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Hippocampal PPARα is involved in the antidepressant-like effects of venlafaxine in mice
Brain Research Bulletin 153 (2019) 171–180
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Hippocampal PPARα is involved in the antidepressant-like effects of venlafaxine in mice
T
Cheng Chena,1, Jian-Hong Shenb,1, Hui Xua, Peng Chena, Fei Chena, Yi-Xiang Guand, Bo Jiangc, , ⁎ Zhong-Hua Wua, ⁎⁎
a
Department of Neurosurgery, The Sixth People’s Hospital of Nantong, Nantong 226011, Jiangsu, China Department of Neurosurgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China c Department of Pharmacology, School of Pharmacy, Nantong University, Nantong 226001, Jiangsu, China d Department of Surgery, Affiliated Haian People’s Hospital of Nantong University, Haian 226600, Jiangsu, China b
ARTICLE INFO
ABSTRACT
Keywords: Chronic unpredictable mild stress Chronic restraint stress Depression Hippocampus Peroxisome proliferator activated receptor α Venlafaxine
Although thought as a serotonin and norepinephrine reuptake inhibitor (SNRI), the antidepressant mechanisms of venlafaxine remain unknown. Previous reports have shown the role of peroxisome proliferator activated receptor α (PPARα) in depression. In this study, we investigated whether the antidepressant-like effects of venlafaxine require PPARα. We first examined whether repeated venlafaxine administration reversed the effects of chronic unpredictable mild stress (CUMS) and chronic restraint stress (CRS) on PPARα in the hippocampus and medial prefrontal cortex (mPFC). Then, the pharmacologcial inhibitors of PPARα, GW6471 and MK886, were used to assay if the protecting effects of venlafaxine against chronic stress were prevented by PPARα blockade. Furthermore, gene knockdown of PPARα by AAV-PPARα-shRNA was also used. It was found that venlafaxine treatment fully restored the decreasing effects of CUMS and CRS on the hippocampal PPARα expression. Pharmacological inhibition of PPARα significantly attenuated the antidepressant-like effects of venlafaxine in mice. Moreover, gene knockdown of hippocampal PPARα also fully abolished the antidepressant-like actions of venlafaxine in mice. Collectively, hippocampal PPARα is an antidepressant target of venlafaxine.
1. Introduction As a prevalent psychiatric disorder, depression affects about onefifth of the world population and will be the second largest global disease burden (Gelenberg, 2010). Current antidepressants used in clinical practice include the selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors (MAOIs), which all target on the monoaminergic system (Mandrioli et al., 2018; Perez-Caballero et al., 2019). However, the exact neurobiology of depression is still unclear. Also, the efficacy of SSRIs, SNRIs and MAOIs is inconsistent, and these antidepressants always produce side effects such as sedation, sleep disturbance, sexual dysfunction and body weight gain (Gorman and Kent, 1999; Masand and Gupta, 2002; Hirschfeld, 2003).
Peroxisome proliferator activated receptor α (PPARα) is a nuclear receptor protein that belongs to the PPAR family and functions as a transcription factor regulating gene expressions (Roy and Pahan, 2015). PPARα is distributed in many tissues like liver, kidney, heart, muscle and small intestine. PPARα is involved in many physiological processes in the central nervous system, such as neurotransmission, neuroinflammation and neurogenesis (Esmaeili et al., 2016; Pérez-Martín et al., 2016; Agarwal et al., 2017). In 2018, Song et al. has found that chronic stress significantly down-regulated the expression and function of PPARα in the hippocampus of mice (Song et al., 2018). Genetic overexpression of hippocampal PPARα induced notable antidepressant-like actions in mice by promoting the cAMP response element-binding protein (CREB)-mediated biosynthesis of brain-derived neurotrophic factor (BDNF). Also, the antidepressant-like effects of fluoxetine (a
Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; CREB, cAMP response element-binding protein; CRS, chronic restraint stress; CUMS, chronic unpredictable mild stress; FST, forced swim test; mPFC, medial prefrontal cortex; PPARα, peroxisome proliferator activated receptor α; SNRIs, serotonin and norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; TST, tail suspension test ⁎ Corresponding author at: Department of Neurosurgery, The Sixth People’s Hospital of Nantong, No. 500 Yonghe Road, Nantong 226011, Jiangsu, China. ⁎⁎ Corresponding author at: Department of Pharmacology, School of Pharmacy, Nantong University, No. 19 Qixiu Road, Nantong 226001, Jiangsu, China. E-mail addresses: [email protected] (B. Jiang), [email protected] (Z.-H. Wu). 1 These authors contributed equally to this paper. https://doi.org/10.1016/j.brainresbull.2019.08.016 Received 28 April 2019; Received in revised form 18 August 2019; Accepted 19 August 2019 Available online 21 August 2019 0361-9230/ © 2019 Elsevier Inc. All rights reserved.
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mostly well-known SSRI) in mice require hippocampal PPARα. Moreover, several agonists of PPARα, WY14643, fenofibrate and gemfibrozil, all have been reported to possess antidepressant-like effects in mice (Jiang et al., 2015, 2017; Ni et al., 2018). These findings indicate that hippocampal PPARα is a potential and novel antidepressant target. As a main SNRI, venlafaxine is a second generation antidepressant. It is widely used to treat major depressive disorder, generalized anxiety disorder, panic disorder and social phobia. Although thought to produce function on the serotonergic and adrenergic systems, more and more pharmacological targets beyond the two systems are being demonstrated for venlafaxine recently (Alaiyed et al., 2019; Jiang et al., 2019). Here we speculated that PPARα in brain may participate in the antidepressant mechanism of venlafaxine. Therefore, the chronic unpredictable mild stress (CUMS) and chronic restraint stress (CRS) models of depression, various behavioral tests, western blotting and the AAV-mediated gene knockdown method were used together to explore our assumption in mice.
2.5. Sucrose preference test In this test, mice were given the choice to drink from two bottles for 6 h (from 09:00 a.m. to 03:00 p.m.) in individual cages. For the two bottles, one contained 1% sucrose solution, while the other contained tap water. To prevent possible effects of side preference in drinking behavior, the positions of two bottles were switched every 2 h. Before the test, all mice were acclimatized for 3 consecutive days to two-bottle choice conditions and deprived of food and water for another 12 h. The consumption of tap water and sucrose solution for each mouse were estimated by weighing the bottles before and after the test. The sucrose preference was measured as a percentage of the consumed sucrose solution relative to the total amount of liquid intake. 2.6. Chronic unpredictable mild stress (CUMS) This model of depression was done according to published reports with some modifications (Forbes et al., 1996; Papp et al., 1996; Li et al., 2007). In this study, the stressed mice were individually housed and exposed to a variable sequence of 8 mild and unpredictable stressors for 7 week. The stressors were 4 °C swimming for 1 min, food deprivation for 24 h, water deprivation for 24 h, damp sawdust for 24 h, cage shaking for 30 min, 45 °C cage tilting, inversion of light/dark cycle and tail clipping for 10 min. All stressors were randomly scheduled in each week to prevent animal habituation. The control mice were left undisturbed in the home cages (5 per cage) and handled daily. The treatments of venlafaxine, GW6471 and MK886 were performed daily during the last 2 weeks. After stress and drugs administration, FST, TST and the sucrose preference test were performed together to assay the depressive-like behaviors in mice.
2. Methods 2.1. Animals Male C57BL/6 J mice (8 weeks old) were bought from SLAC Laboratory Animal Co., Ltd (Shanghai, China), and housed 5 per cage under standard conditions for 1 week with free access to food and water before the experiments. The behavioral experiments were carried out during the light phase. The experiment procedures involving animals and their care were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC), and approved by the Animal Welfare Committee of Nantong University (Approval No. 20170420-013).
2.7. Chronic restraint stress (CRS) The CRS model was performed according to previously described procedures (Ejchel-Cohen et al., 2006; Mendoza et al., 2018; Wang et al., 2019). The stressed mice were individually housed, and received restraint stress which was applied 4 h/d (from 9:00 a.m. to 1:00 p.m.) for 6 weeks using 50 ml conical transparent plastic tubes (Corning Inc.; containing vent holes at one end) that can effectively immobilize the mice. The control mice were left undisturbed in the home cages (5 per cage) and handled daily. The treatments of venlafaxine, GW6471 and MK886 were performed daily during the last 2 weeks. After stress and drugs administration, FST, TST and the sucrose preference test were performed together to assay the depressive-like behaviors in mice.
2.2. Materials Venlafaxine was bought from Sigma (St. Louis, MO, USA). GW6471 and MK886 were bought from Tocris (Bristol, UK). The vehicle for venlafaxine, GW6471 and MK886 was 1% DMSO in 0.9% saline. The doses for venlafaxine (10 mg/kg), GW6471 (1 mg/kg) and MK886 (3 mg/kg) in this study were determined according to published reports (Esposito et al., 2012; Puligheddu et al., 2013; Jiang et al., 2019). All drugs were intraperitoneally (i.p.) injected in a volume of 10 ml/kg. 2.3. Forced swim test (FST)
2.8. Western blotting
The forced swim test was performed according to published protocols with slight modifications (Redrobe and Bourin, 1997; Lamberti et al., 1998). Mice were videotaped in a glass cylinder (45 cm height, 20 cm internal diameter) containing 20 cm height of water at 24 ± 1 °C for 6 min. The videotape was scored for the last 4 min manually by a trained and blinded observer. The water was replaced for every trial. The duration of immobility was measured as the time mice spent without any motion except for single limb paddling to maintain flotation.
Mice were decapitated, and the brain regions were rapidly dissected and homogenized in ice-cold lysis buffer containing Tris-HCl (50 mM, pH 7.4), EDTA (1 mM), NaCl (100 mM), NaF (20 mM), 3 mM Na3VO4 (3 mM), PMSF (1 mM), Nonidet P-40 (1%) and protease inhibitors. The lysates were centrifuged at 12000 × g for 15 min, and the protein concentrations were determined by the Bradford method. The samples were mixed with Laemmli sample buffer (2% SDS) and placed in a boiling water bath for 5 min. Proteins were resolved in SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with antibodies to PPARα (1:500; Abcam, Bristol, UK) and β-actin (1:500; Cell Signaling, Danvers, MA, USA). The immunoblots were developed using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG, followed by detection with enhanced chemiluminescence. The protein bands were quantitated with a densitometer.
2.4. Tail suspension test (TST) The tail suspension test was done according to published protocols with little modifications (Kamei et al., 2003; Ripoll et al., 2003). Mice were suspended by the tail on a horizontal beam 60 cm high. Mice were taped to the bar using adhesive tap placed approximately 1 cm from the tail tip. A 6-min test period was videotaped and scored manually by a trained and blinded observer. The mice were considered immobile only when they hung passively and were completely motionless.
2.9. AAV-mediated gene knockdown This method was done according to published reports with slight modifications (Song et al., 2018; Jiang et al., 2019). Mice were 172
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anesthetized with 0.5% pentobarbital sodium and fixed in stereotaxic frames (Stoelting, USA). For each mouse, its scalp was cut and the skull was exposed using 75% ethanol and 1% H2O2. Microsyringes of 5 μl were used to deliver AAV-Scrambled-EGFP or AAV-PPARα-shRNAEGFP. After making a small drill hole on the skull of each mouse, the microsyringes were positioned at the following coordinates: AP = 2.3 mm, ML = ± 1.8 mm, DV = +2.0 mm. The AAV constructs were injected bilaterally into the hippocampus at a rate of 0.5 μl/min (1.5 μl/ side). The microsyringes were maintained in place for 5 min to limit virus reflux. The incision of each mouse was sutured, and mice were allowed to recover for 3 d before further use. AAV-Scrambled-EGFP and AAV-PPARα-shRNAEGFP were kindly supplied by Dr. Bo Jiang, and the production of these constructs has been provided in Song et al. (Song et al., 2018). The sequences for PPARα-shRNA and Scrambled control-shRNA were 5′-AGAAATTCTTACCTGTGA A-3′ and 5′-TTCTCCGAACGTGTCACGT-3′ respectively. They were diluted to 5 × 109 TU/ml before use. 3 weeks were required for AAVPPARα-shRNA to produce gene knockdown effects.
3. Results 3.1. Repeated venlafaxine administration fully restored the effects of chronic stress on hippocampal PPARα As a first step, the CUMS model of depression was performed in C57BL/6 J mice. As shown in Fig. 1A, 7 weeks of CUMS significantly enhanced the immobility of mice in FST and TST (n = 12, P < 0.01), and decreased the sucrose preference of mice (n = 12, P < 0.01). In contrast, 2 weeks of venlfaxine administration fully reversed these behavioral changes (n = 12, P < 0.01). Afterwards, western blotting was performed to examine the effects of CUMS and venlafaxine on PPARα in the hippocampus and medial prefrontal cortex (mPFC), as the two regions are closely involved in the pathophysiology of depression. Fig. 1B illustrates the hippocampus data. It was found that CUMS induced a 47.1 ± 5.89% decrease of hippocampal PPARα expression compared to the control group (n = 6, P < 0.01), while venlafaxine treatment promoted the level of hippocampal PPARα in the CUMS group by 150.1 ± 13.3% (n = 6, P < 0.01). Interestingly, venlafaxine treatment also enhanced the hippocampal PPARα expression in the control group by 45.7 ± 3.24% (n = 6, P < 0.01). Fig. 1C illustrates the mPFC data. However, it was found that both CUMS exposure and venlafaxine treatment produced no observable effects on the PPARα expression in mPFC (n = 6). Then, the CRS model of depression was also performed in C57BL/6 J mice. Fig. 2A showed that 6 weeks of CRS robustly enhanced the immobility of mice in FST and TST (n = 12, P < 0.01), and down-
2.10. Statistical analysis All data are presented as means ± S.E.M., and analyzed using Sigma Stat10.0. Multiple group comparisons were performed using oneway analysis of variance (ANOVA) followed by Tukey's test, or two-way ANONA followed by Bonferroni’s test. A value of P < 0.05 was considered statistically significant.
Fig. 1. CUMS significantly decreased the expression of hippocampal PPARα in mice, whereas venlafaxine administration reversed it. (A) CUMS induced notable depressive-like behaviors in C57BL/6 J mice, as revealed by FST, TST and the sucrose preference test (n = 12). (B) Representative western blotting images showed the effects of CUMS and venlafaxine administration on PPARα expression in the hippocampus (n = 6). (C) Representative western blotting images showed that CUMS and venlafaxine administration had no effects on PPARα expression in mPFC (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by two-way ANOVA followed by Bonferroni’s test.
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Fig. 2. CRS also down-regulated the expression of hippocampal PPARα in mice, and this was restored by venlafaxine treatment. (A) CRS induced notable depressive-like behaviors in C57BL/6 J mice, as revealed by FST, TST and the sucrose preference test (n = 12). (B) Representative western blotting images revealed the effects of CRS and venlafaxine treatment on PPARα expression in the hippocampus (n = 6). (C) Representative western blotting images indicated that CRS and venlafaxine treatment produced no effects on PPARα expression in mPFC (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by two-way ANOVA followed by Bonferroni’s test.
treated mice displayed 28.1 ± 4.05% more of immobility in FST, 26.8 ± 6.14% more of immobility in TST and 28.5 ± 4.23% less of sucrose preference than the (CUMS + venlafaxine)-treated mice (n = 12, P < 0.01). Similarly, GW6471 co-treatment significantly attenuated the antidepressant-like effects of venlafaxine in the CRS model (Fig. 3C). Detailed analyses revealed that while the (CRS + venlafaxine)-treated mice exhibited 23.4 ± 2.58% less of immobility in FST, 29.4 ± 2.95% less of immobility in TST and 65.8 ± 5.49% more of sucrose preference than the CRS-stressed mice (n = 12, P < 0.01), the (CRS + venlafaxine + GW6471)-treated mice displayed 29.5 ± 3.62% more of immobility in FST, 30.1 ± 5.07% more of immobility in TST and 28.7 ± 5.47% less of sucrose preference than the (CRS + venlafaxine)-treated mice (n = 12, P < 0.01). Meanwhile, Fig. 3B and D indicated that GW6471 co-treatment fully blocked the enhancing effects of venlafaxine on the hippocampal PPARα expression in the stressed mice (n = 6, P < 0.01). Fig. 4 illustrates the MK886 data. As shown in Fig. 4A, MK886 cotreatment fully blocked the antidepressant-like actions of venlafaxine in the CUMS model. Detailed analyses revealed that while the (CUMS + venlafaxine)-treated mice exhibited 23.4 ± 2.73% less of immobility in FST, 26.7 ± 2.44% less of immobility in TST and 45.3 ± 6.12% more of sucrose preference than the CUMS-stressed mice (n = 12, P < 0.01), the (CUMS + venlafaxine + MK886)treated mice displayed 29.4 ± 4.61% more of immobility in FST, 30.2 ± 6.47% more of immobility in TST and 22.7 ± 3.32% less of sucrose preference than the (CUMS + venlafaxine)-treated mice (n = 12, P < 0.01). Also, MK886 co-treatment significantly attenuated the
regulated the sucrose preference of mice (n = 12, P < 0.01), while 2 weeks of venlafaxine treatment fully restored these behavioral changes (n = 12, P < 0.01). Fig. 2B shows the hippocampus results. CRS led to a 59.3 ± 7.03% decrease of hippocampal PPARα expression compared to the control group (n = 6, P < 0.01), while venlafaxine treatment promoted the level of hippocampal PPARα in the CRS group by 164.2 ± 9.56% (n = 6, P < 0.01). Fig. 2C shows the mPFC results. Also, both CRS exposure and venlafaxine treatment produced no significant effects on the PPARα expression in mPFC (n = 6). Taken together, hippocampal PPARα may be necessary for the antidepressantlike effects of venlafaxine in mice. 3.2. The use of PPARα antagonists fully blocked the antidepressant-like actions of venlafaxine in mice Next, the selective PPARα antagonists, GW6471 and MK886, were used to explore whether the antidepressant-like actions of venlafaxine require PPARα. Both the CUMS-stressed and CRS-stressed mice were co-injected with venlafaxine and GW6471/MK886 for 2 weeks, followed by various behavioral tests and western blotting detection. Fig. 3 illustrates the GW6471 results. As shown in Fig. 3A, GW6471 co-treatment significantly attenuated the antidepressant-like effects of venlafaxine in the CUMS model. Detailed analyses revealed that while the (CUMS + venlafaxine)-treated mice exhibited 25.3 ± 4.83% less of immobility in FST, 26 ± 2.62% less of immobility in TST and 37.5 ± 3.77% more of sucrose preference than the CUMS-stressed mice (n = 12, P < 0.01), the (CUMS + venlafaxine + GW6471)174
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Fig. 3. Blockade of PPARα function by GW6471 disturbed the antidepressant-like actions of venlafaxine in mice. (A) GW6471 treatment fully prevented the reversal effects of venlafaxine on the CUMS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data showed that the promoting effects of venlafaxine on hippocampal PPARα in the CUMS-stressed mice were blocked by GW6471 (n = 6). (C) GW6471 treatment also significantly attenuated the restoring effects of venlafaxine on the CRS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data showed that the enhancing effects of venlafaxine on hippocampal PPARα in the CRS-stressed mice were also blocked by GW6471 (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
antidepressant-like effects of venlafaxine in the CRS model (Fig. 4C). Detailed analyses revealed that while the (CRS + venlafaxine)-treated mice exhibited 24.5 ± 5.08% less of immobility in FST, 31.5 ± 3.96% less of immobility in TST and 30.9 ± 3.52% more of sucrose preference than the CRS-stressed mice (n = 12, P < 0.01), the (CRS + venlafaxine + MK886)-treated mice displayed 25.6 ± 2.77% more of immobility in FST, 34.7 ± 5.53% more of immobility in TST and 18.7 ± 3.48% less of sucrose preference than the (CRS + venlafaxine)treated mice (n = 12, P < 0.01). Moreover, Fig. 4B and D indicated that MK886 co-treatment significantly attenuated the enhancing effects of venlafaxine on the hippocampal PPARα expression in the stressed mice (n = 6, P < 0.01).
In addition, repeated treatment of GW6471/MK886 for 2 weeks notably enhanced the immobility of naïve control mice in FST and TST, but not affected the sucrose preference of control mice (n = 10, P < 0.01; Fig. S1A). 3.3. The use of PPARα-shRNA fully abolished the antidepressant-like actions of venlafaxine in mice Furthermore, AAV-PPARα-shRNA was used to selectively knockdown the expression of hippocampal PPARα. The PPARα-knockdown efficacy of AAV-PPARα-shRNA was shown in Fig. 5A and B (n = 5, P < 0.01). Briefly, C57BL/6J mice stereotaxically injected with 175
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Fig. 4. Blockade of PPARα function by MK886 interrupted the antidepressant-like effects of venlafaxine in mice. (A) MK886 treatment notably prevented the reversal effects of venlafaxine on the CUMS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data revealed that the promoting effects of venlafaxine on hippocampal PPARα in the CUMS-stressed mice were blocked by MK886 (n = 6). (C) MK886 treatment also fully attenuated the restoring effects of venlafaxine on the CRS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data indicated that the enhancing effects of venlafaxine on hippocampal PPARα in the CRS-stressed mice were also blocked by MK886 (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
PPARα-shRNA were kept for 3 weeks, and then subjected to CUMS/CRS and venlafaxine administration, followed by various behavioral tests. As shown in Fig. 5C, hippocampal PPARα-knockdown fully abolished the antidepressant-like effects of venlafaxine in the CUMS model. Detailed analyses indicated that while the (CUMS + venlafaxine)treated mice exhibited 26.7 ± 1.84% less of immobility in FST, 27.2 ± 3.06% less of immobility in TST and 45.1 ± 4.53% more of sucrose preference than the CUMS-stressed mice (n = 10, P < 0.01), the (CUMS + venlafaxine + PPARα-shRNA)-treated mice displayed 43.7 ± 7.31% more of immobility in FST, 34.7 ± 4.85% more of
immobility in TST and 25.4 ± 4.63% less of sucrose preference than the (CUMS + venlafaxine)-treated mice (n = 10, P < 0.01). Also, hippocampal PPARα-knockdown significantly prevented the antidepressant-like actions of venlafaxine in the CRS model (Fig. 5D). Detailed analyses showed that while the (CRS + venlafaxine)-treated mice exhibited 22.8 ± 4.37% less of immobility in FST, 24.8 ± 2.66% less of immobility in TST and 47.2 ± 6.84% more of sucrose preference than the CRS-stressed mice (n = 10, P < 0.01), the (CRS + venlafaxine + PPARα-shRNA)-treated mice displayed 25 ± 5.14% more of immobility in FST, 26.7 ± 3.33% more of immobility in TST and 176
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Fig. 5. Hippocampal PPARα knockdown abolished the antidepressant-like efficacy of venlafaxine in mice. (A) Fluorescence of a fixed brain section which expressed AAV-PPARα-shRNA-EGFP in the hippocampus after its stereotactic injection (Scale bar =400 μm). (B) Representative western blotting images showed the knockdown efficacy of AAV-PPARα-shRNA-EGFP in mice (n = 5). (C) The use of AAV-PPARα-shRNA markedly prevented the protective effects of venlafaxineagainst the CUMS-induced depressive-like behaviours in mice, as revealed by FST, TST and the sucrose preference test (n = 10). (D) AAV-PPARα-shRNA pre-infusion also fully abolished the restoring effects of venlafaxine on the CRS-induced helplessness and anhedonia behaviors in mice (n = 10). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
28.4 ± 2.79% less of sucrose preference than the (CRS + venlafaxine)treated mice (n = 10, P < 0.01). Fig. S1B indicated that hippocamapl PPARα-knockdown significantly increased the immobility of naïve control mice in FST and TST (n = 10, P < 0.01). In contrast, Scrambled control-shRNA produced none effects on the mice.
Moreover, the use of PPARα-shRNA fully prevented the promoting effects of venlafaxine on the hippocampal PPARα expression in the stressed mice (n = 5, P < 0.01; Fig. 6). Combined with the GW6471 and MK886 results, it can be concluded that hippocampal PPARα is involved in the antidepressant mechanism of venlafaxine. 177
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Fig. 6. The use of PPARα-shRNA fully prevented the promoting effects of venlafaxine on the hippocampal PPARα expression in the stressed mice. (A) Representative western blotting data showed that the (CUMS + venlafaxine + PPARα-shRNA)-treated mice had notably less expression of hippocampal PPARα than the (CUMS + venlafaxine)-treated mice (n = 5). (B) Representative western blotting results also indicated that the level of hippocampal PPARα in the (CRS + venlafaxine + PPARα-shRNA)-treated mice was significantly lower than that in the (CUMS + venlafaxine)-treated mice (n = 5). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
4. Discussion
et al., 1992; Xu et al., 2017). We firstly reproduced the antidepressantlike effects of venlafaxine in the two models of depression, proving the effectiveness of our models and venlafaxine. It was found that both CUMS and CRS significantly down-regulated the expression of PPARα in the hippocampus, consistent with Song et al. (Song et al., 2018). Importantly, chronic venlafaxine administration fully restored the decreased PPARα expression in the hippocampus. Moreover, the use of GW6471, MK889 and PPARα-shRNA all abolished the antidepressantlike effects of venlafaxine in mice, confirming the speculation of our study. GW6471, MK889 and PPARα-shRNA treatments all enhanced the immobility of naïve control mice in FST and TST, representing a prodepressant-like effect and further indicating the role of PPARα in the pathophysiology of depression. In addition, the finding that chronic stress and venlafaxine produced no effects on PPARα expression in mPFC is interesting. For this region-selective phenomenon, currently there are no convincing explanations, and more profound studies are required in the future. How does venlafaxine produced antidepressant-like effects through hippocampal PPARα activation? As a nuclear receptor protein, PPARα controls the expression of various genes which contain PPREs in the promoter regions. It should be noticed that in 2013, Roy et al. has reported the correlation between hippocampal PPARα and CREB (Roy et al., 2013). It was found that PPARα directly regulates the transcriptional activity of CREB by combining its PPRE (Roy et al., 2013).
Peroxisome proliferator activated receptors (PPARs) are nuclear transcription factors that, in response to the binding of small ligands, regulate the expression of genes involved in many physiological processes (Roy and Pahan, 2015). The family of PPARs comprises three isoforms: PPARα, PPARβ/δ and PPARγ. These isotypes differ from each other in terms of their tissue distributions, ligand specificities and physiological roles (Chinetti et al., 2000; Tyagi et al., 2011). PPARα is activated when bound by endogenous lipid/lipid metabolite ligands or synthetic xenobiotic ligands. Once activated, PPARα heterodimerizes with the retinoid X receptor (RXR), and binds to PPAR response elements (PPREs) in the promoter regions of target genes involved in diverse processes such as energy metabolism, oxidative stress, inflammation, and cell differentiation (Qi et al., 2000). PPARα has beneficial effects in many diseases but also plays a pathological role in some conditions such as the development of insulin resistance (Puligheddu et al., 2013; Esmaeili et al., 2016; Agarwal et al., 2017). Here, this is the first comprehensive in vivo study exploring the role of PPARα in the antidepressant-like actions of venlafaxine. To get a reliable and believable conclusion, both the CUMS and CRS models of depression were used in this study, as they are two widely used and acknowledged animal models which mimic many depressivelike symptoms in human, such as helplessness and anhedonia (Willner 178
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Project in Jiangsu Province (No. BRA2016187), the Provincial Natural Science Foundation of Jiangsu Province (No. BK20161284), and the Science and Technology Projects of Nantong City (No. MS12018076).
PPARα-knockout mice were deficient in CREB and memory-associated proteins. Knockdown of hippocampal PPARα by shRNA suppressed CREB, rendering wild-type mice markedly poor in consolidating spatial memory, whereas introduction of PPARα to the hippocampus of PPARα-knockout mice increased CREB and improved spatial memory (Roy et al., 2013). The role of CREB in the pathophysiology of depression is widely known (Sulser, 2002; Gass and Riva, 2007). Depression is accompanied with significantly down-regulated activity of hippocampal CREB and decreased biosynthesis of BDNF, a well-known downstream protein of CREB (Nestler et al., 2002). In contrast, promotion of hippocampal CREB and BDNF protects against depression in rodents (Nestler et al., 2002). Thus, it is very possible that the antidepressant mechanism of venlafaxine involves a PPARα-CREB-BDNF pathway. This speculation could explain why weeks of administration are always required for venlafaxine to produce curing efficacy in clinical practice, as the PPARα-CREB-BDNF pathway is a long-term process. Another surprising and interesting result is that the use of GW6471 and MK889 significantly down-regulated the expression of hippocampal PPARα in mice treated with chronic stress and venlafaxine, as PPARα antagonists should not affect the biosynthesis of PPARα. Here, we think it is possible that like BDNF, PPARα is also a downstream protein of CREB. In other words, PPARα may have self-regulation on its biosynthesis through combining with PPRE in CREB. Thus, we assume that although GW6471 and MK889 did not influence the promoting effects of venlafaxine on PPARα expression, they significantly interrupted the self-regulation of PPARα on its biosynthesis. This speculation will be further studied in the future. Regarding the mechanism by which venlafaxine affects PPARα, one possibility is that venlafaxine directly binds and activates PPARα, functioning as an exogenous ligand (like WY14643, simvastatin and fibrates), which needs to be further validated using time-resolved FRET, electrospray ionization MS and in silico structural analysis (Roy and Pahan, 2015). The other possibility is that venlafaxine increases the concentration of extracellular 5-HT/NA and thus indirectly activates PPARα via a 5-HT/NA-mediated signaling pathway. However, the connection between PPARα and any 5-HT/NA-mediated signaling has not been confirmed so far. Nevertheless, combined with Song et al. (Song et al., 2018) and this study, both fluoxetine and venlafaxine have been proved to target hippocampal PPARα. If hippocampal PPARα is also involved in the antidepressant mechanisms of other SSRIs and SNRIs, such as paroxetine, citalopram and duloxetine, it will be an exact antidepressant target. Further in-depth studies are ongoing in our group. Besides, this study extends the knowledge of venlafaxine’s pharmacological actions and it is possible that like the fibrates, venlafaxine has protective effects on atherosclerosis by activating hepatic PPARα to reduce serum lipids. Moreover, several studies have demonstrated that PPARβ/δ and PPARγ are also correlated with depression. For example, hippocampal PPARδ overexpression protected against chronic stress-induced depressive-like behaviors in mice (Ji et al., 2015). Decreased PPARδ expression was found in rats subjected to CUMS (Liu et al., 2017). Agonists of PPARγ, such as pioglitazone and rosiglitazone, have potential antidepressant-like effects in rodents (Zhao et al., 2016; Zong et al., 2018). Since PPARβ/δ and PPARγ have similar biological structures to PPARα, they may be also the pharmacological targets of venlafaxine, which needs further exploration. Collectively, our study is new evidence showing the role of hippocampal PPARα in depression and antidepressant responses.
Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.brainresbull.2019.08. 016. References Agarwal, S., Yadav, A., Chaturvedi, R.K., 2017. Peroxisome proliferator-activated receptors (PPARs) as therapeutic target in neurodegenerative disorders. Biochem. Biophys. Res. Commun. 483, 1166–1177. Alaiyed, S., Bozzelli, P.L., Caccavano, A., Wu, J.Y., Conant, K., 2019. Venlafaxine stimulates PNN proteolysis and MMP-9-dependent enhancement of gamma power; relevance to antidepressant efficacy. J. Neurochem. 148, 810–821. Chinetti, G., Fruchart, J.C., Staels, B., 2000. Peroxisome proliferator-activated receptors (PPARs): nuclear receptors at the crossroads between lipid metabolism and inflammation. Inflamm. Res. 49, 497–505. Ejchel-Cohen, T.F., Wood, G.E., Wang, J.F., Barlow, K., Nobrega, J.N., S McEwen, B., Trevor Young, L., 2006. 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Antidepressant-like effects of fenofibrate in mice via the hippocampal brain-derived neurotrophic factor signalling pathway. Br. J. Pharmacol. 174, 177–194. Ji, M.J., Yu, X.B., Mei, Z.L., An, Y.Q., Tang, S.S., Hu, M., Long, Y., Miao, M.X., Hu, Q.H., Sun, H.B., Kong, L.Y., Hong, H., 2015. Hippocampal PPARδ overexpression or activation represses stress-induced depressive behaviors and enhances neurogenesis. Int. J. Neuropsychopharmacol. 19 pii: pyv083. Kamei, J., Miyata, S., Morita, K., Saitoh, A., Takeda, H., 2003. Effects of selective serotonin reuptake inhibitors on immobility time in the tail suspension test in streptozotocin-induced diabetic mice. Pharmacol. Biochem. Behav. 75, 247–254. Lamberti, C., Ipponi, A., Bartolini, A., Schunack, W., Malmberg-Aiello, P., 1998. Antidepressant-like effects of endogenous histamine and of two histamine H1 receptor agonists in the mouse forced swim test. Br. J. Pharmacol. 123, 1331–1336. Li, S., Wang, C., Wang, M., Li, W., Matsumoto, K., Tang, Y., 2007. Antidepressant like effects of piperine in chronic mild stress treated mice and its possible mechanisms. Life Sci. 80, 1373–1381. Liu, K.F., Li, Y., Cheng, K.C., Hsu, C.C., Cheng, J.T., Peng, W.H., 2017. Changes in PPARδ expression in a rat model of stress-induced depression. Clin. Exp. Pharmacol. Physiol. 44, 664–670. Mendoza, C., Perez-Urrutia, N., Alvarez-Ricartes, N., Barreto, G.E., Pérez-Ordás, R., Iarkov, A., Echeverria, V., 2018. Cotinine plus krill oil decreased depressive behavior, and increased astrocytes survival in the Hippocampus of mice subjected to restraint stress. Front. Neurosci. 12, 952. Mandrioli, R., Protti, M., Mercolini, L., 2018. New-generation, non-SSRI antidepressants: therapeutic drug monitoring and pharmacological interactions. Part 1: SNRIs, SMSs, SARIs. Curr. Med. Chem. 25, 772–792. Masand, P.S., Gupta, S., 2002. Long-term side effects of newer-generation antidepressants: SSRIS, venlafaxine, nefazodone, bupropion, and mirtazapine. Ann. Clin. Psychiatry 14, 175–182. Nestler, E.J., Barrot, M., DiLeone, R.J., Eisch, A.J., Gold, S.J., Monteggia, L.M., 2002. Neurobiology of depression. Neuron 34, 13–25. Ni, Y.F., Wang, H., Gu, Q.Y., Wang, F.Y., Wang, Y.J., Wang, J.L., Jiang, B., 2018. Gemfibrozil has antidepressant effects in mice: involvement of the hippocampal brain-derived neurotrophic factor system. J. Psychopharmacol. 32, 469–481.
Declaration of Competing Interest The authors declare no conflict of interests. Acknowledgments This work was supported by grants from the 333 High Level Talent 179
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Roy, A., Pahan, K., 2015. PPARα signaling in the hippocampus: crosstalk between fat and memory. J. Neuroimmune Pharmacol. 10, 30–34. Song, L., Wang, H., Wang, Y.J., Wang, J.L., Zhu, Q., Wu, F., Zhang, W., Jiang, B., 2018. Hippocampal PPARα is a novel therapeutic target for depression and mediates the antidepressant actions of fluoxetine in mice. Br. J. Pharmacol. 175, 2968–2987. Sulser, F., 2002. The role of CREB and other transcription factors in the pharmacotherapy and etiology of depression. Ann. Med. 34, 348–356. Tyagi, S., Gupta, P., Saini, A.S., Kaushal, C., Sharma, S., 2011. The peroxisome proliferator-activated receptor: a family of nuclear receptors role in various diseases. J. Adv. Pharm. Technol. Res. 2, 236–240. Wang, J., Cheng, C., Xin, C., Wang, Z., 2019. The antidepressant-like effect of flavonoids from Trigonella foenum-graecum seeds in chronic restraint stress mice via modulation of monoamine regulatory pathways. Molecules 24 pii: E1105. Willner, P., Muscat, R., Papp, M., 1992. Chronic mild stress-induced anhedonia: a realistic animal model of depression. Neurosci. Biobehav. Rev. 16, 525–534. Xu, P., Wang, K., Lu, C., Dong, L., Chen, Y., Wang, Q., Shi, Z., Yang, Y., Chen, S., Liu, X., 2017. Effects of the chronic restraint stress induced depression on reward-related learning in rats. Behav. Brain Res. 321, 185–192. Zhao, Q., Wu, X., Yan, S., Xie, X., Fan, Y., Zhang, J., Peng, C., You, Z., 2016. The antidepressant-like effects of pioglitazone in a chronic mild stress mouse model are associated with PPARγ-mediated alteration of microglial activation phenotypes. J. Neuroinflamm. 13, 259. Zong, J., Liao, X., Ren, B., Wang, Z., 2018. The antidepressant effects of rosiglitazone on rats with depression induced by neuropathic pain. Life Sci. 203, 315–322.
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Hippocampal PPARα is involved in the antidepressant-like effects of venlafaxine in mice
T
Cheng Chena,1, Jian-Hong Shenb,1, Hui Xua, Peng Chena, Fei Chena, Yi-Xiang Guand, Bo Jiangc, , ⁎ Zhong-Hua Wua, ⁎⁎
a
Department of Neurosurgery, The Sixth People’s Hospital of Nantong, Nantong 226011, Jiangsu, China Department of Neurosurgery, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu, China c Department of Pharmacology, School of Pharmacy, Nantong University, Nantong 226001, Jiangsu, China d Department of Surgery, Affiliated Haian People’s Hospital of Nantong University, Haian 226600, Jiangsu, China b
ARTICLE INFO
ABSTRACT
Keywords: Chronic unpredictable mild stress Chronic restraint stress Depression Hippocampus Peroxisome proliferator activated receptor α Venlafaxine
Although thought as a serotonin and norepinephrine reuptake inhibitor (SNRI), the antidepressant mechanisms of venlafaxine remain unknown. Previous reports have shown the role of peroxisome proliferator activated receptor α (PPARα) in depression. In this study, we investigated whether the antidepressant-like effects of venlafaxine require PPARα. We first examined whether repeated venlafaxine administration reversed the effects of chronic unpredictable mild stress (CUMS) and chronic restraint stress (CRS) on PPARα in the hippocampus and medial prefrontal cortex (mPFC). Then, the pharmacologcial inhibitors of PPARα, GW6471 and MK886, were used to assay if the protecting effects of venlafaxine against chronic stress were prevented by PPARα blockade. Furthermore, gene knockdown of PPARα by AAV-PPARα-shRNA was also used. It was found that venlafaxine treatment fully restored the decreasing effects of CUMS and CRS on the hippocampal PPARα expression. Pharmacological inhibition of PPARα significantly attenuated the antidepressant-like effects of venlafaxine in mice. Moreover, gene knockdown of hippocampal PPARα also fully abolished the antidepressant-like actions of venlafaxine in mice. Collectively, hippocampal PPARα is an antidepressant target of venlafaxine.
1. Introduction As a prevalent psychiatric disorder, depression affects about onefifth of the world population and will be the second largest global disease burden (Gelenberg, 2010). Current antidepressants used in clinical practice include the selective serotonin reuptake inhibitors (SSRIs), serotonin and norepinephrine reuptake inhibitors (SNRIs), and monoamine oxidase inhibitors (MAOIs), which all target on the monoaminergic system (Mandrioli et al., 2018; Perez-Caballero et al., 2019). However, the exact neurobiology of depression is still unclear. Also, the efficacy of SSRIs, SNRIs and MAOIs is inconsistent, and these antidepressants always produce side effects such as sedation, sleep disturbance, sexual dysfunction and body weight gain (Gorman and Kent, 1999; Masand and Gupta, 2002; Hirschfeld, 2003).
Peroxisome proliferator activated receptor α (PPARα) is a nuclear receptor protein that belongs to the PPAR family and functions as a transcription factor regulating gene expressions (Roy and Pahan, 2015). PPARα is distributed in many tissues like liver, kidney, heart, muscle and small intestine. PPARα is involved in many physiological processes in the central nervous system, such as neurotransmission, neuroinflammation and neurogenesis (Esmaeili et al., 2016; Pérez-Martín et al., 2016; Agarwal et al., 2017). In 2018, Song et al. has found that chronic stress significantly down-regulated the expression and function of PPARα in the hippocampus of mice (Song et al., 2018). Genetic overexpression of hippocampal PPARα induced notable antidepressant-like actions in mice by promoting the cAMP response element-binding protein (CREB)-mediated biosynthesis of brain-derived neurotrophic factor (BDNF). Also, the antidepressant-like effects of fluoxetine (a
Abbreviations: ANOVA, analysis of variance; BDNF, brain-derived neurotrophic factor; CREB, cAMP response element-binding protein; CRS, chronic restraint stress; CUMS, chronic unpredictable mild stress; FST, forced swim test; mPFC, medial prefrontal cortex; PPARα, peroxisome proliferator activated receptor α; SNRIs, serotonin and norepinephrine reuptake inhibitors; SSRIs, selective serotonin reuptake inhibitors; TST, tail suspension test ⁎ Corresponding author at: Department of Neurosurgery, The Sixth People’s Hospital of Nantong, No. 500 Yonghe Road, Nantong 226011, Jiangsu, China. ⁎⁎ Corresponding author at: Department of Pharmacology, School of Pharmacy, Nantong University, No. 19 Qixiu Road, Nantong 226001, Jiangsu, China. E-mail addresses: [email protected] (B. Jiang), [email protected] (Z.-H. Wu). 1 These authors contributed equally to this paper. https://doi.org/10.1016/j.brainresbull.2019.08.016 Received 28 April 2019; Received in revised form 18 August 2019; Accepted 19 August 2019 Available online 21 August 2019 0361-9230/ © 2019 Elsevier Inc. All rights reserved.
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mostly well-known SSRI) in mice require hippocampal PPARα. Moreover, several agonists of PPARα, WY14643, fenofibrate and gemfibrozil, all have been reported to possess antidepressant-like effects in mice (Jiang et al., 2015, 2017; Ni et al., 2018). These findings indicate that hippocampal PPARα is a potential and novel antidepressant target. As a main SNRI, venlafaxine is a second generation antidepressant. It is widely used to treat major depressive disorder, generalized anxiety disorder, panic disorder and social phobia. Although thought to produce function on the serotonergic and adrenergic systems, more and more pharmacological targets beyond the two systems are being demonstrated for venlafaxine recently (Alaiyed et al., 2019; Jiang et al., 2019). Here we speculated that PPARα in brain may participate in the antidepressant mechanism of venlafaxine. Therefore, the chronic unpredictable mild stress (CUMS) and chronic restraint stress (CRS) models of depression, various behavioral tests, western blotting and the AAV-mediated gene knockdown method were used together to explore our assumption in mice.
2.5. Sucrose preference test In this test, mice were given the choice to drink from two bottles for 6 h (from 09:00 a.m. to 03:00 p.m.) in individual cages. For the two bottles, one contained 1% sucrose solution, while the other contained tap water. To prevent possible effects of side preference in drinking behavior, the positions of two bottles were switched every 2 h. Before the test, all mice were acclimatized for 3 consecutive days to two-bottle choice conditions and deprived of food and water for another 12 h. The consumption of tap water and sucrose solution for each mouse were estimated by weighing the bottles before and after the test. The sucrose preference was measured as a percentage of the consumed sucrose solution relative to the total amount of liquid intake. 2.6. Chronic unpredictable mild stress (CUMS) This model of depression was done according to published reports with some modifications (Forbes et al., 1996; Papp et al., 1996; Li et al., 2007). In this study, the stressed mice were individually housed and exposed to a variable sequence of 8 mild and unpredictable stressors for 7 week. The stressors were 4 °C swimming for 1 min, food deprivation for 24 h, water deprivation for 24 h, damp sawdust for 24 h, cage shaking for 30 min, 45 °C cage tilting, inversion of light/dark cycle and tail clipping for 10 min. All stressors were randomly scheduled in each week to prevent animal habituation. The control mice were left undisturbed in the home cages (5 per cage) and handled daily. The treatments of venlafaxine, GW6471 and MK886 were performed daily during the last 2 weeks. After stress and drugs administration, FST, TST and the sucrose preference test were performed together to assay the depressive-like behaviors in mice.
2. Methods 2.1. Animals Male C57BL/6 J mice (8 weeks old) were bought from SLAC Laboratory Animal Co., Ltd (Shanghai, China), and housed 5 per cage under standard conditions for 1 week with free access to food and water before the experiments. The behavioral experiments were carried out during the light phase. The experiment procedures involving animals and their care were conducted in compliance with the Guide for the Care and Use of Laboratory Animals (8th edition, Institute of Laboratory Animal Resources on Life Sciences, National Research Council, National Academy of Sciences, Washington DC), and approved by the Animal Welfare Committee of Nantong University (Approval No. 20170420-013).
2.7. Chronic restraint stress (CRS) The CRS model was performed according to previously described procedures (Ejchel-Cohen et al., 2006; Mendoza et al., 2018; Wang et al., 2019). The stressed mice were individually housed, and received restraint stress which was applied 4 h/d (from 9:00 a.m. to 1:00 p.m.) for 6 weeks using 50 ml conical transparent plastic tubes (Corning Inc.; containing vent holes at one end) that can effectively immobilize the mice. The control mice were left undisturbed in the home cages (5 per cage) and handled daily. The treatments of venlafaxine, GW6471 and MK886 were performed daily during the last 2 weeks. After stress and drugs administration, FST, TST and the sucrose preference test were performed together to assay the depressive-like behaviors in mice.
2.2. Materials Venlafaxine was bought from Sigma (St. Louis, MO, USA). GW6471 and MK886 were bought from Tocris (Bristol, UK). The vehicle for venlafaxine, GW6471 and MK886 was 1% DMSO in 0.9% saline. The doses for venlafaxine (10 mg/kg), GW6471 (1 mg/kg) and MK886 (3 mg/kg) in this study were determined according to published reports (Esposito et al., 2012; Puligheddu et al., 2013; Jiang et al., 2019). All drugs were intraperitoneally (i.p.) injected in a volume of 10 ml/kg. 2.3. Forced swim test (FST)
2.8. Western blotting
The forced swim test was performed according to published protocols with slight modifications (Redrobe and Bourin, 1997; Lamberti et al., 1998). Mice were videotaped in a glass cylinder (45 cm height, 20 cm internal diameter) containing 20 cm height of water at 24 ± 1 °C for 6 min. The videotape was scored for the last 4 min manually by a trained and blinded observer. The water was replaced for every trial. The duration of immobility was measured as the time mice spent without any motion except for single limb paddling to maintain flotation.
Mice were decapitated, and the brain regions were rapidly dissected and homogenized in ice-cold lysis buffer containing Tris-HCl (50 mM, pH 7.4), EDTA (1 mM), NaCl (100 mM), NaF (20 mM), 3 mM Na3VO4 (3 mM), PMSF (1 mM), Nonidet P-40 (1%) and protease inhibitors. The lysates were centrifuged at 12000 × g for 15 min, and the protein concentrations were determined by the Bradford method. The samples were mixed with Laemmli sample buffer (2% SDS) and placed in a boiling water bath for 5 min. Proteins were resolved in SDS-polyacrylamide gels, transferred to nitrocellulose, and incubated with antibodies to PPARα (1:500; Abcam, Bristol, UK) and β-actin (1:500; Cell Signaling, Danvers, MA, USA). The immunoblots were developed using horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit IgG, followed by detection with enhanced chemiluminescence. The protein bands were quantitated with a densitometer.
2.4. Tail suspension test (TST) The tail suspension test was done according to published protocols with little modifications (Kamei et al., 2003; Ripoll et al., 2003). Mice were suspended by the tail on a horizontal beam 60 cm high. Mice were taped to the bar using adhesive tap placed approximately 1 cm from the tail tip. A 6-min test period was videotaped and scored manually by a trained and blinded observer. The mice were considered immobile only when they hung passively and were completely motionless.
2.9. AAV-mediated gene knockdown This method was done according to published reports with slight modifications (Song et al., 2018; Jiang et al., 2019). Mice were 172
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anesthetized with 0.5% pentobarbital sodium and fixed in stereotaxic frames (Stoelting, USA). For each mouse, its scalp was cut and the skull was exposed using 75% ethanol and 1% H2O2. Microsyringes of 5 μl were used to deliver AAV-Scrambled-EGFP or AAV-PPARα-shRNAEGFP. After making a small drill hole on the skull of each mouse, the microsyringes were positioned at the following coordinates: AP = 2.3 mm, ML = ± 1.8 mm, DV = +2.0 mm. The AAV constructs were injected bilaterally into the hippocampus at a rate of 0.5 μl/min (1.5 μl/ side). The microsyringes were maintained in place for 5 min to limit virus reflux. The incision of each mouse was sutured, and mice were allowed to recover for 3 d before further use. AAV-Scrambled-EGFP and AAV-PPARα-shRNAEGFP were kindly supplied by Dr. Bo Jiang, and the production of these constructs has been provided in Song et al. (Song et al., 2018). The sequences for PPARα-shRNA and Scrambled control-shRNA were 5′-AGAAATTCTTACCTGTGA A-3′ and 5′-TTCTCCGAACGTGTCACGT-3′ respectively. They were diluted to 5 × 109 TU/ml before use. 3 weeks were required for AAVPPARα-shRNA to produce gene knockdown effects.
3. Results 3.1. Repeated venlafaxine administration fully restored the effects of chronic stress on hippocampal PPARα As a first step, the CUMS model of depression was performed in C57BL/6 J mice. As shown in Fig. 1A, 7 weeks of CUMS significantly enhanced the immobility of mice in FST and TST (n = 12, P < 0.01), and decreased the sucrose preference of mice (n = 12, P < 0.01). In contrast, 2 weeks of venlfaxine administration fully reversed these behavioral changes (n = 12, P < 0.01). Afterwards, western blotting was performed to examine the effects of CUMS and venlafaxine on PPARα in the hippocampus and medial prefrontal cortex (mPFC), as the two regions are closely involved in the pathophysiology of depression. Fig. 1B illustrates the hippocampus data. It was found that CUMS induced a 47.1 ± 5.89% decrease of hippocampal PPARα expression compared to the control group (n = 6, P < 0.01), while venlafaxine treatment promoted the level of hippocampal PPARα in the CUMS group by 150.1 ± 13.3% (n = 6, P < 0.01). Interestingly, venlafaxine treatment also enhanced the hippocampal PPARα expression in the control group by 45.7 ± 3.24% (n = 6, P < 0.01). Fig. 1C illustrates the mPFC data. However, it was found that both CUMS exposure and venlafaxine treatment produced no observable effects on the PPARα expression in mPFC (n = 6). Then, the CRS model of depression was also performed in C57BL/6 J mice. Fig. 2A showed that 6 weeks of CRS robustly enhanced the immobility of mice in FST and TST (n = 12, P < 0.01), and down-
2.10. Statistical analysis All data are presented as means ± S.E.M., and analyzed using Sigma Stat10.0. Multiple group comparisons were performed using oneway analysis of variance (ANOVA) followed by Tukey's test, or two-way ANONA followed by Bonferroni’s test. A value of P < 0.05 was considered statistically significant.
Fig. 1. CUMS significantly decreased the expression of hippocampal PPARα in mice, whereas venlafaxine administration reversed it. (A) CUMS induced notable depressive-like behaviors in C57BL/6 J mice, as revealed by FST, TST and the sucrose preference test (n = 12). (B) Representative western blotting images showed the effects of CUMS and venlafaxine administration on PPARα expression in the hippocampus (n = 6). (C) Representative western blotting images showed that CUMS and venlafaxine administration had no effects on PPARα expression in mPFC (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by two-way ANOVA followed by Bonferroni’s test.
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Fig. 2. CRS also down-regulated the expression of hippocampal PPARα in mice, and this was restored by venlafaxine treatment. (A) CRS induced notable depressive-like behaviors in C57BL/6 J mice, as revealed by FST, TST and the sucrose preference test (n = 12). (B) Representative western blotting images revealed the effects of CRS and venlafaxine treatment on PPARα expression in the hippocampus (n = 6). (C) Representative western blotting images indicated that CRS and venlafaxine treatment produced no effects on PPARα expression in mPFC (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by two-way ANOVA followed by Bonferroni’s test.
treated mice displayed 28.1 ± 4.05% more of immobility in FST, 26.8 ± 6.14% more of immobility in TST and 28.5 ± 4.23% less of sucrose preference than the (CUMS + venlafaxine)-treated mice (n = 12, P < 0.01). Similarly, GW6471 co-treatment significantly attenuated the antidepressant-like effects of venlafaxine in the CRS model (Fig. 3C). Detailed analyses revealed that while the (CRS + venlafaxine)-treated mice exhibited 23.4 ± 2.58% less of immobility in FST, 29.4 ± 2.95% less of immobility in TST and 65.8 ± 5.49% more of sucrose preference than the CRS-stressed mice (n = 12, P < 0.01), the (CRS + venlafaxine + GW6471)-treated mice displayed 29.5 ± 3.62% more of immobility in FST, 30.1 ± 5.07% more of immobility in TST and 28.7 ± 5.47% less of sucrose preference than the (CRS + venlafaxine)-treated mice (n = 12, P < 0.01). Meanwhile, Fig. 3B and D indicated that GW6471 co-treatment fully blocked the enhancing effects of venlafaxine on the hippocampal PPARα expression in the stressed mice (n = 6, P < 0.01). Fig. 4 illustrates the MK886 data. As shown in Fig. 4A, MK886 cotreatment fully blocked the antidepressant-like actions of venlafaxine in the CUMS model. Detailed analyses revealed that while the (CUMS + venlafaxine)-treated mice exhibited 23.4 ± 2.73% less of immobility in FST, 26.7 ± 2.44% less of immobility in TST and 45.3 ± 6.12% more of sucrose preference than the CUMS-stressed mice (n = 12, P < 0.01), the (CUMS + venlafaxine + MK886)treated mice displayed 29.4 ± 4.61% more of immobility in FST, 30.2 ± 6.47% more of immobility in TST and 22.7 ± 3.32% less of sucrose preference than the (CUMS + venlafaxine)-treated mice (n = 12, P < 0.01). Also, MK886 co-treatment significantly attenuated the
regulated the sucrose preference of mice (n = 12, P < 0.01), while 2 weeks of venlafaxine treatment fully restored these behavioral changes (n = 12, P < 0.01). Fig. 2B shows the hippocampus results. CRS led to a 59.3 ± 7.03% decrease of hippocampal PPARα expression compared to the control group (n = 6, P < 0.01), while venlafaxine treatment promoted the level of hippocampal PPARα in the CRS group by 164.2 ± 9.56% (n = 6, P < 0.01). Fig. 2C shows the mPFC results. Also, both CRS exposure and venlafaxine treatment produced no significant effects on the PPARα expression in mPFC (n = 6). Taken together, hippocampal PPARα may be necessary for the antidepressantlike effects of venlafaxine in mice. 3.2. The use of PPARα antagonists fully blocked the antidepressant-like actions of venlafaxine in mice Next, the selective PPARα antagonists, GW6471 and MK886, were used to explore whether the antidepressant-like actions of venlafaxine require PPARα. Both the CUMS-stressed and CRS-stressed mice were co-injected with venlafaxine and GW6471/MK886 for 2 weeks, followed by various behavioral tests and western blotting detection. Fig. 3 illustrates the GW6471 results. As shown in Fig. 3A, GW6471 co-treatment significantly attenuated the antidepressant-like effects of venlafaxine in the CUMS model. Detailed analyses revealed that while the (CUMS + venlafaxine)-treated mice exhibited 25.3 ± 4.83% less of immobility in FST, 26 ± 2.62% less of immobility in TST and 37.5 ± 3.77% more of sucrose preference than the CUMS-stressed mice (n = 12, P < 0.01), the (CUMS + venlafaxine + GW6471)174
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Fig. 3. Blockade of PPARα function by GW6471 disturbed the antidepressant-like actions of venlafaxine in mice. (A) GW6471 treatment fully prevented the reversal effects of venlafaxine on the CUMS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data showed that the promoting effects of venlafaxine on hippocampal PPARα in the CUMS-stressed mice were blocked by GW6471 (n = 6). (C) GW6471 treatment also significantly attenuated the restoring effects of venlafaxine on the CRS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data showed that the enhancing effects of venlafaxine on hippocampal PPARα in the CRS-stressed mice were also blocked by GW6471 (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
antidepressant-like effects of venlafaxine in the CRS model (Fig. 4C). Detailed analyses revealed that while the (CRS + venlafaxine)-treated mice exhibited 24.5 ± 5.08% less of immobility in FST, 31.5 ± 3.96% less of immobility in TST and 30.9 ± 3.52% more of sucrose preference than the CRS-stressed mice (n = 12, P < 0.01), the (CRS + venlafaxine + MK886)-treated mice displayed 25.6 ± 2.77% more of immobility in FST, 34.7 ± 5.53% more of immobility in TST and 18.7 ± 3.48% less of sucrose preference than the (CRS + venlafaxine)treated mice (n = 12, P < 0.01). Moreover, Fig. 4B and D indicated that MK886 co-treatment significantly attenuated the enhancing effects of venlafaxine on the hippocampal PPARα expression in the stressed mice (n = 6, P < 0.01).
In addition, repeated treatment of GW6471/MK886 for 2 weeks notably enhanced the immobility of naïve control mice in FST and TST, but not affected the sucrose preference of control mice (n = 10, P < 0.01; Fig. S1A). 3.3. The use of PPARα-shRNA fully abolished the antidepressant-like actions of venlafaxine in mice Furthermore, AAV-PPARα-shRNA was used to selectively knockdown the expression of hippocampal PPARα. The PPARα-knockdown efficacy of AAV-PPARα-shRNA was shown in Fig. 5A and B (n = 5, P < 0.01). Briefly, C57BL/6J mice stereotaxically injected with 175
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Fig. 4. Blockade of PPARα function by MK886 interrupted the antidepressant-like effects of venlafaxine in mice. (A) MK886 treatment notably prevented the reversal effects of venlafaxine on the CUMS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data revealed that the promoting effects of venlafaxine on hippocampal PPARα in the CUMS-stressed mice were blocked by MK886 (n = 6). (C) MK886 treatment also fully attenuated the restoring effects of venlafaxine on the CRS-induced helplessness and anhedonia behaviors in mice (n = 12). (B) Representative western blotting data indicated that the enhancing effects of venlafaxine on hippocampal PPARα in the CRS-stressed mice were also blocked by MK886 (n = 6). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
PPARα-shRNA were kept for 3 weeks, and then subjected to CUMS/CRS and venlafaxine administration, followed by various behavioral tests. As shown in Fig. 5C, hippocampal PPARα-knockdown fully abolished the antidepressant-like effects of venlafaxine in the CUMS model. Detailed analyses indicated that while the (CUMS + venlafaxine)treated mice exhibited 26.7 ± 1.84% less of immobility in FST, 27.2 ± 3.06% less of immobility in TST and 45.1 ± 4.53% more of sucrose preference than the CUMS-stressed mice (n = 10, P < 0.01), the (CUMS + venlafaxine + PPARα-shRNA)-treated mice displayed 43.7 ± 7.31% more of immobility in FST, 34.7 ± 4.85% more of
immobility in TST and 25.4 ± 4.63% less of sucrose preference than the (CUMS + venlafaxine)-treated mice (n = 10, P < 0.01). Also, hippocampal PPARα-knockdown significantly prevented the antidepressant-like actions of venlafaxine in the CRS model (Fig. 5D). Detailed analyses showed that while the (CRS + venlafaxine)-treated mice exhibited 22.8 ± 4.37% less of immobility in FST, 24.8 ± 2.66% less of immobility in TST and 47.2 ± 6.84% more of sucrose preference than the CRS-stressed mice (n = 10, P < 0.01), the (CRS + venlafaxine + PPARα-shRNA)-treated mice displayed 25 ± 5.14% more of immobility in FST, 26.7 ± 3.33% more of immobility in TST and 176
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Fig. 5. Hippocampal PPARα knockdown abolished the antidepressant-like efficacy of venlafaxine in mice. (A) Fluorescence of a fixed brain section which expressed AAV-PPARα-shRNA-EGFP in the hippocampus after its stereotactic injection (Scale bar =400 μm). (B) Representative western blotting images showed the knockdown efficacy of AAV-PPARα-shRNA-EGFP in mice (n = 5). (C) The use of AAV-PPARα-shRNA markedly prevented the protective effects of venlafaxineagainst the CUMS-induced depressive-like behaviours in mice, as revealed by FST, TST and the sucrose preference test (n = 10). (D) AAV-PPARα-shRNA pre-infusion also fully abolished the restoring effects of venlafaxine on the CRS-induced helplessness and anhedonia behaviors in mice (n = 10). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
28.4 ± 2.79% less of sucrose preference than the (CRS + venlafaxine)treated mice (n = 10, P < 0.01). Fig. S1B indicated that hippocamapl PPARα-knockdown significantly increased the immobility of naïve control mice in FST and TST (n = 10, P < 0.01). In contrast, Scrambled control-shRNA produced none effects on the mice.
Moreover, the use of PPARα-shRNA fully prevented the promoting effects of venlafaxine on the hippocampal PPARα expression in the stressed mice (n = 5, P < 0.01; Fig. 6). Combined with the GW6471 and MK886 results, it can be concluded that hippocampal PPARα is involved in the antidepressant mechanism of venlafaxine. 177
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Fig. 6. The use of PPARα-shRNA fully prevented the promoting effects of venlafaxine on the hippocampal PPARα expression in the stressed mice. (A) Representative western blotting data showed that the (CUMS + venlafaxine + PPARα-shRNA)-treated mice had notably less expression of hippocampal PPARα than the (CUMS + venlafaxine)-treated mice (n = 5). (B) Representative western blotting results also indicated that the level of hippocampal PPARα in the (CRS + venlafaxine + PPARα-shRNA)-treated mice was significantly lower than that in the (CUMS + venlafaxine)-treated mice (n = 5). All results are represented as means ± S.E.M; **P < 0.01. The comparisons were made by one-way ANOVA followed by Tukey’s test.
4. Discussion
et al., 1992; Xu et al., 2017). We firstly reproduced the antidepressantlike effects of venlafaxine in the two models of depression, proving the effectiveness of our models and venlafaxine. It was found that both CUMS and CRS significantly down-regulated the expression of PPARα in the hippocampus, consistent with Song et al. (Song et al., 2018). Importantly, chronic venlafaxine administration fully restored the decreased PPARα expression in the hippocampus. Moreover, the use of GW6471, MK889 and PPARα-shRNA all abolished the antidepressantlike effects of venlafaxine in mice, confirming the speculation of our study. GW6471, MK889 and PPARα-shRNA treatments all enhanced the immobility of naïve control mice in FST and TST, representing a prodepressant-like effect and further indicating the role of PPARα in the pathophysiology of depression. In addition, the finding that chronic stress and venlafaxine produced no effects on PPARα expression in mPFC is interesting. For this region-selective phenomenon, currently there are no convincing explanations, and more profound studies are required in the future. How does venlafaxine produced antidepressant-like effects through hippocampal PPARα activation? As a nuclear receptor protein, PPARα controls the expression of various genes which contain PPREs in the promoter regions. It should be noticed that in 2013, Roy et al. has reported the correlation between hippocampal PPARα and CREB (Roy et al., 2013). It was found that PPARα directly regulates the transcriptional activity of CREB by combining its PPRE (Roy et al., 2013).
Peroxisome proliferator activated receptors (PPARs) are nuclear transcription factors that, in response to the binding of small ligands, regulate the expression of genes involved in many physiological processes (Roy and Pahan, 2015). The family of PPARs comprises three isoforms: PPARα, PPARβ/δ and PPARγ. These isotypes differ from each other in terms of their tissue distributions, ligand specificities and physiological roles (Chinetti et al., 2000; Tyagi et al., 2011). PPARα is activated when bound by endogenous lipid/lipid metabolite ligands or synthetic xenobiotic ligands. Once activated, PPARα heterodimerizes with the retinoid X receptor (RXR), and binds to PPAR response elements (PPREs) in the promoter regions of target genes involved in diverse processes such as energy metabolism, oxidative stress, inflammation, and cell differentiation (Qi et al., 2000). PPARα has beneficial effects in many diseases but also plays a pathological role in some conditions such as the development of insulin resistance (Puligheddu et al., 2013; Esmaeili et al., 2016; Agarwal et al., 2017). Here, this is the first comprehensive in vivo study exploring the role of PPARα in the antidepressant-like actions of venlafaxine. To get a reliable and believable conclusion, both the CUMS and CRS models of depression were used in this study, as they are two widely used and acknowledged animal models which mimic many depressivelike symptoms in human, such as helplessness and anhedonia (Willner 178
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Project in Jiangsu Province (No. BRA2016187), the Provincial Natural Science Foundation of Jiangsu Province (No. BK20161284), and the Science and Technology Projects of Nantong City (No. MS12018076).
PPARα-knockout mice were deficient in CREB and memory-associated proteins. Knockdown of hippocampal PPARα by shRNA suppressed CREB, rendering wild-type mice markedly poor in consolidating spatial memory, whereas introduction of PPARα to the hippocampus of PPARα-knockout mice increased CREB and improved spatial memory (Roy et al., 2013). The role of CREB in the pathophysiology of depression is widely known (Sulser, 2002; Gass and Riva, 2007). Depression is accompanied with significantly down-regulated activity of hippocampal CREB and decreased biosynthesis of BDNF, a well-known downstream protein of CREB (Nestler et al., 2002). In contrast, promotion of hippocampal CREB and BDNF protects against depression in rodents (Nestler et al., 2002). Thus, it is very possible that the antidepressant mechanism of venlafaxine involves a PPARα-CREB-BDNF pathway. This speculation could explain why weeks of administration are always required for venlafaxine to produce curing efficacy in clinical practice, as the PPARα-CREB-BDNF pathway is a long-term process. Another surprising and interesting result is that the use of GW6471 and MK889 significantly down-regulated the expression of hippocampal PPARα in mice treated with chronic stress and venlafaxine, as PPARα antagonists should not affect the biosynthesis of PPARα. Here, we think it is possible that like BDNF, PPARα is also a downstream protein of CREB. In other words, PPARα may have self-regulation on its biosynthesis through combining with PPRE in CREB. Thus, we assume that although GW6471 and MK889 did not influence the promoting effects of venlafaxine on PPARα expression, they significantly interrupted the self-regulation of PPARα on its biosynthesis. This speculation will be further studied in the future. Regarding the mechanism by which venlafaxine affects PPARα, one possibility is that venlafaxine directly binds and activates PPARα, functioning as an exogenous ligand (like WY14643, simvastatin and fibrates), which needs to be further validated using time-resolved FRET, electrospray ionization MS and in silico structural analysis (Roy and Pahan, 2015). The other possibility is that venlafaxine increases the concentration of extracellular 5-HT/NA and thus indirectly activates PPARα via a 5-HT/NA-mediated signaling pathway. However, the connection between PPARα and any 5-HT/NA-mediated signaling has not been confirmed so far. Nevertheless, combined with Song et al. (Song et al., 2018) and this study, both fluoxetine and venlafaxine have been proved to target hippocampal PPARα. If hippocampal PPARα is also involved in the antidepressant mechanisms of other SSRIs and SNRIs, such as paroxetine, citalopram and duloxetine, it will be an exact antidepressant target. Further in-depth studies are ongoing in our group. Besides, this study extends the knowledge of venlafaxine’s pharmacological actions and it is possible that like the fibrates, venlafaxine has protective effects on atherosclerosis by activating hepatic PPARα to reduce serum lipids. Moreover, several studies have demonstrated that PPARβ/δ and PPARγ are also correlated with depression. For example, hippocampal PPARδ overexpression protected against chronic stress-induced depressive-like behaviors in mice (Ji et al., 2015). Decreased PPARδ expression was found in rats subjected to CUMS (Liu et al., 2017). Agonists of PPARγ, such as pioglitazone and rosiglitazone, have potential antidepressant-like effects in rodents (Zhao et al., 2016; Zong et al., 2018). Since PPARβ/δ and PPARγ have similar biological structures to PPARα, they may be also the pharmacological targets of venlafaxine, which needs further exploration. Collectively, our study is new evidence showing the role of hippocampal PPARα in depression and antidepressant responses.
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Declaration of Competing Interest The authors declare no conflict of interests. Acknowledgments This work was supported by grants from the 333 High Level Talent 179
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