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Effect of fluoxetine on the expression of tryptophan hydroxylase and 14-3-3 protein in the dorsal raphe nucleus and hippocampus of rat
Journal of Chemical Neuroanatomy 43 (2012) 96–102
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Effect of fluoxetine on the expression of tryptophan hydroxylase and 14-3-3 protein in the dorsal raphe nucleus and hippocampus of rat Mi Ran Choi a,1, Sejin Hwang b,1, Geu Meum Park a, Kyung Hwa Jung a, Seok Hyeon Kim c, Nando Dulal Das a, Young Gyu Chai a,* a b c
Division of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul, Republic of Korea Department of Neuropsychiatry, College of Medicine & Institute of Mental Health, Hanyang University, Seoul, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2011 Received in revised form 10 January 2012 Accepted 10 January 2012 Available online 20 January 2012
The serotonergic system is one of the major systems targeted in the pharmacological treatment of mood disorders including depression. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been reported to induce the expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin. The 14-3-3 protein family not only activates neuronal enzymes, including TPH, but also plays a role in a wide variety of cell signaling. The aim of the present study was to determine whether fluoxetine regulates both the interaction of TPH and 14-3-3 proteins as well as the increase of those proteins in the dorsal raphe nucleus and the hippocampus. Sprague-Dawley rats were administered fluoxetine or vehicle for 5 and 14 days and sacrificed at 5 and 14 days after initial treatment. The intensity of immunoreactivity for TPH and 14-3-3 proteins in the dorsal raphe nucleus of the midbrain and in the hippocampus was measured, and the colocalization of both proteins was observed with double-labeling immunofluorescence. At 5 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein increased in both the dorsal raphe nucleus and the hippocampus, while that of TPH did not change in either region. In addition, at 14 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein significantly increased in both the dorsal raphe nucleus and hippocampus, while that of TPH showed few changes in either region. Colocalization of TPH and 143-3 proteins was observed in the cell bodies of dorsal raphe nucleus, whereas it was not observed in the hippocampus. These results suggest that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with delayed pharmacological effects of SSRIs. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Dorsal raphe nucleus Fluoxetine Hippocampus TPH 14-3-3 protein
1. Introduction Serotonin (5-HT) is a neurotransmitter involved in a variety of brain functions including the control of mood, anxiety, aggression, pain and cognition. The biosynthesis of 5-HT occurs mainly in serotonergic neurons in the dorsal raphe nucleus but it has a widespread distribution in the central nervous system including the hippocampus and prefrontal cortex (Gingrich and Hen, 2001). Tryptophan hydroxylase (TPH), a member of the superfamily of aromatic amino acid decarboxylases, is composed of two different TPH enzymes. TPH1 is expressed in non-neuronal tissues such as gut, pineal gland, spleen and thymus, while TPH2 is predominantly
* Corresponding author at: Division of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea. Tel.: +82 31 400 5513; fax: +82 31 406 6316. E-mail address: [email protected] (Y.G. Chai). 1 These authors contributed equally. 0891-0618/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2012.01.001
expressed in the brain stem (Walther and Bader, 2003). TPH metabolizes the essential amino acid tryptophan to 5-hydroxytryptophan (5-HTP) in an initial, rate-limiting step (Matthes et al., 2010). Next, 5-HTP is further decarboxylated to 5-HT by aromatic amino acid decarboxylase. However, changes in the level of TPH activity may lead to corresponding alterations in the amount of 5HT released into the synaptic space (Gartside et al., 1992). Changes in the 5-HT system may be associated with the development and pathophysiology of affective disorders, which are a group of psychiatric disorders including major depression and bipolar disorder (Fava and Kendler, 2000; Kessler et al., 2005; Lucki, 1998; Owens and Nemeroff, 1994). Depressed patients having attempted suicide have shown lowered 5-HT and/or 5hydroxyindoleacetic acid, its degradation product, in the cerebrospinal fluid (Asberg, 1997; Mann and Malone, 1997; Nordin, 1988), and the function of serotonergic systems in depressive patients was shown to be downregulated (Mann, 1999). Tryptophan depletion also increases symptom severity in depressed patients, inducing downregulation of 5-HT biosynthesis (Neumeister et al.,
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2004; Ruhe et al., 2007; Smith et al., 1997). However, some studies have reported that antidepressants led to an increase in TPH expression as well as 5-HT synthesis (Briley and Moret, 1993; Kim et al., 2002; Shishkina et al., 2007; Yang et al., 2010). In the study of Yang et al. (2010), animals following antidepressant treatment exhibited an increase in TPH in the dorsal raphe as well as increased swimming in the forced swimming test. On the other hand, Koubi et al. (2001) have reported that in terms of antidepressant mechanisms, electroconvulsive shock therapy led to an increase of TPH in the hippocampus of rats. Taken together, the serotonergic systems, including 5-HT and TPH, may be notable targets for pharmacological intervention in the treatment of neuropsychiatric diseases. However, further studies of the relationship between the serotonergic systems and action mechanisms of antidepressants are needed. The 14-3-3 protein family, which consists of acidic, ubiquitous and highly conserved proteins, was first discovered in the brain (Boston et al., 1982). After the 14-3-3 protein was first found to act as an activator of the neuronal enzymes tyrosine hydroxylase and TPH (Ichimura et al., 1988), its subfamily has been reported to bind to a variety of proteins involved in signal transduction, apoptosis, neurotransmission and cell cycle regulation (Berg et al., 2003; Reuther et al., 1994; Shimizu et al., 1994). Additionally, it is important to identify the nature of binding interactions between 14-3-3 and a diverse numbers of proteins because this could open a gate to identify numerous biological activities related to the 14-3-3 protein. Some studies have not only reported that the 14-3-3 protein binds with and activates phosphorylated TPH but also demonstrated that this activity is mediated through the COOHterminal acidic region of the 14-3-3 molecule (Banik et al., 1997; Ichimura et al., 1988, 1995; Isobe et al., 1991). These results suggest that the 14-3-3 protein is one of the important regulators of phosphorylated TPH function. Therefore, considering that 14-33 protein regulates the activation of TPH, the rate-limiting enzyme in 5-HT biosynthesis, it would also be meaningful to investigate whether antidepressants affect the biological activity of 14-3-3 protein. Stress leads to atrophy and death of CA3 pyramidal neurons in the hippocampus, whereas antidepressant treatment reverses their atrophy and death via an increase of neurotransmitters including 5-HT (Duman et al., 1999). Antidepressants, known as selective serotonin reuptake inhibitors (SSRIs), lead to an increase of 5-HT in the synaptic cleft by inhibiting its reuptake into the presynaptic cell. However, clinical improvements of these drugs are observed after a 2–6-week delay (Fuller and Wong, 1987). The SSRI fluoxetine not only shows a strong antidepressive effect by inhibiting synaptic reuptake of 5-HT in the dorsal raphe nucleus (Marek et al., 1992; Shishkina et al., 2007) but also affects serotonergic terminals in the dentate gyrus of the hippocampus (Silva et al., 2010). In the present study, we hypothesize that the delayed therapeutic action of fluoxetine may be related to interaction of TPH and 14-3-3 proteins as well as an increase in the expression of these proteins in the dorsal raphe nucleus and the hippocampus, the terminals of the serotonergic network. We measured the expression levels of these proteins in the dorsal raphe nucleus and hippocampus of rats after 5- and 14-day administrations of fluoxetine. We then analyzed the expression pattern of and association between TPH and 14-3-3 proteins at each time point. 2. Methods 2.1. Subjects Male specific pathogen free Sprague-Dawley rats (Charles River Breeding Laboratories, Atsugi, Japan) weighing 200–250 g were housed with ad libitum food and water on a 12 h:12 h light–dark cycle throughout the experiments. Animal health was monitored daily. All experimental procedures were approved by the
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institutional animal care and use committee at Hanyang University (HY-IACUC-10007) and were conducted in accordance with local ethical guidelines. 2.2. Fluoxetine treatment Animals were randomly assigned to one of four treatment groups: (1) 5 day control (n = 5); (2) 14 day control (n = 5); (3) 5 day fluoxetine (n = 5); and (4) 14 day fluoxetine (n = 5). Fluoxetine (Eli Lilly, Indianapolis, IN, USA) was dissolved in distilled water to obtain a concentration of 4 mg/ml. Animals in the fluoxetine group were given fluoxetine orally with a gastric sonde via esophagus at a daily oral dose of 10 mg/kg/day for 5 or 14 days. The concentration used in this study was determined by previous studies of dose–effect relationships (Lee, 2009; Mendesda-Silva et al., 2002). Control animals were given a daily oral dose of distilled water (equal to the volume of fluoxetine administered) for 5 or 14 days. 2.3. Immunohistochemistry 2.3.1. 3,30 -Diaminobenzidine immunohistochemistry To avoid the variable expression of TPH upon the circadian rhythm, all experimental animals were sacrificed between 9:00 and 10:00 AM. Animals were anesthetized with ketamine (75 mg/kg, intraperitoneal (IP)) and xylazine (25 mg/ kg, IP) 24 h after the final injection, transcardially perfused with 0.1 M phosphatebuffered saline (PBS) and fixed with a freshly prepared solution consisting of 4% paraformaldehyde in PBS. The brains were then removed, coronally sectioned, and postfixed in the same fixative at 4 8C for 2 h. The coronal sections were cryoprotected in a 20% sucrose solution for 24 h at 4 8C. Coronal sections of 40 mm thickness were taken from rat brains using a Leica Cryostat (Leica, Nussloch, Germany) and processed for 3,30 -diaminobenzidine (DAB) immunohistochemistry. Free-floating brain sections were soaked in 50% ethanol for 45 min, washed with 0.01 M PBS, and incubated with the primary antibody, sheep anti-TPH (1:1000) (AB 1541, Millipore, Billerica, MA, USA), which reacts with human and rat brain tissues by immunohistochemistry, or mouse anti-14-3-3 (1:500) (SC-59413, Santa Cruz Biotechnology, Santa Cruz, CA, USA), which recognizes multiple isoforms of rat origin-derived 14-3-3 protein family, overnight at room temperature. The sections were then incubated with biotinylated donkey anti-mouse IgG (1:200) (Jackson Inc., West Grove, PA, USA) or biotinylated donkey anti-sheep IgG (1:200) (Millipore, Billerica, MA, USA) for 2 h at room temperature, washed with 0.01 M PBS and incubated with extravidin (1:5000) (Sigma–Aldrich, St. Louis, MO, USA) for 20 min at room temperature. The sections were treated with DAB for 5 min at room temperature to visualize labeled cells and were then transferred onto slide glass. The slide glass was air dried, mounted with Permount1 (Thermo Fisher, Waltham, MA, USA), and observed using a light microscope (Leica DMR, Wetzlar, Germany). 2.3.2. Fluorescent immunohistochemistry Coronal sections were obtained by the same method as used for DAB immunohistochemistry. The sections were soaked in 50% ethanol for 45 min, washed with 0.01 M PBS, and incubated with a mixture of primary antibodies, sheep anti-TPH (1:1000) (Millipore, Billerica, MA, USA) and mouse anti-14-3-3 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at room temperature. The sections were then incubated with a mixture of secondary antibodies, fluorescein isothiocyanate-conjugated donkey anti-sheep IgG (1:200) (Jackson Inc., West Grove, PA, USA) and Cy3-conjugated donkey anti-mouse IgG (1:200) (Jackson Inc., West Grove, PA, USA), washed with 0.01 M PBS and mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured from the dorsal raphe nucleus of midbrain and hippocampus in brain sections using a Fluoview FV500 confocal laser scanning microscope (Olympus Optical Co., Tokyo, Japan) interfaced to a personal computer running Adobe Photoshop 8.0 (Adobe Systems, Mountain View, CA, USA) and Corel Draw 10 (Corel Co., Ottawa, Canada). 2.4. Image analysis For the quantitative analysis of the immunoreactivity for TPH and 14-3-3 protein in the midbrain dorsal raphe nucleus and the hippocampus, the optical density was measured with an image analysis software (Analysis Pro.V3.1, SIS, Muenster, Germany). Six coronal sections of the midbrain per rats (5 rats for each time group) with regular intervals (100 mm) at AP levels between bregma 7.5 to 8.3 mm (for dorsal raphe nucleus) and 3.14 mm to 3.8 mm (for hippocampus) (Paxinos and Watson, 1996) were chosen. The images of the midbrain and hippocampus sections were acquired by a cooled CCD camera (Fluoview II, SIS, Muenster, Germany) attached to a microscope under a constant exposure time and digital gain at the frame size of 898 mm 600 mm. The average intensity of the each image was measured with image analysis software. The images stained with DAB were inverted before the measurement of intensity to express stronger reaction as higher gray value. The mean intensity of 6 images from each rat was averaged. 2.5. Statistical analysis All of the data were expressed as the mean standard error of the mean (SEM). All statistical analyses were conducted using Prism software 5.0 (GraphPad Software, San
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Fig. 1. Effect of fluoxetine on expression of TPH in the dorsal raphe nucleus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, the brains were obtained from rats of both groups. (A) Immunohistochemistry for the TPH in the dorsal raphe nucleus. The 6 midbrain sections (coordination: bregma 7.5 to 8.3 mm) were chosen and subjected to immunohistochemistry. (B) Densitometric analysis for the relative intensity of TPH immunoreaction in the dorsal raphe nucleus. Intensity of TPH was measured with computer-aided image analyzing software at 5 and 14 days after fluoxetine treatment. The data are represented as the mean SEM of 5 rats/group. Con, control; Flux, fluoxetine. Scale bar = 250 mm.
Diego, CA, USA). The individual protein intensities between fluoxetine-treated group and control group according to treatment time were compared by using an unpaired Student’s t-test (http://www.physics.csbsju.edu/stats/t-test_NROW_form.html). Pvalues <0.05 were considered significant.
significantly greater than 20% increase compared with controls (t = 5.71, P = 0.005). 3.2. Colocalization of TPH and 14-3-3 in the dorsal raphe nucleus (fluorescent immunohistochemistry)
3. Results 3.1. Expression of TPH and 14-3-3 in the dorsal raphe nucleus (DAB immunohistochemistry) TPH was mainly observed in the cell bodies, and TPH-positive cells were apparently localized to the dorsal raphe nucleus (Fig. 1A). Immunoreactivity of TPH showed no difference between the control and fluoxetine-treated groups after 5 (t = 0.537, P = 0.620) and 14 (t = 0.702, P = 0.521) days of daily fluoxetine administration (Fig. 1B). As shown in by the TPH immunoreactivity pattern, 14-3-3 protein was also observed in the cell bodies of the dorsal raphe nucleus (Fig. 2A). Immunoreactivity of 14-3-3 protein after 5 days of fluoxetine treatment was increased compared with controls (t = 3.53, P = 0.024) (Fig. 2B). After administration of fluoxetine for 14 days, immunoreactivity of 14-3-3 protein showed a
To investigate whether TPH and 14-3-3 proteins are expressed in the dorsal raphe nucleus at the same time, the colocalization of TPH and 14-3-3 proteins was analyzed. As already mentioned in DAB immunohistochemistry data, immunoreactivity of TPH did not increase after 5 and 14 days of daily fluoxetine administration (Fig. 3A), while immunoreactivity of 14-3-3 protein increased after 5 and 14 days of daily fluoxetine administration compared with controls (Fig. 3B). However, colocalization of TPH and 14-3-3 proteins was observed in the cell bodies of dorsal raphe nucleus (Fig. 3C), whereas it was not observed in the hippocampus (Fig. 4A). 3.3. Expression of TPH and 14-3-3 in the hippocampus (fluorescent immunohistochemistry) TPH immunoreactivity in both controls and fluoxetine-treated groups was mainly observed in the nerve endings of the
Fig. 2. Effect of fluoxetine on expression of 14-3-3 in the dorsal raphe nucleus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, the brains were obtained from rats of both groups. (A) Immunohistochemistry for the 14-3-3 protein in the dorsal raphe nucleus. The 6 midbrain sections (coordination: bregma 7.5 to 8.3 mm) were chosen and subjected to immunohistochemistry. (B) Densitometric analysis for the relative intensity of 14-3-3 immunoreaction in the dorsal raphe nucleus. Intensity of TPH was measured with computer-aided image analyzing software at 5 and 14 days after fluoxetine treatment. The data are represented as the mean SEM of 5 rats/group. *Significantly different between control and fluoxetine-treated groups by an unpaired Student’s t-test (*P < 0.05). Con, control; Flux, fluoxetine. Scale bar = 250 mm.
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Fig. 3. Double immunofluorescence for TPH and 14-3-3 proteins in the dorsal raphe nucleus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, the midbrain sections (coordination: bregma 7.5 to 8.3 mm) obtained from rats of both groups were subjected to fluorescent immunohistochemistry. (A) Immunofluorescence for TPH in the dorsal raphe nucleus. Representative photomicrographs show immunofluorescent staining TPH (green). (B) Immunofluorescence for 143-3 protein in the dorsal raphe nucleus. Representative photomicrographs show immunofluorescent staining 14-3-3 protein (red). (C) Double immunofluorescence for TPH and 14-3-3 proteins in the dorsal raphe nucleus. Both TPH and 14-3-3 proteins were colocalized (yellow) in the majority of cells in the dorsal raphe nucleus (arrow) whereas some cells were single stained for TPH (arrowhead). Con, control; Flux, fluoxetine. Scale bar = 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
hippocampal CA3 regions (Fig. 4A). However, there were no differences in TPH immunoreactivity between controls and fluoxetine-treated groups at both 5 (t = 1.04, P = 0.359) and 14 (t = 1.31, P = 0.261) days (Fig. 4B). As shown in the dorsal raphe nucleus, the expression pattern of 14-3-3 protein was affected by fluoxetine treatment time even in the hippocampus (Fig. 4A). After 5 days of daily drug administration, 14-3-3 immunoreactivity did not exhibit significant difference compared with controls (t = 0.972, P = 0.386) (Fig. 4C). In particular, 14-3-3 in 14-day fluoxetine-treated groups exhibited significantly higher immunoreactivity than 14 day controls (t = 3.07, P = 0.037), suggesting an increase in 14-3-3 protein by chronic fluoxetine treatment (Fig. 4C). 4. Discussion Antidepressants that interact with the serotonin system are commonly used in the treatment of depression. Nevertheless, as the innervations from serotonin terminals influence almost all regions of the brain and there exist a number of serotonin receptor subtypes, it has been difficult to understand the mechanisms of action of these drugs. To identify the mutual association of proteins regulated by antidepressants and suggest their possibility as a new
target for pharmacological intervention in the treatment of depression, we observed the interaction between TPH and 14-33 proteins after fluoxetine treatment in vivo. TPH is a rate-limiting enzyme that plays a vital role in the biosynthesis of 5-HT (Matthes et al., 2010). In the present study, we showed that TPH-positive cells were mainly localized to the dorsal raphe nucleus, the largest serotonergic nucleus, in rats. Considering that TPH2, one of the two TPH isoforms, is mainly expressed in the brain stem and anti-TPH used in this study reacts with rat brain tissue, the TPH mainly detected in the dorsal raphe nucleus is likely TPH2. However, any differences in the signal intensity of TPH between fluoxetine-treated groups and controls did not reach statistical significance. As shown in the dorsal raphe nucleus, the expression of TPH in the hippocampus also did not change upon treatment with fluoxetine. Previous studies have observed the effects of SSRIs on TPH expression in the brain stem raphe nuclei including dorsal raphe nucleus (Abumaria et al., 2007; Dygalo et al., 2006; Kim et al., 2002; MacGillivray et al., 2010; Maciag et al., 2006; Saitoh et al., 2007) and other regions (Spurlock et al., 1994; Zusso et al., 2008) in rats. MacGillivray et al. (2010) observed a decrease in TPH-positive cells in the dorsal raphe nucleus of rats exposed to 5 mg/kg/day of citalopram and 5 mg/kg/day of fluoxetine. In addition, TPH mRNA expression in the dorsal raphe
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Fig. 4. Effect of fluoxetine on expression of TPH and 14-3-3 in the hippocampus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, CA3 regions in the hippocampus were subjected to fluorescent immunohistochemistry. (A) Double immunofluorescence for TPH and 14-3-3 in the hippocampus. Representative photomicrographs show double immunofluorescent staining TPH (green) and 14-3-3 (red) in the CA3 region of the hippocampus. (B) and (C) show the effect of immunoreactivity for TPH and 14-3-3 proteins in the CA3 region of the hippocampus, respectively. Intensities of TPH and 14-3-3 proteins were measured with computeraided image analyzing software at 5 and 14 days after fluoxetine treatment. The data are represented as the mean SEM of 5 rats/group. *Significantly different between control and fluoxetine-treated groups by an unpaired Student’s t-test (*P < 0.05). Con, control; Flux, fluoxetine. Scale bar = 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
nucleus was significantly decreased after citalopram (Abumaria et al., 2007) or fluoxetine administration (Dygalo et al., 2006). Unlike these results, Kim et al. (2002) reported an increase in TPH level in the dorsal raphe nucleus after sertraline treatment (treating 10 mg/kg/day of sertraline for 14 days). However, Spurlock et al. (1994) reported that there were no changes in the TPH mRNA level in whole brain in rats treated with citalopram and fluvoxamine. This result was essentially consistent with our observation that fluoxetine did not or slightly increased the TPH protein levels in the dorsal raphe nucleus and hippocampus. On the other hand, administration of SSRIs in neonatal rats not only resulted in a decrease in TPH-positive cells in the dorsal raphe nucleus (exposure to 10 mg/kg/day of citalopram) that persisted into adulthood (Maciag et al., 2006) but also affected the development of serotonergic neurons in the dorsal raphe nucleus and serotonergic terminals in the hippocampal dentate gyrus (exposure to 10 mg/kg/day of fluoxetine) (Silva et al., 2010). These results suggest that early exposure to antidepressants can obstruct the normal maturation of the serotonergic system. Based on previous studies and our results, these discrepancies in the expression of TPH in brain regions, mainly the dorsal raphe nucleus, may be due to the experimental conditions used or concentrations and types of antidepressants, even though the antidepressants mentioned above are SSRIs. Therefore, further research is needed to determine whether SSRIs mediate the activation of TPH as well as the expression of TPH in vivo after optimizing treatment time and dosage of SSRI antidepressants. In terms of neuronal plasticity, protein phosphorylation, such as protein tyrosine and serine/threonine phosphorylation, is an important mechanism through which molecular switches between the phosphorylated and the unphosphorylated state occur in response to stimuli (Ramser et al., 2010). Accordingly, it is worthwhile to investigate whether phosphoserine/phosphothreonine binding proteins such as 14-3-3 protein are involved in neural plasticity or neuroprotection through antidepressant mechanisms.
The 14-3-3 protein binds to and stabilizes enzymes such as TPH and inhibitors of protein kinase C after phosphorylation, resulting in activation or inactivation (Bridges and Moorhead, 2005). In the present study, we found that the expression of the 14-3-3 protein was significantly increased in the dorsal raphe nucleus in rats after fluoxetine treatment compared with controls. As already mentioned, the serotonergic neurons are located in the dorsal raphe nucleus. Additionally, considering that the 14-3-3 protein has been known to have various functions in addition to regulating the activation of TPH, many of their functions may be unknown. The upregulation of the 14-3-3 protein in the dorsal raphe nucleus by fluoxetine implies that there may be a new mechanism of 14-3-3 protein associated with an antidepressive effect. In addition, we demonstrated that 14-3-3 protein was highly expressed in the hippocampus, one of the regions regulated by the serotonergic system, after fluoxetine treatment. As a result of depression, CA3 pyramidal neurons in the hippocampus exhibit dendritic atrophy, death or endangerment, whereas antidepressants reverse this damage in the hippocampus through mechanisms including the cyclic adenosine monophosphate-response element binding protein pathway (Duman et al., 2000). Therefore, based on our results and the data from Duman et al. (2000), it would be valuable to study whether 14-3-3 protein plays a specific role in the neuroprotective functions of fluoxetine. Recently, some studies observed an association between fluoxetine and 14-3-3 protein (Baik et al., 2005; Cecconi et al., 2007). In a study by Baik et al. (2005), it was reported that the mRNA and protein levels of 14-3-3 z, one of the seven known isoforms, was upregulated after fluoxetine treatment for 2 and 3 days in rat basophilic RBL-2H3 cells, which have an active 5-HT system. Their results implied that 14-3-3 protein, especially the z isoform, might be involved in the action mechanism of fluoxetine. However, Cecconi et al. (2007) reported that fluoxetine treatment for 3 days led to the downregulation of 14-3-3 protein z/d, a brain-specific protein, in rat cortical neurons. They hypothesized that the opposite results of
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14-3-3 protein z expression between the two research groups (Baik et al., 2005; Cecconi et al., 2007) may be caused by differences in analysis methods and target cells (RBL-2H3 cells versus cortical neurons). Furthermore, while Cecconi et al. (2007) used 1 mM of fluoxetine as the concentration proportional to the lowest efficacious dose in behavioral models (Muscat et al., 1992), Baik et al. (2005) used 10 mM of fluoxetine. It is believed that a 10-fold difference in fluoxetine concentration between Baik et al. (2005) and Cecconi et al. (2007) groups may be enough to lead to the opposite effect of a drug in both in vitro and in vivo. Our result is meaningful regarding that the upregulation of 14-3-3 protein in the dorsal raphe nucleus and the hippocampus after fluoxetine treatment is reported in an in vivo model for the first time. When we measured the colocalization of TPH and 14-3-3 proteins in both the dorsal raphe nucleus and the hippocampus after fluoxetine treatment for 5 and 14 days, their colocalization was detected only in the dorsal raphe nucleus. The 14-3-3 protein binds to phosphorylated TPH (the active form of TPH), increases its activity and inhibits the dephosphorylation of TPH (Banik et al., 1997). Furthermore, based on the study showing the exact distribution of six 14-3-3 protein isoforms in mouse brains (Baxter et al., 2002), 14-3-3 protein expressed in the both dorsal raphe nucleus and hippocampus was shown to have the same pattern as the h isoform. However, more studies are needed to clarify and better explain what type of 14-3-3 protein isoforms in the dorsal raphe nucleus are regulated by fluoxetine and whether the phosphorylation of TPH in the dorsal raphe nucleus is regulated by fluoxetine. 5. Conclusions We have identified that as time went on, fluoxetine led to the upregulation of the 14-3-3 protein in the dorsal raphe nucleus, suggesting that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with the delayed pharmacological effects of SSRIs. In addition, it has been found that over time, fluoxetine leads to the upregulation of 14-3-3 protein rather than TPH in the hippocampus. Therefore, our present results may demonstrate a new mechanism of antidepressant action, suggesting a possible way to induce faster pharmacological effects of the drugs. Although the present results are the first to demonstrate the upregulation of 14-3-3 protein in the dorsal raphe nucleus in response to fluoxetine, more studies are needed to clarify and better explain what type of 14-3-3 protein isoforms in the dorsal raphe nucleus are regulated by fluoxetine and whether the phosphorylation of TPH in the dorsal raphe nucleus is regulated by fluoxetine. Acknowledgments We thank Professor Adam Turner for kind reviewing of our paper. This work was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101712). References Abumaria, N., Rygula, R., Hiemke, C., Fuchs, E., Havemann-Reinecke, U., Ruther, E., Flugge, G., 2007. Effect of chronic citalopram on serotonin-related and stressregulated genes in the dorsal raphe nucleus of the rat. Eur. Neuropsychopharmacol. 17, 417–429. Asberg, M., 1997. Neurotransmitters and suicidal behavior. The evidence from cerebrospinal fluid studies. Ann. N. Y. Acad. Sci. 836, 158–181. Baik, S.Y., Jung, K.H., Choi, M.R., Yang, B.H., Kim, S.H., Lee, J.S., Oh, D.Y., Choi, I.G., Chung, H., Chai, Y.G., 2005. Fluoxetine-induced up-regulation of 14-3-3zeta and tryptophan hydroxylase levels in RBL-2H3 cells. Neurosci. Lett. 374, 53–57. Banik, U., Wang, G.A., Wagner, P.D., Kaufman, S., 1997. Interaction of phosphorylated tryptophan hydroxylase with 14-3-3 proteins. J. Biol. Chem. 272, 26219– 26225.
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Journal of Chemical Neuroanatomy journal homepage: www.elsevier.com/locate/jchemneu
Effect of fluoxetine on the expression of tryptophan hydroxylase and 14-3-3 protein in the dorsal raphe nucleus and hippocampus of rat Mi Ran Choi a,1, Sejin Hwang b,1, Geu Meum Park a, Kyung Hwa Jung a, Seok Hyeon Kim c, Nando Dulal Das a, Young Gyu Chai a,* a b c
Division of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do, Republic of Korea Department of Anatomy and Cell Biology, College of Medicine, Hanyang University, Seoul, Republic of Korea Department of Neuropsychiatry, College of Medicine & Institute of Mental Health, Hanyang University, Seoul, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 July 2011 Received in revised form 10 January 2012 Accepted 10 January 2012 Available online 20 January 2012
The serotonergic system is one of the major systems targeted in the pharmacological treatment of mood disorders including depression. Fluoxetine, one of the selective serotonin reuptake inhibitors (SSRIs), has been reported to induce the expression of tryptophan hydroxylase (TPH), the rate-limiting enzyme in the biosynthesis of serotonin. The 14-3-3 protein family not only activates neuronal enzymes, including TPH, but also plays a role in a wide variety of cell signaling. The aim of the present study was to determine whether fluoxetine regulates both the interaction of TPH and 14-3-3 proteins as well as the increase of those proteins in the dorsal raphe nucleus and the hippocampus. Sprague-Dawley rats were administered fluoxetine or vehicle for 5 and 14 days and sacrificed at 5 and 14 days after initial treatment. The intensity of immunoreactivity for TPH and 14-3-3 proteins in the dorsal raphe nucleus of the midbrain and in the hippocampus was measured, and the colocalization of both proteins was observed with double-labeling immunofluorescence. At 5 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein increased in both the dorsal raphe nucleus and the hippocampus, while that of TPH did not change in either region. In addition, at 14 days after initial treatment with fluoxetine, immunoreactivity of 14-3-3 protein significantly increased in both the dorsal raphe nucleus and hippocampus, while that of TPH showed few changes in either region. Colocalization of TPH and 143-3 proteins was observed in the cell bodies of dorsal raphe nucleus, whereas it was not observed in the hippocampus. These results suggest that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with delayed pharmacological effects of SSRIs. ß 2012 Elsevier B.V. All rights reserved.
Keywords: Dorsal raphe nucleus Fluoxetine Hippocampus TPH 14-3-3 protein
1. Introduction Serotonin (5-HT) is a neurotransmitter involved in a variety of brain functions including the control of mood, anxiety, aggression, pain and cognition. The biosynthesis of 5-HT occurs mainly in serotonergic neurons in the dorsal raphe nucleus but it has a widespread distribution in the central nervous system including the hippocampus and prefrontal cortex (Gingrich and Hen, 2001). Tryptophan hydroxylase (TPH), a member of the superfamily of aromatic amino acid decarboxylases, is composed of two different TPH enzymes. TPH1 is expressed in non-neuronal tissues such as gut, pineal gland, spleen and thymus, while TPH2 is predominantly
* Corresponding author at: Division of Molecular and Life Science, Hanyang University, Ansan, Gyeonggi-do 426-791, Republic of Korea. Tel.: +82 31 400 5513; fax: +82 31 406 6316. E-mail address: [email protected] (Y.G. Chai). 1 These authors contributed equally. 0891-0618/$ – see front matter ß 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.jchemneu.2012.01.001
expressed in the brain stem (Walther and Bader, 2003). TPH metabolizes the essential amino acid tryptophan to 5-hydroxytryptophan (5-HTP) in an initial, rate-limiting step (Matthes et al., 2010). Next, 5-HTP is further decarboxylated to 5-HT by aromatic amino acid decarboxylase. However, changes in the level of TPH activity may lead to corresponding alterations in the amount of 5HT released into the synaptic space (Gartside et al., 1992). Changes in the 5-HT system may be associated with the development and pathophysiology of affective disorders, which are a group of psychiatric disorders including major depression and bipolar disorder (Fava and Kendler, 2000; Kessler et al., 2005; Lucki, 1998; Owens and Nemeroff, 1994). Depressed patients having attempted suicide have shown lowered 5-HT and/or 5hydroxyindoleacetic acid, its degradation product, in the cerebrospinal fluid (Asberg, 1997; Mann and Malone, 1997; Nordin, 1988), and the function of serotonergic systems in depressive patients was shown to be downregulated (Mann, 1999). Tryptophan depletion also increases symptom severity in depressed patients, inducing downregulation of 5-HT biosynthesis (Neumeister et al.,
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2004; Ruhe et al., 2007; Smith et al., 1997). However, some studies have reported that antidepressants led to an increase in TPH expression as well as 5-HT synthesis (Briley and Moret, 1993; Kim et al., 2002; Shishkina et al., 2007; Yang et al., 2010). In the study of Yang et al. (2010), animals following antidepressant treatment exhibited an increase in TPH in the dorsal raphe as well as increased swimming in the forced swimming test. On the other hand, Koubi et al. (2001) have reported that in terms of antidepressant mechanisms, electroconvulsive shock therapy led to an increase of TPH in the hippocampus of rats. Taken together, the serotonergic systems, including 5-HT and TPH, may be notable targets for pharmacological intervention in the treatment of neuropsychiatric diseases. However, further studies of the relationship between the serotonergic systems and action mechanisms of antidepressants are needed. The 14-3-3 protein family, which consists of acidic, ubiquitous and highly conserved proteins, was first discovered in the brain (Boston et al., 1982). After the 14-3-3 protein was first found to act as an activator of the neuronal enzymes tyrosine hydroxylase and TPH (Ichimura et al., 1988), its subfamily has been reported to bind to a variety of proteins involved in signal transduction, apoptosis, neurotransmission and cell cycle regulation (Berg et al., 2003; Reuther et al., 1994; Shimizu et al., 1994). Additionally, it is important to identify the nature of binding interactions between 14-3-3 and a diverse numbers of proteins because this could open a gate to identify numerous biological activities related to the 14-3-3 protein. Some studies have not only reported that the 14-3-3 protein binds with and activates phosphorylated TPH but also demonstrated that this activity is mediated through the COOHterminal acidic region of the 14-3-3 molecule (Banik et al., 1997; Ichimura et al., 1988, 1995; Isobe et al., 1991). These results suggest that the 14-3-3 protein is one of the important regulators of phosphorylated TPH function. Therefore, considering that 14-33 protein regulates the activation of TPH, the rate-limiting enzyme in 5-HT biosynthesis, it would also be meaningful to investigate whether antidepressants affect the biological activity of 14-3-3 protein. Stress leads to atrophy and death of CA3 pyramidal neurons in the hippocampus, whereas antidepressant treatment reverses their atrophy and death via an increase of neurotransmitters including 5-HT (Duman et al., 1999). Antidepressants, known as selective serotonin reuptake inhibitors (SSRIs), lead to an increase of 5-HT in the synaptic cleft by inhibiting its reuptake into the presynaptic cell. However, clinical improvements of these drugs are observed after a 2–6-week delay (Fuller and Wong, 1987). The SSRI fluoxetine not only shows a strong antidepressive effect by inhibiting synaptic reuptake of 5-HT in the dorsal raphe nucleus (Marek et al., 1992; Shishkina et al., 2007) but also affects serotonergic terminals in the dentate gyrus of the hippocampus (Silva et al., 2010). In the present study, we hypothesize that the delayed therapeutic action of fluoxetine may be related to interaction of TPH and 14-3-3 proteins as well as an increase in the expression of these proteins in the dorsal raphe nucleus and the hippocampus, the terminals of the serotonergic network. We measured the expression levels of these proteins in the dorsal raphe nucleus and hippocampus of rats after 5- and 14-day administrations of fluoxetine. We then analyzed the expression pattern of and association between TPH and 14-3-3 proteins at each time point. 2. Methods 2.1. Subjects Male specific pathogen free Sprague-Dawley rats (Charles River Breeding Laboratories, Atsugi, Japan) weighing 200–250 g were housed with ad libitum food and water on a 12 h:12 h light–dark cycle throughout the experiments. Animal health was monitored daily. All experimental procedures were approved by the
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institutional animal care and use committee at Hanyang University (HY-IACUC-10007) and were conducted in accordance with local ethical guidelines. 2.2. Fluoxetine treatment Animals were randomly assigned to one of four treatment groups: (1) 5 day control (n = 5); (2) 14 day control (n = 5); (3) 5 day fluoxetine (n = 5); and (4) 14 day fluoxetine (n = 5). Fluoxetine (Eli Lilly, Indianapolis, IN, USA) was dissolved in distilled water to obtain a concentration of 4 mg/ml. Animals in the fluoxetine group were given fluoxetine orally with a gastric sonde via esophagus at a daily oral dose of 10 mg/kg/day for 5 or 14 days. The concentration used in this study was determined by previous studies of dose–effect relationships (Lee, 2009; Mendesda-Silva et al., 2002). Control animals were given a daily oral dose of distilled water (equal to the volume of fluoxetine administered) for 5 or 14 days. 2.3. Immunohistochemistry 2.3.1. 3,30 -Diaminobenzidine immunohistochemistry To avoid the variable expression of TPH upon the circadian rhythm, all experimental animals were sacrificed between 9:00 and 10:00 AM. Animals were anesthetized with ketamine (75 mg/kg, intraperitoneal (IP)) and xylazine (25 mg/ kg, IP) 24 h after the final injection, transcardially perfused with 0.1 M phosphatebuffered saline (PBS) and fixed with a freshly prepared solution consisting of 4% paraformaldehyde in PBS. The brains were then removed, coronally sectioned, and postfixed in the same fixative at 4 8C for 2 h. The coronal sections were cryoprotected in a 20% sucrose solution for 24 h at 4 8C. Coronal sections of 40 mm thickness were taken from rat brains using a Leica Cryostat (Leica, Nussloch, Germany) and processed for 3,30 -diaminobenzidine (DAB) immunohistochemistry. Free-floating brain sections were soaked in 50% ethanol for 45 min, washed with 0.01 M PBS, and incubated with the primary antibody, sheep anti-TPH (1:1000) (AB 1541, Millipore, Billerica, MA, USA), which reacts with human and rat brain tissues by immunohistochemistry, or mouse anti-14-3-3 (1:500) (SC-59413, Santa Cruz Biotechnology, Santa Cruz, CA, USA), which recognizes multiple isoforms of rat origin-derived 14-3-3 protein family, overnight at room temperature. The sections were then incubated with biotinylated donkey anti-mouse IgG (1:200) (Jackson Inc., West Grove, PA, USA) or biotinylated donkey anti-sheep IgG (1:200) (Millipore, Billerica, MA, USA) for 2 h at room temperature, washed with 0.01 M PBS and incubated with extravidin (1:5000) (Sigma–Aldrich, St. Louis, MO, USA) for 20 min at room temperature. The sections were treated with DAB for 5 min at room temperature to visualize labeled cells and were then transferred onto slide glass. The slide glass was air dried, mounted with Permount1 (Thermo Fisher, Waltham, MA, USA), and observed using a light microscope (Leica DMR, Wetzlar, Germany). 2.3.2. Fluorescent immunohistochemistry Coronal sections were obtained by the same method as used for DAB immunohistochemistry. The sections were soaked in 50% ethanol for 45 min, washed with 0.01 M PBS, and incubated with a mixture of primary antibodies, sheep anti-TPH (1:1000) (Millipore, Billerica, MA, USA) and mouse anti-14-3-3 (1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at room temperature. The sections were then incubated with a mixture of secondary antibodies, fluorescein isothiocyanate-conjugated donkey anti-sheep IgG (1:200) (Jackson Inc., West Grove, PA, USA) and Cy3-conjugated donkey anti-mouse IgG (1:200) (Jackson Inc., West Grove, PA, USA), washed with 0.01 M PBS and mounted using Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA). Images were captured from the dorsal raphe nucleus of midbrain and hippocampus in brain sections using a Fluoview FV500 confocal laser scanning microscope (Olympus Optical Co., Tokyo, Japan) interfaced to a personal computer running Adobe Photoshop 8.0 (Adobe Systems, Mountain View, CA, USA) and Corel Draw 10 (Corel Co., Ottawa, Canada). 2.4. Image analysis For the quantitative analysis of the immunoreactivity for TPH and 14-3-3 protein in the midbrain dorsal raphe nucleus and the hippocampus, the optical density was measured with an image analysis software (Analysis Pro.V3.1, SIS, Muenster, Germany). Six coronal sections of the midbrain per rats (5 rats for each time group) with regular intervals (100 mm) at AP levels between bregma 7.5 to 8.3 mm (for dorsal raphe nucleus) and 3.14 mm to 3.8 mm (for hippocampus) (Paxinos and Watson, 1996) were chosen. The images of the midbrain and hippocampus sections were acquired by a cooled CCD camera (Fluoview II, SIS, Muenster, Germany) attached to a microscope under a constant exposure time and digital gain at the frame size of 898 mm 600 mm. The average intensity of the each image was measured with image analysis software. The images stained with DAB were inverted before the measurement of intensity to express stronger reaction as higher gray value. The mean intensity of 6 images from each rat was averaged. 2.5. Statistical analysis All of the data were expressed as the mean standard error of the mean (SEM). All statistical analyses were conducted using Prism software 5.0 (GraphPad Software, San
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Fig. 1. Effect of fluoxetine on expression of TPH in the dorsal raphe nucleus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, the brains were obtained from rats of both groups. (A) Immunohistochemistry for the TPH in the dorsal raphe nucleus. The 6 midbrain sections (coordination: bregma 7.5 to 8.3 mm) were chosen and subjected to immunohistochemistry. (B) Densitometric analysis for the relative intensity of TPH immunoreaction in the dorsal raphe nucleus. Intensity of TPH was measured with computer-aided image analyzing software at 5 and 14 days after fluoxetine treatment. The data are represented as the mean SEM of 5 rats/group. Con, control; Flux, fluoxetine. Scale bar = 250 mm.
Diego, CA, USA). The individual protein intensities between fluoxetine-treated group and control group according to treatment time were compared by using an unpaired Student’s t-test (http://www.physics.csbsju.edu/stats/t-test_NROW_form.html). Pvalues <0.05 were considered significant.
significantly greater than 20% increase compared with controls (t = 5.71, P = 0.005). 3.2. Colocalization of TPH and 14-3-3 in the dorsal raphe nucleus (fluorescent immunohistochemistry)
3. Results 3.1. Expression of TPH and 14-3-3 in the dorsal raphe nucleus (DAB immunohistochemistry) TPH was mainly observed in the cell bodies, and TPH-positive cells were apparently localized to the dorsal raphe nucleus (Fig. 1A). Immunoreactivity of TPH showed no difference between the control and fluoxetine-treated groups after 5 (t = 0.537, P = 0.620) and 14 (t = 0.702, P = 0.521) days of daily fluoxetine administration (Fig. 1B). As shown in by the TPH immunoreactivity pattern, 14-3-3 protein was also observed in the cell bodies of the dorsal raphe nucleus (Fig. 2A). Immunoreactivity of 14-3-3 protein after 5 days of fluoxetine treatment was increased compared with controls (t = 3.53, P = 0.024) (Fig. 2B). After administration of fluoxetine for 14 days, immunoreactivity of 14-3-3 protein showed a
To investigate whether TPH and 14-3-3 proteins are expressed in the dorsal raphe nucleus at the same time, the colocalization of TPH and 14-3-3 proteins was analyzed. As already mentioned in DAB immunohistochemistry data, immunoreactivity of TPH did not increase after 5 and 14 days of daily fluoxetine administration (Fig. 3A), while immunoreactivity of 14-3-3 protein increased after 5 and 14 days of daily fluoxetine administration compared with controls (Fig. 3B). However, colocalization of TPH and 14-3-3 proteins was observed in the cell bodies of dorsal raphe nucleus (Fig. 3C), whereas it was not observed in the hippocampus (Fig. 4A). 3.3. Expression of TPH and 14-3-3 in the hippocampus (fluorescent immunohistochemistry) TPH immunoreactivity in both controls and fluoxetine-treated groups was mainly observed in the nerve endings of the
Fig. 2. Effect of fluoxetine on expression of 14-3-3 in the dorsal raphe nucleus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, the brains were obtained from rats of both groups. (A) Immunohistochemistry for the 14-3-3 protein in the dorsal raphe nucleus. The 6 midbrain sections (coordination: bregma 7.5 to 8.3 mm) were chosen and subjected to immunohistochemistry. (B) Densitometric analysis for the relative intensity of 14-3-3 immunoreaction in the dorsal raphe nucleus. Intensity of TPH was measured with computer-aided image analyzing software at 5 and 14 days after fluoxetine treatment. The data are represented as the mean SEM of 5 rats/group. *Significantly different between control and fluoxetine-treated groups by an unpaired Student’s t-test (*P < 0.05). Con, control; Flux, fluoxetine. Scale bar = 250 mm.
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Fig. 3. Double immunofluorescence for TPH and 14-3-3 proteins in the dorsal raphe nucleus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, the midbrain sections (coordination: bregma 7.5 to 8.3 mm) obtained from rats of both groups were subjected to fluorescent immunohistochemistry. (A) Immunofluorescence for TPH in the dorsal raphe nucleus. Representative photomicrographs show immunofluorescent staining TPH (green). (B) Immunofluorescence for 143-3 protein in the dorsal raphe nucleus. Representative photomicrographs show immunofluorescent staining 14-3-3 protein (red). (C) Double immunofluorescence for TPH and 14-3-3 proteins in the dorsal raphe nucleus. Both TPH and 14-3-3 proteins were colocalized (yellow) in the majority of cells in the dorsal raphe nucleus (arrow) whereas some cells were single stained for TPH (arrowhead). Con, control; Flux, fluoxetine. Scale bar = 50 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
hippocampal CA3 regions (Fig. 4A). However, there were no differences in TPH immunoreactivity between controls and fluoxetine-treated groups at both 5 (t = 1.04, P = 0.359) and 14 (t = 1.31, P = 0.261) days (Fig. 4B). As shown in the dorsal raphe nucleus, the expression pattern of 14-3-3 protein was affected by fluoxetine treatment time even in the hippocampus (Fig. 4A). After 5 days of daily drug administration, 14-3-3 immunoreactivity did not exhibit significant difference compared with controls (t = 0.972, P = 0.386) (Fig. 4C). In particular, 14-3-3 in 14-day fluoxetine-treated groups exhibited significantly higher immunoreactivity than 14 day controls (t = 3.07, P = 0.037), suggesting an increase in 14-3-3 protein by chronic fluoxetine treatment (Fig. 4C). 4. Discussion Antidepressants that interact with the serotonin system are commonly used in the treatment of depression. Nevertheless, as the innervations from serotonin terminals influence almost all regions of the brain and there exist a number of serotonin receptor subtypes, it has been difficult to understand the mechanisms of action of these drugs. To identify the mutual association of proteins regulated by antidepressants and suggest their possibility as a new
target for pharmacological intervention in the treatment of depression, we observed the interaction between TPH and 14-33 proteins after fluoxetine treatment in vivo. TPH is a rate-limiting enzyme that plays a vital role in the biosynthesis of 5-HT (Matthes et al., 2010). In the present study, we showed that TPH-positive cells were mainly localized to the dorsal raphe nucleus, the largest serotonergic nucleus, in rats. Considering that TPH2, one of the two TPH isoforms, is mainly expressed in the brain stem and anti-TPH used in this study reacts with rat brain tissue, the TPH mainly detected in the dorsal raphe nucleus is likely TPH2. However, any differences in the signal intensity of TPH between fluoxetine-treated groups and controls did not reach statistical significance. As shown in the dorsal raphe nucleus, the expression of TPH in the hippocampus also did not change upon treatment with fluoxetine. Previous studies have observed the effects of SSRIs on TPH expression in the brain stem raphe nuclei including dorsal raphe nucleus (Abumaria et al., 2007; Dygalo et al., 2006; Kim et al., 2002; MacGillivray et al., 2010; Maciag et al., 2006; Saitoh et al., 2007) and other regions (Spurlock et al., 1994; Zusso et al., 2008) in rats. MacGillivray et al. (2010) observed a decrease in TPH-positive cells in the dorsal raphe nucleus of rats exposed to 5 mg/kg/day of citalopram and 5 mg/kg/day of fluoxetine. In addition, TPH mRNA expression in the dorsal raphe
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Fig. 4. Effect of fluoxetine on expression of TPH and 14-3-3 in the hippocampus. After fluoxetine (for Flux groups) and distilled water (for Con groups) treatments for 5 and 14 days, CA3 regions in the hippocampus were subjected to fluorescent immunohistochemistry. (A) Double immunofluorescence for TPH and 14-3-3 in the hippocampus. Representative photomicrographs show double immunofluorescent staining TPH (green) and 14-3-3 (red) in the CA3 region of the hippocampus. (B) and (C) show the effect of immunoreactivity for TPH and 14-3-3 proteins in the CA3 region of the hippocampus, respectively. Intensities of TPH and 14-3-3 proteins were measured with computeraided image analyzing software at 5 and 14 days after fluoxetine treatment. The data are represented as the mean SEM of 5 rats/group. *Significantly different between control and fluoxetine-treated groups by an unpaired Student’s t-test (*P < 0.05). Con, control; Flux, fluoxetine. Scale bar = 100 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)
nucleus was significantly decreased after citalopram (Abumaria et al., 2007) or fluoxetine administration (Dygalo et al., 2006). Unlike these results, Kim et al. (2002) reported an increase in TPH level in the dorsal raphe nucleus after sertraline treatment (treating 10 mg/kg/day of sertraline for 14 days). However, Spurlock et al. (1994) reported that there were no changes in the TPH mRNA level in whole brain in rats treated with citalopram and fluvoxamine. This result was essentially consistent with our observation that fluoxetine did not or slightly increased the TPH protein levels in the dorsal raphe nucleus and hippocampus. On the other hand, administration of SSRIs in neonatal rats not only resulted in a decrease in TPH-positive cells in the dorsal raphe nucleus (exposure to 10 mg/kg/day of citalopram) that persisted into adulthood (Maciag et al., 2006) but also affected the development of serotonergic neurons in the dorsal raphe nucleus and serotonergic terminals in the hippocampal dentate gyrus (exposure to 10 mg/kg/day of fluoxetine) (Silva et al., 2010). These results suggest that early exposure to antidepressants can obstruct the normal maturation of the serotonergic system. Based on previous studies and our results, these discrepancies in the expression of TPH in brain regions, mainly the dorsal raphe nucleus, may be due to the experimental conditions used or concentrations and types of antidepressants, even though the antidepressants mentioned above are SSRIs. Therefore, further research is needed to determine whether SSRIs mediate the activation of TPH as well as the expression of TPH in vivo after optimizing treatment time and dosage of SSRI antidepressants. In terms of neuronal plasticity, protein phosphorylation, such as protein tyrosine and serine/threonine phosphorylation, is an important mechanism through which molecular switches between the phosphorylated and the unphosphorylated state occur in response to stimuli (Ramser et al., 2010). Accordingly, it is worthwhile to investigate whether phosphoserine/phosphothreonine binding proteins such as 14-3-3 protein are involved in neural plasticity or neuroprotection through antidepressant mechanisms.
The 14-3-3 protein binds to and stabilizes enzymes such as TPH and inhibitors of protein kinase C after phosphorylation, resulting in activation or inactivation (Bridges and Moorhead, 2005). In the present study, we found that the expression of the 14-3-3 protein was significantly increased in the dorsal raphe nucleus in rats after fluoxetine treatment compared with controls. As already mentioned, the serotonergic neurons are located in the dorsal raphe nucleus. Additionally, considering that the 14-3-3 protein has been known to have various functions in addition to regulating the activation of TPH, many of their functions may be unknown. The upregulation of the 14-3-3 protein in the dorsal raphe nucleus by fluoxetine implies that there may be a new mechanism of 14-3-3 protein associated with an antidepressive effect. In addition, we demonstrated that 14-3-3 protein was highly expressed in the hippocampus, one of the regions regulated by the serotonergic system, after fluoxetine treatment. As a result of depression, CA3 pyramidal neurons in the hippocampus exhibit dendritic atrophy, death or endangerment, whereas antidepressants reverse this damage in the hippocampus through mechanisms including the cyclic adenosine monophosphate-response element binding protein pathway (Duman et al., 2000). Therefore, based on our results and the data from Duman et al. (2000), it would be valuable to study whether 14-3-3 protein plays a specific role in the neuroprotective functions of fluoxetine. Recently, some studies observed an association between fluoxetine and 14-3-3 protein (Baik et al., 2005; Cecconi et al., 2007). In a study by Baik et al. (2005), it was reported that the mRNA and protein levels of 14-3-3 z, one of the seven known isoforms, was upregulated after fluoxetine treatment for 2 and 3 days in rat basophilic RBL-2H3 cells, which have an active 5-HT system. Their results implied that 14-3-3 protein, especially the z isoform, might be involved in the action mechanism of fluoxetine. However, Cecconi et al. (2007) reported that fluoxetine treatment for 3 days led to the downregulation of 14-3-3 protein z/d, a brain-specific protein, in rat cortical neurons. They hypothesized that the opposite results of
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14-3-3 protein z expression between the two research groups (Baik et al., 2005; Cecconi et al., 2007) may be caused by differences in analysis methods and target cells (RBL-2H3 cells versus cortical neurons). Furthermore, while Cecconi et al. (2007) used 1 mM of fluoxetine as the concentration proportional to the lowest efficacious dose in behavioral models (Muscat et al., 1992), Baik et al. (2005) used 10 mM of fluoxetine. It is believed that a 10-fold difference in fluoxetine concentration between Baik et al. (2005) and Cecconi et al. (2007) groups may be enough to lead to the opposite effect of a drug in both in vitro and in vivo. Our result is meaningful regarding that the upregulation of 14-3-3 protein in the dorsal raphe nucleus and the hippocampus after fluoxetine treatment is reported in an in vivo model for the first time. When we measured the colocalization of TPH and 14-3-3 proteins in both the dorsal raphe nucleus and the hippocampus after fluoxetine treatment for 5 and 14 days, their colocalization was detected only in the dorsal raphe nucleus. The 14-3-3 protein binds to phosphorylated TPH (the active form of TPH), increases its activity and inhibits the dephosphorylation of TPH (Banik et al., 1997). Furthermore, based on the study showing the exact distribution of six 14-3-3 protein isoforms in mouse brains (Baxter et al., 2002), 14-3-3 protein expressed in the both dorsal raphe nucleus and hippocampus was shown to have the same pattern as the h isoform. However, more studies are needed to clarify and better explain what type of 14-3-3 protein isoforms in the dorsal raphe nucleus are regulated by fluoxetine and whether the phosphorylation of TPH in the dorsal raphe nucleus is regulated by fluoxetine. 5. Conclusions We have identified that as time went on, fluoxetine led to the upregulation of the 14-3-3 protein in the dorsal raphe nucleus, suggesting that the time-dependent regulation of 14-3-3 protein may be one of the various factors associated with the delayed pharmacological effects of SSRIs. In addition, it has been found that over time, fluoxetine leads to the upregulation of 14-3-3 protein rather than TPH in the hippocampus. Therefore, our present results may demonstrate a new mechanism of antidepressant action, suggesting a possible way to induce faster pharmacological effects of the drugs. Although the present results are the first to demonstrate the upregulation of 14-3-3 protein in the dorsal raphe nucleus in response to fluoxetine, more studies are needed to clarify and better explain what type of 14-3-3 protein isoforms in the dorsal raphe nucleus are regulated by fluoxetine and whether the phosphorylation of TPH in the dorsal raphe nucleus is regulated by fluoxetine. Acknowledgments We thank Professor Adam Turner for kind reviewing of our paper. This work was supported by a grant of the Korean Health Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101712). References Abumaria, N., Rygula, R., Hiemke, C., Fuchs, E., Havemann-Reinecke, U., Ruther, E., Flugge, G., 2007. Effect of chronic citalopram on serotonin-related and stressregulated genes in the dorsal raphe nucleus of the rat. Eur. Neuropsychopharmacol. 17, 417–429. Asberg, M., 1997. Neurotransmitters and suicidal behavior. The evidence from cerebrospinal fluid studies. Ann. N. Y. Acad. Sci. 836, 158–181. Baik, S.Y., Jung, K.H., Choi, M.R., Yang, B.H., Kim, S.H., Lee, J.S., Oh, D.Y., Choi, I.G., Chung, H., Chai, Y.G., 2005. Fluoxetine-induced up-regulation of 14-3-3zeta and tryptophan hydroxylase levels in RBL-2H3 cells. Neurosci. Lett. 374, 53–57. Banik, U., Wang, G.A., Wagner, P.D., Kaufman, S., 1997. Interaction of phosphorylated tryptophan hydroxylase with 14-3-3 proteins. J. Biol. Chem. 272, 26219– 26225.
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