Akebia saponin D acts via the PPAR-gamma pathway to reprogramme a pro-neurogenic microglia that can restore hippocampal neurogenesis in mice exposed to chronic mild stress

DOI: https://doi.org/10.21203/rs.3.rs-1838854/v1

Abstract

Using drugs to modulate microglial function may be an effective way to treat disorders such as depression that involve impaired neurogenesis. Akebia saponin D (ASD) can cross the blood-brain barrier and exert anti-inflammatory and neuroprotective effects, so we wondered whether it might influence adult hippocampal neurogenesis to treat depression. We exposed C57BL/6 male mice to chronic mild stress (CMS) as a model of depressive-like behaviors, then gave them ASD (40 mg/kg) intraperitoneally once daily for 3 weeks. We investigated the effects of ASD on microglial phenotype, hippocampal neurogenesis, and animal behavior. We found that CMS upregulated pro-inflammatory factors and downregulated anti-inflammatory factors in dentate gyrus of mice, while decreasing sucrose preference and prolonging immobility time in the forced swimming test. CMS also inhibited hippocampal neurogenesis. ASD treatment induced Arg-1+ microglia and increases brain-derived neurotrophic factor (BDNF) expression in the dentate gyrus of CMS mice, and partially reversed all these stress effects. Conditioned medium from microglia treated with ASD and lipopolysaccharide (LPS) strongly increased levels of phospho-TrkB in neural stem/progenitor cells (NSPCs), while promoting their proliferation, survival and neuronal differentiation. An antagonist of TrkB blocked the pro-neurogenic, anti-depressant effects of ASD. Furthermore, ASD activated peroxisome proliferator-activated receptor-gamma (PPAR-γ) in dentate gyrus of CMS mice as well as in primary microglial cultures treated with LPS. Blocking the PPAR-γ using GW9962 suppressed the ASD-reprogrammed Arg-1+ microglia and BDNF expression in dentate gyrus of CMS mice. Such blockade abolished the promoted effects of ASD-treated microglia on NSPC proliferation, survival and neurogenesis. The pro-neurogenic and anti-depressant effects of ASD were blocked by GW9962. These results suggested that ASD acts via the PPAR-γ pathway to induce a pro-neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neurogenesis. Our results justify further studies of ASD as a potential treatment for depression and may inspire new lines of research targeting the PPAR-γ pathway in disorders involving impaired neurogegammnesis.

1. Introduction

Major depressive disorder affects millions of people worldwide and has multifactorial causes, leading to heterogeneous clinical presentations [1, 2]. Current anti-depressant medications often lack efficacy [3, 4], highlighting the need for further studies of how depression and related diseases can be treated.

The occurrence and development of depression have been linked to decreased neurogenesis in the adult hippocampus [5–7]. Restoring hippocampal neurogenesis can enhance stress resistance and mitigate depressive symptoms [8, 9], so this may be an effective strategy for treating depression [10, 11]. During neurogenesis in adult hippocampus, neural stem/progenitor cells (NSPCs) proliferate and differentiate into neurons [12]. In neurogenic niches, NSPCs produce neurons that support learning, memory, and behavior, and this production continues through adulthood [13]. Efficient neurogenesis depends on a supportive “neurogenic niche” in the hippocampus [9].

An important modulator of neurogenesis in this niche are microglia [14–16]. Pro-inflammatory microglia suppress adult hippocampal neurogenesis, while anti-inflammatory microglia do the opposite [9, 17, 18]. Chronic stress hyperactivates microglial cells, which secrete inflammatory mediators that impair neuroplasticity and neurogenesis [19–21]. Nevertheless, microglia are endowed with phenotypic plasticity to regulate physiological responses and behavioral outcomes during stress [22–25]. In particular, the Arg-1+ microglia were considered as a neuroprotective microglial phenotype [26, 27]. Our previous research found that this subgroup of microglia can secrete brain-derived neurotrophic factor (BDNF) to promote hippocampal neurogenesis in responding to chronic stress, helping protect against depressive-like symptoms [9]. Thus, pharmacological modulation of the microglial phenotype may allow control of NSPC proliferation and differentiation to treat disorders associated with neurogenic dysfunction.

Several natural products show promise for modulating microglial phenotype [20]. For example, the akebia saponin D (ASD), from the herb Dipsacus asper Wall., efficiently crosses the blood-brain barrier and exerts anti-inflammatory and neuroprotective effects [28–30]. We have shown that ASD regulates microglial function to ameliorate neuroinflammation and depression-like behaviors of mice exposed to lipopolysaccharide (LPS) [31, 32]. However, we are unaware of studies exploring whether ASD influences hippocampal neurogenesis, which may thereby help explain its antidepressant effects.

Thus, here we investigated the effects of ASD on microglial phenotypes, hippocampal neurogenesis and depressive-like behaviors in mice exposed to chronic mild stress (CMS). To gain greater mechanistic insights, we also examined the effects of conditioned medium from ASD-induced microglia on NSPC proliferation, survival and neuronal differentiation.

2. Materials And Methods

2.1 Animals

Male C57BL/6J mice (7–8 weeks old) were purchased from Changsha Tianqin Biotechnology (Changsha, China), caged individually, and assigned unique numbers. The mice were habituated to their new environment for two weeks. The mice were then habituated to a 1% sucrose solution for 48 h. Sucrose preference and body weight were determined weekly as described in section 2.4.1. Body weight and sucrose preference during the first four weeks served as the pretreatment baseline, and animals were allocated into five groups as described in section 2.3.1. All experiments were approved by the Institutional Animal Care and Use Committee at the Guizhou University of Traditional Chinese Medicine.

2.2 Chronic mild stress (CMS)

Animals were exposed to CMS for 7 weeks as described [9]. Everyday, animals were exposed to three of the following stressors in random order: empty water bottles (12 h), food deprivation (12 h), tail clipping (10 min), restraint (2 h), lights-off for 3 h during the daylight phase, cage shaking (1 h), cage tilting (45°, 24 h), reversal of the light–dark cycle (24 h), strobe lighting (12 h), damp bedding (24 h) and a soiled cage (24 h). The schedule is detailed in supplementary Table 1.

2.3 Pharmacological treatments in vivo

2.3.1 Treatment with ASD and imipramine

Akebia saponin D (99.92% pure) was purchased from Chengdu Alfa Biotechnology (Chengdu, China), and dissolved to a concentration of 4 mg/mL in 0.9% saline. After 4 weeks of CMS, the mice were allocated into five groups such that the groups did not differ significantly in sucrose preference or body weight: control + saline (Ctrl), control + ASD (ASD), CMS + saline (CMS), CMS + ASD (CMS + ASD), and CMS + imipramine (CMS + IMI). The mice were intraperitoneally administered saline, ASD (40 mg/kg/d), or imipramine (10 mg/kg/d; Sigma-Aldrich, MO, USA) once daily (at 16:00 h) for 3 weeks. The doses of ASD and imipramine were chosen based on previous studies [20, 31].

2.3.2 Treatment with temozolomide

The potential role of the neurogenesis in the anti-depressant was analyzed using the neurogenesis inhibitor temozolomide (TMZ, Sigma-Aldrich, MO, USA). TMZ was dissolved in 0.9% saline containing 5% dimethyl sulfoxide (DMSO) at a concentration of 2.5µg/mL. After 4 weeks of CMS, four animal groups with similar sucrose preference and body weight were formed: control + saline + DMSO (Ctrl), CMS + saline + DMSO (CMS), CMS + ASD + DMSO (CMS + ASD), CMS + ASD + TMZ (CMS + ASD + TMZ). The mice were intraperitoneally administered saline, DMSO, ASD (40 mg/kg/d), and TMZ (25 µg/kg/d) once daily (at 16:00 h) for 3 weeks.

2.3.3 Treatment with K252a or GW9662

To investigate the role of peroxisome proliferator-activated receptor-gamma (PPAR-γ) pathway in ASD regulation of microglia phenotype, we used the PPAR-γ inhibitor GW9662 (Sigma-Aldrich, MO, USA). To investigate the potential role of the BDNF-tropomyosin receptor kinase B (TrkB) pathway in pro-neurogenic effects of ASD, the K252a (Sigma-Aldrich, MO, USA) was used to block the TrkB. GW9662 or K252a was dissolved in 0.9% saline containing 5% dimethyl sulfoxide (DMSO) at a concentration of 1 mg/mL and 2.5µg/mL respectively. After 4 weeks of CMS, five animal groups with similar sucrose preference and body weight were formed: control + saline + DMSO (Ctrl), CMS + saline + DMSO (CMS), CMS + ASD + DMSO (CMS + ASD), CMS + ASD + K252a (CMS + ASD + K252a) and CMS + ASD + GW9662 (CMS + ASD + GW9662). The mice were intraperitoneally administered saline, DMSO, ASD (40 mg/kg/d), and K252a (25 µg/kg/d) or GW9662 (1 mg/kg/d) once daily (at 16:00 h) for 3 weeks. The doses of K252a and GW9662 were chosen based on previous studies [9, 33].

2.4 Behavioral testing

2.4.1 Sucrose preference test (SPT)

The sucrose preference test was performed as described [34]. Mice were individually housed, deprived of food and water for 12 h, then given access to 1% sucrose solution (A) and water (B) for 2 h. The bottle positions were switched daily to avoid a side bias. The sucrose preference was calculated each week for each mouse using the formula: 100 × [VolA / (VolA + VolB)]. The sucrose consumption was normalized to body weight for each mouse.

2.4.2 Forced swimming test (FST)

At 24 h before the test, each mouse was placed individually for 10 min in a glass cylinder (height, 25 cm; diameter, 15 cm) that was filled with water to a depth of 15 cm at 26 ℃. The next day, the mice were placed again in the same situation for 6 min. An observer masked to treatment conditions recorded the latency between suspension and first abandonment of struggle as well as the time spent immobile during the 6-min period.

2.4.3 Open field test (OFT)

Mice were placed into an open field (50 × 50 cm2) and allowed to explore freely for 15 min. Total distance and time spent in the center (25 × 25 cm2) were quantified using video tracking software (OFT100, Taimeng Tech, Chengdu, China).

2.5 Primary culture of microglia and treatments

Primary microglia were isolated from brains of neonatal C57BL/6 mice (P0–P3) as described [20]. The purified microglial cells were cultured at 37°C in DMED–F12 medium (Gibco, CA, USA) containing 10% fetal bovine serum (Gibco, CA, USA). After seven days, microglia were pre-treated with 10, 50 or 100 µM ASD (Alfabiotech, Chengdu, China) or pioglitazone (10 µM, Sigma-Aldrich, MO, USA) [35]. After 30 min, microglia were treated for 24 or 48 h with either 50 ng/mL LPS (Sigma-Aldrich, MO, USA) or phosphate-buffered saline (PBS; BOSTER, Wuhan, China). Experimental groups were as follows: control group (Ctrl), not treated with ASD or LPS; LPS group (LPS), treated with LPS but not ASD; LPS + ASD (10, 50, 100 µM) group, treated with LPS and ASD at the indicated concentrations; LPS + pioglitazone group, treated with LPS and 10 µM pioglitazone. At each time-point, microglia were collected and transferred to new plates for further experiments.

2.6 Culture of NSPCs with conditioned medium from pre-treated microglia

NSPCs were obtained from the hippocampus of eight-week-old male C57BL/6J mice as described [36]. Microglia were plated at a density of 5 × 105 cells/cm2, treated with either LPS or PBS for 24 h in the presence or absence of ASD, washed twice with PBS, and then cultured for another 24 h in DMEM-F12 + GlutaMax medium (Gibco, CA, USA). The microglial medium was collected and used as conditioned medium (CM) to stimulate NSPC differentiation and proliferation.

NSPCs were cultured in CM from microglia treated with PBS, LPS, or both ASD and LPS. Experimental groups were as follows: PBS-M-CM group, incubated in CM from PBS-treated microglia; LPS-M-CM group, incubated in CM from LPS-treated microglia; ASD-M-CM group, incubated in CM from microglia treated with LPS and 50 µM ASD; and the Piog-M-CM group, incubated in CM from microglia treated with LPS and pioglitazone. NSPC proliferation, survival and neuronal differentiation were evaluated using immunofluorescence as described in sections 2.13.

2.7 Treatment with K252a or GW9662 in vitro

In some experiments involving blockade of PPAR-γ signalling pathway in ASD-treated primary microglia, microglia were treated with ASD (50 µM) and PPAR-γ inhibitor GW9662 (10 µM) [33]. After 30 min, microglia were treated for 24 h with either 100 ng/mL lipopolysaccharide (LPS; Sigma-Aldrich, MO, USA) or phosphate-buffered saline (PBS; BOSTER, Wuhan, China). Following the immunocytochemistry, RT-PCR analysis, western blot analysis and conditional culture of NSPC were performed.

In some experiments involving blockade of BDNF-TrkB signalling pathway in NSPCS, BDNF receptor antagonist K252a (100 ng/mL) [9] were added to the conditioned medium from the microglia treated by ASD and LPS. Then the conditioned medium was used for proliferation culture or differentiation culture of NSPC.

2.8 BrdU incorporation

To determine cell proliferation in the brain, mice received intraperitoneal injections of 5’-bromo-2’deoxyuridine (BrdU; Sigma-Aldrich, MO, USA; 50 mg/kg/d) for 3 days [9]. To examine NSPC proliferation and differentiation, mice were euthanized at 7 days after the last injection. To examination neuronal survival in the granular layer, animals were injected with a double dose of BrdU and euthanized at 7 weeks after injection.

To determine the NSPCs and newborn neurons survival during culture in conditioned medium from microglia activated with LPS in the presence or absence of ASD, the NSPCs were incubated with BrdU (100 ng/mL) for 24 h in proliferation medium [9]. After that, these NSPCs were allowed to grow for 7 days in differentiation medium. Half of the volume of culture medium for induced differentiation was replaced with the microglia-conditioned medium (M-CM). After three days, the survival of the newborn neurons was measured using immunofluorescence as described in sections 2.13.

2.9 Animal perfusion

Mice were anesthetized with 10% pentobarbital and transcardially perfused with phosphate-buffered saline (PBS) containing heparin. Brains were removed, fixed in 4% paraformaldehyde for 48 h, washed with PBS, and dehydrated in 30% sucrose as previously described [20]. Coronal sections containing the hippocampus were obtained using a sliding vibratome (CM1900; Leica Microsystems, Wetzlar, Germany). Six sequential slices were collected into each well of a 12-well plate containing PBS with 0.02% sodium azide and stored at 4℃. The 20 micron thick slices were used for immunofluorescence and the 100 micron thick slices were used for protein and RNA extraction.

2.10 RNA extraction and real-time PCR (RT-PCR)

The dentate gyrus was isolated form slices containing the hippocampus. Total RNA was isolated from dentate gyrus or cultured cells using Trizol (Invitrogen Life Technologies, CA, USA) according to the manufacturer's instructions. RT-PCR was performed using the First Strand cDNA Synthesis Kit (TaKaRa, Shiga, Japan) according to the manufacturer’s instructions. RT-PCR amplification was performed using a Bio-Rad CFX 96 system (Bio-Rad Laboratories, CA, USA) and the primers in supplementary Table 2. Each sample was tested in triplicate. The threshold cycle (Ct) number was determined from the linear phase of the amplification plot using the -ΔΔCt method, and values were normalized against the housekeeping gene β-actin.

2.11 Enzyme-linked immunosorbent assay (ELISA)

The dentate gyrus was dissociated from slices containing the hippocampus, flash-frozen in liquid nitrogen, and homogenized. Primary microglia were cultured in six-well plates at 5 × 105 cells/cm2, then treated for 24 or 48 h with LPS or PBS in the presence or absence of ASD. The culture medium was collected, microglia were lysed in cell lysis buffer (Solarbio, Beijing, China), and the lysates were centrifuged at 1,000 g for 30 min. The concentration of total protein in the supernatant was determined using the BCA kit (BOSTER, Wuhan, China), and each sample was diluted to 1 g/mL. Then samples were assayed using commercial ELISAs against the following signaling factors: interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-10, IL-4, insulin-like growth factor (IGF)-1 and brain-derived neurotrophic factor (BDNF) (BOSTER); inducible nitric oxide synthase (iNOS) and arginase (Arg-1) (Elabscience, Shanghai, China); transforming growth factor (TGF)-β (4A Biotech, Beijing, China); as well as basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) (ColorfulGene Biotech, Wuhan, China). The manufacturer-specified detection limit of all kits was 4 pg/mL.

2.12 Western blotting (WB)

Mice were perfused with PBS, hippocampi were removed and homogenized. Cultures of proliferative NSPCs were sonicated in RIPA buffer containing protease and phosphorylase inhibitors (Solarbio, Beijing, China), lysates were centrifuged at 1,000 g for 30 min, and equal amounts of soluble protein were fractionated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were washed with Tris-buffered saline containing 0.1% Tween-20 (TBST), incubated with skim milk for 30 min, then incubated on a shaker at 4°C overnight with primary antibodies as list in supplementary Table 3. Membranes were washed three times with TBST, incubated for 30 min with secondary antibody (1:10,000; Abcam), washed three times with TBST, then developed for 1–2 min using enhanced chemiluminescence (Millipore, MMAS, USA). Blots were visualized using the ChemiDoc Touch system (Bio-Rad, CA, USA) and band intensity was quantified using Alpha software (version 1.45J; National Institutes of Health, Bethesda, MD, USA).

2.13 Immunohistochemistry and immunocytochemistry

The following types of cells were plated separately at a density of 105 cells/cm2 and cultured for 24 h: microglia, neurospheres, proliferative NSPCs, and differentiated NSPCs. Cells were fixed with 4% paraformaldehyde (pH 7.2) for 30 min. Slices containing the hippocampus and culture cells were permeabilized with 0.5% Triton X-100 in PBS for 15 min, blocked in 10% donkey serum (Solarbio, Beijing, China) for 2 h, then incubated overnight at 4°C with the following primary antibodies as list in in supplementary Table 4. Slices or cells were washed three times with PBS, then incubated for 2 h at room temperature with DyLight 549- or DyLight 488-conjugate secondary antibodies (both 1:300; Jackson ImmunoResearch, West Grove, PA, USA). Finally, cells were incubated for 5 min with 4',6-diamidino-2-phenylindole (DAPI; 1:10000, Roche, Basel, Swizerland) and imaged using a fluorescence microscope (Olympus IX 73, Tokyo, Japan).

2.14 Imaging and statistical analyses

Images were analysed as described [9]. Statistical analyses were performed using GraphPad Prism (version 8.0, SPSS Inc., Chicago, USA). Experimental data were expressed as mean ± SEM. Differences between mean values were assessed for significance using a paired Student’s t-test for independent samples or, where appropriate, one- or two-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison analysis. Differences were considered statistically significant if P < 0.05.

3. Results

3.1 Akebia saponin D ameliorates depressive-like behaviors of CMS mice

We first evaluated the anti-depressant efficacy of ASD in mice (Fig. 1A). CMS reduced sucrose preference, which was reversed by 3-week treatment with ASD or IMI (Fig. 1B and 1C). Similarly, CMS shortened latency and prolonged immobility in the FST, which ASD reversed as well as IMI (Fig. 1D). ASD also reversed the CMS-induced decrease in body weight (Fig. S1). In contrast, neither ASD nor IMI affected the distance travelled or immobility time in the OFT (Fig. 1E and Fig. S1). These results suggest that ASD ameliorates depressive-like behaviors in CMS mice.

3.2 Akebia saponin D rescues CMS-induced deficits in hippocampal neurogenesis

NSPCs in the dentate gyrus (DG) of the hippocampus proliferate and differentiate into neurons even in adulthood, and this neurogenesis is negatively associated with depression and positively associated with the efficacy of anti-depressants [9, 20]. Consistent with this, CMS strikingly reduced the number of newborn neurons (DCX+) in the hippocampus of our mice (Fig. 2A). In fact, CMS reduced numbers of Ki67+ cells and DCX+-BrdU+ cells in the subgranular zone (SGZ) of the hippocampus, suggesting inhibition of NSPC proliferation and neuronal differentiation (Fig. 2B and 2C). Conversely, CMS strongly increased the number of GFAP+-BrdU+ cells in hippocampus, suggesting the induction of NSPC differentiation into astrocytes (Fig. 2D). ASD reversed these effects of CMS (Fig. 2A-2D).

To examine the effects of ASD on the survival and maturation of proliferative cells, BrdU was injected into mice before CMS exposure (Fig. 2E). CMS reduced the numbers of surviving newborn neurons (BrdU+-NeuN+) in the DG, which ASD reversed (Fig. 2E and 2F). CMS slowed rates of neuronal differentiation and maturation, while accelerating NSPC differentiation into astrocytes, and ASD reversed these effects (Fig. 2G). These results indicate that ASD improves NSPC proliferation, survival, as well as neuronal differentiation and maturation in the hippocampus of CMS mice. 

To investigate the role of neurogenesis in antidepressant effects of ASD, we used the temozolomide (TMZ) to ablate neurogenesis in ASD-treated CMS mice (Fig. 2H and 2I). TMZ treatment abolished the anti-depressant effects of ASD in the SPT and FST (Fig. 2J-2L). These results suggest that the anti-depressant effects of ASD depend in part on promoting hippocampal neurogenesis.

3.3 Akebia saponin D reprogrammes a pro-neurogenic microglia in dentate gyrus of CMS mice

Microglia control the neurogenic microenvironment, and the Arg-1+ microglia contributes to hippocampal neurogenesis [9]. Therefore we examined the effects of ASD on Arg-1+ microglia in dentate gyrus of CMS-exposed mice. The results showed that ASD significantly increased the percentage of Arg-1+ microglia in dentate gyrus of CMS-exposed mice (Fig. 3A and 3B). The immobility time in forced swimming test was negatively correlated with Arg-1+ microglia in dentate gyrus of mice (Fig. 3C). ASD also reversed the CMS-induced increases in the pro-inflammatory factors TNF-α, iNOS and IL-1β and decrease in the anti-inflammatory factors IL-4 and Arg-1 in dentate gyrus of mice (Fig. 3D). Analogously, CMS substantially reduced the levels of IGF-1, TGF-β and BDNF, while ASD significantly increased BDNF in dentate gyrus of mice (Fig. 3D). The BDNF levels were positively correlated with Arg-1 levels in dentate gyrus of mice (Fig. 3E). The results from immunofluorescent staining showed that ASD upregulated the BDNF in Arg-1+ microglia of in dentate gyrus (Fig. 3F). Considering that ASD increases microglial secretion of BDNF, which in turn promotes neurogenesis from NSPCs, we examined the levels of phosphorylation of the BDNF-specific receptor TrkB in hippocampus of mice. The results showed that CMS reduced the levels of p-TrkB in the SGZ of hippocampus, which ASD reversed (Fig. 3G).

To confirm that ASD directly regulates microglial function, we examined the effects of ASD on primary cultures of microglia that were treated with LPS as a model of neuroinflammation (Fig. S2). LPS shifts microglia toward a pro-inflammatory phenotype that inhibits NSPC proliferation, survival and differentiation [20]. Pretreatment with ASD at 50 or 100 μM, but not 10 μM, prevented LPS from upregulating iNOS and TNF-α, and increased the expression of IL-10, Arg-1 and BDNF at 24 and 48 h (Fig. S2 and S3). 

To confirm that the ASD-induced changes in microglia in turn influence NSPCs, we treated primary cultures of microglia in different ways, then transferred the culture medium to NSPC cultures and observed their proliferation, survival and neuronal differentiation (Fig. 3H and Fig. S4). We first examined levels of p-TrkB in primary NSPCs cultured in conditioned medium from microglia. The conditioned medium from microglia treated with 50 μM ASD + LPS (ASD-M-CM) increase the pTrkB in NSPCs (Fig. 3I). Compared with conditioned medium from PBS-treated microglia (PBS-M-CM), the conditioned medium from LPS-treated microglia (LPS-M-CM) decreased the size of NSPC neurospheres. ASD-M-CM increased the size of NSPC neurospheres when compared with PBS-M-CM or LPS-M-CM. (Fig. 3J). LPS-M-CM inhibited NSPC differentiation into neurons (DCX+ cells), but ASD-M-CM promoted such differentiation (Fig. 3K). Overall, the effects of ASD were similar to those of pioglitazone, an agonist of PPAR-γ pathway, which reprogrammes a pro-neurogenic microglial phenotype. 

3.4 The PPAR-γ plays a critical role in reprogramming of pro-neurogenic microglia by akebia saponin D in dentate gyrus of CMS mice

Since mammalian target of rapamycin (mTOR) / PPAR-γ signaling plays a key role in induction of anti-inflammatory microglial phenotypes [37], we asked whether ASD acts via such signaling to exert its “microglial reprogramming” effect. Indeed, CMS reduced expression of mTOR and PPAR-γ as well as their phosphorylated levels in dentate gyrus (Fig. 4A). After treatment with ASD, p-mTOR and p-PPAR-γ were significantly increased in CMS-exposed mice (Fig. 4A). Activation of PPAR-γ with ASD reversed the CMS-induced decrease in BDNF level in dentate gyrus of mice (Fig. 4A). The results from immunofluorescent staining showed that PPAR-γ localized in cytoplasm and nucleus of Arg-1+ microglia in the dentate gyrus of mice that were exposed to CMS and then treated with ASD (Fig. 4B). 

To confirm the role of PPAR-γ in induction of the pro-neurogenic microglia in dentate gyrus of ASD-treated mice, we repeated the above experiments in the presence of the PPAR-γ antagonist GW9662 (Fig. 4C), which effectively blocked the PPAR-γ pathway in dentate gyrus (Fig. 4D). Such blockade abolished the ability of ASD to increase numbers of Arg-1+ microglia in the dentate gyrus of CMS mice (Fig. 4E). Blockade of PPAR-γ signaling in ASD-treated primary microglia also abolished the ability of ASD-M-CM to stimulate NSPC proliferation and neuronal differentiation (Fig. 4F-4I). GW9662 treatment abolished the ability of ASD to increase the BNDF and pTrkB levels in dentate gyrus of CMS mice (Fig. 4D). These results suggest that soluble microglial factors such as BDNF activate the TrkB of NSPC to promote NSPC proliferation, survival and neurogenesis. Consistent with this, the TrkB inhibitor K252a prevented ASD-M-CM from stimulating NSPC proliferation and neuronal differentiation (Fig. S5). 

Blockade of PPAR-γ or TrkB signaling abolished the ability of ASD to promote hippocampal neurogenesis in CMS mice (Fig. 5A and 5B). Either GW9662 or K252a also blocked the anti-depressant effects of ASD in the sucrose preference test and forced swimming test (Fig. 5C and 5D). These results suggest that ASD reprogrammes the pro-neurogenic microglia in dentate gyrus of CMS mice via the PPAR-γ signaling pathway. 

4. Discussion

Our previous research revealed that modulation of microglial phenotype and function may be an effective neurotherapy for depression [9]. Consistent with reports that natural products can be effective modulators of microglial phenotype and promoters of neurogenesis [20], here we demonstrate in vivo and in vitro that ASD, the major active ingredient in the traditional Chinese medicine Dipsacus asper Wall., can induce a pro-neurogenic microglial phenotype in a PPAR-γ-dependent manner, which activates the BDNF-TrkB pathway in NSPCs to promote their proliferation and neuronal differentiation. The resulting neurogenesis can ameliorate depressive-like behaviors in CMS mice.

We previously reported that ASD at 40 mg/kg significantly ameliorated depressive-like behaviors in LPS-treated mice [31]. Here we found similar effects of ASD in mice exposed to CMS, a classical model of depression [38]. In fact, ASD exerted similar anti-depressant effects as imipramine, a commonly used clinical antidepressant [39]. Decreased neurogenesis in the hippocampus is involved in the pathogenesis of depression and Alzheimer's disease [6, 40–42] and has been associated with depressive-like behaviors [10, 43]. CMS decreased hippocampal neurogenesis in our mice, which ASD reversed, leading to increases in the numbers of DCX+, Ki67+, BrdU+, BrdU+-DCX+ and BrdU+-NeuN+ cells in the DG, where NSPCs undergo proliferation and neuronal differentiation [20, 44, 45]. The resulting neurons are known to participate in mood and behavior [46, 47]. These results indicate that ASD rescues CMS-induced deficits in hippocampal neurogenesis by promoting NSPC proliferation, survival, as well as neuronal differentiation and maturation.

Psychological stress activates microglia to secrete pro-inflammatory factors that impair neuroplasticity, especially in the dentate gyrus of hippocampus [48]. Consistent with this, CMS strongly increased the number and area of microglia in the hippocampus of our mice, and it upregulated pro-inflammatory TNF-α and IL-1β, while downregulating the anti-inflammatory factors IL-4 and Arg-1 and neurotrophic factors IGF-1, TGF-β and BDNF. ASD reversed all these changes, exerting a neuroprotective microglial phenotype in response to CMS. This result is consistent with our previous work [31], where we implicated the nuclear transcription factor PPAR-γ in the effects of ASD on microglial phenotype. Consistent with that study, we found here that the PPAR-γ agonist pioglitazone, like ASD, induced an anti-inflammatory microglial phenotype. Our findings here may help explain how ASD can attenuate microglia-mediated inflammation in animal models of depression [31, 32].

The strong ability of NSPCs to proliferate and differentiate makes them promising targets for repairing nerve injury [49]. However, adverse changes in the microenvironment of the CNS, including signals from microglia [50], can induce NSPCs to differentiate into astrocytes at the expense of neurons, which increases the risk of glial scar formation [20]. A pro-inflammatory phenotype of microglia, which can be induced in animal models using CMS or LPS [51], is thought to suppress adult NSPC proliferation [20]. Here we also found that factors secreted by microglia exposed to LPS inhibited differentiation of adult NSPCs into neurons. Pretreating microglia with ASD, in contrast, enabled microglia to promote NSPC proliferation, survival, and neuronal neurogenesis. Thus, ASD appears to induce secretion of neurogenic factors from microglia, which then influence NSPCs. This mechanism may explain how ASD can exert anti-depressant effects and mitigate cognitive impairment in animal models of depression [30, 31].

One of the neurogenic factors secreted by ASD-treated microglia appears to be BDNF, which plays a neuroprotective and neurotrophic role [52–54]. We found that ASD treatment upregulated BDNF in hippocampal microglia of CMS mice. Pretreating microglia with ASD before LPS increased the secretion of BDNF into the medium, such that this conditioned medium promoted NSPC proliferation and neuronal differentiation. ASD also activated the BDNF receptor, TrkB, in the SGZ of CMS mice. Consistent with a role of BDNF as mediator of ASD-induced neurogenesis, we found that NSPC proliferation and differentiation correlated with an increase in levels of p-TrkB, while the TrkB inhibitor K252a blocked ASD-induced neurogenesis and anti-depressant effects.

We found that the ability of ASD to reprogramme pro-neurogenic microglial phenotype are mediated by PPAR-γ, a ligand-dependent transcription factor belonging to the nuclear hormone receptor superfamily [55]. PPAR-γ regulates the expression of anti-inflammatory cytokines [56], and the PPAR-γ agonists pioglitazone or rosiglitazone can switch activated microglia cells from a pro-inflammatory to anti-inflammatory state [57]. Our previous research showed that ASD acts via PPAR-γ to switch activated microglia from a pro-inflammatory to anti-inflammatory phenotype in vitro [32]. In present study, we further demonstrated that ASD acts via PPAR-γ to induce a pro-neurogenic microglial phenotype in dentate gyrus of CMS-exposed mice and mitigate depressive-like mouse behaviors. Conversely, blocking the PPAR-γ signaling pathway abolished the Arg-1+ microglia and BDNF expression induced by ASD in dentate gyrus of CMS-exposed mice, as well as the pro-neurogenic and antidepressant effects of ASD.

Taken together, these experiments strongly suggest that ASD acts via the PPAR-γ pathway to reprogramme a pro-neurogenic microglia in dentate gyrus of CMS mice that can increase BDNF expression and promote NSPC proliferation, survival, and neuronal differentiation. This neurogenesis then mitigates CMS-induced deficits in hippocampal neurogenesis and depressive-like behaviors. Our results justify further studies of ASD as a potential treatment for depression and may inspire new lines of research targeting the PPAR-γ pathway in disorders involving impaired neurogenesis.

Abbreviations

Arg-1, Arginase-1, ASD, Akebia saponin D, BDNF, brain-derived neurotrophic factor, BrdU, 5’-bromo-2’deoxyuridine, CMS, chronic mild stress, DAPI, 4’,6-diamidino-2-phenylindole, DCX, doublecortin, DG, dentate gyrus, ELISA, enzyme-linked immunosorbent assay, FST, forced swimming test, GCL, granular cell layer, GFAP, glial fibrillary acidic protein, Iba1, ionized calcium binding adapter molecule 1, IL-4, interleukin 4, IL-10, interleukin 10, iNOS, inducible nitric oxide synthase, IMI, imipramine, IHC, immunocytochemistry, LPS, lipopolysaccharide, MAP2, microtubule-associated protein 2, mTOR, Mammalian target of rapamycin, NeuN, neuron-specific nucleoprotein, NSPC, neural stem/progenitor cell, OFT, open field test, p-mTOR, phospho-mammalian target of rapamycin, PBS, phosphate-buffered saline, PPAR-γ, Peroxisome proliferator-activated receptor-gamma, p-PPAR-γ, phospho-peroxisome proliferator-activated receptor-gamma, qPCR, real-time quantitative PCR, LPS, lipopolysaccharide, SGZ, subgranular zone, TNF-α, tumor necrosis factor-α, TrkB, tyrosine kinase receptor B, WB, western blotting.

Declarations

Acknowledgments

We are grateful to Creaducate Consulting GmbH for help in revising the manuscript. 

Funding

This work was supported by the National Natural Science Foundation of China (82060726), the Department of Science and Technology of Guizhou High-level Innovative Talents ([2018]5638-2), Sichuan Science and Technology Program (2020YJ0225), Guizhou Science and Technology Plan Project ([2019]5611), the Department of Science and Technology of Guizhou basic research ([2019]1026) and the PhD Start-up Fund of the Guizhou University of Traditional Chinese Medicine ([2019]31).

Availability of data and materials

Please contact the corresponding author for data requests.

Author contributions

JZ and TZ conceived and designed the study. JZ and QL wrote the manuscript, which was revised by TZ and ZY, approved by all the authors. QL, CX and WJ performed behavioral tests and immunostaining. LL cultured NSPCs and performed immunofluorescence and cytokine assays. DS performed statistical analyses of the data. 

Ethics approval

All experiments were approved by the Institutional Animal Care and Use Committee of the Guizhou University of Traditional Chinese Medicine.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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