Differential regulation of synaptic vesicle proteins by antidepressant ...

The Pharmacogenomics Journal (2004) 4, 110–113 & 2004 Nature Publishing Group All rights reserved 1470-269X/04 $25.00 www.nature.com/tpj

ORIGINAL ARTICLE

Differential regulation of synaptic vesicle proteins by antidepressant drugs S Rapp1 M Baader1 M Hu1 C Jennen-Steinmetz1 FA Henn1 J Thome1 1 Central Institute of Mental Health, Mannheim, Germany

Correspondence: Dr J Thome, Central Institute of Mental Health, Mannheim 68159, J5, Germany. Tel: þ 49 621 1703 955 Fax: þ 49 621 1703 760 E-mail: [email protected]

ABSTRACT

Synaptic vesicle proteins (SVP) play a critical role in neurotransmitter release and neural plasticity, and have been implicated in the pathophysiology of psychiatric disorders such as depression. Antidepressant drugs not only alter the level of neurotransmitters, but also modulate de novo gene transcription and synthesis of proteins involved in neural plasticity. In order to investigate the effects of antidepressant compounds on SVP-mRNA levels, the expressions of synaptophysin, synaptotagmin, VAMP, and synapsin-I were analysed by in situ hybridization in rats which had been treated with desipramine, fluoxetine, tranylcypromine, or saline. The results demonstrate that chronic treatment with fluoxetine and tranylcypromine leads to an increased expression of synaptophysin, but decreased expression of synaptotagmin and VAMP in the hippocampus and cerebral cortex. Additionally, synapsin ImRNA levels in the hippocampus and cerebral cortex are significantly reduced in tranylcypromine-treated animals. This identifies SVP genes as target genes of antidepressant treatment. The Pharmacogenomics Journal (2004) 4, 110–113. doi:10.1038/sj.tpj.6500229 Published online 6 January 2004 Keywords: depression; psychopharmacology; psychosis; SSRI; synapsin; synaptophysin

INTRODUCTION Synaptic vesicle proteins (SVP) such as synapsin I–III, synaptophysin, synaptotagmin, and synaptobrevin (VAMP – vesicle-associated membrane protein) play a critical role in synaptic plasticity, and are required for vesicle fusion and neurotransmitter release.1–6 Hence, changes in the expression of these proteins may contribute to the molecular effects of antidepressant treatment and associated behavioral and cognitive alterations. Previous studies have shown that antidepressants modulate de novo gene transcription and synthesis of proteins involved in neural and synaptic plasticity; in a recent transcriptomic study, a decreased expression of VAMP was found with imipramine and sertraline.7 In order to investigate the effects of antidepressant compounds on synaptic plasticity, the expression of SVP was analysed by in situ hybridization in rats which had been treated with desipramine, fluoxetine, tranylcypromine, or saline (control group) for 2 weeks.

Published online 6 January 2004

RESULTS The results of the in situ hybridization data revealed a significant increase of synaptophysin I expression in both hippocampus and cerebral cortex after chronic treatment with fluoxetine or tranylcypromine (Figures 1 and 3).

Synaptic vesicle proteins and antidepressants S Rapp et al

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CON

FLU

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TCP

Figure 1 Representative in situ hybridization of synaptophysin mRNA. Compared to controls (CON), synaptophysin expression is increased in animals chronically treated with fluoxetine (FLU) or tranylcypromine (TCP), respectively.

CON

FLU

CON

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Figure 3 Expression of synaptophysin in hippocampus and cortex is increased by antidepressants. Synaptophysin mRNA expression in hippocampus (dentate gyrus (DG), CA1, CA3) and cerebral cortex (CC) in rats from the DES (desipramine), FLU (fluoxetine), TCP (tranylcypromine), and CON (control, saline) groups: Synaptophysin expression in hippocampus and cerebral cortex was significantly enhanced in the FLU and TCP groups relative to the CON group (*Po0.05). The differences in the values of the DES and CON groups were not statistically significant. In situ hybridization using 35Sradiolabeled ribonucleotide probes complimentary to specific mRNA sequences was quantified from autoradiographic images by densitometry. The Wilcoxon–Mann–Whitney test was used for statistical analysis.

Figure 2 Representative in situ hybridization of synaptotagmin mRNA. In contrast to synaptophysin, synaptotagmin expression is decreased in animals treated with fluoxetine (FLU) or tranylcypromine (TCP) when compared to controls (CON).

Desipramine treatment did not lead to higher synaptophysin-I mRNA levels than the controls. Synaptotagmin III, in contrast, showed a significant decrease of expression in the area CA1 of hippocampus and cerebral cortex after chronic antidepressant treatment with fluoxetine or tranylcypromine, respectively (Figures 1–4). Desipramine showed a similar trend, but did not reach significance. Synapsin-I mRNA expression in CA1, CA3, dentate gyrus, and cerebral cortex (frontal area, 3.3–3.6 mm posterior to bregma) was significantly lower in the tranylcypromine group than in the control group (Figure 5). Also, VAMP 5 was significantly reduced by tranylcypromine treatment in all areas investigated. The same was true for fluoxetine treatment, with the exception of the cerebral cortex (Figure 6). The influence of desipramine administration on levels of VAMP-5 mRNA was not determined.

Figure 4 Expression of synaptotagmin in hippocampus and cortex is decreased by antidepressants. Expression of synaptotagmin was significantly decreased in the hippocampal area CA1 and the CC in the fluoxetine (FLU) and tranylcypromine (TCP) groups relative to CON rats (*Po0.05). In the other areas and in the DES (desipramine) group, similar tendencies were observed. However, these differences did not reach the level of significance in the Wilcoxon– Mann–Whitney test, which was used for statistical analysis.

DISCUSSION It has been shown that SVP are involved in the pathophysiology of psychiatric disorders such as schizophrenic psychoses and depression.8–10 Interestingly, the expression of SVP mRNA is modified by stress exposure. Stress leads to a

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Figure 5 Treatment with tranylcypromine (TCP) leads to a significant (*Po0.05) reduction in hippocampal (dentate gyrus (DG), CA1, CA3) and cortical (CC) synapsin-I mRNA expression. Treatment with DES (desipramine) and FLU (fluoxetine) has no significant influence on synapsin-I expression compared to controls (CON).

Figure 6 Expression of VAMP (synaptobrevin) in hippocampus and cortex is decreased by the antidepressants. VAMP mRNA expression in hippocampus (dentate gyrus (DG), CA1, CA3) and CC of rats from the DES (desipramine) and FLU (fluoxetine) groups was significantly reduced relative to the CON group (*Po0.05).

reduced synaptophysin expression, but increases synaptotagmin-mRNA levels.11 These changes, opposite to those seen for fluoxetine and tranylcypromine here, may be part of the neural adaptation to stress. These plastic changes may play a role in the hippocampal alterations observed in stressrelated disorders such as depression and post-traumatic stress disorder.12,13 The increase in hippocampal synaptophysin expression after antidepressant treatment and the decrease of synaptotagmin expression reported here are opposite to the SVP changes observed under such stress exposure. Together with the altered synapsin-I and VAMP expression, this provides evidence that synaptic integral membrane proteins are involved in altered synaptic plasticity in the adult CNS, following treatment with psychotropic compounds. It is

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important to note, however, that changes in proteins may not correspond to changes in respective mRNAs. Nonetheless, Yamada et al7 reported a differential expression of VAMP 2 in the frontal cortex after antidepresssant and electroconvulsive treatment using Western blot analysis, which suggests that changes of SVP concentrations on the protein level do occur; interestingly, the authors reported an induction of VAMP 2 in the rat frontal cortex after chronic antidepressant treatment, whereas, in our study, VAMP-5 mRNA was reduced in the cortex after treatment with tranylcypromine. Previous studies have shown that antidepressants cause metaplastic effects in the CNS, modulate long-term potentiation, facilitate the induction of long-term potentiation, and alter synaptic terminal structure.14–17 Furthermore, antidepressant agents exhibit rapid and powerful interactions with the mechanisms controlling the persistence of the block of LTP by inescapable stress, an important animal model of depression.18 Here, we demonstrate the differential regulation of the expression of SVP by antidepressants, indicating that SVP genes are targeted by such drugs, and that the regulation of these proteins is possibly a molecular correlate of hippocampal metaplastic and electrophysiological changes, as well as of behavioral and cognitive alterations after treatment with antidepressant drugs in vivo. With respect to the modification of SVP expression, this study revealed interesting differences between different classes of antidepressants. Whereas the mainly noradrenergic tricyclic desipramine induced almost no changes, the SSRI fluoxetine and the nonselective, nonreversible MAOI tranylcypromine exhibited strong effects, which suggest that effects on SVP may be mediated via the 5-HT system. Synaptic function is modulated by the phosphorylation of synaptic proteins by protein kinases,19,20 by changes in the ratio of their different isoforms,21 and by Ca2 þ -dependent mechanisms.22 Such regulatory processes are part of the CNS’s response to stress, a major risk factor of depression. These effects are modified by the increased expression of synaptophysin and decreased expression of synaptotagmin, synapsin I, and VAMP after antidepressant treatment, as described above. Alterations in the expression pattern of synaptic vesicle integral membrane proteins may also be associated with neural plasticity in the hippocampus including modifications in neural connectivity, and modulation of synaptic density that occur during antidepressant treatment. In summary, our findings demonstrate that synaptophysin, synaptotagmin, synapsin I, and VAMP are drugresponsive genes, and raise the possibility that alterations in the expression of these or other SVP might be important in producing some of the molecular and cellular effects of antidepressants in the limbic system.

MATERIALS AND METHODS Desipramine (15 mg/kg, i.p.), fluoxetine (10 mg/kg, i.p.), or tranylcypromine (10 mg/kg, i.p.) was administered daily for


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14 days. Control animals were administered 0.9% saline (i.p.) for 14 days. The animals (male Sprague–Dawley rats) were maintained under standard conditions (12 h light/dark cycle, food and water ad libitum). To investigate hippocampal gene expression following chronic treatment with antidepressant drugs, the animals were killed 3 h after the last i.p. injection, and their brains processed for in situ hybridization. Synaptophysin riboprobes were designed to hybridize with nucleotides 1513– 1991 of rat synaptophysin RNA, according to the nucleotide counting by Bargon and Leube.23 Synaptotagmin riboprobes corresponded to nucleotides 62–299 of rat synaptotagmin III RNA, according to the nucleotide counting by Mizuta et al.24 Synapsin-I and VAMP 5 riboprobes corresponded to nucleotides 2103–2224 and 1–295, respectively.25,26 The expression of SVP was quantified by measuring the optical density of autoradiographic film images in the hippocampal dentate gyrus (DG), CA1 and CA3 areas as well as in the cerebral cortex (CC), and standardized to the measurement results of the control group (CO) for each region. The equipment used for image acquisition and densitometry consisted of a ScanJX 250 system from Sharp Corp., Japan, and IPLab Spectrum 3.1.2 from Scanalytics Inc., USA. For statistical evaluation, the Wilcoxon–Mann–Whitney test was applied for pairwise comparisons of each treatment group with the control group, using a Bonferroni correction for multiple testing. A level of Po0.05 was accepted as significant. DUALITY OF INTEREST None declared. REFERENCES 1 Matthew WD, Tsavaler L, Reichardt LF. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue. J Cell Biol 1981; 91: 257–269. 2 Jahn R, Schiebler W, Ouiment C, Greengard P. A 38,000-dalton membrane protein (p38) present in synaptic vesicles. Proc Natl Acad Sci USA 1985; 82: 4137–4141. 3 Rehm H, Wiedenmann B, Betz H. Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane. EMBO J 1986; 5: 535–541. 4 Su¨dhof TC, Lottspeich F, Greengard P, Mehl E, Jahn R. A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions. Science 1987; 238: 1142–1144. 5 Greengard P, Valtorta F, Czernik AJ, Benfenati F. Synaptic vesicle phosphoproteins and regulation of synaptic function. Science 1993; 259: 780–785. 6 Janz R, Sudhof TC, Hammer RE, Unni V, Siegelbaum SA, Bolshakov VY. Essential roles in synaptic plasticity for synaptogyrin I and synaptophysin I. Neuron 1999; 24: 687–700.

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