Sigma-1 receptor activation mediates the sustained antidepressant effect of ketamine in mice via increasing BDNF levels

Abstract

Sigma-1 receptor (S1R) is a unique multi-tasking chaperone protein in the endoplasmic reticulum. Since S1R agonists exhibit potent antidepressant-like activity, S1R has become a novel target for antidepression therapy. With a rapid and sustained antidepressant effect, ketamine may also interact with S1R. In this study, we investigated whether the antidepressant action of ketamine was related to S1R activation. Depression state was evaluated in the tail suspension test (TST) and a chronic corticosterone (CORT) procedure was used to induce despair-like behavior in mice. The neuronal activities and structural changes of pyramidal neurons in medial prefrontal cortex (mPFC) were assessed using fiber-optic recording and immunofluorescence staining, respectively. We showed that pharmacological manipulation of S1R modulated ketamine-induced behavioral effect. Furthermore, pretreatment with an S1R antagonist BD1047 (3 mg·kg⁻¹·d⁻¹, i.p., for 3 consecutive days) significantly weakened the structural and functional restoration of pyramidal neuron in mPFC caused by ketamine (10 mg·kg⁻¹, i.p., once). Ketamine indirectly triggered the activation of S1R and subsequently increased the level of BDNF. Pretreatment with an S1R agonist SA4503 (1 mg·kg⁻¹·d⁻¹, i.p., for 3 consecutive days) enhanced the sustained antidepressant effect of ketamine, which was eliminated by knockdown of BDNF in mPFC. These results reveal a critical role of S1R in the sustained antidepressant effect of ketamine, and suggest that a combination of ketamine and S1R agonists may be more beneficial for depression patients.

Introduction

Sigma-1 receptor (S1R) is a Ca²⁺-sensitive and ligand-operated chaperone protein that highly clusters at the mitochondria-associated endoplasmic reticulum membrane (MAM) [1]. Different from classic receptors, it has a powerful capability to interact with various proteins and thereby fine tune cell functions, including gene transcription, intracellular calcium homeostasis, ion channel function, and electrical activity [2–6]. Most notably, S1R has long been considered a potential pharmacological target for depression therapy, as antidepressants have a relatively high affinity for S1R [7, 8]. Indeed, both endogenous and exogenous ligands that acts as S1R agonists demonstrate antidepressant-like activity [9–11].

Ketamine, a glutamate N-methyl-D-aspartate (NMDA) receptor blocker, is one of the most promising drugs for depression treatment in humans due to its powerful ability to rapidly ameliorate behavioral despair and alleviate severe suicidal ideation [12, 13]. Previous studies have explored the mechanisms underlying the antidepressant effect of ketamine, and led to several hypotheses [14]. It has also been reported that ketamine has micromolar affinity for S1R (K_i of 139.6 μmol/L) [15], but whether its antidepressant effect is related to S1R has not been completely investigated. Moreover, the effect of ketamine persists long even after its metabolism, which indicates sustained adaptations in key brain regions after ketamine treatment.

The mPFC is a high-order brain region responsible for the regulation of depression disorder, and the delicate functional balance of pyramidal (Pyr) neurons and GABAergic interneurons in the mPFC might be required for the onset of antidepressant [16]. The disinhibition hypothesis postulates that ketamine preferentially inhibits GABAergic interneuron activity, leading to “indirect” disinhibition of mPFC^Pyr neurons [17, 18]; another hypothesis proposes that ketamine directly acts on mPFC^Pyr neurons and causes a cascade of intracellular signaling events [19]. However, the underlying mechanisms through which the mPFC is involved in the sustained antidepressant effect of ketamine are not fully clear.

In the present study, we investigated whether the antidepressant action of ketamine was related to S1R activation and found that pharmacological manipulation of S1R could modulate ketamine-induced behavioral effect. Pretreatment with the S1R antagonist BD1047 significantly inhibited the restoration of mPFC^Pyr neuron structure and function by ketamine. Ketamine indirectly triggered the activation of S1R and subsequently increased the level of BDNF, and pretreatment with the S1R agonist SA4503 enhanced the sustained antidepressant effect of ketamine, which was eliminated by knockdown of BDNF in mPFC. In summary, our study elucidates a previously unknown interaction between ketamine and S1R, providing new insights for the development of combined antidepressant strategies.

Materials and methods

Animals and behavioral tests

Young adult (6 to 8 week of age) male C57BL/6 mice were purchased from Beijing SPF Biotechnology (Beijing, China). The mice were group-housed on a 12 h light-dark cycle and provided ad libitum access to food and water, while the animals subjected to the chronic corticosterone (CORT) procedure were given CORT in place of drinking water for 21 d. For tail suspension test (TST), the mouse was hung upside down by the tail on a hook in a suspension chamber for 6 min. The immobility duration was recorded during the last 5 min. All procedures were approved by the Institutional Animal Care and Use Committee of the Beijing Institute of Basic Medical Sciences.

Drugs and virus

The drugs used in our experiments were as follows: ketamine (Ket; Fort Dodge Animal Health; 5 or 10 mg·kg⁻¹, i.p., once); SA4503 (SA; 165377-43-5, MCE; 0.1 ~ 10 mg·kg⁻¹·d⁻¹, i.p., for 3 consecutive days); BD1047 (BD; 138356-21-5, Sigma–Aldrich; 1 ~ 10 mg·kg⁻¹·d⁻¹, i.p., for 3 consecutive days) and corticosterone hemisuccinate (CORT; Q1562-000, Steraloids; 32.22 mg in 1 L drinking water for 25 μg·mL⁻¹ solution) [20]. The following viruses used in our experiments were purchased from BrainVTA Technology and OBiO Technology: AAV2/9-CaMKIIα-GCaMp6s (5.21 × 10¹²V.G.·mL⁻¹); AAV2/9-CaMKIIα-EGFP (5.63 × 10¹²V.G.·mL⁻¹); AAV-CaMKIIα-miR30shRNA(S1R) (target-sequence: 5′-GACTATTATCGCAGTGCTGAT-3′, 6.30 × 10¹²V.G.·mL⁻¹); AAV-CaMKIIα-miR30shRNA(NC) (target-sequence: 5′-CTCGCTTGGGCGAGAGTAA-3′, 8.29 × 10¹²V.G.·mL⁻¹); AAV-CaMKIIα-miR30shRNA(BDNF) (target-sequence:  5′-GGTGATGCTCAGCAGTCAAGT-3′, 5.28 × 10¹²V.G.·mL⁻¹) and AAV-CaMKIIα-miR30shRNA(NC) (target-sequence: 5′-CCTAAGGTTAAGTCGCCCTCG-3′, 5.11 × 10¹²V.G.·mL⁻¹). The injection volume was 0.3 μL per site.

Immunofluorescence staining

Mice were anesthetized with 1% pentobarbital sodium (50 mg·kg⁻¹, i.p.) 90 min after TST and transcardially perfused. The brains were removed and coronal sections (30 µm thick) were cut using a cryostat microtome (model 2050, Leica). The sections were incubated with primary antibody overnight at 4 °C and secondary antibodies for 1.5 h at room temperature. Finally, the sections were mounted with anti-fade medium (including DAPI; C1211-10, Applygen), and immunofluorescence staining was assessed by an FV1000 confocal laser-scanning microscope (Olympus Corporation). All images were acquired at 1024 × 1024 resolution using 3× Kalman frame averaging. For counting of double-positive cells, 2 ~ 3 sections were selected in mPFC from an individual mouse and counted in a double-blinded manner. For counting spines, three secondary or tertiary dendritic branch segments longer than 30 μm were randomly selected from individual neurons. Mushroom spines were defined as a head diameter larger than 0.6 μm and a head-to-neck diameter ratio greater than 1:1. The following antibodies were used in our experiments: rabbit anti-c-Fos (1:500; sc-52, Santa Cruz Biotechnology); mouse anti-NeuN (1:800; MAB377, Millipore); mouse anti-CaMKIIα (1:500; sc-13141, Santa Cruz Biotechnology); mouse anti-PV (1:500; PV235, Swant); rabbit anti-SOM (1:600; PA5-85759, Invitrogen); guinea pig anti-c-Fos (1:500; 325005, Synaptic Systems); rabbit anti-S1R (1:500; 61994, Cell Signaling Technology); rabbit anti-MAP-2 (1:500; ab32454, Abcam); Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:500; ab150077, Abcam); Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:500; ab150080, Abcam); Alexa Fluor 488-conjugated goat anti-mouse IgG (1:500; ab150113, Abcam); Alexa Fluor 594-conjugated goat anti-mouse IgG (1:500; ab150116, Abcam) and Alexa Fluor 488-conjugated goat anti-guinea pig IgG (1:500; ab150185, Abcam).

Microinjection

The mice were anesthetized with 1% pentobarbital sodium (50 mg·kg⁻¹, i.p.) and head-fixed in a stereotaxic apparatus (RWD Life Science). The virus was delivered with a syringe pump (Harvard Apparatus) at a rate of 0.05 µL·min⁻¹, and the syringe was left in place for an additional 5 min before being slowly withdrawn to allow virus diffusion. The coordinates were measured from bregma according to the mouse atlas [21] (AP, +1.54 to +1.98 mm, ML, ±0.20 to ±0.40 mm, DV, −2.00 to −3.00 mm).

Fiber-optic recording

Mice were injected with AAV2/9-CaMKIIα-GCaMp6s into the mPFC and then an optical ceramic insert (1.25 mm in diameter) was implanted 0.3 mm above the virus injection site. The mice were allowed to recover for 4 weeks before the TST. After the CORT procedure and drug application, the mice were hung upside down by the tail on a hook in a suspension chamber, and neuronal Ca²⁺ signaling and animal behavior were simultaneously recorded using a two-color multichannel optical fiber recording system (410&470, RWD Life Science). The data obtained were analysed with RWD analysis software.

Molecular docking

The 3D structure of human S1R was obtained from the RCSB Protein Data Bank (PDB ID: 5HK1, resolution: 2.51 Å) [22]. The cocrystallized structure was prepared using the QuickPrep module of MOE version 2016.08 to correct the structure issues, add hydrogens and calculate the partial charges [23]. The 2D structure of ketamine was downloaded from the DrugBank database in SD file format and converted to 3D in MOE through energy minimization [24]. The binding site of the cocrystallized ligand was chosen as the binding pocket for docking. Classical triangle matching was selected for the placement method, and the number of placement poses was set to 300. The output docking poses were evaluated by the default London dG score. Then, the rigid receptor method, which keeps the ligand-binding groove rigid, was employed for the refinement step. The number of final docking poses was set to 20, and then the top 20 ranked poses were subjected to minimization using the Amber10:EHT force field in MOE. The GBVI/WSA dG score was used to evaluate the binding of ketamine with S1R.

Binding assays

Sprague Dawley rat liver membrane aliquots were diluted in Krebs solution (NaCl 118 mM, KCl 4.7 mM, CaCl₂ 2.5 mM, MgSO₄ 1.2 mM, KH₂PO₄ 1.2 mM, NaHCO₃ 25 mM, glucose 11.1 mM; pH 7.2 ~ 7.4) to a final concentration of 3 mg·mL⁻¹. The binding assay buffer consisted of 100 μL membrane aliquots, 100 μL [³H]-(+)-pentazocine (a final concentration of 3 nmol), and 100 μL ketamine or 100 μL Krebs solution. [³H]-(+)-pentazocine was employed as a highly selective probe for S1R. The bound and free radioligands were separated by rapid filtration with a Brandel harvester through Whatman GF/B glass fiber filters after incubation for 2.5 h at 35 °C. PerkinElmer liquid scintillation spectrometers (PerkinElmer) were used to measure radioactivity.

Immunoprecipitation (IP)

Flag-tagged S1R stably transfected HEK293T cells (293T-S1R-OE) were purchased from Genechem. When the cell density reached 60% –70%, drugs were added as appropriate. The lysed cells were prepared in IP buffer (50 mM Tris, 150 mM NaCl, 0.2% CHAPS, 0.5 mM EDTA, 10% glycerin, pH 7.4) [1] and centrifuged at 4 °C at 14000 r/min for 15 min, and the precipitate was discarded. Then, 100 μL of supernatant was added to 20 μL of 6× SDS buffer as the input. The remaining supernatant was added to Flag beads and incubated at 4 °C overnight. After centrifugation and removal of the unbound beads, the samples were boiled to obtain protein for subsequent Western blotting.

Immunoprecipitation-mass spectrum (IP-MS)

Flag-tagged 293T-S1R-OE and normal control HEK293T cells (293T-NC) were seeded on 10-cm dishes in DMEM-F12 (C11995500BT, Invitrogen) containing 10% fetal bovine serum (FBS, Gibco) and 1% antibiotics (P1400, Solarbio). After ketamine or vehicle incubation for 24 h, the cells were lysed and immunoprecipitated with anti-S1R (1:1000, 61994, Cell Signaling Technology). The immunoprecipitation was verified using silver staining and then the immunoprecipitates were subjected to liquid chromatography with tandem mass spectrometry for proteomics analysis by the National Center for Protein Sciences (Beijing, China).

Cell culture and transfection

Primary cortical neurons were isolated from newborn C57BL/6 mice within 24 h. The culture procedure was modified from a previous report [25]. The siRNAs used in our experiments were purchased from GenePharma as follows: siRNA(S1R)1#, sense (5′-3′), GGCAUGGAUCACCCUGAUUTT, antisense (5′-3′), AAUCAGGGUGAUCCAUGCCTT; siRNA(S1R)2#, sense (5′-3′), GCAGCUUGCUCGACAGUAUTT, antisense (5′-3′), AUACUGUCGAGCAAGCUGCTT; siRNA(S1R)3#, sense (5′-3′), UCUGGCACCUUCCACCAAUTT, antisense (5′-3′), AUUGGUGGAAGGUGCCAGATT; siRNA(NC), sense (5′-3′), UUCUCCGAACGUGUCACGUTT, antisense (5′-3′), ACGUGACACGUUCGGAGAATT. siRNAs were transfected into primary cortical neurons using the transfection reagent Lipofectamine RNAiMAX (Thermo Fisher Scientific). First, 2.5 μL siRNA (20 μmol) and 2.5 μL RNAiMAX transfection reagent were respectively added to 25 μL Opti-MEM (Gibco) and incubated for 5 min. Then, the reagents were fully mixed and allowed to stand for the next 15 min. A total of 50 μL of the mixture was added to each well, and the plate was shaken sideways to mix well. After 48 h, ketamine or vehicle was added to the culture plate for 24 h.

Quantitative RT-PCR

Total RNA was isolated with TRIzol reagent (Invitrogen), and cDNA was then synthesized using MonScript™ RTIII All-in-One Mix with dsDNase (Monad). qRT-PCR was performed with 2×RealStar GreenFast Mixture with ROXII (GenStar). The following primers were used: Bdnf (forward: 5′-TCATACTTCGGTTGCATGAAGG-3′; reverse: 5′-AGACCTCTCGAACCTGCCC-3′) and β-actin (forward: 5′-GGCTGTATTCCCCTCCATCG-3′; reverse: 5′-CCAGTTGGTAACAATGCCATGT-3′). β-actin quantification was used as an internal control for normalization. Relative mRNA levels were calculated using the 2^^(-ΔΔCt) method.

Western blotting

Total protein was isolated with RIPA buffer (R0278, Sigma–Aldrich) and determined by the BCA protein assay (Pierce, Rockford). Equal amounts of proteins were loaded on an SDS-PAGE gel. The blots were blocked in TBST (TBS with 0.1% Tween-20) containing 5% nonfat milk for 1 h at room temperature. Then the blots were incubated with primary antibody overnight at 4 °C and secondary antibodies for 1 h at room temperature, and washed three times after each incubation with TBST. The following antibodies were used in our experiments: rabbit anti-Bip (1:1000, ab21685, Abcam); mouse anti-Flag (1:10000, F1804, Sigma-Aldrich); rabbit anti-S1R (1:1000, 61994, Cell Signaling Technology) [26]; mouse anti-BDNF (1:100, sc-65514, Santa Cruz) and rabbit anti-β-actin (1:2000, 4970, Cell Signaling Technology). The protein bands were detected using a chemiluminescence detection kit (WBKLS0500, Millipore) and visualized by exposure to Kodak film. β-actin was used as an internal control, and the relative expression for each protein was normalized to the mean value in the control group.

Statistical analysis

Statistical analysis was performed using GraphPad Prism version 6.0. The significance of differences was calculated using one-way or two-way ANOVA followed by a post hoc test for comparisons among multiple groups, or unpaired Student’s t test for comparisons between two groups.

Results

Pharmacological manipulation of S1R modulates the sustained antidepressant effect of ketamine

S1R function can be regulated by highly specific agonists and antagonists. Here, two days after intraperitoneal administration of different doses of BD1047 (a selective S1R antagonist) or SA4503 (a selective S1R agonist) for three consecutive days, normal mice were subjected to the TST to assess the elicited behavioral effects (Fig. 1a). There were no changes in immobility time during the TST in mice treated with BD1047 compared to the control group (Fig. 1b). However, we found a significant reduction in immobility time in mice treated with the two higher doses of SA4503 (Fig. 1c). These results indicate that the antidepressant effect of the S1R agonist was dose dependent. To explore whether S1R and ketamine interact to exert their antidepressant effects, mice were administered saline, ketamine (10 mg·kg⁻¹) or ketamine in combination with a subeffective dose of BD1047 (1 or 3 mg·kg⁻¹) (Fig. 1d). As expected, mice treated with ketamine showed a significant decrease in immobility time during the TST compared to the control group, suggesting that ketamine alone can exert a prominent antidepressant effect; however, the aforementioned effect was abolished in mice treated with the combination of ketamine and 3 mg·kg⁻¹ BD1047 (Fig. 1e). We then utilized a similar strategy to examine whether subeffective doses of SA4503 (0.3 and 1 mg·kg⁻¹) could amplify the antidepressant effect of ketamine. Our data showed that ketamine in combination with SA4503 (1 mg·kg⁻¹) exerted a significantly stronger effect than ketamine alone (Fig. 1f). These results suggest that pharmacological manipulation of S1R modulates the sustained antidepressant effect of ketamine.

Fig. 1. Pharmacological manipulation of S1R modulates the sustained antidepressant effect of ketamine.

a, d Experimental design. Mice were treated with vehicle, BD1047 (1 to 10 mg·kg⁻¹) or SA4503 (0.1 to 10 mg·kg⁻¹) (a); mice were treated with vehicle, ketamine (10 mg·kg⁻¹) alone, or a combination of ketamine and BD1047 (1 or 3 mg·kg⁻¹) or SA4503 (0.3 and 1 mg·kg⁻¹) (d). S1R, Sigma-1 receptor; BD BD1047, SA SA4503, Ket ketamine, TST tail suspension test. b, c, e, f Immobility time in TST. One-way ANOVA with Dunnett’s post hoc test, n = 9–13, *P < 0.05, **P < 0.01. Data are presented as the mean ± SEM.

S1R is involved in ketamine-induced restoration of neuronal structure and function

Since the mPFC is one of the most critical brain regions underlying depression pathophysiology [27], we assessed neuronal activity by measuring c-Fos expression in the mPFC after a chronic CORT procedure, which is used to induce despair-like behavior in mice [28]. Fewer c-Fos positive neurons (marked by NeuN) were detected in the mPFC of CORT mice after TST compared with the control group (Supplementary Fig. 1a, b), and there was a negative correlation between the c-Fos level in mPFC and the immobility time in TST (Supplementary Fig. 1c). To further verify the identity of the activated neurons, costaining for the Pyr neuronal marker, CaMKIIα, or the GABAergic neuronal marker, PV or SOM with c-Fos was performed. Major coexpression between c-Fos and CaMKIIα was detected, while only a small fraction of cells expressed both c-Fos and PV or SOM (Supplementary Fig. 1e, f). These results suggest that the activation of mPFC^Pyr neurons elicits active struggling during the TST. Given that S1R-positive signal was detected in mPFC^Pyr neurons (Supplementary Fig. 1g, h), we next monitored the real-time activity of mPFC^Pyr neurons via CaMKIIα promoter-driven GCaMP6s in adeno-associated virus (AAV) after the combined administration of ketamine (10 mg·kg⁻¹) and BD1047 (3 mg·kg⁻¹) (Fig. 2a, b). As shown in Fig. 2c–e, the calcium signal (dF/F) synchronized well with the start of active struggling in the TST, and the area under curve (AUC) of dF/F (0–5 s) in the CORT group was lower than that in the control group (Fig. 2f), which indicates decreased neuronal activation in the CORT mice. Ketamine alone reversed the CORT-induced decline in calcium signal, while BD1047 dramatically blocked the effect of ketamine when administered simultaneously (Fig. 2f). Interestingly, the immobility time in TST exhibited the opposite change (Fig. 2g), and there was a negative correlation between the AUC and the immobility time (Fig. 2h). These results demonstrate that S1R inhibition decreases ketamine-induced activity of mPFC^Pyr neurons. To further explore the structural changes in mPFC^Pyr neurons after CORT exposure and combined administration of ketamine and BD1047, we analysed the morphology of specific cell type using AAV-CaMKIIα-EGFP (Fig. 2i) and observed a dramatic decrease in the density of mature spines in the CORT group; ketamine alone reversed this decrease, while BD1047 blocked the effect of ketamine (Fig. 2j, k). These results indicate that S1R plays a crucial role in the ketamine-induced restoration of mPFC^Pyr neuron structure and function.

Fig. 2. S1R is involved in ketamine-induced structural and functional restoration of mPFC^Pyr neurons.

a Experimental design. Ctrl control, CORT corticosterone, FPR Fiber photometry recording, TST tail suspension test. b Representative immunostaining image of GCaMP6s (green) and DAPI (blue) in mPFC. c Representative trace depicting the Ca²⁺ signals during TST (1–6 min). d The mean of Ca²⁺ signals (dF/F) from mPFC^Pyr neuron (−2–6 s, time0 = onset of struggling, n = 50 trails from 10 mice). e Representative heatmaps of each group. The colored bars indicate dF/F. f, g Area under curve (AUC) of dF/F (0–5 s) and immobility time in TST. Unpaired Student’s t tests, n = 9–10, *P < 0.05, ***P < 0.001. h Relationship between AUC and immobility time. Pearson’s correlation, r = −0.6535, P < 0.001. i Representative immunostaining image of CaMKIIα-EGFP (green) injection in mPFC. j Representative images of spines of each group. k Quantification of mushroom spines. Unpaired Student’s t test, n = 15 dendritic segments from 5 mice, *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± SEM.

Ketamine triggers the activation of S1R

To verify whether there is a direct interaction between ketamine and S1R, we performed a molecular docking simulation using MOE Dock. The binding conformation with the highest docking score is shown in Fig. 3a, which had the lowest conformation energy of 92.03 kcal·mol⁻¹. Ketamine can form three H-π interactions with the Tyr103 in S1R, and the binding free energy was −7.30 kcal·mol⁻¹. The distances of the three H-π interactions were 4.66 Å, 4.11 Å and 2.41 Å, respectively. The benzene ring of ketamine can form another H-π interaction with Ala185 in the binding pocket with a distance of 3.98 Å. In addition, ketamine can also form van der Waals interactions with Met93 in the receptor pocket. The above interactions are shown in a 2D image in Fig. 3b. Next, we carried out a radioligand assay using [³H]-(+)-pentazocine-induced inhibition to quantify the K_i value of ketamine [29]. As shown in Fig. 3c, d, the average K_i value of SA4503 was 110.2 nmol/L (K_d: 6.13 nmol/L [30]; IC₅₀: 164.1 nmol/L; 95%IC₅₀: 122.1–220.7 nmol/L), indicating a high affinity for S1R; however, ketamine hardly bound directly to S1R under our conditions. As dissociation from BiP is an indicator of the activation of S1R [1], we then performed IP with S1R-overexpressing HEK293T cells to examine whether ketamine has an indirect effect on S1R. Interestingly, our data showed that S1R activation became more obvious with increasing ketamine concentrations and incubation times (Fig. 3e–h). These results suggest that ketamine triggers S1R activation without direct interaction with S1R. Incubation with 10 μmol ketamine for 24 h was thus used in subsequent studies.

Fig. 3. Analysis of interaction between ketamine and S1R.

a The binding mode of the ketamine and S1R. Ketamine is shown as green, and the pocket residues as yellow. b The 2D interactions diagram of the ketamine and S1R. c, d Displacement curves of SA4503 and ketamine for [³H]-(+)-pentazocine binding to S1R. e, g Representative Western blotting images of S1R-BiP association after ketamine treatment. f, h Quantification for the effects of ketamine on the S1R-BiP association. One-way ANOVA with Dunnett’s post hoc test, n = 4, *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± SEM.

S1R plays an important role in ketamine-induced increase in BDNF levels

As a molecular chaperone, S1R functions by interacting with various proteins. To explore the subsequent effects of ketamine-induced activation of S1R, we next employed IP-MS to identify candidate proteins that interact with S1R after ketamine administration (Supplementary Figs. 2~3). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the “genetic information processing” was significantly enriched, such as “DNA replication”, “RNA transport”, “mRNA surveillance pathway” and “protein processing in endoplasmic reticulum” (Fig. 4a). Considering that BDNF is crucial for the antidepressant action of ketamine and the restoration of mPFC^Pyr neuron structure and function [14, 31], we thus investigated the impact of S1R on the regulatory effect of ketamine on BDNF levels. We employed a small interfering RNA (siRNA) to knock down S1R in primary cortical neurons, and the control group received an equal volume of siRNA(NC) (Fig. 4b, c). As shown in Fig. 4d, e, there was a marked increase in the mRNA level of BDNF after ketamine incubation, which was reversed by S1R knockdown; knocking down S1R also weakened the effects of ketamine on the levels of pro-BDNF and mature BDNF. We next injected a short hairpin RNA (shRNA) via CaMKIIα promoter-driven AAV into the mPFC to specifically knock down S1R in mPFC^Pyr neurons. The control group received AAV-CaMKIIα-shRNA(NC), which carried only the independent sequence. Immunofluorescence staining showed that shRNA expression was confined to the mPFC (Fig. 4f). Ketamine treatment resulted in reduced passive immobility (Fig. 4g) and increased the levels of BDNF mRNA, pro-BDNF and mature BDNF in control mice, while these effects were weakened in S1R knockdown mice (Fig. 4h, i). These results indicate that S1R plays an important role in ketamine-induced increase of BDNF.

Fig. 4. S1R plays an important role in ketamine-induced increase of BDNF.

a KEGG enrichment analysis. S1R-OE, S1R overexpression; Ket, ketamine. Veh, vehicle. b Experimental design and representative immunostaining image of MAP-2 (red) and DAPI (blue) in primary cortical neurons. c Verification of siRNA knockdown effect. d, e Quantification and representative Western blotting images of S1R and BDNF in primary cortical neurons. Two-way ANOVA with Sidak’s post hoc test, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001. f Experimental design and representative immunostaining images of shRNA(green) and DAPI (blue) in mPFC. g Immobility time in TST. Two-way ANOVA with Sidak’s post hoc test, n = 10–11, *P < 0.05. h, i Quantification and representative Western blotting images of S1R and BDNF in mPFC. Two-way ANOVA with Sidak’s post hoc test, n = 4–6, *P < 0.05, **P < 0.01, ***P < 0.001. Data are presented as the mean ± SEM.

Pretreatment with an S1R agonist enhances the sustained effect of ketamine via BDNF

Finally, we examined the synergistic effects of ketamine and S1R agonists. Mice were treated with ketamine at a subeffective dose (1 or 5 mg·kg⁻¹) or an effective dose (10 mg·kg⁻¹) or ketamine in combination with SA4503 (1 mg·kg⁻¹) and then the TST test was performed two days after drug administration (Fig. 5a). Our data showed that there was a significant decline in immobility time during the TST only in mice treated with 10 mg·kg⁻¹ ketamine, while the lower doses failed to induce active struggling to escape (Fig. 5b). Notably, both 5 and 10 mg·kg⁻¹ ketamine produced antidepressant-like effects in mice when administered in combination with SA4503 (Fig. 5b). These results suggest that pretreatment with an S1R agonist enhances the antidepressant-like effect of ketamine. To further clarify whether the synergistic effect of ketamine with the SIR agonist was related to BDNF, we used an shRNA to knockdown BDNF specifically in mPFC^Pyr neurons as previously mentioned (Fig. 5c). The shRNA was detected in the mPFC and suppressed the expression of BDNF (Fig. 5d, e). The results showed that the combined application of ketamine and the S1R agonist effectively reduced the immobility time during TST in the shRNA(NC) groups, but this reduction was significantly blocked in the shRNA(BDNF) groups (Fig. 5f). Our findings demonstrate that an increase of BDNF is necessary for S1R agonists to enhance the sustained antidepressant effect of ketamine.

Fig. 5. Pretreatment with S1R agonists augments the effects of ketamine via BDNF.

a Experimental design. b Immobility time in TST after combination treatment. One-way ANOVA with Tukey’s post hoc test, n = 8–10, *P < 0.05, **P < 0.01. c, d Representative images of injection sites in mPFC. e Verification of shRNA knockdown effect. f Immobility time in TST after gene manipulation of BDNF. Unpaired Student’s t test, n = 10–11, *P < 0.05. Data are presented as the mean ± SEM.

Discussion

S1R is a type II membrane protein comprised of at least one transmembrane domain. It is highly clustered at the MAM and has a long C-terminus residing in the endoplasmic reticulum (ER) lumen, which forms a ligand-binding site [32]. S1R is not a classic receptor in the conventional sense but rather a chaperone protein. Mechanistically speaking, S1R acts more like a signaling modulator between organelles; functionally speaking, it is versatile [33]. At the MAM, the physiologic function of S1R is achieved through another ER chaperone immunoglobulin heavy chain binding protein (BiP). Normally, S1R forms a Ca²⁺-sensitive complex with BiP; upon ER Ca²⁺ depletion or agonist stimulation, S1R dissociates from BiP and translocates to other parts of the ER, thereby maximizing its chaperone activity. In contrast, S1R antagonists strengthen the binding between S1R and BiP, blocking the effect of S1R agonists [1].

S1R was identified as a pharmacological target of antidepressants in the 1990s [34]. The evidence supporting this finding is the high affinity of serotonin reuptake inhibitors (SSRIs), such as fluvoxamine and sertraline, for S1R [7, 8, 35, 36]. Indeed, both endogenous and exogenous ligands that act as S1R agonists demonstrate antidepressant-like activity [9–11, 37]. Ketamine, which produces a rapid antidepressant effect and is widely recognized as an NMDA receptor antagonist [38], has also been reported to have micromolar affinity for S1R [15]. Our data demonstrate that S1R activity is involved in the sustained behavioral effect of ketamine (Fig. 1). The contradictory finding that BD1047 did not affect the antidepressant-like action of ketamine (40 mg·kg⁻¹) in forced swimming test [15] is thought to be due to differences in the mouse species, drug regimen, and observation time point used. S1R is highly expressed in neurons and glial cells in the brain, among which mPFC^Pyr neurons have been strongly implicated in depression [39]. Inhibition of S1R inhibited the restoration of mPFC^Pyr neuron structure and function (Fig. 2). Although our molecular docking results show that ketamine binds well with S1R, ketamine could not displace a labeled form of the S1R agonist [³H]-(+)-pentazocine from the binding site even when the dose reached 1 mmol (Fig. 3). However, ketamine strongly agonized S1R according to the S1R/Bip association assay (Fig. 3). Our findings suggest that ketamine indirectly activates S1R, possibly through the induction of Ca²⁺ influx [14] (Supplementary Fig. 4), resulting in calcium dyshomeostasis and ER stress [40]. The underlying mechanisms need to be further investigated in future studies.

S1R is redistributed from the MAM to the entire ER network after activation and dissociation from BiP [1]. Several studies have even suggested that S1R translocated to various cellular compartments, where it can interact with various ion channels, G-protein coupled receptors, and plasma and nuclear proteins [41–47]. Notably, whether S1R undergoes translocation in this manner is controversial; S1R has been reported to be an ER resident protein in HEK293 cells that does not translocate to the plasma membrane in either the absence or presence of S1R agonists or ER stress [32]. Although the author cannot exclude that in some cell types, S1R might exit the ER in complex with other proteins, the previous findings are undoubtedly worth revisiting. However, it is certain that S1R acts as a multifunctional molecular chaperone.

S1R is involvedt in controlling protein translation and suppressing the misfolded proteins, which cause the unfolded protein response [1]. Our results show that S1R participated in ketamine-induced BDNF upregulation (Fig. 4). BDNF/TrkB signaling pathway and spine formation are key cellular responses that underlie the sustained antidepressant effect of ketamine and long-lasting behavioral recovery [28, 48–53]. Further investigation demonstrates that pretreatment with an S1R agonist enhanced the sustained antidepressant effect of a lower dose of ketamine, but this effect was abolished after specific knockdown of BDNF (Fig. 5). In addition, S1R has been reported to promote neurite outgrowth via direct interaction with the TrkB cytosolic domain and phosphorylation at Y515 of TrkB [54]. The presence of both S1R and BDNF is necessary for the activation of TrkB signaling and spine formation, which can be blocked by pharmacological inhibition of S1R [55, 56]. The potential mechanism by which S1R mediates the sustained antidepressant effect of ketamine needs further exploration.

In summary, we have demonstrated that S1R is involved in the behavioral effect of ketamine and the restoration of mPFC^Pyr neuron structure and function; that ketamine triggers the activation of S1R and then increases the level of BDNF; and that in the presence of BDNF, pretreatment with an S1R agonist can enhance the sustained antidepressant effect of ketamine. Thus, combination treatment with S1R agonists and ketamine may be a promising approach for depression.

Author contributions

HM, YFL, and YZ designed the study; HM, JFL, XQ, YZ, XJH, HXC, and HLC performed the research; HM, JFL, and XQ wrote the manuscript; YFL and YZ revised the manuscript. All authors approved the submitted version.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1038/s41401-023-01201-8.