Role of Gsk3ß in Behavioral Abnormalities Induced by Serotonin

Role of GSK3␤ in behavioral abnormalities induced by serotonin deficiency

Jean-Martin Beaulieu*†, Xiaodong Zhang*‡, Ramona M. Rodriguiz§, Tatyana D. Sotnikova*, Michael J. Cools§, William C. Wetsel*§¶, Raul R. Gainetdinov*, and Marc G. Caron*¶ʈ**

Departments of *Cell Biology, ʈMedicine, ¶Neurobiology, and §Psychiatry and Behavioral Sciences, Mouse Behavioral and Neuroendocrine Analyses Core Facility, Duke University Medical Center, Durham, NC 27710

Communicated by L. L. Iversen, University of Oxford, Oxford, United Kingdom, December 6, 2007 (received for review November 6, 2007)

Dysregulation of brain serotonin (5-HT) neurotransmission is pharmacological stimulation of 5-HT1 receptors enhances phos- thought to underlie mental conditions as diverse as depression, phorylation of the Ser-9 residue, thus leading to inhibition of the anxiety disorders, bipolar disorder, autism, and schizophrenia. kinase (7). In contrast, 5-HT2 receptor signaling reduces GSK3␤ Despite treatment of these conditions with serotonergic drugs, the phosphorylation thus leading to kinase activation (7). Hence, a molecular mechanisms by which 5-HT is involved in the regulation general 5-HT deficiency may exert differential effects on GSK3␤ of aberrant emotional behaviors are poorly understood. Here, we activity in brain regions expressing diverse sets of 5-HT generated knockin mice expressing a mutant form of the brain receptors. 5-HT synthesis enzyme, tryptophan hydroxylase 2 (Tph2). This Several lines of evidence indicate that the rate-limiting en- mutant is equivalent to a rare human variant (R441H) identified in zyme for 5-HT synthesis in adult brain is tryptophan hydroxylase few individuals with unipolar major depression. Expression of 2 (Tph2) (15, 16). This enzyme catalyzes the conversion of the mutant Tph2 in mice results in markedly reduced (Ϸ80%) brain amino acid tryptophan to 5-hydroxytryptophan (5-HTP) that is 5-HT production and leads to behavioral abnormalities in tests subsequently decarboxylated to 5-HT by L-aromatic amino acid assessing 5-HT-mediated emotional states. This reduction in brain decarboxylase. Recent investigations of naturally occurring ge- 5-HT levels is accompanied by activation of glycogen synthase netic polymorphisms in humans and mice have identified func- kinase 3␤ (GSK3␤), a signaling molecule modulated by many tional mutations in Tph2 that lead to pronounced reduction in psychiatric therapeutic agents. Importantly, inactivation of GSK3␤ enzyme activity (15, 17–19). A rare R441H TPH2 variant has NEUROSCIENCE in Tph2 knockin mice, using pharmacological or genetic ap- been identified in individuals from a small cohort of elderly proaches, alleviates the aberrant behaviors produced by 5-HT patients with major unipolar depression. This mutation causes a deficiency. These findings establish a critical role of Tph2 in the reduction of Ϸ80% of enzyme activity when expressed in maintenance of brain serotonin homeostasis and identify GSK3␤ transfected PC12 cells (18). To investigate more directly the signaling as an important pathway through which brain 5-HT effects of this mutation on in vivo 5-HT synthesis, cellular deficiency induces abnormal behaviors. Targeting GSK3␤ and re- signaling, and behavior, we generated mice where the R441H lated signaling events may afford therapeutic advantages for the TPH2 mutation was engineered at the equivalent R439H amino management of certain 5-HT-related psychiatric conditions. acid residue of the mouse Tph2. Here we show that this mutation recapitulates in the mouse the neurochemical changes predicted GSK-3 ͉ mood disorders ͉ serotonin ͉ Tph2 ͉ functional polymorphism from cellular studies of the human mutant Tph2 and reveals an important role of the GSK3 signaling pathway in the behavioral erotonin (5-HT) is involved in multiple aspects of normal abnormalities associated with reduced brain 5-HT levels. brain functions ranging from the regulation of mood to the S Results control of appetite and social interactions (1–3). Several studies have suggested a contribution of abnormal 5-HT transmission in Generation of R439H Tph2 Knockin Mice. R439H Tph2 knockin mice various human psychiatric conditions and drugs acting on 5-HT were generated by using a homologous recombination strategy neurotransmission are commonly used for the management of that resulted in the insertion of the mutation in exon 11 and of major depression, anxiety disorder, obsessive-compulsive disor- a residual loxP site in the ninth intron of the gene (Fig. 1A). der, autism, and schizophrenia (1–3). Although drugs that Homozygous (HO) and heterozygous (HET) mutant mice were influence the 5-HT system can affect histone acetylation, mod- first identified using a PCR genotyping protocol detecting the ulate production of brain-derived neurotrophic factor, increase presence of the LoxP insertion in intron 9 (Fig. 1B). Subse- neural progenitor cell proliferation, and inhibit glycogen syn- quently, the presence of the mutated tph2 alleles in both HO and thase kinase 3␤ (GSK3␤) in certain brain areas (4, 5), the HET knockin mice was confirmed (Fig. 1C) by ARMS-PCR (15, mechanisms underlying the regulation of behavior by 5-HT are 20). Mice carrying the R439H mutation were viable, developed still obscure. without overt phenotype, and reproduced normally. RT-PCR There are indications that pharmacologic manipulations of confirmed the integrity of the mutated R439H Tph2 transcript 5-HT levels by different classes of drugs can affect distinct neuronal signaling mechanisms (6–9). For instance, multiple Author contributions: J.-M.B. and X.Z. contributed equally to this work; J.-M.B., X.Z., R.M.R., classes of 5-HT drugs, including selective 5-HT reuptake inhib- W.C.W., R.R.G., and M.G.C. designed research; J.-M.B., X.Z., R.M.R., T.D.S., M.J.C., and R.R.G. itors (SSRIs), tricyclic antidepressants, monoamine oxidase in- performed research; J.-M.B., X.Z., R.M.R., T.D.S., and R.R.G. analyzed data; and J.-M.B., X.Z., hibitors, and atypical antipsychotics (5, 7, 9), inhibit brain W.C.W., R.R.G., and M.G.C. wrote the paper. GSK3␤ signaling. GSK3␤ is also inhibited in vivo by lithium (6, The authors declare no conflict of interest. 10–12), which is often used in combination with antidepressants †Present address: CRULRG, Research Institute, Universite´Laval, Que´bec City, Canada. for the management of certain mood disorders (13). However, ‡Present address: Duke–National University of Singapore Graduate Medical School, whether these changes in GSK3␤ are incidental or contribute to Singapore. the regulation of 5-HT-related abnormal behaviors is unex- **To whom correspondence should be addressed. E-mail: [email protected]. plored (5). GSK3␤ is a constitutively active kinase that is This article contains supporting information online at www.pnas.org/cgi/content/full/ inhibited following the phosphorylation of the Ser-9 residue 0711496105/DC1. located in its amino-terminal domain (14). In mouse brain, © 2008 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711496105 PNAS ͉ January 29, 2008 ͉ vol. 105 ͉ no. 4 ͉ 1333–1338 Downloaded by guest on September 25, 2021 Fig. 2. Neurochemical measurements of 5-HT synthesis rates and contents in R439H Tph2 knockin mice. (A) 5-HTP synthesis rates in striatum [ANOVA, FGenotype(2,21) ϭ 5.412, P Ͻ 0.012], frontal cortex [ANOVA, FGenotype(2,21) ϭ 11.791, P Ͻ 0.003], and hippocampus [ANOVA, FGenotype(2,21) ϭ 17.426, P Ͻ 0.001] from WT, HET and HO Tph2 mice. (B) 5-HT tissue contents in striatum [ANOVA, FGenotype(2,21) ϭ 19.236, P Ͻ 0.001], frontal cortex [ANOVA, FGenotype(2,21) ϭ 17.675, P Ͻ 0.001], and hippocampus [ANOVA, FGenotype(2,21) ϭ 15.319, P Ͻ 0.001]. (C) 5-HIAA tissue contents in striatum [ANOVA, FGenotype(2,21) ϭ 9.678, P Ͻ 0.001], frontal cortex [ANOVA, FGenotype(2,21) ϭ 6.238, P Ͻ 0.007], and hippocampus [ANOVA, FGenotype(2,21) ϭ8.112, PϽ0.002] of WT, HET and HO Tph2 mice (nϭ4–11 mice per group). Data are means Ϯ SEM. Bonferroni corrected pair-wise comparisons: *, P Յ 0.05; ***, P Յ 0.005 from the WT control.

hydroxybenzylhydrazine, provides a direct assessment of Tph2 activity in vivo (15). Synthesis rates in the striatum, frontal cortex (FC), and hippocampus were reduced by Ϸ40% and Ϸ80% in Fig. 1. Generation of mice expressing the R439H allele of Tph2.(A) Diagram HET and HO R439H Tph2 knockin mice, respectively (Fig. 2A). of the Tph2 locus and the targeting vector. The floxed PGK-NEO/HSV-TK Due to decreased synthesis, tissue contents of 5-HT were also cassette, flanked by the PGK-DT cassette and targeting arms, is illustrated. The substantially reduced in these same brain regions of HO R439H G1449A coding mutation was engineered in exon 11 of the mouse Tph2 gene. mice (Fig. 2B). However, in HET mice, a significant reduction After homologous recombination, ES cell clones carrying the mutated exon 11 in 5-HT contents was only apparent in the FC, suggesting that were selected using ARMS-PCR and transfected with a CRE recombinase homeostatic regulation of 5-HT levels may differ among various expression vector to remove the PGK-NEO/HSV-TK cassette resulting in a Tph2 mutant allele carrying a mutated exon 11 and a residual LoxP site in intron 9. brain regions. Finally, no differences between WT and HET The WT and mutant (Mut) alleles are shown; the location of PCR primers is R439H mice were evident in tissue levels of the 5-HT degrada- indicated, red arrows: LoxP primers, green arrows: ARMS-PCR external prim- tion product 5-hydroxyindoleacetic acid (5-HIAA), whereas the ers, blue or purple arrows: ARMS-PCR allele-specific primers. (B and C) PCR metabolite was reduced to almost undetectable levels in all brain genotyping of R439H Tph2 knockin mice. Two genotyping protocols were regions examined in HO Tph2 knockin mice (Fig. 2C). Taken used, one detected the residual loxP element in intron 9 (B) and the other together, these neurochemical data indicate that the R439H detected the G1449A mutation in exon 11 (C). W, G-allele; M, A-allele; the mutation markedly reduces Tph2 activity in vivo and they molecular weight product sizes for the G- and A-alleles are noted. WT, wild provide further direct evidence for the critical role of this type; HET, heterozygote; HO, homozygous mutant. (D) Semiquantitative RT-PCR of Tph2 and SERT normalized to ␤-actin, and GADPH mRNA levels in enzyme in the regulation of 5-HT synthesis in brain (15). the brainstem of WT and HO R439H Tph2 mice (n ϭ 4 mice per group). (E) Densitometric Western blot analyses of SERT expression in different brain Activation of GSK3␤ in the Frontal Cortex of R439H Tph2 Knockin Mice. areas of WT, HET, and HO Tph2 knockin mice. Data are normalized to optical Because GSK3 activity is modulated by pharmacological ma- densities for WT animals, ␤-actin was used as a loading control (n ϭ 4 mice per nipulations of 5-HT levels (5, 7, 22), we assessed the effects of group). Data are means Ϯ SEM. 5-HT deficiency on GSK3␤. Ser-9 phosphorylation of GSK3␤ was measured in the FC, hippocampus, and striatum of WT and R439H Tph2 knockin mice (Fig. 3 A–C). Although overall and semiquantiative RT-PCR analyses showed no differences in GSK3␤ levels were similar in these brain regions for all geno- expression of Tph2 between wild type (WT) and HO R439H types, levels of phospho-Ser-9-GSK3␤ were specifically reduced Tph2 mice (Fig. 1D). Importantly, expression of the 5-HT in the FC of Tph2 knockin mice, suggesting activation of the transporter (SERT), a target for most antidepressants and a enzyme (Fig. 3 A–C). Moreover, in FC, decreases in GSK3␤ potential contributing factor to the regulation of behavior by phosphorylation were directly proportional to levels of 5-HT 5-HT (1, 2, 21), was also unaffected in these mice as determined synthesis as compared across WT, HET, and HO R439H Tph2 by RT-PCR and Western blots (Fig. 1 D and E). knockin mice, with the latter having the lowest phospho-GSK3␤ levels and 5-HT synthesis (Figs. 2A and 3B). These data suggest 5-HT Deficiency in R439H Tph2 Knockin Mice. The effect of the that GSK3␤ activity within the FC is directly regulated by the R439H mutation on 5-HT brain tissue contents and synthesis in availability of 5-HT. To verify this hypothesis, a kinase assay (11) vivo was evaluated. Monitoring the accumulation of the 5HT was performed to directly measure GSK3 activities within the precursor and Tph2 product, 5HTP, following treatment of mice FC of WT and HO R439H Tph2 knockin mice. This assay with the L-aromatic amino acid decarboxylase inhibitor, m- confirmed that reduced GSK3␤ phosphorylation resulted in an

1334 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711496105 Beaulieu et al. Downloaded by guest on September 25, 2021 Thus, R439H Tph2 knockin mice were bred with GSK3␤ϩ/Ϫ animals (24) to produce six genotypes: WT, HET, and HO R439H Tph2 knockin mice, as well as each of the same Tph2 genotypes displaying Ϸ50% reduction in GSK3␤ expression. These mice were evaluated on a battery of behavioral tests designed to analyze 5-HT-related emotional states in rodents (Fig. 4). To control for possible developmental differences in haploinsufficient GSK3␤ mice, some tests were conducted also with WT, HET, and HO R439H Tph2 knockin mice treated acutely with the brain permeable GSK3␤ inhibitor thiadiazo- lidinone (TDZD-8) (11, 25). Reduction of immobility times in the tail suspension test, is a paradigm commonly used to evaluate effects of antidepressants in rodents (26). It is well-known that behavioral responses in this test are affected by acute reductions in 5-HT synthesis or its synaptic release (27). Immobility times were prolonged for both HET and HO Tph2 knockin mice (Fig. 4A Left). Strikingly, whereas GSK3␤ haploinsufficiency and TDZD-8 had no significant effect in animals carrying WT Tph2 alleles, both genetic and pharmacological inhibition of GSK3␤ activity significantly reversed immobility in HET and HO Tph2 knockin mice (Fig. 4A). The dark–light emergence test is a 5-HT receptor-responsive behavioral assay used to assess anxiolytic efficacies of drugs such as SSRIs and benzodiazepines in rodents (28, 29). In this test, mice are placed in a darkened compartment and allowed free exploration of the darkened and adjoining lighted compart-

ments. HET and HO Tph2 knockin mice displayed longer NEUROSCIENCE latencies to cross to the lighted compartment (Fig. 4B Left), as well as reduced activity in this compartment (Fig. 4D Left). Furthermore, HO Tph2 knockin mice also exhibited more transitions between the two compartments (Fig. 4C). Together, these results indicate that expression of R439H Tph2 results in enhanced anxiogenesis in mice. In contrast, both genetic and ␤ Fig. 3. Regulation of cortical GSK3 activity and levels by 5-HT and ﬂuox- pharmacological reductions in GSK3␤ activity antagonized etine. (A–C) Western blot (A) and densitometric analyses (B and C) of phospho- GSK3␤ (Ser-9) in the frontal cortex (A and B) and hippocampus and striatum ‘‘anxiety-like’’ responses induced by the expression of R439H (C) of WT and HO R439H Tph2 mice (n ϭ 5 mice per group). Densitometic data Tph2, and they restored behaviors essentially to levels compa- were normalized to the average optical density in WT mice, and total GSK3 rable to those of the WT controls (Fig. 4 B–D). levels in extracts were used as loading controls for measurement of phospho- Because changes in general activity can affect results from protein levels [ANOVA frontal cortex, FGenotype (2,12) ϭ 9.951, P Ͻ 0.003]. (D) these tests (26, 30), basal locomotor activity in the open field was Kinase activity assays performed after immunoprecipitation of GSK3 from monitored. No differences in basal locomotion were discerned protein extracts prepared from the frontal cortex of WT and HO R439H Tph2 among mice from the six genotypes (Fig. 4E). These findings ϭ Ͻ mice [t test, t (1,8) 4.883, P 0.006]. Kinase assays were performed using indicate that reduced 5-HT synthesis in R439H Tph2 mice and recombinant inhibitor 2 as a substrate and kinase activity in the assay was GSK3␤ haploinsufficiency do not affect general motor activity, sensitive to the GSK3 inhibitor kenpaullone (11). (E and F) Phospho-GSK3␤ levels in the frontal cortex of WT (E)[t test, t ϭ 2.768, P Ͻ 0.012] and HO whereas 5-HT deficiency selectively modulates behavioral re- (1,8) sponses in the tail suspension and the dark–light emergence tests R439H Tph2 knockin mice (F)[t test, t (1,8) ϭ 2.639, P Ͻ 0.029] (n ϭ 5 mice per group) 30 min after administration of vehicle or 5 mg/kg ﬂuoxetine (s.c.). Data that are rescued by genetic or pharmacologic inhibition of are normalized to the average signal obtained from vehicle-treated mice. GSK3␤. Data are means Ϯ SEM. *, P Յ 0.05; ***, P Յ 0.005 from the WT or vehicle Besides exerting multiple effects on the behaviors described control; one-way ANOVA with Bonferroni corrected pair-wise comparisons or above, 5-HT-mediated mechanisms are also associated with t test. changes in more complex behavioral paradigms involving the regulation of emotionality and sociability (31, 32). To assess ␤ Ϸ the impact of reduced 5-HT synthesis and enhanced GSK3 2-fold increase in kinase activity in the FC of HO R439H signaling on emotional and social functioning, males from the knockin mice (Fig. 3D). To further evaluate the contribution of ␤ six genotypes were examined in a test of social interaction in 5-HT to the regulation of GSK3 , the SSRI fluoxetine was used a neutral arena (33). Levels of physical attacks against a (23) to augment extracellular 5-HT levels. Administration of nonaggressive male were dramatically enhanced in HO Tph2 fluoxetine at a dose (5 mg/kg) that normally induces at least a knockin males (Fig. 4F). Although HET Tph2 mice did not 2-fold increase in extracellular 5-HT levels in the FC (23) display increased attacks, these animals did show a significant resulted in enhanced GSK3␤ phosphorylation in FC of WT and increase in threatening postures (Fig. 4G), suggesting that HO R439H Tph2 knockin mice (Fig. 3 E and F); thus, further aggressive behavior is also enhanced in these mice, albeit to a supporting a role of 5-HT in the inhibition of GSK3␤ in this brain lesser degree than in the homozygous mutants. Importantly, region. attacks were completely suppressed in HO Tph2 knockin mice that were also haploinsufficient for GSK3␤ (Fig. 4F). Reduced GSK3␤ Contributes to Behavioral Changes in 5-HT-Deficient R439H GSK3␤ expression also resulted in decreased threatening Tph2 Knockin Mice. The availability of haploinsufficient GSK3␤ postures in HET Tph2 knockin mice (Fig. 4G). Interestingly, mice (24) allowed us to assess the contribution of GSK3␤ GSK3␤ haploinsufficient HO R439H Tph2 mice displayed activation to behavioral changes induced by 5-HT deficiency. markedly enhanced social investigation (Fig. 4H), suggesting

Beaulieu et al. PNAS ͉ January 29, 2008 ͉ vol. 105 ͉ no. 4 ͉ 1335 Downloaded by guest on September 25, 2021 Fig. 4. Reduction of GSK3␤ activity antagonizes behavioral changes in R439H Tph2 mice. (A) Immobility times in tail suspension for vehicle-treated mice from the six genotypes (Left) and for Tph2 knockin mice treated with 10 mg/kg TDZD-8 (i.p.) the GSK3␤ inhibitor (Right). Data are presented as total immobility time for the 5-min test. [ANOVA, FGenotype(5,52) ϭ 11.481, P Ͻ 0.001; TDZD ANOVA, FGenotype(2,41) ϭ 21.775, P Ͻ 0.001; FDrug ϭ ns; FGenotypexDrug(2,41) ϭ 11.196, P Ͻ 0.001]. (B–D) Dark–light emergence test evaluating latency to the ﬁrst cross to the lighted chamber [ANOVA, FGenotype(5,49) ϭ 13.564, P Ͻ 0.001; TDZD ANOVA, FGenotype(2.41) ϭ 4.595, P Ͻ 0.016; FDrug(1.41) ϭ 10.986, P Ͻ 0.002; FGenotypexDrug(2,41) ϭ 3.601, P Ͻ 0.036] (B), number of crosses to lighted chamber [ANOVA, FGenotype(5,49) ϭ 6.301, P Ͻ 0.001; TDZD ANOVA, FGenotype(2.41) ϭ 3.477, P Ͻ 0.040; FDrug(1.41) ϭ 14.920, P Ͻ 0.001; FGenotypexDrug(2.41) ϭ 4.980, P Ͻ 0.012] (C), and locomotor activity in the lighted chamber [ANOVA, FGenotype(5,49) ϭ 6.493, P Ͻ 0.001; TDZD ANOVA, FGenotype(2.41) ϭ 5.456, P Ͻ 0.008; FDrug(1.41) ϭ 3.982, P Ͻ 0.049; FGenotypexDrug(2,41) ϭ 4.794, P Ͻ 0.013] (D) in vehicle-treated mice from the six genotypes (Left) and for Tph2 knockin mice treated with 10 mg/kg TDZD-8 (i.p.) (Right). (E) Basal locomotor activities in vehicle-treated mice from the six genotypes. Mice were placed into the open ﬁeld, and the distance traveled was monitored over 30 min. (F–H) Parameters of social interaction in the dyadic test. (F) Attacks [ANOVA, FGenotype(5,45) ϭ 8.101 P Ͻ 0.001]. (G) Threatening postures [ANOVA, FGenotype(5,45) ϭ 2.875 P Ͻ 0.025]. (H) Mild exploratory behaviors [ANOVA, FGenotype(5,45) ϭ 24.722 P Ͻ 0.001]. For all behavioral tests, balanced groups of male and female mice were used, with the exception of the social interaction test, where only males where used. Data are shown as means Ϯ SEM. *, P Ͻ 0.05; ***, P Ͻ 0.005, as compared with WT mice; #, P Ͻ 0.05; ###, P Ͻ 0.005, as compared with vehicle-treated mice from the same genotype, ANOVA with Bonferoni corrected pair-wise comparisons. Numbers of animals for each condition (n) are indicated.

that their reduction in numbers of attacks was not due to Discussion disruption of investigative social behaviors. Collectively, these The results presented here show that a functional Tph2 gene results suggest that the well known effects of 5-HT on aggres- mutation produces profound reductions of brain 5-HT synthesis in sion may be controlled at least in part through regulation of mice. Furthermore, reduced 5-HT levels lead to an activation of GSK3␤ activity. cortical GSK3␤ and the development of behavioral abnormalities Taken together, our results identify multiple abnormal behaviors in tests used to model endophenotypes of 5-HT associated disorders in R439H Tph2 mice that are consistent with a generalized reduc- in rodents. These observations provide direct functional evidence tion in brain 5-HT function. Furthermore, the rescue of these for a contribution of GSK3␤ mediated signaling in 5-HT functions behavioral aberrations by genetic or pharmacologic inhibition of and further establish Tph2 as an important determinant of brain GSK3␤ indicates that control of the phosphorylation/activity status 5-HT synthesis and mood regulation. of this enzyme constitutes an important, and previously unrecog- Although further genetic studies will be needed to firmly link nized, mechanism of 5-HT action in brain. this functional R441H Tph2 polymorphism with human depres-

1336 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0711496105 Beaulieu et al. Downloaded by guest on September 25, 2021 sion (34, 35), large-scale genetic analyses have associated several and Tph2. These observations provide a basis for a cell signaling noncoding polymorphisms in the human TPH2 gene with de- mechanism by which a reduction of 5-HT synthesis leads to the pression, bipolar disorder, and suicidality (36–41). Furthermore, expression of behavioral abnormalities in the animals. Further- several naturally occurring functional polymorphisms in the more, these results directly implicate a functional Tph2 gene Tph2 gene have also been identified in mice (15), rhesus monkeys polymorphism identified in humans in the expression of these (42), chimpanzees (43), and humans (17–19). The existence of behavioral changes and suggest a potential role of Tph2 variants multiple rare variants in a single gene leading to the development in the etiology of 5-HT related brain disorders. Investigations of of human pathologies is not a rare occurrence. For example, the function and mechanisms of GSK3␤ mediated signaling in mainly because of the availability of a readily measurable 5-HT synaptic transmission should provide research avenues to biological marker in phenylketonuria, as many as 307 missense understand and potentially manage human disorders such as mutations have been reported in the Tph2-related enzyme mood disorders and schizophrenia. phenylalanine hydroxylase (PAH) (34, 44). This suggests that, although the human R441H mutation may contribute to the Methods development of major depression in some individuals, multiple Generation of R439H Tph2 Knockin Mice. Knockin mice carrying the R439H Tph2 other functional TPH2 mutations can also exist and potentially allele (GenBank accession no. NM࿝173391), equivalent to the human R441H play a role in the etiology of this and other psychiatric condi- TPH2 allele (GenBank accession no. NM࿝173353) identiﬁed in major unipolar tions (34). depression patients (18), were derived as follows. A 4.6-kb ‘‘long arm’’ and a We previously identified a naturally occurring P447R variant 2.0-kb ‘‘short arm’’ were cloned by PCR with EXL DNA polymerase (Stratagene, of Tph2 in specific inbred mouse lines. This variant results in a La Jolla, CA) using genomic DNA obtained from 129S6/SvEv mice as template. reduction of enzyme activity by Ϸ50% when expressed in PC12 The long arm corresponded to sequences from Tph2 intron 9, whereas the cells. Furthermore, expression of this Tph2 variant in these short arm comprised intron 9 sequences as well as exon 10, intron 10, exon 11, Ϸ Ј inbred mouse lines is associated with reduced brain 5-HT and 1kbofthe3UTR of the mouse Tph2 gene. To engineer the R439H mutation, guanine 1449 in exon 11 was changed to an adenosine using synthesis (15) and differences in aggressive behavior (45), pre- site-directed mutagenesis. The long and short arms were then subcloned into mRNA editing of the 5-HT2C receptor (46), as well as respon- a targeting vector (51) resulting in the insertion of a ﬂoxed herpes virus siveness to SSRIs (47, 48). However, these studies can only be thymidine kinase/neomicin (TK/NEO) selection cassette in the ninth intron of viewed as correlative because they were all conducted by com- the gene. The targeting construct was transfected into 129S6/SvEv mouse ES paring different strains of inbred mice and the potential contri- cells. Clones carrying recombinant Tph2 alleles were selected using a standard

bution of other genetic variations in these mice cannot be ruled diphtheria toxin (DT)/G418 double selection protocol. Positive ES cell clones NEUROSCIENCE out. In contrast, the results presented here provide direct in vivo were subsequently karyotyped, and the occurrence of homologous recombi- evidence that a Tph2 genetic variation is sufficient to affect brain nation was confirmed by PCR. Four recombinant ES cell lines were transiently 5-HT synthesis and induce biochemical and behavioral changes transfected with a CRE recombinase expression construct to remove the associated with reduced 5-HT neurotransmission. As such, TK/NEO selection cassette (Fig. 1A). Ganciclovir-negative selection followed R439H Tph2 knockin mice represent a unique animal model to by PCR amplification using primers (5Ј-CACCCAATTTGCCTGCCGTAGGGA-3Ј; Ј Ј study the biological functions of Tph2 and brain 5-HT. 5 -GCTGCAAAACATATCACAGAACTCATTCAAGACCA-3 ) flanking the TK/ NEO cassette were used to select clones lacking the selection cassette. These In addition, the R439H Tph2 knockin mice have allowed a (LoxP) primers were also used for routine genotyping of Tph2 knockin mice. direct assessment of the contribution of GSK3␤ signaling in the ␤ Moreover, a G-allele/A-allele-specific ARMS-PCR protocol was used to confirm regulation of behavior by 5-HT. GSK3 is an established target the presence of the G1449A mutation in the selected ES cell clones. The of lithium, which inhibits this kinase through direct and indirect following primers were used for ARMS-PCR: mOuter/Forward primer (5Ј- mechanisms (10, 11). GSK3␤ has also recently been identified as TGGTCTTGAATGAGTTCTGTGATATGTTTTGCA-3Ј); mOuter/Reverse primer a common target for SSRIs, tricyclic antidepressants, and anti- (5Ј-TCATGCTGACAATAACTCTGGTTCTAGGC-3Ј); G-allele specific primer (5Ј- psychotics (5, 7, 9). Furthermore, reduction of GSK3␤ phos- TAGGGGTTGAAGTATACCGAGAAGGCAC-3Ј); A-allele specific primer (5Ј- phorylation, resulting in kinase activation, in the prefrontal TAGGGGTTGAAGTATACCGAGAAGGCAT-3Ј). Chimeras generated from se- cortex has been associated with major depressive disorders in a lected ES cells were intercrossed with C57BL6/J mice; offspring (F1) that cohort of suicide victims (49). Although these observations inherited the mutant Tph2 allele were identified by PCR analysis (Fig. 1B). suggest an involvement of GSK3␤ signaling in the etiology of Finally, expression and integrity of the mutant Tph2 transcripts in F1 mice various disorders and the action of psychoactive drugs, a direct carrying the engineered A-allele was confirmed by RT-PCR cloning of brain- role for GSK3␤ in the regulation of 5-HT related behaviors or stem Tph2 full-length mRNA followed by sequencing as described in ref. 15. the effect of drugs enhancing 5-HT neurotransmission has Additional Experimental Animals. GSK3␤ mice were described in ref. 24. HET remained unclear. R439H TpH2 mice were mated following a two-step breeding protocol (52) Here we show that 5-HT deficiency leads to a dose- with GSK3␤-HET animals to obtain littermate WT, HET, or HO R439H TpH2 ␤ dependent activation of GSK3 in the frontal cortex of mice mice having one or two functional copies of the GSK3␤ gene. C3H/HeJ mice for carrying the R439H Tph2 mutation. In addition, either phar- dyadic testing were obtained from The Jackson Laboratory (Bar Harbor, MA). macological or genetic inhibition of this kinase prevents the Experiments were conducted with an approved protocol from the Duke expression of behavioral changes induced by 5-HT deficiency University Institutional Animal Care and Use Committee according to National in these mice. These results provide direct in vivo evidence for Institutes of Health guidelines. a role of GSK3␤ activation in behavioral changes associated with low 5-HT synthesis and suggest that drugs that enhance Neurochemistry. Methods used to analyze levels of 5-HT and 5-HTP using HPLC 5-HT neurotransmission may exert some of their actions by and electrochemical detection are described in ref. 15. 5-HT synthesis in vivo inhibiting this kinase. Furthermore, the involvement GSK3␤ in was measured in mice treated with 100 mg/kg (i.p.) m-hydroxybenzylhydr- the regulation of 5-HT-related behavior suggests that inhibi- azine (NSD-1015) for 1 h (15). tion of GSK3␤ may also contribute to the enhancing effect of lithium and antipsychotics, which are often used as augmen- Western Blot Analysis and Kinase Activity Assay. Briefly, mice were killed by decapitation, after which the heads of the animals were immediately tation therapies to increase the efficacy of antidepressants (13, cooled by immersion in liquid nitrogen for 6 s. The right hemisphere FC, 50). Further studies using tissue specific deletion of GSK3 or striatum, and hippocampus were rapidly dissected (within 60 s) on an other signaling components may be useful to address some of ice-cold surface and frozen in liquid nitrogen before protein extraction. these interesting issues. Western blot analysis and kinase activity assays were carried out as de- In conclusion, our results directly demonstrate the involve- scribed in ref. 11. Results were normalized to respective control conditions ment of GSK3␤ in the regulation of behavior controlled by 5-HT and presented as means Ϯ SEM.

Beaulieu et al. PNAS ͉ January 29, 2008 ͉ vol. 105 ͉ no. 4 ͉ 1337 Downloaded by guest on September 25, 2021 Measurement of Locomotor Activity. Locomotion was monitored for 30 min Statistical Analysis. Data from the neurochemical and biochemical studies were under illuminated conditions in an automated Omnitech Digiscan apparatus analyzed by univariate ANOVA or two-tailed t tests. ANOVA with Bonferroni (AccuScan Instruments, Columbus, OH). Activity was measured in terms of the corrected pair-wise comparisons were applied to behavioral studies using the total distance traveled (horizontal activity) (11). Statistical Package for the Social Sciences (SPSS), Version 11.0 (Chicago, IL). Detailed methods are available in supporting information (SI) Text. Tail Suspension Test. Mice were tested in a tail suspension apparatus (Med- Associates, St. Albans, VT) as described (26). Behavior was scored as time spent ACKNOWLEDGMENTS. We thank Cheryl B. Bock from the Duke Comprehen- in immobility (sec). sive Cancer Center Transgenic Facility for transgenic services, Wendy Roberts for assistance in the maintenance of mouse colonies, and Yuxin Ma for his unstinting assistance with behavioral testing. GSK3␤ haploinsufﬁcient mice Dark–Light Emergence Test. The test was performed over a period of 5 min with were a kind gift from James R. Woodgett (Samuel Lunenfeld Research Insti- mice initially placed in the center of the darkened chamber (29). Tests were tute, Toronto, Canada). This work was supported in part by National Institutes conducted using a passive-avoidance chamber with activity monitors (Med- of Health Grants MH-79201, MH-73853, MH-60451, and NS19576 (to M.G.C.). Associates). The scored behaviors were: initial latency to enter the lighted Support from the Lennon Family Foundation is also greatly appreciated. compartment, number of compartment entries, and total locomotor activity M.G.C. is the NARSAD Lattner Foundation Distinguished Investigator. J-M.B. is in each compartment. NARSAD Southwest Florida Investigator and the recipient of a fellowship from the Canadian Institutes of Health Research (CIHR), and X.Z. has a NARSAD Social Interaction Test. Dyadic tests were conducted as described in ref. 33. Young Investigator award.

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