Molecular mechanisms associated with the antidepressant effects of the class I histone deacetylase inhibitor MS-275 in the rat ventrolateral orbital cortex

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Research Report

Molecular mechanisms associated with the antidepressant effects of the class I histone deacetylase inhibitor MS-275 in the rat ventrolateral orbital cortex Huali Lina,⁎, Xiaoying Gengb , Wei Danga , Bin Wua , Zunxiao Daia , Yongchun Lia , Yani Yanga , Haiyan Zhanga , Jianguo Shia,⁎ a

Department of Psychiatry, Xi'an Mental Health Center, No. 15 Yanyin Road, Xi'an, Shannxi, China Department of Neurology, Fourth Hospital of Xi'an, No. 21 Jiefang Road, Xi'an, Shannxi, China

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Histone modifications mediated by histone acetylation are thought to play an important

Accepted 23 January 2012

role in the pathogenesis and treatment of depression. Recent studies have revealed that

Available online 27 January 2012

histone deacetylase inhibitors (HDACis), such as sodium valproate (VPA) and MS-275, may be involved in the pathogenesis of depression and in the underpinnings of antidepressant

Keywords:

therapeutic action in several brain regions, including the ventrolateral orbital cortex

MS-275

(VLO). In the present study, we investigated whether the class I histone deacetylase inhibi-

Ventrolateral orbital cortex

tor MS-275 exerts antidepressant-like effects when infused bilaterally into the VLO of a rat,

Histone acetylation

using the forced swimming test (FST) and tail suspension test (TST) as behavioral measures.

Antidepressant

We found that chronic intra-VLO infusion of MS-275 significantly reduced immobility time in the FST and TST compared with vehicle-treated controls, similar to the effects of systemically administered fluoxetine. These antidepressant-like effects of MS-275 are associated with an increase in H3 acetylation and elevated CREB and BDNF levels in the VLO. Our findings suggest the possibility that alterations in gene expression due to chromatin remodeling, including upregulation of CREB and BDNF, may be involved in the antidepressant-like effect of HDACis in the VLO. © 2012 Elsevier B.V. All rights reserved.

1.

Introduction

Depression is one of the most common mental illnesses and is a major cause of disability worldwide but has largely unknown etiology (Krishnan and Nestler, 2008). Although current treatments, mostly targeting the brain's monoamine systems, are highly effective for some sufferers with depression, about half of all patients remain symptomatic despite receiving standard antidepressant medications. Considering

the enormous burden on society, it is a high priority to identify the biological underpinnings of depression and develop more effective treatments. Aside from monoamine disturbances, recent studies in humans and animal models indicate that epigenetic regulation of gene function, a mechanism known to regulate longlasting behavioral responses to environmental stimuli, may play an important role in the pathogenesis of depression and in the mechanisms underlying the therapeutic action of

⁎ Corresponding authors at: Department of Psychiatry, Xi'an Mental Health Center, No. 15 Yanyin Road (710061), Xi'an, China. Fax: +86 29 85551506. E-mail addresses: [email protected] (H. Lin), [email protected] (J. Shi). 0006-8993/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2012.01.053

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Fig. 1. Only injection sites in the VLO were included in the data analysis (DMSO, n = 5; MS-275 (10 μM), n = 6; MS-275 (50 μM), n = 6; MS-275 (100 μM), n = 6).

antidepressants (Hobara et al., 2010). Histone acetylation contributes to the transcriptional activation process by relaxing a repressive chromatin state, which facilitates the sequestration of the basal transcriptional machinery. Therefore, histone acetylation may represent a key target for antidepressant action. For example, it has been shown that histone deacetylase inhibitors (HDACis) such as sodium butyrate (NaBt), which can increase levels of brain-derived neurotrophic factor (BDNF) expression in the frontal cortex (Schroeder et al., 2007), and exert an antidepressant-like effect when administered systemically. Chronic social defeat stress induces a selective lasting decrease in BDNF transcription, which can be reversed by chronic antidepressant treatment via increased histone acetylation at the promoter of the BDNF gene (Tsankova et al., 2006). Recently, Xing et al. (2011) have shown an antidepressant-like effect of sodium valproate (VPA), a HDAC inhibitor traditionally used as a mood stabilizer, when microinjected into the ventrolateral orbital cortex (VLO). It has been suggested that the VLO, a sub-region of the orbitofrontal cortex (OFC), is part of the limbic–thalamic– cortical circuit that is highly involved in the pathogenesis of depression (Drevets, 2000). It is possible that the action of VPA and NaBt on CREB, BDNF, or other gene transcriptions and beneficial behavioral effects on the animal models of depression are mediated by mechanisms that are independent of the inhibition of histone acetylation. However, VPA is an inhibitor of HDACs with possible non-specific effects such as elevation of GABA concentration, which is also associated with antidepressant-like action. Experiments with more potent and specific HDACi should help to clarify the underlying molecular mechanisms the action of VPA and NaBt on the regulation of CREB and BDNF expression in the VLO. Studies have shown that the 2′-aminophenyl-benzamide derivative MS-275 is a potent brain region-selective HDACi. It is 100-fold more potent than VPA for increasing the content of acetylated histone H3 (acH3) in the frontal cortex, and its action is longer lasting than that of VPA (Simonini et al., 2006). When MS-275 is infused into the nucleus accumbens (NAc), it produces an antidepressant-like effect by counteracting learned helplessness and social defeat stress (Covington et al., 2009). Moreover, as revealed by microarray analysis, the changes in gene expression patterns in the NAc induced by chronic defeat stress can be counteracted by intra-NAc infusion, similar to the effects of the antidepressant fluoxetine (Covington et al., 2009). Based on those findings, the aim of this study was to further investigate whether HDACi MS-275 could produce an antidepressant-like effect when infused into the VLO, and to explore changes in gene expression after intra-VLO infusion in rats.

Because there was a significant effect of repeated intra-VLO MS-275 treatment in the TST and FST, we performed Western blot analyses to investigate possible protein acetylation and expression changes contributing to depression-like behaviors. Western blot analysis revealed a significant enhancement of histone H3 acetylation in MS-275 (50 and 100 μM) groups [F(3,19) = 7.044, p = 0.0022; posthoc, p < 0.05 and 0.01, respectively] (Fig. 3). Since it is well known that the sustained acetylation of histones by HDAC inhibitors leads to upregulation of gene transcription, we used Western blotting to examine whether repeated intra-VLO administration of MS-275 affects the total protein expression of CREB and BDNF. MS-275 (100 μM) robustly increased BDNF expression approximately 1.75-fold [F(3,19) = 8.06, p = 0.0011; posthoc, p < 0.01] (Fig. 3) and increased CREB expression approximately 1.96-fold [F(3,19) = 5.593, p = 0.0064; posthoc, p < 0.0001] (Fig. 3).

2.

Results

2.4. Effect of repeated intra-VLO MS-275 treatment on CREB and BDNF mRNA levels

2.1.

Determination of microinjection sites

The injection sites were visually confirmed and marked on diagrams from the atlas (Paxinos et al., 1980). Photomicrographs of coronal brain sections depicting bilateral microinjection sites in the VLO of representative animals are presented in

2.2. Behavioral effects of bilateral intra-VLO administration of MS-275 The effects of MS-275 at different concentrations were assessed behaviorally with the open-field test, the TST and FST and compared with the effect of a known antidepressant, Fluoxetine (FLX). There was no significant effect of FLX or repeated MS-275 treatment, regardless of concentration, on the locomotor activity in the open-field test (Fig. 2A). In the TST, repeated FLX treatment significantly reduced immobility time in the 6-min test period compared with the saline controls [F(5,29) = 16.41, p < 0.0001; posthoc, p < 0.05] (Fig. 2B). When compared with the DMSO vehicle group, repeated MS-275 treatment with 50 and 100 μM, but not 10 μM, significantly decreased the duration of immobility in the TST [F(5,29) = 16.41, p < 0.0001; posthoc, p < 0.05 and p < 0.0001, respectively]. In the FST, the immobility time was significantly reduced in repeated FLX-treated rats [F(5,29) = 20.02, p < 0.0001; posthoc, p < 0.05]. Conversely, there was no significant effect of FLX in climbing or swimming time compared with the saline group. A significant immobility decrease and climbing time increase were observed in rats receiving MS-275 (100 μM) versus DMSO controls [F(5,29) = 20.02, p < 0.0001; posthoc, p < 0.0001 for both]. In addition, MS-275 (50 μM) showed a limited behavioral effect in climbing time only [F(5,29) = 20.02, p < 0.0001; posthoc, p < 0.05] (Figs. 2C–E).

2.3. Effect of repeated intra-VLO MS-275 treatment on protein levels of acH3, CREB and BDNF

We subsequently investigated the levels of CREB and BDNF mRNA during MS-275 treatment. Consistent with the Western blot analysis, a significant elevation of CREB mRNA was observed in MS-275 (50 and 100 μM) animals compared with DMSO controls [F(3,22) = 51.14, p < 0.0001; posthoc, p < 0.01 and 0.0001, respectively] (Fig. 4). In addition, BDNF mRNA levels

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Fig. 1 – Locations of microinjection cannula tips in the VLO of the rats included in the data analyses. Only rats with injection cannula tips located bilaterally in this site were included. A: Photomicrographs of a coronal brain section from a representative rat showing bilateral microinjection sites into the VLO. The section is 30 μm thick and stained with Cresyl Violet. B: Schematic representation showing the approximate location of microinjections into the VLO. The line drawings are adapted from Paxinos et al. (1980).

Fig. 2 – Behavioral measures of anxiety and despair after standard FLX treatment or chronic bilateral intra-VLO administration of MS-275. FLX and MS-275 were administered for 7 days. MS-275 (10, 50, 100 μM/0.5 μl per side) was bilaterally infused into the VLO 30 min before rats were tested. A: Open-field test: the distances traveled in 10 min were determined. B: Tail suspension test: The duration of total immobility in 6 min was determined. The data shown are the mean ± SD for 5–6 rats/group. *or # p < 0.05; *** or ###p < 0.001, analysis of variance, posthoc Dunnett. C–E: Forced swimming test: Time spent immobile, climbing, and swimming in 5 min was determined. The data shown are the mean ± SD for 5–6 rats/group. *p < 0.05, ** or ##p < 0.01 and ***, ###p < 0.001, one-way ANOVA, posthoc Bonferroni.

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Fig. 3 – Effect of MS-275 (10, 50, 100 μM/0.5 μl per side) on the changes in acH3, CREB and BDNF protein expression in the VLO assessed by Western blotting. The expression of acH3, CREB and BDNF in the VLO was significantly increased following chronic microinjection of MS-275 at a higher dose, compared with the DMSO-treated group. The results were normalized to H1 or β-actin. The data are expressed as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 versus DMSO controls. were significantly increased in all three MS-275 (10, 50 and 100 μM) treated groups [F(3,22) = 129.6, p < 0.0001; posthoc, p < 0.01, 0.0001 and 0.0001, respectively] (Fig. 4).

3.

Discussion

Despite advances in research studying the molecular and cellular basis for depression, progress in the development of

effective antidepressant agents has been relatively disappointing. Accumulating evidence suggests a potential role for histone modifying enzymes as promising pharmacologic targets for novel antidepressant treatments due to their effect on gene transcription, which ultimately influences the response to environmental stressors. The present study reveals a significant antidepressant-like effect of HDACi MS-275 when microinjected into the VLO in classic tests of behavioral despair. Our results also indicate that chronic intra-VLO

Fig. 4 – Effect of MS-275 (10, 50, 100 μM/0.5 μl per side) on the changes in CREB and BDNF mRNA expression in the VLO assessed by RT-PCR. Levels of GAPDH mRNA were used to normalize the relative expression levels of CREB and BDNF mRNA. The data are expressed as the mean ± SD. **p < 0.01, ***p < 0.001 versus DMSO controls.

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administration of MS-275 elevates BDNF and CREB expression in the VLO, which could contribute to its antidepressant-like action. There are many experimental procedures to evaluate the stress-related behavior in rodents such as FST, TST, sucrose preference, elevated plus-maze, dark-light box, etc. In the present study, we used FST and TST to evaluate the depressivelike behaviors after the antidepressant treatment. Of all the tests used in preclinical depression research, the FST (a.k.a. Porsolt's test; behavioral despair test) is probably the most widely and most frequently used (Cryan et al., 2002; Porsolt et al., 1977). It is effective and reliable to detect a broad spectrum of antidepressants after acute administration which allows for rapid screening of novel drugs (Rupniak, 2003). Like the FST, the TST is based on the observation that rodents will develop an immobile posture when placed in an inescapable situation. An obvious advantage of this test is its ability to detect a broad spectrum of antidepressants, inexpensive and methodologically unsophisticated. Although both the FST and TST are similar in the constructs that they purport to assess they are probably different in terms of the biological substrates that underlie the observed behavior although they often offer converging data on a potential antidepressant (Renard et al., 2003). The OFC is thought to be involved in motivational (such as feeding and drinking), emotional, and social behavior (Rolls, 2004). Depressed patients often exhibit anhedonia along with motivational and arousal deficits, suggesting a role for the OFC in mediating these symptoms. Neuroimaging, neuropathology, and lesion analysis techniques show that the OFC is important in the pathophysiology of depression (Drevets, 2007). However, there is evidence indicating that some subdivisions of the OFC function to inhibit, while others enhance, emotional expression (Drevets, 2007). There is a need to clarify the functions of different subdivisions of the OFC and their involvement in the pathophysiology of mood disorders. In this study, chronic intra-VLO infusion of higher concentrations of MS-275 was associated with antidepressantlike responses in the FST and TST. These behavioral results were accompanied by upregulation of BDNF and CREB mRNA and protein levels in the VLO. Elevated acH3 protein levels were also observed in the VLO, which further demonstrates the relevance of chromatin remodeling in antidepressant-like behavior. It is thought that increased BDNF in the VLO is important for treatment response. This hypothesis is consistent with postmortem studies showing decreased levels of prefrontal cortical BDNF in depressed individuals and suicide victims but not those with a history of antidepressant therapy (Dwivedi et al., 2003; Karege et al., 2005). In animal models, NaBt-related improved immobility scores in the TST are associated with a transient, at least 50%, increase in BDNF mRNA levels in the frontal cortex in mice (Schroeder et al., 2007). However, social defeat stress can increase BDNF levels in the NAc, and infusion of BDNF into the ventral tegmental area (VTA) produces depressionlike behavior (Berton et al., 2006; Eisch et al., 2003). These results suggest that the alteration of BDNF signaling, as it relates to depression, is region-specific. In addition, increased CREB levels were found in the present study, suggesting a role for this transcription factor in mediating the

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antidepressant effects of HDACis. These results are in line with previous observations that depressed patients without antidepressant therapy exhibit reduced expression of CREB protein in the OFC (Yamada et al., 2003), and antidepressant treatment increases CREB levels in rats. CREB-deficient mice lack antidepressant-induced changes in BDNF expression, indicating that activation of the transcription regulator CREB is essential for the induction of BDNF by antidepressants (Conti et al., 2002; Krishnan and Nestler, 2008). It is hypothesized that the antidepressant-like effect of MS-275 is due to changes in gene expression mediated by BDNF via activation of the CREB pathway. In summary, we report that chronic infusion of the HDACi MS-275 into the VLO improves behavioral despair in rats. This antidepressant-like effect could have been due, at least in part, to increased expression of CREB and BDNF mediated by increased acetylation of histone H3. Further studies aiming to clarify the mechanism of HDACis as antidepressants should evaluate other sub-regions of the OFC in the pathogenesis of depression and in HDACi-induced alterations in the functional balance between the OFC and other moodregulating brain regions such as the hippocampus and NAc.

4.

Experimental procedures

4.1.

Animals

All experiments were performed with male Sprague–Dawley rats, weighing 220–265 g at the beginning of the experiment. Animals were single-housed and maintained on a 12-h light– dark cycle (lights on at 07:00 a.m.) with access to food and water ad libitum. Animals were conditioned and tested during the light phase of the cycle. All experimental procedures conformed to the Institutional Animal Care and Use Committee (IACUC) guidelines, and all efforts were made to minimize animal suffering. 4.2.

Stereotaxic surgery and drug treatment

After 7 days of handling, the rats were treated with atropine methyl nitrate (0.4 mg/kg, i.p.) and penicillin (1.5 × 105 U/rat), anesthetized with chloral hydrate (35 mg/kg, i.p.), and mounted on a stereotaxic apparatus (RWD Life Science Co., Ltd, Shenzhen, China). Bilateral cannulae (21 gauge) were inserted into the VLO (3.7 mm anterior to the bregma, 2.0 mm lateral to the midline and 4.5 mm from the top of the skull) (Drevets, 2000). Guide cannulae were anchored to the skull with sterile stainless steel screws and acrylic dental cement. After surgery, two stainless steel obturators were inserted into the guides in order to prevent cannula occlusion. The obturators were removed and replaced every other day during the 5-day recovery period. For drug treatment, MS-275 (Santa Cruz, CA, USA) was dissolved in DMSO, and fluoxetine hydrochloride (Sigma-Aldrich, MO, USA) was dissolved in 0.9% saline immediately before use. Animals were microinjected for 7 consecutive days until 1 day before the behavioral tests. Obturators were removed and injection needles (27 gauge) connected to 1.0 μl Hamilton microsyringes were inserted, extending

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2.0 mm beyond the tip of the guide cannulae. Bilateral microinjections of MS-275 (10, 50 or 100 μM, 0.5 μl per side) or DMSO were administered into the VLO while the rat was gently held. The microinjectors were left in place for an additional 1 min to allow for drug diffusion. The standard antidepressant fluoxetine (FLX) (10 mg/kg, i.p., daily) or saline was administered to surgery-free rats for 7 days until 1 day before the behavioral tests. At the end of the behavioral tests, the animals were sacrificed and the brains were quickly removed. The brains were sliced into 1.0 mm sections using the adult rat brain matrix, and the injection sites were identified and marked on diagrams. Punches from the VLO regions were collected and immediately frozen in liquid nitrogen until subsequent experiments. 4.3.

Behavioral assessments

After 7 days of drug treatment, the following behavioral assessments were administered: Open-field test: To assess locomotor activity (distance traveled, cm), rats were placed at the center of a Plexiglas box (100 × 100 × 45 cm) and allowed 10 min of exploration. Each rat's performance was recorded by a video-tracking system (ANY-maze Ver4.7, Stoelting Co., IL, USA). The test room was dimly lit with indirect white lighting. Animals were habituated to the testing room for 20 min before the start of the session. Tail suspension test (TST): Rats were suspended by bands on the edge of a shelf 50 cm above the floor. Time spent immobile during a 6-min testing period was measured. Immobility time was defined as lack of all movement except whisker movement and respiration (Steru et al., 1985). Forced swim test (FST): Rats were placed into a clear acrylic cylinder (diameter 24 cm; height 50 cm; containing 30 cm of water at 25 ± 1 °C). Time spent immobile during a 5-min testing period was measured. Three behaviors were scored, as defined in previous studies (Shirayama et al., 2002): immobility (when the rat stopped all active behaviors and remained floating in the water with minimal movements, with its head just above the water); swimming (movements throughout the swim cylinder, including crossing into another quadrant); and climbing (upward-directed movements of the forepaws along the cylinder walls). 4.4.

Protein extraction and Western blotting

For protein extraction, frozen VLO tissues were homogenized in a lysis buffer containing Hepes 10 mM, EGTA 0.1 mM, sucrose 0.28 M, and Na4P2O7 5 mM, in the presence of a PMSF protease inhibitor (Sigma-Aldrich). A fraction of the lysate was collected, and the remaining fraction was centrifuged at 1000 × g for 5 min at 4 °C. The pellets were resuspended in a buffer containing NaCl 120 mM, Hepes 20 mM, EGTA 0.1 mM, Dithiothreitol 0.1 mM, Sodium Pyrophosphate 5 mM, NaF 20 mM, Na3VO4 1 mM, and PMSF 0.1 mg/ml (nuclear enriched extract). Protein concentration was determined with a standard protocol using a Quant-iT protein assay kit (Invitrogen). For the Western blotting, all of the extraction procedures were carried out at 4 °C. Protein extracts (30–60 μg) were run

on a 14% SDS-PAGE gel and subsequently blotted onto a PVDF membrane (BioRad, Hercules, CA). The membranes were blocked for 1 h with 5% non-fat dry milk in TBS-Tween 20, followed by overnight incubation at 4 °C with the following primary antibodies: anti-AcH3 (Lys 9/14) 1:1000 (Santa Cruz, sc-8655), anti-Histone H1 1:1000 (Histone H1, sc-34464), antiCREB 1:1000(Cell Signaling #9192), anti-BDNF 1:1000 (Santa Cruz, sc-33905) and β-actin 1:5000 (Santa Cruz, sc-130657). Membranes were then washed three times, incubated with the appropriate secondary HRP-linked antibodies, and washed three times again. Bands were detected using an enhanced chemiluminescence (ECL) reagent (Millipore). Immunoreactive protein bands were quantified by densitometry using Quantity One (BioRad). Ratios of acH3 over total H1 were calculated. The signals of CREB and BNDF were normalized to the density of β-actin. 4.5. RNA isolation, reverse transcription, and quantitative PCR Total RNA from VLO punches was isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer's instructions. Quantification was carried out by determining absorption at 260 nm. The mRNA in 2 μg of total RNA was reverse transcribed to cDNA in 50 μl of reaction mix using a High Capacity cDNA Kit (Applied Biosystems). PCR was performed in ABIPRISM 7900 HT (Applied Biosystems) using SYBR Green JumpStart Taq Ready Mix (Sigma-Aldrich). Target cDNA was amplified with the following cycling parameters: 94 °C for 2 min, 94 °C for 15 s, 60 °C for 1 min, for 40 cycles. Primers used for quantitative PCR are as follows: Total BDNF: forward 5′-GGA CAT ATC CAT GAC CAG AAA GAA A3, reverse 5′-GCA ACA AAC CAC AAC ATT ATC GAG-3′; CREB: forward 5′-CCG CCA GCA TGC CTT C-3′, reverse 5′-TGC AGC CCA ATG ACC AAA-3′; GAPDH: forward 5′-ATG GGG AAG GTG AAG GTC G-3′, reverse 5′-GGG GTC ATT GAT GGCAAC AA-3′. All measurements were performed in duplicate and at least two independent experiments per primer set were conducted. Levels of GAPDH mRNA were used to normalize the relative expression levels of target mRNA. 4.6.

Statistical analysis

Data analysis was performed using the commercial software GraphPad Prism 5.0. All data are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was utilized to estimate the statistical differences among different treatments’ effects on behavior in the openfield test, TST and FST. Where appropriate, Bonferroni or Dunnett's posthoc tests were used to determine group differences. For Western blotting and quantitative PCR, saline or DMSO controls were set at 1. In all cases, p values were twotailed, and comparisons were considered statistically significant at p < 0.05.

Acknowledgment This work was supported by the National Natural Science Foundation of China (31100900).

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