-HT 2A internalization signaling may be separated into hallucinogenic and antipsychotic specific pathways, because hallucinogenic and nonhallucinogenic 5-HT 2A ligands induce distinct immediate early gene expression patterns (38)(39)(40)(41). Hallucinogenic DOI-induced 5-HT 2A internalization is independent on b-arrestins and antipsychotic clozapine-mediated internalization is independent on PKC (16,42). Urs et al. (43) reported that b-arrestin-biased D2 ligands exert unique brain region-specific antipsychotic actions (43). The present observation that PACAP-PAC1 signaling regulates 5-HT 2A internalization in a PKC-and b-arrestin2dependent manner provides a new molecular mechanism for this peptidergic signaling that cross-talks with serotonergic signaling in the brain.
In the present study, we observed, in our HEK293T cell cultures, expression of PAC1 transcript, maxadilan-induced cAMP elevation, PACAP-induced 5-HT 2A internalization as well as inhibition of the PACAP-induced 5-HT 2A internalization by PACAP 6-38 and shRNA-mediated PAC1 silencing. In addition, we observed that the HEK293T cells which was newly obtained from RIKEN BRC Cell Bank expressed PAC1 mRNA at a similar level with our HEK293T cell cultures used in the present 5-HT 2A internalization study. However, previous studies have shown that HEK293T cells did not express PAC1 (3,28,48,49) and therefore PAC1 was exogenously expressed to investigate the signal transduction system. In contrast, it was also reported that HEK293T cells expressed the PAC1 protein as observed by western blot analysis (50,51). The reason for the disagreement in PAC1 expression in HEK293T cells is currently unknown but might be related with passage number and culture conditions. Serotonin syndrome is caused by adverse side effects of serotonergic drugs and is associated with increased serotoninergic activity (52). By indirectly antagonizing 5-HT 2A function, PACAP signaling may have the potential to ameliorate serotonin syndrome. Accumulating evidence suggests that PACAP-PAC1 signaling in the brain provides clues to elucidating the pathomechanisms of neurological and psychiatric disorders (53)(54)(55). The present study furthers understanding of PACAP-PAC1 signaling and shows that this pathway is a promising target for the development of neurotherapeutics.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors.

ETHICS STATEMENT
This animal study was reviewed and approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences, Osaka University.

ACKNOWLEDGMENTS
We are grateful to Dr. Atsuro Miyata at the Graduate School of Medical and Dental Sciences, Kagoshima University for his indispensable support. We are also grateful to the Center for Medical Research and Education, Graduate School of Medicine, Osaka University, for confocal microscopy analyses.