Abstract
Serotonin is implicated in stress-related psychopathologies. Two isoforms of the rate-limiting enzyme of serotonin biosynthesis, tryptophan hydroxylase, TPH1 and TPH2, are known. We show here that in the rat dorsal raphe nucleus (DRN), the nucleus that contains the highest number of 5-HT neurons in the brain, TPH1 mRNA reveals a low level of expression but is detectable both by quantitative real-time PCR and in situ hybridization whereas in the pineal gland (PiG), TPH1 mRNA is strongly expressed. To examine effects of stress on TPH expression we exposed male Wistar rats to daily restraint stress for 1 week. As shown by quantitative real-time PCR, TPH1 mRNA is 2.5-fold upregulated by the stress in DRN but not in PiG. Using 3′-RACE, we identified two TPH2 mRNA splice variants in the rat DRN which differ in the length of their 3′-untranslated regions (UTRs). TPH2b (with a short 3′-UTR) is the predominant variant in the DRN, whereas TPH2a (with a longer 3′-UTR) shows a low abundance in this nucleus. In the PiG, only TPH2b is detectable revealing a low level of expression. Expression of both TPH2 splice variants is not affected by stress, neither in DRN nor in the PiG. These data indicate that TPH1 in the serotonergic neurons of the DRN might be relevant for stress-induced psychopathologies.
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Abbreviations
- 3′-RACE:
-
3′-Rapid amplification of cDNA ends
- 5-HT:
-
5-hydroxytryptamine or serotonin
- DRN:
-
Dorsal raphe nucleus
- PiG:
-
Pineal gland
- RT:
-
Room temperature
- TPH:
-
Tryptophan hydroxylase
- UTR:
-
Untranslated region
References
Abumaria N, Rygula R, Havemann-Reinecke U, Rüther E, Bodemer W, Roos C, Flügge G (2006) Identification of genes regulated by chronic social stress in the rat dorsal raphe nucleus. Cell Mol Neurobiol 26:145–162
Abumaria N, Rygula R, Hiemke C, Fuchs E, Havemann-Reinecke U, Rüther E, Flügge G (2007) Effect of chronic citalopram on serotonin-related and stress-regulated genes in the dorsal raphe nucleus of the rat. Eur Neuropsychopharmacol 17:417–429
Arango V, Underwood MD, Mann JJ (2002) Serotonin brain circuits involved in major depression and suicide. Prog Brain Res 136:443–453
Austin MC, O’Donnell SM (1999) Regional distribution and cellular expression of tryptophan hydroxylase messenger RNA in postmortem human brainstem and pineal gland. J Neurochem 72:2065–2073
Azmitia EC Jr, McEwen BS (1974) Adrenalcortical influence on rat brain tryptophan hydroxylase activity. Brain Res 78:291–302
Azmitia EC, McEwen BS (1976) Early response of rat brain tryptophan hydroxylase activity to cycloheximide, puromycin and corticosterone. J Neurochem 27:773–778
Bach-Mizrachi H, Underwood MD, Kassir SA, Bakalian MJ, Sibille E, Tamir H, Mann JJ, Arango V (2006) Neuronal tryptophan hydroxylase mRNA expression in the human dorsal and median raphe nuclei: major depression and suicide. Neuropsychopharmacol 31:814–824
Boadle-Biber MC, Corley KC, Graves L, Phan TH, Rosecrans J (1989) Increase in the activity of tryptophan hydroxylase from cortex and midbrain of male Fischer 344 rats in response to acute or repeated sound stress. Brain Res 482:306–316
Boularand S, Darmon MC, Mallet J (1995a) The human tryptophan hydroxylase gene. An unusual splicing complexity in the 5′-untranslated region. J Biol Chem 270:3748–3756
Boularand S, Darmon MC., Ravassard P, Mallet J (1995b) Characterization of the human tryptophan hydroxylase gene promoter. Transcriptional regulation by cAMP requires a new motif distinct from the cAMP-responsive element. J Biol Chem 270:3757–3764
Brown SM, Peet E, Manuck SB, Williamson DE, Dahl RE, Ferrell RE, Hariri AR (2005) A regulatory variant of the human tryptophan hydroxylase-2 gene biases amygdala reactivity. Mol Psychiatry 10: 805, 884–888
Chamas F, Sabban EL (2002) Role of the 5′ untranslated region (UTR) in the tissue-specific regulation of rat tryptophan hydroxylase gene expression by stress. J Neurochem 82:645–654
Chamas F, Serova L, Sabban EL (1999) Tryptophan hydroxylase mRNA levels are elevated by repeated immobilization stress in rat raphe nuclei but not in pineal gland. Neurosci Lett 267:157–160
Chamas FM, Underwood MD, Arango V, Serova L, Kassir SA, Mann JJ, Sabban EL (2004) Immobilization stress elevates tryptophan hydroxylase mRNA and protein in the rat raphe nuclei. Biol Psychiatry 55:278–283
Chaouloff F (2000) Serotonin, stress and corticoids. J Psychopharmacol 14:139–151
Chen GL, Novak MA, Hakim S, Xie Z, Miller GM (2006) Tryptophan hydroxylase-2 gene polymorphisms in rhesus monkeys: association with hypothalamic-pituitary-adrenal axis function and in vitro gene expression. Mol Psychiatry 11:914–928
Christensen AK, Kahn LE, Bourne CM (1987) Circular polysomes predominate on the rough endoplasmic reticulum of somatotropes and mammotropes in the rat anterior pituitary. Am J Anat 178:1–10
Clark JA, Pai LY, Flick RB, Rohrer SP (2005) Differential hormonal regulation of tryptophan hydroxylase-2 mRNA in the murine dorsal raphe nucleus. Biol Psychiatry 57:943–946
Clark JA, Flick RB, Pai LY et al (2007) Glucocorticoid modulation of tryptophan hydroxylase-2 protein in raphe nuclei and 5-hydroxytryptophan concentrations in frontal cortex of C57/Bl6 mice. Mol Psychiatry; doi: 10.1038/sj.mp.4002041
Cote F, Thevenot E, Fligny C et al (2003) Disruption of the nonneuronal tph1 gene demonstrates the importance of peripheral serotonin in cardiac function. Proc Natl Acad Sci USA 100:13525–13530
de Kloet ER (1997) Why dexamethasone poorly penetrates in brain. Stress 2:13–20
de Kloet ER, Reul JM (1987) Feedback action and tonic influence of corticosteroids on brain function: a concept arising from the heterogeneity of brain receptor systems. Psychoneuroendocrinology 12:83–105
Dumas S, Darmon MC, Delort J, Mallet J (1989) Differential control of tryptophan hydroxylase expression in raphe and in pineal gland: evidence for a role of translation efficiency. J Neurosci Res 24:537–547
Dygalo NN, Shishkina GT, Kalinina TS, Yudina AM, Ovchinnikova ES (2006) Effect of repeated treatment with fluoxetine on tryptophan hydroxylase-2 gene expression in the rat brainstem. Pharmacol Biochem Behav 85:220–227
Geijer T, Frisch A, Persson ML, et al (2000) Search for association between suicide attempt and serotonergic polymorphisms. Psychiatr Genet 10:19–26
Gizatullin R, Zaboli G, Jonsson EG, Asberg M, Leopardi R (2006) Haplotype analysis reveals tryptophan hydroxylase (TPH) 1 gene variants associated with major depression. Biol Psychiatry 59:295–300
Gundlah C, Alves SE, Clark JA, Pai LY, Schaeffer JM, Rohrer SP (2005) Estrogen receptor-beta regulates tryptophan hydroxylase-1 expression in the murine midbrain raphe. Biol Psychiatry 57:938–942
Hamon M, Bourgoin S, Artaud F, Nelson D (1981) Regulatory properties of neuronal tryptophan hydroxylase. Adv Exp Med Biol 133:231–251
Huh SO, Park DH, Cho JY, Joh TH, Son JH (1994) A 6.1 kb 5′ upstream region of the mouse tryptophan hydroxylase gene directs expression of E. coli lacZ to major serotonergic brain regions and pineal gland in transgenic mice. Mol Brain Res 24:145–152
Kendler KS, Karkowski LM, Prescott CA (1999) Causal relationship between stressful life events and the onset of major depression. Am J Psychiatry 156:837–841
Lowry CA, Rodda JE, Lightman SL, Ingram CD (2000) Corticotropin-releasing factor increases in vitro firing rates of serotonergic neurons in the rat dorsal raphe nucleus: evidence for activation of a topographically organized mesolimbocortical serotonergic system. J Neurosci 20:7728–7736
Magarinos AM, McEwen BS (1995) Stress-induced atrophy of apical dendrites of hippocampal CA3c neurons: comparison of stressors. Neuroscience 69:83–88
Mayr B, Montminy M (2001) Transcriptional regulation by the phosphorylation-dependent factor CREB. Nat Rev Mol Cell Biol 2:599–609
McEwen BS (1998) Stress, adaptation, and disease. Allostasis and allostatic load. Ann N Y Acad Sci 840:33–44
McKinney J, Knappskog PM, Haavik J (2005) Different properties of the central and peripheral forms of human tryptophan hydroxylase. J Neurochem 92:311–320
Meyer H, Palchaudhuri M, Scheinin M, Flügge G (2000) Regulation of alpha(2A)-adrenoceptor expression by chronic stress in neurons of the brain stem. Brain Res 880:147–158
Nakamura K, Sugawara Y, Sawabe K, et al (2006) Late developmental stage-specific role of tryptophan hydroxylase 1 in brain serotonin levels. J Neurosci 26:530–534
Nielsen DA, Virkkunen M, Lappalainen J, Eggert M, Brown GL, Long JC, Goldman D, Linnoila M (1998) A tryptophan hydroxylase gene marker for suicidality and alcoholism. Arch Gen Psychiatry 55:593–602
Palkovits M (1973) Isolated removal of hypothalamic or other brain nuclei of the rat. Brain Res 59:449–450
Patel PD, Pontrello C, Burke S (2004) Robust and tissue-specific expression of TPH2 versus TPH1 in rat raphe and pineal gland. Biol Psychiatry 55:428–433
Peirson SN, Butler JN, Foster RG (2003) Experimental validation of novel and conventional approaches to quantitative real-time PCR data analysis. Nucleic Acids Res 31:e73
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160
Sachs AB, Buratowski S (1997) Common themes in translational and transcriptional regulation. Trends Biochem Sci 22:189–192
Sapolsky RM, Romero LM, Munck AU (2000) How do glucocorticoids influence stress responses? Integrating permissive, suppressive, stimulatory, and preparative actions. Endocr Rev 21:55–89
Singh VB, Corley KC, Phan TH, Boadle-Biber MC (1990) Increases in the activity of tryptophan hydroxylase from rat cortex and midbrain in response to acute or repeated sound stress are blocked by adrenalectomy and restored by dexamethasone treatment. Brain Res 516:66–76
Sugden D (2003) Comparison of circadian expression of tryptophan hydroxylase isoform mRNAs in the rat pineal gland using real-time PCR. J Neurochem 86:1308–1311
Tenner K, Walther D, Bader M (2007) Influence of human tryptophan hydroxylase 2 N- and C-terminus on enzymatic activity and oligomerization. J Neurochem 102:1887–1894
Walther DJ, Peter JU, Bashammakh S, Hortnagl H, Voits M, Fink H, Bader M (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76
Wang GA, Coon SL, Kaufman S (1998) Alternative splicing at the 3′-cDNA of human tryptophan hydroxylase. J Neurochem 71:1769–1772
Wiklund L, Bjorklund A (1980) Mechanisms of regrowth in the bulbospinal serotonin system following 5,6-dihydroxytryptamine induced axotomy. II. Fluorescence histochemical observations. Brain Res 191:109–127
Zaboli G, Jonsson EG, Gizatullin R, Asberg M, Leopardi R (2006) Tryptophan hydroxylase-1 gene variants associated with schizophrenia. Biol Psychiatry 60:563–569
Zhang X, Beaulieu JM, Sotnikova TD, Gainetdinov RR, Caron MG (2004) Tryptophan hydroxylase-2 controls brain serotonin synthesis. Science 305:217
Zhang X, Gainetdinov RR, Beaulieu JM, Sotnikova TD, Burch LH, Williams RB, Schwartz DA, Krishnan KR, Caron MG (2005) Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression. Neuron 45:11–16
Zhou Z, Roy A, Lipsky R, Kuchipudi K, Zhu G, Taubman J, Enoch MA, Virkkunen M, Goldman D (2005) Haplotype-based linkage of tryptophan hydroxylase 2 to suicide attempt, major depression, and cerebrospinal fluid 5-hydroxyindoleacetic acid in 4 populations. Arch Gen Psychiatry 62:1109–1118
Zill P, Buttner A, Eisenmenger W, Bondy B, Ackenheil M (2004a) Regional mRNA expression of a second tryptophan hydroxylase isoform in postmortem tissue samples of two human brains. Eur Neuropsychopharmacol 14:282–284
Zill P, Buttner A, Eisenmenger W, Möller HJ, Bondy B, Ackenheil M (2004b) Single nucleotide polymorphism and haplotype analysis of a novel tryptophan hydroxylase isoform (TPH2) gene in suicide victims. Biol Psychiatry 56:581–586
Zill P, Buttner A, Eisenmenger W, Möller HJ, Ackenheil M, Bondy B (2007) Analysis of tryptophan hydroxylase I and II mRNA expression in the human brain: a post-mortem study. J Psychiatr Res 41:168–173
Acknowledgments
Nashat Abumaria was in part funded by the DFG-Research Center Molecular Physiology of the Brain (CMPB) at the University of Göttingen. Adema Ribic is supported by a European Community fellowship for Neuroscience Early Stage Research Training (NEUREST), Göttingen, and is enrolled in the MSc/PhD Program for Molecular Biology at the University of Göttingen. Christoph Anacker is a fellow and grant recipient of the MSc/PhD Program Neurosciences at the University of Göttingen. We are grateful to Dr. B. Czeh for helpful discussions and to the excellent technical assistance of A. Hoffmann.
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Abumaria, N., Ribic, A., Anacker, C. et al. Stress Upregulates TPH1 but not TPH2 mRNA in the Rat Dorsal Raphe Nucleus: Identification of Two TPH2 mRNA Splice Variants. Cell Mol Neurobiol 28, 331–342 (2008). https://doi.org/10.1007/s10571-007-9259-5
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DOI: https://doi.org/10.1007/s10571-007-9259-5