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J Neural Transm DOI 10.1007/s00702-008-0096-6 BASIC NEUROSCIENCES, GENETICS AND IMMUNOLOGY - RAPID COMMUNICATION Deficiency of brain 5-HT synthesis but serotonergic neuron formation in Tph2 knockout mice Lise Gutknecht Æ Jonas Waider Æ Stefanie Kraft Æ Claudia Kriegebaum Æ Bettina Holtmann Æ Andreas Reif Æ Angelika Schmitt Æ Klaus-Peter Lesch Received: 30 June 2008 / Accepted: 6 July 2008 Ó Springer-Verlag 2008 Abstract The relative contribution of the two tryptophan hydroxylase (TPH) isoforms, TPH1 and TPH2, to brain serotonergic system function is controversial. To investigate the respective role of TPH2 in neuron serotonin (5-HT) synthesis and the role of 5-HT in brain development, mice with a targeted disruption of Tph2 were generated. The preliminary results indicate that in Tph2 knockout mice raphe neurons are completely devoid of 5-HT, whereas no obvious alteration in morphology and fiber distribution are observed. The findings confirm the exclusive specificity of Tph2 in brain 5-HT synthesis and suggest that Tph2-synthesized 5-HT is not required for serotonergic neuron formation. Keywords Tryptophan hydroxylase 2 Tph2 Knockout Serotonin Development Depression Attention-deficit/hyperactivity disorder L. Gutknecht (&) J. Waider S. Kraft C. Kriegebaum A. Reif A. Schmitt K.-P. Lesch (&) Molecular and Clinical Psychobiology, Department of Psychiatry, Psychosomatics, and Psychotherapy, University of Wuerzburg, Fuechsleinstrasse 15, 97080 Wuerzburg, Germany e-mail: [email protected] K.-P. Lesch e-mail: [email protected] B. Holtmann Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Wuerzburg, Versbacherstrasse 9, 97078 Wuerzburg, Germany Introduction The two tryptophan hydroxylase (TPH) isoforms, TPH1 and TPH2, act as rate-limiting enzymes in serotonin (5-hydroxytryptamine, 5-HT) synthesis (Walther and Bader 2003; McKinney et al. 2005). Based on evidence supporting exclusive brain specificity of TPH2, particularly this isoform has been implicated in cognition and emotion regulation in the general population (Gutknecht et al. 2007; Canli et al. 2005; Herrmann et al. 2007) as well as in the pathophysiology of syndromal dimensions of various neuropsychiatric conditions, such as depression (Zhou et al. 2005), bipolar disorder (Harvey et al. 2004) and attentiondeficit/hyperactivity disorder (ADHD) (Walitza et al. 2005; Baehne et al. 2008). However, there is persisting controversy regarding the respective roles of the two TPH isoforms in brain 5-HT synthesis and the role of 5-HT in neuron formation and brain development. While the two initial studies revealing the existence of TPH2 (Walther et al. 2003; Côté et al. 2003) concluded that the expression of the two isoforms is mutually exclusive, designating TPH1 as the peripheral form, extremely abundant in the pineal gland and enterochromaffine cells, and TPH2 representing the neuronal subtype, specifically expressed in serotonergic neurons of the raphe nuclei, several subsequent differential expression studies reported detectable TPH1 expression in raphe neurons of the rat (Patel et al. 2004; Malek et al. 2005), the mouse (Gundlah et al. 2005) and humans (Zill et al. 2007). Nakamura et al. (2006), who described preferential expression of Tph1 mRNA at postnatal day 21 (P21) in the murine raphe, assumed that Tph1 plays a critical role during late developmental stages of the brain. Recently, we have conducted a systematic analysis of Tph1 and Tph2 expression in adult mouse and human brain, as well as during pre- and 123 L. Gutknecht et al. postnatal development of the murine brain (L. Gutknecht et al., submitted). Four complementary approaches (qRTPCR, in situ hybridization, immunohistochemistry, and Western blot) did not reveal significant Tph1 expression in the raphe of both species. These results indicate exclusive Tph2 expression in raphe neurons in adulthood as well as during development. To further confirm the role of TPH2 in raphe neuron 5-HT synthesis and eventually the role of 5-HT in brain development and function, we inactivated Tph2 in mice by homologous recombination and Cre-assisted deletion resulting in constitutive Tph2 null mutants with a complete loss of 5-HT immunodetection in raphe serotonergic neurons. Methods Generation of Tph2 knockout mice Genomic contigs of Tph2 encompassing exon 5 and flanking sequence were obtained by screening of a 129/Ola mouse Cosmid library (library number 121, RZPD, Berlin, Germany). Positive clones were further characterized by restriction mapping and Southern blot analysis. For the gene targeting construct, a *4.4 kb XhoI-XhoI fragment located upstream of exon 5 was selected as the 50 flank, while a *3.4 kb XhoI-XhoI fragment containing exon 5 was projected to induce the targeted deletion (Fig. 1a). The elimination of Tph2 exon 5, which codes for an amino acid sequence at the start of the catalytic domain and ends with a partial codon, was predicted to create a shift in the reading frame resulting in a truncated non-functional Tph2 protein. A LoxP site was inserted between these two fragments by cloning into pKSLox (modified pKS bluescript including one LoxP site). A *2.0 kb XhoI-XmnI fragment, serving as 30 flank, was cloned into pKSLNL Fig. 1 Generation and genotyping of Tph2 null mutants. a Targeting strategy. Top: targeting vector designed to remove exon 5 and flanking sequences (XhoI-XhoI deletion, pink) by homologous recombination in wildtype (WT) genomic DNA (middle). Exons 2– 6 are depicted by vertical black boxes. XhoI-XhoI 50 and XhoI-XmnI 30 flanks are indicated in green and blue, respectively. Bottom: predicted structure of the Tph2 null allele (KO) after excision by Nestin-Cre 123 (modified pKS bluescript including a neomycine-resistance (NEO) cassette flanked by two LoxP sites) downstream of the NEO cassette. Then, the NotI-ApaI fragment comprising 50 flank-LoxP-deletion was excised from the pKSLox vector and inserted 50 of the NEO cassette of 30 flankcontaining pKSLNL. After verification by restriction analysis and sequencing, the targeting construct was linearized with NotI and electroporated into 129 R1 embryonic stem (ES) cells which were subjected to G418 selection. Targeted homologous recombination was confirmed by PCR and Southern blot analysis. The NEO cassette was removed by transient expression of Cre recombinase in the recombinant ES cells resulting in a Tph2 allele with exon 5 flanked by LoxP sites. After verification of NEO cassette excision by PCR, Southern blot and sequencing, an ES clone was injected into C57BL/6 blastocysts and implanted into pseudopregnant mice. A chimeric male displaying germ-line transmission was then used to propagate the floxed Tph2 allele on a C57BL/6 background for several generations. Tph2+/- were obtained by breeding Tph2+/flox mice with Nestin-Cre transgenics (Tronche et al. 1999) maintained on a C57BL/6 background. Several intercrossing of Tph2+/flox mice with Tph2+/flox/cre eventually resulted in Tph2 inactivation in the germline with a transmittable Tph2 null allele in Cre transgene-devoid Tph2-/mice, equivalent to constitutive knockout. Control wildtype (WT) and null alleles were detected by duplex PCR using the oligonucleotide 50 - tggggcctgcc gatagtaacac as reverse primer located in the 50 side of the 30 flank (position of the primers are indicated in Fig. 1a). Paired to this reverse primer, the forward primer 50 -tggggcatctcaggacgtagtagt, located in the 30 end of the targeted deletion led to the amplification of a 437 bp product in the presence of the WT allele, while the forward primer 50 -caccccaccttgcagaaatgttta, located in the 30 side of the 50 flank permits amplification of a 387 bp amplicon indicative of the Tph2 null allele (Fig. 1b). The reliability recombinase of the sequence flanked by LoxP sites (red triangles). Horizontal lines illustrate the position of primers used for genotyping. b Duplex PCR genotyping using 2 forward and 1 reverse primers indicated in a. Size of the amplicons from homozygous KO (387 bp, Tph2-/-) and WT (437 bp, Tph2+/+) mice are shown. SM DNA size marker Tph2 knockout mouse model of the PCR-based genotyping procedure was verified by Southern blot analysis using standard procedures with genomic DNA derived from ES cells or tail cuts using cDNA probes (data not shown). Generation of Tph2-specific antibodies Tph2-specific antibodies were generated as described in detail (L. Gutknecht et al., submitted). Briefly, for antiTph2 specific antibodies generation, two peptides were selected within the murine Tph2 protein based on their internal characteristics and specificity for this isoform. The peptides comprising the murine amino acids 458–472 (TRSIENVVQDLRSDL) and 476–488 (CDALNKMNQYLGI) corresponding to the C-terminal portion were chosen as immunogens. Peptide synthesis, coupling to keyhole limpet haemocyanin (KLH) and immunization of rabbits were carried out at Eurogentec (Seraing, Belgium) following their AS-DOUB-LX protocol. Hyperimmune serum samples were tested for reactivity and specificity using Western blot analysis and immunohistochemistry. Immunohistochemistry for Tph2, 5-HT and 5-HT transporter Dissected brains with attached pineal gland from Tph2-/mice and WT littermates (3.5 months of age, 6th generation, with an estimated *94% C57BL/6 background) were fixed by immersion in 4% PFA in PBS, cryoprotected in 20% sucrose, frozen in dry ice-cooled 2-methylbutane and stored at -80°C until 12 lm cryostat sections were prepared and mounted on glass slides. Immunohistochemistry of Tph2 was performed using the avidin-biotin method whereas a polymer-based detection system (EnVision + SystemHRP, DAKO, Glostrup, Denmark) was used for 5-HT and 5-HT transporter (Sert), with diaminobenzidine (DAB) as chromogen. Frozen sections were allowed to dry at room temperature (RT) and heat-induced unmasking of epitope were performed for each antibody at 96°C for 10 min in 10 mM citrate buffer (pH 6.0) containing 0.05% Tween. Washings were performed between each step 3 9 5 min in Tris-buffered saline (TBS, pH 7.5). After 40 min of subsequent cooling at RT, and blocking of endogenous peroxidase activity for 30 min with 0.6% H2O2 in TBS in case of Tph2, and 10 min using peroxidase blocking solution (DAKO) for 5-HT and Sert, sections were preincubated for 1 h at RT in blocking solution (BL) containing 5% normal goat serum (NGS), 2% BSA and 0.25% Triton X-100 diluted in TBS to prevent non-specific binding. Tissue sections were then exposed to primary antibodies diluted 1:900 for Tph2 in TBST for 2 h at RT and overnight at 4°C, 1:10,000 for 5-HT (rabbit anti-5-HT, Incstar, Stillwater, USA) in BL for 90 min at RT and 1:750 for Sert (rabbit anti-Sert, Calbiochem, Darmstadt, Germany) overnight at 4°C. For negative control of Tph2 immunostaining, primary antibodies were omitted from the incubation and 1:900 dilution of the rabbit preimmune serum was used. The detection of bound anti-Tph2 antibodies was carried out with biotinylated secondary antibodies (goat anti-rabbit IgG, 1:900; Vector Laboratories, Wiesbaden, Germany) diluted in 2% NGS, 2% BSA and 0.25% Triton-X100 in TBS for 90 min, followed by incubation in streptavidinbiotin-peroxidase complex (Vector Laboratories, Wiesbaden, Germany) for 1 h at RT. Anti-5-HT and anti-Sert detection was achieved with secondary anti-rabbit antibody coupled to horse radish peroxidase (DAKO) for 40 min at RT. Peroxidase activity was visualized using DAB substrate (metal enhanced; Roche, Penzberg, Germany) or DAB substrate (DAKO) for 4 min. Results Tph2 null mutant mice (Tph2-/-) are viable and do not show apparent increase in premature lethality during development or adulthood. Despite a tendency toward a lower body weight compared to their WT littermates, Tph2-/- mice do not display obvious developmental defects including overall brain structure. While Tph2-specific antibodies detect the expected expression pattern of Tph2 in the soma and fibers of raphe serotonergic neurons of WT control mice, complete absence of Tph2 immunostaining was observed in Tph2-/- mice (Fig. 2a, b). Immunohistochemistry of 5-HT in raphe serotonergic neurons revealed that constitutive gene inactivation is associated with a loss of Tph2 enzymatic activity. Typical distribution of 5-HT positive neurons was observed in the WT control brain, whereas no trace of 5-HT immunoreactivity was detected in the raphe nuclei of Tph2-/- mice (Fig. 2c, d). In contrast, a comparably strong 5-HT-specific signal was observed in the pineal gland of both WT and null mutant mice (Fig. 2, inserts in c and d). Immunodetection of the Sert, located on the soma, dendrites and terminals of serotonergic neurons, failed to reveal obvious differences in serotonergic neuron morphology including fiber distribution in Tph2-/- mice compared to WT controls. A similar pattern of innervation by Sert-positive soma, dendrites and terminal was observed in the raphe nuclei as well as across several target regions of the brain, including the hippocampus. (Fig. 2, e and f, inserts). Discussion Tph2 was inactivated in mice using the Cre/LoxP strategy resulting in viable Tph2 null mutants. The preliminary 123 L. Gutknecht et al. Fig. 2 Immunohistochemical analysis of homozygous WT (top panels) and KO (bottom panels) mice. Representative pictures from raphe nuclei (a–d) and hippocampus (e, f) coronal sections are displayed. a, b Detection of Tph2 with isoformspecific Tph2 antibody at the level of dorsal (DR) and median (MnR) raphe. c, d Detection of 5-HT at the level of DR, MnR and pineal gland (PG, inserts). e, f Detection of Sert in different layers of the hippocampus (H hilus; GL granule cell layer; ML molecular cell layer; LM lacunosum-molecular layer; RL radiatum layer) and DR (inserts). All scales bars represent 200 lm results indicate that raphe serotonergic neurons in mice with a targeted disruption of Tph2 are completely devoid of 5-HT immunoreactivity, whereas no alterations in morphology and fiber distribution appear to occur. Our findings therefore confirm the exclusiveness of Tph2 in raphe neuron 5-HT synthesis in mice and provide preliminary evidence that Tph2-synthesized 5-HT may not be required for raphe serotonergic neuron development, maturation, maintenance, and survival. Our recent systematic analysis of Tph1 and Tph2 expression in adult mouse and human brain, as well as during pre- and postnatal development of the murine brain, did not reveal significant Tph1 expression in the raphe of both species, whereas the pineal gland displayed high concentrations of this isoform. Based on these findings, we concluded that Tph1 is unlikely to contribute to brain 5-HT synthesis neither in adulthood nor during development (L. Gutknecht et al., submitted). This view of exclusive Tph2 specificity in brain 5-HT synthesis is strongly supported by the complete absence of 5-HT immunostaining in serotonergic neurons of the raphe nuclei of Tph2-/mice as compared to WT controls and the unchanged strong Tph1-dependent 5-HT immunoreactivity in the pineal gland of both the Tph2-/- and WT control mice. Moreover, the lack of Tph2-dependent 5-HT synthesis during early brain development does not seem to reverse 123 repression of Tph1 transcription as an adaptive mechanism to initiate and maintain physiological 5-HT system function. The apparent failure of Tph1 to compensate for Tph2related 5-HT deficiency in brain also underscores the relevance of allelic variation in TPH2 function resulting in alterations of brain 5-HT system function associated with cognitive and emotional processes as well as the pathophysiology of affective disorders and ADHD. Although Tph2 inactivation leads to stalled 5-HT synthesis in the brain, the virtually unaltered immunostaining of the serotonergic cell-specific marker Sert on the soma, dendrites, and fibers of raphe neural cells suggests the machinery of specification and other differentiation process, including neurite outgrowth towards projection areas of serotonergic neurons is not dependent on the synthesis or release of 5-HT. Extensive evidence supports a morphogenic effect of 5-HT on proliferation, differentiation, migration, and survival of neural cells (Di Pino et al. 2004; Gaspar et al. 2003). During ontogeny, 5-HT appears long before maturation of raphe serotonergic neurons suggesting a fundamental role in embryonic brain development as well as on the development and maturation of the serotonergic system itself. Although in vivo studies generally underscore this notion (Gaspar et al. 2003; Vitalis et al. 2007), several recently generated mouse models for 5-HT deficiency lead to mice with no apparent developmental abnormalities. Tph2 knockout mouse model Among these models are Nkx2.2 knockout mice in which 5-HT neurons are preserved only in the dorsal raphe (Briscoe et al. 1999), conditional Lmx1b knockout mice lacking most 5-HT neuron in adult brain (Ding et al. 2003; Zhao et al. 2006), Pet1-deficient mice displaying a 70% loss of differentiated raphe serotonergic neurons (Hendricks et al. 2003), BALB/cJ mouse strain which present a *40% decreased 5-HT concentration, compared to 129X1/ SvJ strain, in frontal cortex and striatum linked to proline substitution at amino acid position 447 of the Tph2 protein (Zhang et al. 2004), and mutant Tph2 mice, carrying the rare human variant R441H, which show a *80% reduced brain 5-HT (Beaulieu et al. 2008). In contrast to Tph2-/mice however, none of these mouse models permit investigation of the consequences of a complete loss of brain 5-HT synthesis, either during development or across the life span. In Lmx1b and Pet1 knockout mice 5-HT neurons seem to be generated but fail to further differentiate. Remaining 5-HT-positive neurons in Pet1-deficient mice display reduced expression of Tph2 and Sert, whereas in conditional Lmx1b knockouts, early transient raphe neuron 5-HT synthesis up to embryonic day E14.5 is not maintained in later developmental stages. Taken together, these and our findings suggest that differentiation and maintenance of a serotonergic phenotype is independent on the production of serotonin by the maturating neurons. Nevertheless, further detailed studies are required to evaluate to which degree the serotonergic phenotype of these neurons is complete, specifically how the expression pattern and function of markers, such as 5-HT1A and 5-HT1B autoreceptors as well as the function of these neurons and their electrophysiological properties such as firing rate, among numerous other parameters that reflect 5-HT system function, are altered by a deficiency of 5-HT synthesis. Conclusion The Tph2 knockout mouse represents a novel rodent model which will be useful for investigations of the developmental, adaptive, and functional consequences of altered brain-specific 5-HT synthesis and their role in cognition, emotion regulation, and neuropsychiatric disease risk. Acknowledgments The authors are grateful to S. Wiese for providing cloning vectors and construct advise as well as to M. Sendtner, C. Gross and P. Gaspar for helpful discussions and continuing support. The authors also thank N. 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