Involvement of the agmatinergic system in the depressive-like phenotype of the Crtc1 knockout mouse model of depression

Abstract

Recent studies implicate the arginine-decarboxylation product agmatine in mood regulation. Agmatine has antidepressant properties in rodent models of depression, and agmatinase (Agmat), the agmatine-degrading enzyme, is upregulated in the brains of mood disorder patients. We have previously shown that mice lacking CREB-regulated transcription coactivator 1 (CRTC1) associate behavioral and molecular depressive-like endophenotypes, as well as blunted responses to classical antidepressants. Here, the molecular basis of the behavioral phenotype of Crtc1 ^-/- mice was further examined using microarray gene expression profiling that revealed an upregulation of Agmat in the cortex of Crtc1^-/- mice. Quantitative polymerase chain reaction and western blot analyses confirmed Agmat upregulation in the Crtc1^-/- prefrontal cortex (PFC) and hippocampus, which were further demonstrated by confocal immunofluorescence microscopy to comprise an increased number of Agmat-expressing cells, notably parvalbumin- and somatostatin-positive interneurons. Acute agmatine and ketamine treatments comparably improved the depressive-like behavior of male and female Crtc1 ^-/- mice in the forced swim test, suggesting that exogenous agmatine has a rapid antidepressant effect through the compensation of agmatine deficit because of upregulated Agmat. Agmatine rapidly increased brain-derived neurotrophic factor (BDNF) levels only in the PFC of wild-type (WT) females, and decreased eukaryotic elongation factor 2 (eEF2) phosphorylation in the PFC of male and female WT mice, indicating that agmatine might be a fast-acting antidepressant with N-methyl-D-aspartate (NMDA) receptor antagonist properties. Collectively, these findings implicate Agmat in the depressive-like phenotype of Crtc1 ^-/- mice, refine current understanding of the agmatinergic system in the brain and highlight its putative role in major depression.

OPEN

Citation: Transl Psychiatry (2016) 6, e852; doi:http://dx.doi.org/10.1038/tp.2016.116

Web End =10.1038/tp.2016.116

http://www.nature.com/tp

Web End =www.nature.com/tp

ORIGINAL ARTICLE

Involvement of the agmatinergic system in the depressive-like phenotype of the Crtc1 knockout mouse model of depression

EM Meylan1,2, L Breuillaud1,2,7, T Seredenina3,8, PJ Magistretti1,4,5, O Halfon2, R Luthi-Carter3,6 and J-R Cardinaux1,2

Recent studies implicate the arginine-decarboxylation product agmatine in mood regulation. Agmatine has antidepressant properties in rodent models of depression, and agmatinase (Agmat), the agmatine-degrading enzyme, is upregulated in the brains of mood disorder patients. We have previously shown that mice lacking CREB-regulated transcription coactivator 1 (CRTC1) associate behavioral and molecular depressive-like endophenotypes, as well as blunted responses to classical antidepressants. Here, the molecular basis of the behavioral phenotype of Crtc1/ mice was further examined using microarray gene expression proling that revealed an upregulation of Agmat in the cortex of Crtc1/ mice. Quantitative polymerase chain reaction and western blot analyses conrmed Agmat upregulation in the Crtc1/ prefrontal cortex (PFC) and hippocampus, which were further demonstrated by confocal immunouorescence microscopy to comprise an increased number of Agmat-expressing cells, notably parvalbumin- and somatostatin-positive interneurons. Acute agmatine and ketamine treatments comparably improved the depressive-like behavior of male and female Crtc1/ mice in the forced swim test, suggesting that exogenous agmatine has a rapid antidepressant effect through the compensation of agmatine decit because of upregulated Agmat. Agmatine rapidly increased brain-derived neurotrophic factor (BDNF) levels only in the PFC of wild-type (WT) females, and decreased eukaryotic elongation factor 2 (eEF2) phosphorylation in the PFC of male and female WT mice, indicating that agmatine might be a fast-acting antidepressant with N-methyl-D-aspartate (NMDA) receptor antagonist properties. Collectively, these ndings implicate Agmat in the depressive-like phenotype of Crtc1/ mice,

rene current understanding of the agmatinergic system in the brain and highlight its putative role in major depression.

Translational Psychiatry (2016) 6, e852; doi:http://dx.doi.org/10.1038/tp.2016.116

Web End =10.1038/tp.2016.116 ; published online 12 July 2016

INTRODUCTIONMajor depressive disorder (MDD) is a complex neuropsychiatric disease comprising one of the leading causes of disability worldwide, with an estimated lifetime prevalence of 16%.1 However, the etiological mechanisms underlying MDD are not clearly established. Studies over the past decades have suggested that altered neuroplasticity is a cardinal feature of MDD,2 leading to the network hypothesis of depression. This latter proposes that impaired neuroplasticity related to problems in activity-dependent neuronal communication might alter information processing in the affected neural networks, and ultimately cause MDD.3 In line with this hypothesis, antidepressants have been shown to promote synaptogenesis, neurogenesis and dendritic growth in the hippocampus (HIP) of rodents.4,5 These neurotrophic effects correlate with positive behavioral responses to antidepressants and are thought to rely, at least partly, on the activation of cAMP-response element-binding protein (CREB)-regulated genes, including increased signaling of the brain-derived neurotrophic factor (BDNF)-TrkB pathway.4 We and others have previously shown that CREB-dependent Bdnf expression requires CREB-regulated transcription coactivator 1 (CRTC1).68 CRTC1 has been shown to act as a neuronal calcium- and cAMP-sensitive coincidence

detector and to promote CREB-dependent transcription.6,9 In

addition to its important role in Bdnf expression, CRTC1 has also been shown to be critical for specic aspects of neuroplasticity, as evidenced by its role in dendritic growth of developing cortical neurons10,11 and its requirement for maintenance of long-term potentiation in the HIP.6,8

To further understand the role and function of CRTC1, we generated a Crtc1-decient mouse line.12 These mice present behavioral and molecular features mirroring mood disorders, such as increased behavioral despair, anhedonia, increased irritability/ aggressiveness, decreased sexual motivation, social withdrawal, decreased dopamine and serotonin turnover in the prefrontal cortex (PFC), as well as decreased HIP and PFC expression in several neuroplasticity-related genes including Bdnf and its receptor TrkB.13

Furthermore, Crtc1/ mice exhibit a blunted antidepressant response to the selective serotonin reuptake inhibitor uoxetine and to the tricyclic antidepressant desipramine in a behavioral despair paradigm.13,14 Taken together, these ndings suggest an important role for CRTC1 in the etiology of MDD and a possible involvement in treatment-resistant depression.

Substantial evidence supports the involvement of the argininedecarboxylation product agmatine in MDD. This metabolite is

1Center for Psychiatric Neuroscience, Department of Psychiatry, University Medical Center, University of Lausanne, Prilly, Switzerland; 2Service of Child and Adolescent Psychiatry, Department of Psychiatry, University Medical Center, University of Lausanne, Lausanne, Switzerland; 3Laboratory of Functional Neurogenomics, Brain Mind Institute, Ecole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland; 4Division of Biological and Environmental Sciences and Engineering, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia; 5Laboratory of Neuroenergetics and Cellular Dynamics, Brain Mind Institute, Ecole Polytechnique Fdrale de Lausanne, Lausanne, Switzerland and 6Department of Neuroscience, Psychology and Behaviour, University of Leicester, Leicester, UK. Correspondence: Dr J-R Cardinaux, Center for Psychiatric Neuroscience, Department of PsychiatryCHUV, Hospital of Cery, CH-1008 Prilly, Switzerland.

E-mail: mailto:[email protected]

Web End [email protected]

7Present address: Department of Pharmacology and Therapeutics, McGill University, Montral, Canada.

8Present address: Department of Pathology and Immunology, Medical School, University of Geneva, Geneva, Switzerland.

Received 17 November 2015; revised 27 April 2016; accepted 29 April 2016

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widely expressed in several mammalian organs, including the brain. Agmatine synthesis competes with other arginine-dependent pathways, such as the urea cycle and nitric oxide (NO) synthesis.15 It is degraded by the enzyme agmatinase (Agmat) into putrescine, a key precursor for polyamine synthesis.15,16 Accumulating evidence suggests that polyamines and their precursors have a role in the etiology and pathology of mental disorders, notably in mood disorders and suicidal behavior.17,18 Agmatine has also been proposed to function as a neurotransmitter: it is stored in synaptic vesicles and released upon depolarization, followed by selective reuptake or degradation.19 In addition, agmatine has the ability to bind a wide range of receptors, including nicotinic receptors, imidazoline I1 and

I2 receptors, 2-adrenergic receptors and serotoninergic 5HT-2A and 5HT-3 receptors.2023 Remarkably, agmatine also acts as a glutamate N-methyl-D-aspartate receptor (NMDAR) antagonist.24

This property is particularly interesting in light of the recent and growing interest in glutamate-based rapid-acting antidepressants, whose prototype is the NMDAR antagonist ketamine.2529

Importantly, humans affected by depression show altered blood levels of agmatine, and post-mortem studies have shown increased Agmat levels in brain tissues from depressed individuals.30,31 In

rodents, cortical and hippocampal agmatine levels are decreased by restraint stress, and agmatine demonstrates neuroprotection against acute and chronic stress effects.3235 Furthermore, acute agmatine treatment has rapid antidepressant activities in depression-related paradigms such as the forced swim test (FST) and tail suspension test.36 These effects have been attributed to agmatine actions on monoaminergic and opioid systems, imidazoline and 2-adrenergic receptors, and NMDAR blockade.3639 Moreover, agmatine has the ability to modulate pro- and anti-oxidative balance in the HIP, which might also underlie its behavioral effects.34 Finally, a recent study has shown that in parallel to its antidepressant activity, agmatine increases HIP CREB phosphorylation and BDNF levels, and induces cell survival pathways.40 Altogether, these data suggest that agmatine stimulates several endogenous mood-regulating mechanisms known to be altered in MDD, leading to the conclusion that dysregulation of the agmatinergic system could play a role in the etiopathogenesis of MDD and agmatine supplementation might have a positive outcome on the disease.

In this study, we investigated the molecular basis for the depressive-like phenotype of Crtc1/ mice, which led us by differential expression analysis to discover a cortical upregulation of Agmat expression. Immunohistochemical studies revealed that mutant mice have an increased number of Agmat-expressing cells in the PFC and HIP, particularly parvalbumin (PV)- and somatostatin (Sst)-positive interneurons. Based on this result, we hypothesized that increased Agmat levels would result in reduced agmatine bioavailability in the brains of Crtc1/ mice, and that supplementation with exogenous agmatine would improve their depressive-like behavior. Indeed, we found that acute agmatine administration had a rapid antidepressant effect both in wild-type (WT) and Crtc1/ mice, the latter requiring a higher dose than WT, in accordance with their increased brain Agmat levels. Finally, we also investigated the molecular mechanisms underlying the rapid antidepressant effects of exogenous agmatine in WT and Crtc1/

mice. We found that agmatine induced BDNF translation in the PFC of WT mice, paralleled by dephosphorylation of eukaryotic elongation factor 2 (eEF2), suggesting NMDAR-mediated anti-depressant mechanisms.

MATERIALS AND METHODS Animals

Crtc1/ mice and WT littermates were generated and genotyped as previously described.12 Mice were housed under a 12-h lightdark cycle with ad libitum access to water and standard rodent chow diet. All animal experiments were conducted in accordance with the Swiss Federal

Veterinary Ofces guidelines and were approved by the Cantonal Veterinary Service. All behavioral tests were carried out in the dark phase of the reverse light cycle according to the standard procedures. Male and female mice were weaned at 21 days and housed in same-sex sibling groups until being isolated at 5 weeks of age in order to avoid wounding of cage mates by aggressive Crtc1/ male mice.13 At the age of 8 weeks, animals were randomly assigned into treatment groups and either killed for molecular experiments or used for behavioral assessments.

Brain microdissectionMale and female mice were killed by cervical dislocation, and the brain was rapidly placed in a stainless steel adult mouse brain slicer matrix with 1-mm coronal section slice intervals. A rst cut included the PFC from which the olfactory bulbs and associated structures were removed. Total hippocampi were unrolled from the cortex. All the structures were sequentially quick-frozen in dry ice for mRNA and protein extraction and stored at 80 C until further processing.

Gene expression analysisTotal RNA was extracted and puried from the PFC and HIP using the

RNAeasy Plus Minikit (Qiagen, Hombrechtikon, Switzerland) according to the manufacturers instructions. RNA concentrations were measured by Ultraviolet spectophotometry with a NanoDrop Lite (Thermo Scientic, Wilmington, DE, USA). Complementary DNA was prepared in a 50-l reaction by reverse transcription, using 200 ng of RNA with Taqman Reagents and random hexamers (Applied Biosystems, Foster City, CA, USA). Complementary DNA (0.8 l) was amplied on a 96-well plate using the SYBR Green PCR Master Mix (Applied Biosystem). Amplication was performed with an ABIPRISM 7500 real-time PCR system (Applied Biosystem). The program was 2 min at 50 C, 10 min at 95 C, followed by 45 cycles of 15 s at 95 C and 1 min at 60 C. Relative gene expression was quantied using the comparative Ct method and normalized with -actin transcript levels.

The following primers were used at a concentration of 250 nM: -actin forward 5-GCTTCTTTGCAGCTCCTTCGT-3, -actin reverse 5-ATATCGTCAT

CCATGGCGAAC-3, Agmat forward 5-TGGACAGCAAGCGAGTGGTACA-3, Agmat reverse 5-GGACCAGTGACTTCATCCAACAG-3.

Affymetrix gene expression arraysGene expression levels were evaluated using DNA microarrays (GeneChip

Mouse Genome 430 version 2.0, Affymetrix, Santa Clara, CA, USA) and RNA from the cerebral cortices of female mice (n = 5 for WT and n = 5 for Crtc1/). Biotinylated cRNAs were prepared from 300 ng total RNA using the GeneChip 3 IVT Express Kit (Affymetrix) following the manufacturers instructions. cRNA (15 g) was hybridized to GeneChip arrays and processed, stained and scanned according to the manufacturers recommendations. The quality of input RNAs and cRNAs was veried with the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) before use. Microarray quality control was performed using the software package provided on RACE.41 Chips with a median-normalized unscaled s.e. greater than 1.05 were excluded. Affymetrix annotations (version 3.0) were used for probeset-to-gene assignments. Mod t-statistics and false discovery rate corrections for multiple testing with a signicance threshold of Po0.05 were used as criteria for differential expression, as described in Hochberg and Benjamini.42 Microarray data have been deposited in NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE80633 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80633

Web End =http://www.ncbi.nlm.nih.gov/geo/query/ http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE80633

Web End =acc.cgi?acc = GSE80633 ).

Western blotPFC and HIP samples were manually homogenized with a microtube pestle in RIPA buffer (50 mM HEPES (pH 7.6), 150 mM NaCl, 1 mM EDTA (pH 7.5),2.5 mM EGTA (pH 8.0), 10% glycerol, 1% NP-40, 1% deoxycholate, 0.1% SDS, with a protease inhibitor cocktail (Sigma, St Louis, MO, USA) and a phosphatase inhibitor cocktail (PhosSTOP, Roche, Rotkreuz, Switzerland) and extracted for 20 min at 4 C. Protein quantication was performed with the Pierce BCA Protein Assay Kit (Thermo Scientic). Samples with low-protein extract (o2 mg ml1) were excluded. Fifty g of tissue homogenates were diluted 1:1 with sample buffer (50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, 2% SDS, 9% glycerol, 1% bromophenol blue), separated on a 12% SDS-polyacrylamide gel and proteins were transferred to polyvinylidene diuoride membranes with a semi-dry blotting system

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(Bio-Rad, Hercules, CA, USA). Blots were blocked for 1 h at room temperature (RT) in TBST (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.1% Tween-20), supplemented with 5% skim milk powder. Blots were subsequently incubated with a primary antibody in TBST plus 5% bovine serum albumin overnight at 4 C. Finally, polyvinylidene diuoride membranes were incubated for 1 h at RT with horseradish peroxidase (HRP)-conjugated secondary antibodies in TBST plus 5% skim milk powder, and were developed using a Pierce ECL Western Chemiluminescence Detection Kit (Thermo Scientic). The following antibodies and dilutions were used: rabbit -Agmat 1:400 (sc-98802, Santa Cruz Biotechnology, Dallas, TX, USA), rabbit -BDNF 1:500 (sc-546, Santa Cruz Biotechnology), rabbit -phospho-eEF2 1:1000 (#2331, Cell Signaling, Danvers, MA, USA), rabbit -eEF2 1:1000 (#2332, Cell Signaling), mouse --actin 1:10 000 (Ab-6276, Abcam, Cambridge, UK), donkey HRP--rabbit 1:2000 (#NA934, GE Healthcare, Little Chalfont, UK) and sheep HRP--mouse 1:2000 (#NA931, GE Healthcare). Quantication of band intensity was performed with the Image J software (National Institute of Health, Bethesda, MD, USA). Agmat and BDNF band intensities were normalized with -actin signals; phosphoeEF2 band intensities were normalized with total eEF2 signals.

ImmunouorescenceEight-week-old male mice were deeply anesthetized using sodium pentobarbital and intracardially perfused with saline followed by 4% buffered paraformaldehyde. Brains were dissected out, postxed for 1 h in 4% paraformaldehyde and cryoprotected in 30% sucrose. Brain sections of 35 m were cut with a freezing microtome (Microm, Thermo Fisher Scientic, Waltham, MA, USA) and stored at 20 C in a cryoprotectant solution. Blocking (1 h, RT) as well as primary (overnight, 4 C) and secondary antibody incubation (1 h, RT) were performed in phosphate-buffered saline+0.3% Triton X-100+2% normal horse serum+0.2% bovine serum albumin. Slices were washed three times in phosphate-buffered saline+0.3% Triton X-100 after each incubation. The following antibodies and dilutions were used: rabbit -Agmat 1:100 (sc-98802, Santa Cruz Biotechnology), mouse -Parvalbumin 1:2500 (PV235, Swant, Marly, Switzerland), mouse -Calretinin 1:2500 (CR7697, Swant), goat -Somatostatin 1:500 (sc-7819, Santa Cruz Biotechnology), Cy3-conjugated donkey -rabbit 1:500 (#711-165-152, Jackson Immunoresearch, West Grove, PA, USA), Alexa Fluor 488-conjugated goat -mouse 1:500 (A-21121, Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-conjugated donkey -goat 1:500 (A11055, Invitrogen, Carlsbad, CA, USA). After the secondary antibody incubation, slices were washed, stained with 4,6-diamidino-2-phenylindole 1:30 000 (Invitrogen), mounted on glass slides with the antifade Vectashield medium (Vector Laboratories, Burlingame, CA, USA) and analyzed with a Zeiss LSM 710 Quasar Confocal Microscope (Carl Zeiss, Oberkochen, Germany). Image processing and cell counting were carried out with the Image J Software (National Institute of Health).

Agmatine and ketamine treatmentAgmatine sulphate salt and ketamine (Ketanarkon) were, respectively, purchased from Sigma and Streuli Pharma (Uznach, Switzerland) and dissolved in saline solution. Male and female mice were intraperitoneally injected with 10 ml kg1 of agmatine (10 or 50 mg kg1) or ketamine (3 mg kg1). Controls were injected with saline. Injections were performed 30 min before the FST for two consecutive days.

FSTA 2-day test was performed (days 1 and 2). Mice were put during 5 min in a 5-l glass beaker (26 cm tall, 18 cm) lled to a depth of 22 cm with tap water (25 1 C). Sessions were videotaped from above and manually analyzed non-blindly by the experimenter with the Ethovision 3.1 Software (Noldus Information Technology, Wageningen, The Netherlands) for immobility and climbing time. Mice were judged immobile when no detectable movement was observed, except for minor movements to keep their head above the water. The experiment was conducted in a room with a light intensity of ~ 35 lux. Immediately after the test on day 2, mice were killed for BDNF and phospho-eEF2 measurements.

Statistical analysesThe number of animals tested in each group is specied in the gure legends. Owing to the small number of WT and Crtc1/ mice per litter and a limited breeding cage space, all experiments were performed sequentially with several batches of mice, and their data were combined. Sample sizes were determined based on power analysis and common practice in behavioral experiments (~10 animals per group). Statistical analyses (other than those employed for microarray analyses (see above)) were performed using the Statistica 8.0 Software (StatSoft, Tulsa, OK, USA). All data are presented as mean s.e.m. Po0.05 were considered statistically signicant. A ShapiroWilk test and a Levene test were rst performed to assess data normality and variance homogeneity. All results were found to follow normal distribution and to display similar variance. For immunouorescence, quantitative PCR and western blot data, a two-tailed Student's t-test was performed when only two groups were compared (WT versus Crtc1/ mice). For behavioral data, BDNF and phospho-eEF2 data, a two-way analysis of variance (ANOVA; with genotype and treatment as independent variables) was performed, followed by a Fisher's Least Signicant Difference (LSD) post hoc test.

RESULTSMale and female Crtc1/ mice exhibit increased levels of Agmat mRNA and protein in the PFC and HIP

To identify gene expression changes associated with Crtc1 deciency, we performed genome-wide transcriptomic proling analyses of cortical samples from Crtc1/ and WT female mice using oligonucleotide microarrays (Table 1). Among the down-regulated genes were CREB target genes that we previously showed to have a decreased expression in the PFC and HIP of Crtc1/ male mice.13 Interestingly, a few genes were upregulated in Crtc1/ mice, and amidst them Agmat, whose expression was increased by 1.67-fold. To follow up and conrm this nding, we measured Agmat messenger RNA (mRNA) and protein levels in male and female Crtc1/ mice (Figure 1). We focused our investigations on the PFC and HIP, as these two regions are widely implicated in mood disorders and are known to have high levels of agmatine.43 Quantitative PCR analyses of Agmat mRNA levels found a signicant (t = 3.31, degree of freedom (df) = 9, P = 0.013)1.5-fold increase of Agmat mRNA in the HIP and a threefold increase (t = 6.72, df = 8, Po0.001) in the PFC of male Crtc1/

Table 1. Selection of genes differentially expressed in the cortex of Crtc1/ mice

Symbol Gene GenBank Fold change FDR P

Cartpt CART prepropeptide NM_013732 0.42 0.0086 Nr4a1 Nuclear receptor subfamily 4, group A, 1 NM_010444 0.50 0.0181 Nr4a3 Nuclear receptor subfamily 4, group A, 3 NM_015743 0.52 0.0103 Crem cAMP responsive element modulator NM_001110859 0.59 0.0028 Bdnf Brain-derived neurotrophic factor NM_007540 0.61 0.0131 Nr4a2 Nuclear receptor subfamily 4, group A, 2 NM_013613 0.70 0.0476 Ntrk2 Neurotrophic tyrosine kinase receptor, 2 (TrkB) NM_008745 0.81 0.0108 Agmat Agmatine ureohydrolase (agmatinase) NM_001081408 1.67 0.0099

Abbreviation: FDR P, false discovery rate corrected P-value.

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Male Female

Figure 1. Increased levels of agmatinase (Agmat) in Crtc1/ mice. (a) Real-time quantitative PCR measurements showed an increased expression of Agmat in the hippocampus (HIP) and prefrontal cortex (PFC) of male Crtc1/ mice (n = 5) compared with wild-type (WT) littermates (n = 5). (b) Agmat was also overexpressed in the HIP and PFC of female Crtc1/ mice (n = 6) compared with WT control mice (n = 5). Representative western blot of Agmat and -actin is shown in c for male mice and in e for female mice. Quantitative analyses of western blot showed increased protein levels of Agmat in the HIP of male Crtc1/ mice (n = 4) compared with WT mice (n = 5; d). Protein levels of Agmat were also increased in the HIP of female Crtc1/ mice (n = 6) compared with WT mice (n = 5; f). Results are presented as ratio between Agmat and -actin signals. Data are mean s.e.m. *Po0.5, ***Po0.001.

mice (Figure 1a). In female Crtc1/ mice, a signicant (t = 2.65, df = 9, P = 0.029) threefold increase in Agmat mRNA was observed in the HIP and a 2.5-fold increase (t = 5.45, df = 9, Po0.001) in the

PFC (Figure 1b). Western blot analysis of extracts from these same structures showed an Agmat protein band at the expected size of ~ 35 kDa (Figures 1c and e), which was subsequently quantied (normalized to -actin signal). Crtc1/ male mice had increased levels of Agmat protein in the HIP (t = 2.72, df = 6, P = 0.036), whereas a nonsignicant trend (t = 1.51, df = 9, P = 0.152) of increased Agmat was observed in the PFC (Figure 1d). In female Crtc1/ mice, a similar increase was found in the HIP (t = 2.58, df = 9, P = 0.027), whereas no change in Agmat protein content could be seen in the PFC (t = 0.61, df = 9, P = 0.553; Figure 1f). Taken together, these results conrm an upregulation of Agmat gene expression in the HIP and PFC of Crtc1/ mice, independently of gender. Although Agmat protein levels only partially correlated with the gene expression data (which might reect translational regulation or a complex subcellular protein localization), these results strongly suggest that Crtc1/ mice have an altered agmatinergic system.

Crtc1/ mice have an increased number of Agmat-expressing cells in the PFC and in several regions of the HIP

To determine whether the increased Agmat expression in Crtc1/

mice was because of higher Agmat levels or an increased number of Agmat-expressing cells, we visualized Agmat protein expression using immunouorescence in the PFC and in the CA1, CA3 and dentate gyrus (DG) subregions of the HIP (Figure 2). Staining revealed numerous cells in the PFC (Figure 2a), and in the DG (Figure 2b), CA1 (Figure 2c) and CA3 (Figure 2d) regions of the HIP, and its subcellular localization appeared mainly perinuclear within those structures. In the HIP, staining could be observed in the pyramidal cell layer, but appeared stronger in interneuron-like cells. Agmat-expressing cells were counted and results were normalized to total numbers of cells, counted with 4,6-diamidino-2-phenylindole staining (Figure 2e). Agmat-positive cells counting revealed a signicant increased number of cells in the PFC of Crtc1/ mice (+60%, t = 4.23, df = 9, P = 0.001) as compared with WT littermates. This could also be observed in the DG (+45%, t = 2.55, df = 9, P = 0.029) and CA1 regions of the HIP (+70%,

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Figure 2. Increased number of agmatinase (Agmat)-expressing cells in male Crtc1/ mice. Representative immunouorescence staining of Agmat-expressing cells (red) and total cells (4,6-diamidino-2-phenylindole (DAPI) staining) in the prefrontal cortex (PFC; a), and in the dentate gyrus (DG; b), CA1 (c) and CA3 (d) regions of the hippocampus (HIP). Cell counting resulted in an increased number of Agmat-expressing cells in the PFC, DG and CA1 regions of male Crtc1/ mice (n = 6) compared with wild-type (WT) littermates (n = 5; e). No difference in number of Agmat-expressing cells was found in the CA3 region. Results are expressed as ratio between number of Agmat-expressing cells and total number of cells. Data are mean s.e.m. *Po0.5, **Po0.01. Scale bar, 50 m.

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t = 2.28, df = 9, P = 0.049). No differences in the numbers of Agmat-expressing cells could be seen in the CA3 region of the HIP (t = 0.40, df = 9, P = 0.698). Overall, these data suggest that the increased Agmat expression found in Crtc1/ mice would be the result of a higher number of Agmat-expressing cells in the PFC and selected HIP subregions. Moreover, the morphology and localization of the hippocampal cells expressing higher Agmat levels indicated that they could be GABAergic interneurons.

Characterization of Agmat-expressing cells in the PFC and HIPAs hippocampal Agmat-expressing cells have an interneuron-like morphology, we characterized Agmat-expressing cells with markers for specic GABAergic interneuron types. Bernstein et al.44 studied regional and cellular expression of Agmat in the rat brain and found that Agmat colocalized with calretinin (CR)-expressing interneurons in the cortex and CA1 region of the HIP.44

We therefore performed double immunolabeling of CR and Agmat in the PFC and HIP of WT mice (Figure 3 and Supplementary Figure S1). In the PFC, we observed very little colocalization between CR and Agmat staining (Figure 3a, double-labeled cells indicated with arrows). Little colocalization was also seen in the CA3 region of the HIP (Figure S1b). Moreover, no colocalization was observed in the DG and CA1 regions of the HIP (Figure 3b and Supplementary Figure S1a, respectively). In order to further investigate which type of interneurons expressed Agmat, we performed double immunostaining of Agmat- and PV-expressing cells. We observed high colocalization of Agmat and PV cells in all studied structures: PFC, DG, CA1 and CA3 regions of the HIP (Figures 3c and d and Supplementary Figure S1c and d, respectively). Indeed, nearly all PV-expressing cells were also Agmat-positive. On the other hand, there were some Agmat-expressing cells that were not PV-positive, therefore suggesting that other types of cells express Agmat.

Several lines of evidence involved somatostatin (Sst) in mood disorders,45 and we previously observed that Sst was down-regulated in the brain of Crtc1/ mice.13 Therefore, we investigated whether Agmat might be expressed in somatostatinergic interneurons by performing double immunostaining of Agmat- and Sst-expressing cells. As for PV staining, most of Sst-expressing cells were colocalized with Agmat in all regions observed: PFC, DG, CA1 and CA3 regions of the HIP (Figures 3e and f and Supplementary Figure S1e and f, respectively). Colocalization was, however, less extended in the PFC; as some Sst-expressing cells were not colocalized with Agmat staining.

Altogether, these data conrm the expression of Agmat in specic GABAergic interneuron subpopulations, with apparent high expression in PV and Sst interneurons, and slight colocalization with CR interneurons.

Rapid ketamine-like antidepressant effect of acute agmatine in male and female WT and Crtc1/ mice

Given the possible role of an agmatine decit in depression and the observed increase in Agmat levels in the brain of Crtc1/

mice, we postulated that their depressive-like behavior is due, at least in part, to a dysregulated agmatinergic system. To test this hypothesis, we treated WT and Crtc1/ mice with acute intraperitoneal (IP) injections of agmatine and tested antidepressant effects in the FST, a classical test for rodent depression-related behavior. We hypothesized that restoring agmatine levels by exogenous supplementation would normalize behavioral response to the helplessness-inducing effects of FST. We rst treated the animals with agmatine at 10 mg kg1 and compared their depressive-like behavior in the FST with saline-injected control animals. This protocol was repeated a second time on the next day. The antidepressant effects of agmatine were assessed by measuring the oating (immobility) time of the mice. Agmatine had a signicant antidepressant effect on WT mice but failed to

signicantly reduce Crtc1/ mouse immobility time (data not shown). However, a tendency to decrease the immobility time of Crtc1/ mice prompted us to repeat this experiment with an increased dose of agmatine (50 mg kg1, IP; Figures 4a and b). For male mice (Figure 4a), a signicant effect of genotype on immobility could be seen on both days of test, as shown by two-way ANOVA (Day 1: F(1,29) = 6.74, P = 0.014; Day 2: F(1,29) = 19.4,

Po0.001). Post hoc analyses revealed that vehicle-treated Crtc1/

mice presented higher immobility time than WT mice (Day 1: +13%, P = 0.031, Day 2: +22%, P = 0.004). A signicant treatment effect could be observed on the second day of test (F(1,29) = 12.38,

P = 0.001) as agmatine signicantly decreased the immobility time of WT and Crtc1/ mice (P = 0.012 and P = 0.026, respectively). For female mice (Figure 4b), the analysis of immobility time by twoway ANOVA revealed a signicant treatment effects for both days of test (Day 1: F(1,28) = 10.28, P = 0.003; Day 2: F(1,28) = 23.46,

Po0.001). Post hoc analyses showed that agmatine signicantly reduced immobility time in WT female mice on day 1 (30%, P = 0.014) and of both genotypes on day 2 (WT: 30%, P = 0.003; Crtc1/: 30%, P = 0.001). Thus, 50 mg kg1of agmatine reduced the immobility time of male and female Crtc1/ mice. Overall, these data conrm the antidepressant effect of an acute agmatine treatment in WT animals. Crtc1/ mice also respond to the antidepressant effect of agmatine, but require a higher dose (50 mg kg1).

Ketamine and other NMDAR antagonists have been shown to have rapid and long-lasting antidepressant effects in behavioral despair paradigms such as the FST.27 As agmatine also acts as a NMDAR antagonist,24 we assessed the antidepressant effect of ketamine in male and female WT and Crtc1/ mice

(Supplementary Figure S2). Interestingly, ketamine (3 mg kg1,

IP) signicantly decreased the depressive-like behavior of WT and Crtc1/ mice of both sexes in a very similar way as agmatine did, which suggested that the rapid antidepressant action of agmatine and ketamine may involve the same molecular pathways.

Characterization of pathways involved in agmatine antidepressant effect

Although agmatine antidepressant effects have been established, the underlying molecular mechanisms remain unclear. A recent study suggested that chronic agmatine treatment induces an increase of BDNF protein levels, as well as an increased phosphorylation of CREB, PKA and other kinases involved in pathways associated with neuroplasticity.40 Although the underlying cellular and molecular mechanisms of ketamines antidepressant action are not completely understood, they involve the rapid induction of BDNF translation via activation of the mammalian target of rapamycin (mTOR) pathway as seen by dephosphorylation of eEF2.27 Therefore, we investigated whether agmatines rapid antidepressant effect involved NMDAR blockade-associated changes in BDNF levels and eEF2 phosphorylation. We measured the levels of phospho-eEF2 and BDNF proteins in the PFC and HIP of male and female mice treated with 50 mg kg1

(Figures 4c-j). Western blot for phospho-eEF2 and total eEF2 showed a single band at the expected size of 95 kDa (Figures 4c and d). Phospho-eEF2 signal was quantied and normalized over total eEF2 signal. In male mice (Figure 4e), a two-way ANOVA showed no effect of genotype or treatment in the HIP. A signi-cant effect of genotype could be seen in the PFC (F(1,23) = 13.34,

P = 0.001) as both groups of Crtc1/ mice presented lower levels of phospho-eEF2 (P = 0.001 and P = 0.002). Agmatine treatment had no effect on p-eEF2 levels of Crtc1/ mice, but it signicantly reduce p-eEF2 in WT mice (P = 0.04). In female mice (Figure 4f), a two-way ANOVA showed no effect of genotype or treatment in the HIP. A trend to decreased levels of phosphorylated eEF2 was present in agmatine-treated WT mice and in both groups of Crtc1/ animals. In the PFC, signicant effects of

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Figure 3. Characterization of GABAergic interneuron subpopulations expressing agmatinase (Agmat) in the prefrontal cortex (PFC) and hippocampus (HIP) of wild-type (WT) male mice. Double immunouorescence labeling of (a, b) Agmat and calretinin (CR; c, d) Agmat and parvalbumin (PV), and (e, f) Agmat and somatostatin (Sst) in (a, c, e) PFC and (b, d, f) dentate gyrus (DG) of the HIP. Total cells were identied by nuclear 4,6-diamidino-2-phenylindole (DAPI) staining. Merged images showed few colocalization of Agmat and CR staining in the PFC (a) as indicated by arrows. No colocalization could be observed in the DG (b). All PV and Sst interneurons also expressed Agmat in the PFC (c, e) and DG (d, f). Scale bar, 50 m.

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genotype (F(1,13), P = 0.011), treatment (F(1,13), P = 0.043) and

genotype treatment (F(1,13), P = 0.011) were observed. Post hoc

analyses revealed that agmatine signicantly decreased the phosphorylation of eEF2 in WT mice (P = 0.003). Both groups of Crtc1/ mice also presented decreased levels of phospho-eEF2 as compared with vehicle-treated WT mice (Crtc1/ Vehicle: P = 0.001; Crtc1/ Agmatine: P = 0.002). Agmatine had no effect on the phosphorylation of eEF2 in Crtc1/ mice.

Western blot for BDNF revealed a single band at the expected size of 14 kDa (Figures 4gh). BDNF signal was quantied and normalized with -actin signal. In male mice (Figure 4i), no effect of genotype or treatment could be observed in the HIP. In the PFC, a two-way ANOVA showed an effect of genotype (F(1, 23) = 29.12,

Po0.001). Post hoc analysis revealed that both vehicle- and agmatine-treated Crtc1/ mice presented lower levels of BDNF protein than WT mice (Po0.001 for both groups). Agmatine

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treatment had no effect on BDNF levels of WT and Crtc1/ mice. In female mice (Figure 4j), a two-way ANOVA revealed a signicant effect of genotype in both structures (PFC: F(1,22) = 6.78, P = 0.016;

HIP: F(1,22) = 6.27, P = 0.02). Post hoc analyses showed that both

groups of Crtc1/ mice had signicantly lower levels of BDNF than WT animals in the HIP, independently of the treatment (P = 0.01). No effect of agmatine treatment could be seen in the HIP, whereas it was signicant in the PFC (F(1,22) = 4.63, P = 0.042).

Post hoc analyses indicated that agmatine signicantly increased BDNF protein levels in the PFC, however, only in WT mice (P = 0.024). Taken together, these results provide evidence that agmatine induces eEF2 dephosphorylation in WT male and female mice, thus suggesting activation of the mTOR pathway, through its NMDAR antagonist property. Agmatine effects on BDNF levels are gender-dependent, as agmatine rapidly induces BDNF translation in WT female mice, but not in WT male mice.

DISCUSSIONIn this study, we showed for we believe the rst time a link between downregulation of the CRTC1CREB pathway and alteration of the agmatinergic system in the context of a rodent model of depression. We found that CRTC1-decient mice exhibit increased mRNA and protein levels of Agmat in the HIP and PFC. We also determined that these higher Agmat levels are mainly because of an increased number of Agmat-expressing cells in the PFC and HIP of Crtc1/ mice. These ndings suggest that Crtc1/ mice have a dysregulated agmatinergic system resulting in increased Agmat expression and ensuing decreased agmatine levels. Hence, the depressive-like phenotype of these animals would be in keeping with the protective and antidepressant role of endogenous agmatine.

Immunouorescent detection of Agmat revealed that this enzyme is mainly expressed in interneurons in accordance with a previous characterization in the rat brain.44 As Crtc1/ mice have more Agmat-expressing cells, these mice might also have overall GABAergic system alteration. In line with this, agmatine and GABA seem to be closely related because agmatine is degraded into putrescine, whose derived polyamines can be used as GABA precursors.16 Therefore, we hypothesize that dysregulation of agmatine metabolism might lead to abnormal GABA regulation and ultimately to overall impaired interneuronal circuitry.

A characterization of the subpopulations of GABAergic inter-neurons expressing Agmat showed that it mainly colocalizes with PV and Sst interneurons, whereas no or little colocalization with CR interneurons was observed. These ndings are in contradiction with the study of Bernstein et al.,44 which showed that Agmat was mainly found in CR interneurons. The reason for this discrepancy is unclear. Future studies should focus on a deeper characterization of Agmat-expressing cells, as Agmat staining revealed that it is present in many cells in the mouse brain and thus probably in a wide range of cell types.

The depressive-like behavior of Crtc1/ mice was successfully normalized by acute agmatine treatment as efciently as ketamines effect. This suggests that their altered agmatinergic system contributes to their phenotype. It is noteworthy that only a higher dose of agmatine (50 mg kg1) was effective, suggesting that the 10 mg kg1 dose was not sufcient to compensate for a possible decrease in agmatine levels and to restore normal agmatinergic functions.

When looking at the molecular effects of agmatine, we found that it was able to induce an increase in BDNF protein levels in the PFC of WT females. This agmatine-induced BDNF upregulation was paralleled by eEF2 dephosphorylation, which stimulates protein translation. This mechanism has been shown to underlie the rapid antidepressant effect of NMDAR antagonists such as ketamine and MK-801.26,27,46 Therefore, our results suggest that agmatine acts as an antidepressant, possibly through this pathway. This is in line with the ability of agmatine to block NMDAR and the involvement of this function in its antidepressant effects.36,47,48 In contrast, the

behavioral effects of agmatine in WT males were apparently not mediated by BDNF because agmatine did not increase its levels in HIP and PFC. The mechanisms that underlie these sex differences are still unclear, but not completely unexpected. These gender-specic effects are actually of much interest in the light of the female preponderance in major depression. Sex differences have been reported in animal models of depression. For instance, the impact of BDNF signaling on depression-like behavior is different in male and female mice.49 Moreover, it has been shown that hippocampal NO may contribute to sex difference in depressive-like behaviors.50 This study showed that stress promotes hippocampal NO production in male mice, whereas stress suppresses it in female ones. Worthy of note, both NO excess in male mice and shortage in female mice resulted in depressive-like behaviors through affecting CREB activation.

Figure 4. Behavioral and molecular effects of agmatine treatment on male and female wild-type (WT) and Crtc1/ mice. (a, b) Effects of acute agmatine treatment (50 mg kg1, intraperitoneal (IP)) 30 min before a forced swim test (FST) during two consecutive days. In male mice (a) vehicle-treated Crtc1/ mice (n = 7) showed higher immobility levels than vehicle-treated WT mice (n = 9) on both days of test (+Po0.05,

++Po0.01). On Day 2, agmatine signicantly decreased the immobility time of WT mice (*Po0.05) and Crtc1/ mice (#Po0.05; n = 10 and n = 7, respectively). In female mice (b) WT mice treated with agmatine (n = 8) showed signicantly decreased immobility time (*Po0.05, **Po0.01) compared with vehicle-treated WT mice (n = 9) on both days of test. On day 2, agmatine-treated Crtc1/ mice (n = 8) also presented signicantly decreased immobility time (##Po0.01) than vehicle-treated Crtc1/ mice (n = 8). (cf) Effects of acute agmatine treatment (50 mg kg1, IP) on eukaryotic elongation factor 2 (eEF2) phosphorylation. (c, d) A representative western blot for phospho-eEF2 and total eEF2 in the hippocampus (HIP) and prefrontal cortex (PFC) of WT and Crtc1/ mice (V, vehicle-treated; A, agmatine-treated) in male (c) and female (d) mice. (e, f) Quantitative analyses of p-eEF2 western blot in male (e) and female (f) mice. In male mice, no effect of agmatine could be seen in the HIP of WT and Crtc1/ mice. In the PFC, agmatine-treated WT mice (n = 8) presented lower levels of p-eEF2 than vehicle-treated WT mice (n = 7; *Po0.05). Both vehicle- and agmatine-treated Crtc1/ mice (n = 6 for both) displayed lower p-eEF2 levels than WT mice (++Po0.01); agmatine treatment had no effect on p-eEF2 levels in these animals (e). In female mice, quantication showed no effect of agmatine treatment in the HIP of WT and Crtc1/ mice (f). In the PFC, agmatine-treated WT mice (n = 4) presented lower levels of eEF2 phosphorylation compared with vehicle-treated WT animals (n = 4; **Po0.01). Crtc1/ mice treated with vehicle (n = 5) or agmatine (n = 5)

also displayed lower levels of eEF2 phosphorylation than WT mice (**Po0.01). Agmatine treatment had no effect on Crtc1/ mice. Results are presented as ratio between phospho-eEF2 and total eEF2 signals. (gj) Effects of acute agmatine treatment (50 mg kg1, IP) on brain-derived neurotrophic factor (BDNF) protein level. (g, h) A representative western blot for BDNF and -actin in the HIP and PFC of WT and Crtc1/ mice in male (g) and female (h) mice. (i, j) Quantitative analyses of BDNF western blot in male (i) and female (j) mice. In male mice, no effect of agmatine could be seen in the HIP of WT and Crtc1/ mice. In the PFC, both vehicle- and agmatine-treated Crtc1/ mice (n = 6 for both) displayed lower BDNF levels than vehicle- and agmatine-treated WT mice (n = 7 and n = 8, respectively; +++Po0.001) (i). In female mice, quantitative analyses of western blot showed decreased levels of BDNF in the HIP of all Crtc1/ animals (n = 14) compared with WT animals (n = 13), independently of the treatment (+Po0.05). In the PFC, agmatine-treated WT mice (n = 7) presented higher levels of BDNF compared with vehicle-treated WT animals (n = 5; *Po0.05). Crtc1/ mice treated with agmatine (n = 8) did not present different BDNF levels than those treated with saline (n = 6; j). Results are presented as ratio between BDNF and -actin signals. Data are mean s.e.m.

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Interestingly, the effects of agmatine on BDNF and eEF2 were restricted to WT animals. Crtc1/ male and female mice displayed basal lower levels of phospho-eEF2 in the PFC, and agmatine did not decrease them further nor did it increase BDNF levels, which suggest a dysregulation of this pathway and the involvement of alternative mechanisms underlying agmatines antidepressant effects in these animals.

In conclusion, our results provide evidence for the involvement of the agmatinergic system in the Crtc1/ mouse model of depression, and lend support to previous reports of the antidepressant properties of agmatine. The comparable rapid antidepressant effects of agmatine and ketamine in WT and Crtc1/ mice, as well as the molecular effects that acute agmatine treatment causes in the brain of WT mice suggest that agmatine possibly functions as a fast-acting antidepressant through NMDAR blockade. The relationship between the CRTC1CREB pathway and agmatine regulation merits further investigation, as it will bring better knowledge of these systems and their contribution to MDD etiology.

CONFLICT OF INTEREST

The authors declare no conict of interest.

ACKNOWLEDGMENTS

We thank Suzanne Badoux for her initial work with an agmatinase antibody that was not used in this study. This work was funded by a grant from the Swiss National Science Foundation (31003A-135692) and partly supported by the National Centre of Competence in Research (NCCR) Synapsy.

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