The Prefrontal Dectin-1/AMPA Receptor Signaling Pathway Mediates The Robust and Prolonged Antidepressant Effect of Proteo-β-Glucan from Maitake

Proteo-β-glucan from Maitake (PGM) is a strong immune regulator, and its receptor is called Dectin-1. Cumulative evidence suggests that AMPA receptors are important for the treatment of depression. Here, we report that PGM treatment leads to a significant antidepressant effect in the tail suspension test and forced swim test after sixty minutes of treatment in mice. After five consecutive days of PGM treatment, this antidepressant effect remained. PGM treatment did not show a hyperactive effect in the open field test. PGM significantly enhanced the expression of its receptor Dectin-1, as well as p-GluA1(S845) and GluA1, but not GluA2 or GluA3 in the prefrontal cortex (PFC) after five days of treatment. The Dectin-1 inhibitor Laminarin was able to block the antidepressant effect of PGM. At the synapses of PFC, PGM treatment significantly up-regulated the p-GluA1(S845), GluA1, GluA2, and GluA3 levels. Moreover, PGM’s antidepressant effects and the increase of p-GluA1(S845)/GluA1 lasted for 3 days after stopping treatment. The AMPA-specific antagonist GYKI 52466 was able to block the antidepressant effect of PGM. This study identified PGM as a novel antidepressant with clinical potential and a new antidepressant mechanism for regulating prefrontal Dectin-1/AMPA receptor signalling.


PGM demonstrated a robust antidepressant effect in the tail suspension test (TST) and forced swim test (FST).
Seven-week-old CD-1 mice were intraperitonially (i.p.) injected with a low (5 mg/kg), medium (8 mg/kg), and high (12.5 mg/kg) doses of PGM for 60 minutes or 5 days before testing. Sixty minutes after the treatment, the mice were subjected to either TST or FST (Fig. 1). The data showed that the duration of immobility in the PGM-treated groups was significantly lower than that of the controls (103.0 ± 12.0 sec) in a dose-dependent manner, as low as 60.3 ± 9.2 sec (for 5 mg/kg of PGM), 55.3 ± 10.5 sec (for 8 mg/kg of PGM), and 42.8 ± 11.3 sec (for 12.5 mg/kg of PGM) in the TST (ANOVA, F(4,53) = 6.991, p < 0.01) ( Fig. 2A). The positive control imipramine (33.2 ± 8.0 sec) also demonstrated an antidepressant effect ( Fig. 2A). To confirm these data, FST was performed under similar conditions. After 60 minutes of treatment with PGM, the data showed that the duration of immobility in the PGM-treated groups was significantly lower than that of the controls (103.8 ± 14.5 sec), as low as 51.0 ± 9.5 sec (for 5 mg/kg of PGM), 52.3 ± 10.0 sec (for 8 mg/kg of PGM), and 50.5 ± 9.1 sec (for 12.5 mg/kg of PGM) for FST, similar to the traditional antidepressant imipramine (51.5 ± 12.2 sec) (ANOVA, F(4,54) = 4.348, p < 0.01) (Fig. 2B). To further examine the antidepressant effect of PGM after long-term treatment, we treated mice with the same doses as mentioned above for 5 consecutive days, Figure 1. Experimental procedures. Mice were acclimatized for at least 1 week. Four independent cohorts of animals were used to test the antidepressant effects of proteo-β -glucan from Maitake (PGM). The animal behavioural tests were performed 60 minutes (mins) after drugs or vehicle were administered. The first independent cohort of animals underwent the tail suspension test (TST) after 60 minutes treatment, open field test (OFT) on the third day, and forced swim test (FST) on the fifth day. To confirm the antidepressant effects of PGM, the second independent cohort of animals was subjected to the FST after 60 minutes of treatment and to the TST on the fifth day under similar conditions. The third independent cohort of animals was used to test the Dectin-1 receptor-specific blocking reagent Laminarin (Lam) in the antidepressant effect of PGM in the TST. The fourth independent cohort of animals was used to test whether the α -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor-specific antagonist GYKI 52466 (GYKI) was involved in the antidepressant effect of PGM in the TST.
Scientific RepoRts | 6:28395 | DOI: 10.1038/srep28395 and TST and FST were performed after the treatment (Fig. 1). The data showed that the immobility times in the PGM-treated groups were significantly lower than those of the controls (107.3 ± 14.0 sec) in a dose-dependent . CD-1 mice were i.p. injected with a low dose of PGM (5 mg/kg/day, PGM-L), a medium dose of PGM (8 mg/kg/day, PGM-M), a high dose of PGM (12.5 mg/kg/day, PGM-H), imipramine (15 mg/kg/day, IMI) or saline (Sal). After PGM treatment for sixty minutes (mins) or five days, mice were subjected to the TST or FST. The number (N) of mice per group is indicated in each individual graph. Any experimental data value greater than mean plus 2 × standard deviations (SDs) from a group was considered an outlier and was not considered in the analysis. Data were analysed by one-way ANOVA and presented as the mean ± SE (post hoc Tukey's test, * p < 0.05, * * p < 0.01, * * * p < 0.001). (A,B) Sixty minutes after the injection, PGM significantly reduced the immobility time in the TST or FST. (C,D) After 5 consecutive days of injection, PGM significantly reduced the immobility time in the TST or FST.

PGM did not show locomotor hyperactivity in the open field test (OFT).
We performed OFT after 3 consecutive days of treatment with a low, medium, or high doses of PGM (Fig. 1). The total distance travelled showed no significant difference in the PGM-treated groups compared with the controls, which suggested that PGM does not cause locomotor hyperactivity in mice (ANOVA, F(4,35) = 0.4653, p = 0.7607) (Fig. 3A). In addition, the distance travelled in the centre area also showed no significant difference in the PGM-treated groups compared with the control groups (ANOVA, F(4,35) = 0.8818, p = 0.4848) (Fig. 3B). The weights of mice after 5 consecutive days of treatment did not show any significant differences (ANOVA, F(4,35) = 2.448, p = 0.0797) (Fig. 3C).
The antidepressant effect of PGM was blocked by the AMPA receptor-specific antagonist GYKI 52466. We hypothesized that the increase in AMPA receptor signalling is critical for the antidepressant effect of PGM. To test this hypothesis, the AMPA receptor-specific antagonist GYKI 52466 was used to determine whether the antidepressant effect of PGM could be blocked by GYKI 52466. CD-1 mice were i.p. injected with PGM (8 mg/kg) for 60 minutes. GYKI 52466 (10 mg/kg) was administered 30 minutes prior to behavioural testing (Fig. 1). Sixty minutes after the PGM injection, the mice were subjected to TST. The treatment with GYKI 52466 almost completely blocked the decrease of immobility time caused by PGM (Sal: 127.6 ± 11.4 sec; Sal + GYKI: 135.8 ± 12.5 sec; PGM + GYKI: 115.1 ± 11.5 sec; PGM: 62.3 ± 12.8 sec) (ANOVA, F(3,28) = 7.534, p < 0.01) (Fig. 9), which suggested that enhanced AMPA receptor excitability at the synapses might play an important role in the antidepressant effect of PGM.

Discussion
This study sought to study the robust and prolonged antidepressant effects of PGM and its underlying mechanisms in regulating Dectin-1/AMPA receptor signalling. We found that: (1) PGM had a robust antidepressant effect in the TST and FST after 60 minutes of treatment, and this effect remained after 5 consecutive days of treatment in the TST and FST; (2) PGM had no hyperactive effect in the OFT; (3) PGM enhanced the expression Figure 7. Proteo-β-glucan from Maitake (PGM) produced a prolonged antidepressant effect. CD-1 mice were i.p. injected with a high dose of PGM (12.5 mg/kg/day) or imipramine (15 mg/kg/day, IMI) or saline (Sal) for 5 days. Then, the treatment with PGM or imipramine was stopped for 3 days or 5 days. As shown in the figure, the PGM-S and IMI-S indicated the groups that stopped treatment with PGM or imipramine; PGM-C and IMI-C indicated the groups that continued the treatment. The number (N) of mice per group was indicated in each individual graph. Any experimental data value greater than mean plus 2 × standard deviations (SDs) from a group was considered an outlier and was not considered in the analysis. Data were analysed by one-way ANOVA and presented as the mean ± SE (post hoc Tukey's test, * p < 0.05, * * p < 0.01, * * * p < 0.001). (A,B) After stopping treatment for 3 days, the animals were subjected to TST or FST. (C) After stopping treatment for 5 days, mice were subjected to FST.

. Proteo-β-glucan from Maitake (PGM) evoked a prolonged increase of p-GluA1(S845) (P-GluA1)
and GluA1 levels in the prefrontal cortex (PFC). CD-1 mice were i.p. injected with a high dose of PGM (12.5 mg/kg/day) or imipramine (15 mg/kg/day, IMI) or saline (Sal) for 5 days. Then, the treatment with PGM or imipramine was stopped for 3 days. As shown in the figure, the PGM-S and IMI-S indicated the groups that stopped treatment with PGM or imipramine; PGM-C and IMI-C indicated the groups that continued the treatment. Western blot analyses of the proteins from the PFC were performed with anti-p-GluA1(S845), anti-GluA1, anti-GluA2 or anti-GluA3 antibodies. The number (N) of mice per group is indicated in each individual graph. Data were analysed by one-way ANOVA and presented as the mean ± SE (post hoc Tukey's test, * p < 0.05, * * p < 0.01, * * * p < 0.001). The expression levels of (A) p-GluA1(S845), (B) GluA1, (C) GluA2, or (D) GluA3 in the PFC were determined.
Scientific RepoRts | 6:28395 | DOI: 10.1038/srep28395 synapses in the PFC, PGM enriched p-GluA1(S845) expression after 60 minutes of treatment and p-GluA1(S845), GluA1, GluA2, and GluA3 levels after 5 days of treatment, similar to imipramine; (7) the antidepressant effect of PGM lasted for 3 days, but not for 5 days after stopping the treatment; and (8) the AMPA receptor-specific antagonist GYKI 52466 was able to block the robust antidepressant effect of PGM.
The Dectin-1/AMPA receptor signalling pathway demonstrated a novel mechanism that is closely related to the neuroimmune system. Currently, the most famous rapid antidepressant is ketamine, a NMDA receptor antagonist that elicits a rapid antidepressant response in patients with depression 23,24 and bipolar depression [25][26][27] . Evidence suggests that ketamine's antidepressant properties rely on blocking NMDA receptors, increased AMPA signalling, rapidly induced synaptogenesis 1,10 and lasted for a few days to weeks 28 . Here, we reported a replacement for ketamine, PGM, which showed a robust and prolonged antidepressant effect mediated through the Dectin-1/AMPA receptor signalling pathway. Both PGM and imipramine showed an antidepressant effect in mice ( Fig. 2A-D); however, clinical trials are warranted to determine the effective times in humans. Although similar results were found for the 5-day treatment after acute treatment for 60 minutes, a prolonged antidepressant result was observed after stopping the drug treatment for up to three days (Fig. 7A,B). Although PGM showed a prolonged antidepressant effect for 3 days, it was shorter than the weeks-long antidepressant effect of ketamine 10 .
Other psycho-stimulants, such as cocaine and amphetamine, may also have an antidepressant effect, but these psycho-stimulants usually induce excitatory neural toxicity and drug addiction and lead to the development of depressive symptoms during withdrawal [29][30][31] . Although PGM demonstrated a strong antidepressant effect, it did not elicit hyperactivity like some other psycho-stimulants (Fig. 3A,B). This is different from the psycho-stimulant amphetamine, which significantly increased the total distance travelled in rats during the OFT 32 .
PGM is a proteo-β -glucan. It has been shown that glucan can pass through the blood brain barrier via transporters or during the inflammatory process 5,33,34 . Here, we reported that PGM within a specific dose range (5-12.5 mg/kg) might bind to its receptor Dectin-1 in the brain to exert a robust antidepressant effect. The fact that the Dectin-1 receptor levels were significantly increased after PGM treatment was an indicator of the activation of the Dectin-1 receptors (Fig. 4A). Dectin-1 inhibition almost completely blocked the antidepressant effect of PGM (Fig. 4B). Therefore, the Dectin-1/AMPA pathway could be a newly discovered pathway for the treatment of depression. Binding to Dectin-1 could lead to the activation of Syk/NFκ b signalling pathways and regulation of the neuroimmune system 35 . Targeting Dectin-1 may become a novel strategy for the treatment of mood disorders.
PGM regulated the p-GluA1(S845), GluA1, GluA2, and GluA3 levels at the synapses in the PFC, which suggested a critical role of the AMPA signalling pathway. GluA1 phosphorylation at S845, which is a PKA site, is often viewed as an indicator of GluA1 membrane insertion in neurons and wide channel opening 20,[36][37][38] . Previous studies have shown that the levels of AMPA GluA1 in the PFC in depressed patients were decreased 39,40 , which was consistent with previous animal experiments [41][42][43] . AMPA receptors may serve as a common mechanism for the treatment of mood disorders 21,[44][45][46][47] . Consistent with previous findings, we found that PGM was able to enhance p-GluA1(S845) in the total protein preparations or in the synaptic fractions from the PFC within 60 minutes (Figs 5A and 6B). After 5 days of treatment, PGM was able to enhance both the p-GluA1(S845) and GluA1
As to the mechanisms of regulating AMPA receptors, PGM may go through either direct or indirect mechanism(s). PGM may directly regulate neuronal synapse through the Dectin-1 receptor on the neurons followed by the activation of the signalling cascades (e.g., Syk/NFκ b signalling pathway) 35 . PGM may also modulate cytokine expression, leading to the production of IL-2, TNF-α , and IL-18 48,49 . It was also reported that cytokines could regulate AMPA receptor signalling 50 . Further exploration of these mechanisms will be one of our future directions.
Previous studies showed that lithium, cordycepin, and dextromethorphanall exerted a rapid antidepressant effect in mice in the TST and FST via up-regulating the AMPA receptor subunits, and an AMPA inhibitor was able to block the antidepressant effects 47,51,52 . Recent clinical trials have shown that Org 26576 (ionotropic AMPA-type glutamate receptor enhancer) significantly improved symptoms in depressed patients as revealed by the Montgomery-Asberg Depression Rating Scale 53 . In addition, the biaryl-propyl-sulphonamide ARPs (LY392098 and LY451616, AMPA receptor potentiators) have antidepressant effects in animal models of depression, in learned-helplessness models of depression, and in animals exposed to chronic mild stress 17,54 , which suggests that the enhancement of AMPA function was sufficient for the antidepressant effect. We found that PGM up-regulated AMPA receptors at the synapse (Fig. 6G-I), and this effect lasted for 3 days after stopping the treatment (Fig. 8B). The AMPA antagonist GYKI 52466 was able to block the PGM-induced antidepressant effect (Fig. 9), which suggested that enhanced AMPA synaptic transmission is essential for the antidepressant effect.
In this paper, we identified PGM as a strong antidepressant with clinical potential for the treatment of depression. It acts by enhancing novel prefrontal Dectin-1/AMPA receptor signalling. This study suggested that the novel target Dectin-1 may be significant in the development of effective antidepressants with novel mechanism(s) for the treatment of major depression.

Methods
Animals. All animal procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (ISBN: 0-309-05377-3) and were approved by the Institutional Animal Care and Use Committee at Yunnan University School of Medicine (IACUC: MS201402). Male CD-1 mice (6 weeks; starting weight, 22-26 g; Vital River, Beijing, China) were group housed (N = 4/cage) in an animal room with a constant temperature (22 ± 1 °C) and maintained on a 12-hour light/dark cycle (lights on/off at 9:00 A.M./9:00 P.M.), with constant humidity (55 ± 10%) and free access to water and food. After a one-week acclimatization period, the mice were treated with drugs or vehicle in a volume of 10 μ l/g by intraperitoneal (i.p.) injection and tested between 9 A.M. and 12 A.M.

Bioactive Proteo-β-Glucan from Maitake (PGM). The Mataike D-fraction was extracted from
Maitake mushrooms, which were prepared in a standardized procedure as described previously by Mushroom Wisdom, Inc. 3,55 . The D-fraction obtained using this standard method showed a single peak with a MW of 1200-2000 kDa by HPLC analysis and consisted of 98% polysaccharide and 2% peptides. We purchased the supplies from Mushroom Wisdom Inc (East Rutherford, NJ, USA). They labeled their product as containing 30% of the D-fraction solution in water and glycerol. We performed a triple volume of 95% ethanol precipitation procedure to remove the water and glycerol. After incubating the mixture for 60 minutes at room temperature, the samples were centrifuged at 12000rcf/min at 4 °C for 60 minutes 35 . The precipitate was dried with air and re-dissolved in saline for the animal i.p. injections. After ethanol extraction, the resulting proteo-β -glucan was defined as the proteo-β -glucan from Maitake (PGM).

Animal behavioural studies.
To examine the antidepressant effects from PGM, mice were randomly assigned to five treatment groups: saline (sterile 0.9% sodium chloride solution), low dose of PGM (5 mg/kg, dissolved in saline), medium dose of PGM (8 mg/kg, dissolved in saline), high dose of PGM (12.5 mg/kg, in saline), and imipramine (15 mg/kg, in saline; Sigma, St. Louis, MO, USA). The animal behavioural tests were performed 60 minutes after drugs or vehicle were administered, and the TST was performed after 60 minutes, OFT on the third day, and FST on the fifth day. To confirm the antidepressant effects of PGM, another group of mice was subjected to the FST after 60 minutes and to the TST on the fifth day, respectively, under similar conditions.
To examine whether the antidepressant effect of PGM could be blocked by Laminarin (Lam, a specific Dectin-1 blocking reagent, Sigma, St. Louis, MO, USA), CD-1 mice were i.p. injected with Laminarin (10 mg/kg) 2 hours prior to the behavioural testing, followed by the PGM injection one hour later. Sixty minutes after the PGM injection, the mice were subjected to the TST 56 . The AMPA-specific inhibitor GYKI 52466 (a selective non-competitive AMPA receptor antagonist, TOCRIS Bioscience, R&D, Minneapolis, USA) was used to determine the AMPA effect. CD-1 mice were i.p. injected with PGM (60 minutes before the TST) followed by a GYKI 52466 injection (10 mg/kg in 8%DMSO/92%saline 30 minutes before the TST) 47,57 . For each drug treatment, the control mice received the vehicle alone via i.p. injection.
Tail suspension test (TST). The TST was performed according to a previously described procedure 58 with minor modifications. A piece of tape 7 cm in length and 2 cm in width was positioned with approximately 2 mm of the tail protruding. Each mouse was individually suspended by the tail from a bar (30 cm high) and videotaped during a 6-minute test session. Immobility time was quantified by a naive observer for the last 4 minutes. Open field test (OFT). An activity chamber (60 × 60 × 30 cm) with a black floor was divided into 16 squares of equal area (15 × 15 cm) by white lines and used to study PGM-induced locomotor hyperactivity. Three days after i.p. injection with various concentrations of drugs, the mice were placed in the centre of the chamber and their behaviours were recorded for 60 minutes. The total distance travelled and the distance travelled in the centre area (the 4-square area in the middle of the chamber) were analysed by the ANY-maze system (Stoelting, Wood Dale, USA).

Forced swim test (FST).
The FST was carried out according to previously described procedures 59 with minor modifications. Mice were placed in a cylinder (Φ = 20 cm) with water (temperature between 23 ± 1 °C) 20 cm in depth. Mice were videotaped during a 6-minute test session and were later analysed for mobility for the last 4 minutes. Mobility was defined as any movement beyond what was necessary to maintain their head above water. Immobility time was quantified by a naive observer.