Ludwig Czibere

Identification of Glyoxalase-I as a Protein Marker in a Mouse Model of Extremes in Trait Anxiety

For >15 generations, CD1 mice have been selectively and bidirectionally bred for either high-anxiety-related behavior (HAB-M) or low-anxiety-related behavior (LAB-M) on the elevated plus-maze. Independent of gender, HAB-M were more anxious than LAB-M animals in a variety of additional tests, including those reflecting risk assessment behaviors and ultrasound vocalization, with unselected CD1 “normal” control (NAB-M) and cross-mated (CM-M) mice displaying intermediate behavioral scores in most cases. Furthermore, in both the forced-swim and tail-suspension tests, LAB-M animals showed lower scores of immobility than did HAB-M and NAB-M animals, indicative of a reduced depression-like behavior. Using proteomic and microarray analyses, glyoxalase-I was identified as a protein marker, which is consistently expressed to a higher extent in LAB-M than in HAB-M mice in several brain areas. The same phenotype-dependent difference was found in red blood cells with NAB-M and CM-M animals showing intermediate expression profiles of glyoxalase-I. Additional studies will examine whether glyoxalase-I has an impact beyond that of a biomarker to predict the genetic predisposition to anxiety- and depression-like behavior.

Candidate genes of anxiety-related behavior in HAB/LAB rats and mice: focus on vasopressin and glyoxalase-I.

Two animal models of trait anxiety, HAB/LAB rats and mice, are described, representing inborn extremes in anxiety-related behavior. The comprehensive phenotypical characterization included basal behavioral features, stress-coping strategies and neuroendocrine responses upon stressor exposure with HAB animals being hyper-anxious, preferring passive coping, emitting more stressor-induced ultrasonic vocalization calls and showing typical peculiarities of the hypothalamic-pituitary-adrenocortical axis and line-specific patterns of Fos expression in the brain indicative of differential neuronal activation. In most cases, unselected Wistar rats and CD1 mice, respectively, displayed intermediate behaviors. In both HAB/LAB rats and mice, the behavioral phenotype has been found to be significantly correlated with the expression of the neuropeptide arginine vasopressin (AVP) at the level of the hypothalamic paraventricular nucleus (PVN). Additional receptor antagonist approaches in HABs confirmed that intra-PVN release of AVP is likely to contribute to hyper-anxiety and depression-like behavior. As shown exemplarily in HAB rats and LAB mice, single nucleotide polymorphisms (SNPs) in regulatory structures of the AVP gene underlie AVP-mediated phenotypic phenomena; in HAB rats, a SNP in the promoter of the AVP gene leads to reduced binding of the transcriptional repressor CBF-A, thus causing AVP overexpression and overrelease. Conversely, in LAB mice, a SNP in the AVP gene seems to cause an amino acid exchange in the signal peptide, presumably leading to a deficit in bioavailable AVP likely to underlie the total hypo-anxiety of LAB mice in combination with signs of central diabetes insipidus. Another feature of LAB mice is overexpression of glyoxalase-I. The functional characterization of this enzyme will determine its involvement in anxiety-related behavior beyond that of a reliable biomarker. The further identification of quantitative trait loci, candidate genes (and their products) and SNPs will not only help to explain inter-individual variation in emotional behavior, but will also reveal novel targets for anxiolytic and antidepressive interventions.

TMEM132D, a new candidate for anxiety phenotypes: evidence from human and mouse studies

The lifetime prevalence of panic disorder (PD) is up to 4% worldwide and there is substantial evidence that genetic factors contribute to the development of PD. Single-nucleotide polymorphisms (SNPs) in TMEM132D, identified in a whole-genome association study (GWAS), were found to be associated with PD in three independent samples, with a two-SNP haplotype associated in each of three samples in the same direction, and with a P-value of 1.2e−7 in the combined sample (909 cases and 915 controls). Independent SNPs in this gene were also associated with the severity of anxiety symptoms in patients affected by PD or panic attacks as well as in patients suffering from unipolar depression. Risk genotypes for PD were associated with higher TMEM132D mRNA expression levels in the frontal cortex. In parallel, using a mouse model of extremes in trait anxiety, we could further show that anxiety-related behavior was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex, central to the processing of anxiety/fear-related stimuli, and that in this animal model a Tmem132d SNP is associated with anxiety-related behavior in an F2 panel. TMEM132D may thus be an important new candidate gene for PD as well as more generally for anxiety-related behavior.

A hypomorphic vasopressin allele prevents anxiety-related behavior

Background To investigate neurobiological correlates of trait anxiety, CD1 mice were selectively bred for extremes in anxiety-related behavior, with high (HAB) and low (LAB) anxiety-related behavior mice additionally differing in behavioral tests reflecting depression-like behavior. Methodology/ Principal Findings In this study, microarray analysis, in situ hybridization, quantitative real-time PCR and immunohistochemistry revealed decreased expression of the vasopressin gene (Avp) in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei of adult LAB mice compared to HAB, NAB (normal anxiety-related behavior) and HABxLAB F1 intercross controls, without detecting differences in receptor expression or density. By sequencing the regions 2.5 kbp up- and downstream of the Avp gene locus, we could identify several polymorphic loci, differing between the HAB and LAB lines. In the gene promoter, a deletion of twelve bp Δ(−2180–2191) is particularly likely to contribute to the reduced Avp expression detected in LAB animals under basal conditions. Indeed, allele-specific transcription analysis of F1 animals revealed a hypomorphic LAB-specific Avp allele with a reduced transcription rate by 75% compared to the HAB-specific allele, thus explaining line-specific Avp expression profiles and phenotypic features. Accordingly, intra-PVN Avp mRNA levels were found to correlate with anxiety-related and depression-like behaviors. In addition to this correlative evidence, a significant, though moderate, genotype/phenotype association was demonstrated in 258 male mice of a freely-segregating F2 panel, suggesting a causal contribution of the Avp promoter deletion to anxiety-related behavior. Discussion Thus, the identification of polymorphisms in the Avp gene promoter explains gene expression differences in association with the observed phenotype, thus further strengthening the concept of the critical involvement of centrally released AVP in trait anxiety.

Proteomic-based genotyping in a mouse model of trait anxiety exposes disease-relevant pathways

In our biomarker identification efforts, we have reported earlier on a protein that differs in its electrophoretic mobility between mouse lines bred either for high or low trait anxiety. The altered electrophoretic behavior of enolase phosphatase (EP) is now identified to be caused by two single-nucleotide polymorphisms. In both cases, the genetic polymorphism introduces an amino acid change in the proteins sequence resulting in differential mobility on SDS gels. This was shown by recombinantly expressing the two EP isoforms. Functional studies indicate that the EP isoform from the high anxiety mouse line has a lower enzymatic activity than does its low anxiety mouse counterpart. EP is a member of the methionine salvage pathway that is responsible for the synthesis of S-adenosyl-L-methionine, a natural compound with potential antidepressant activities. In addition, it is linked to the polyamine pathway whose members have functions in anxiety/depression-related behaviors. In a freely-segregating F2 panel, both single-nucleotide polymorphisms were significantly associated with locomotion-independent trait anxiety, further supporting a functional role of EP for this phenotype. The study shows that proteomic analysis can reveal genotypic differences relevant for the phenotype. The identified protein alterations, in turn, can expose metabolic pathways pertinent to the behavioral phenotype.

Diabetes insipidus and, partially, low anxiety‐related behaviour are linked to a SNP‐associated vasopressin deficit in LAB mice

Following secretion from the posterior pituitary, the neuropeptide vasopressin (AVP) stimulates the kidney to retain water, and when released centrally it can contribute to anxiety‐ and depression‐like behaviours. We hypothesized that CD1 mice bred for low trait anxiety (LAB) suffer from a deficit in AVP. Both osmotically stimulated peripheral secretion and intra‐paraventricular nucleus (PVN) release of AVP were found decreased in LAB animals compared with normal anxiety (NAB) or high anxiety (HAB) controls. Consequently, in addition to their extreme non‐anxiety, LAB mice showed signs of central diabetes insipidus (cDI), including increased fluid intake and reduced urine osmolality, as well as a pathological increase in plasma osmolality upon water deprivation. These cDI symptoms were attenuated by administration of a selective AVP V2 receptor agonist. A single nucleotide polymorphism (SNP) in exon 1 (C(+ 40)T) of the Avp gene of LAB animals causes an amino acid substitution in the signal peptide of the AVP precursor, and is likely to impair processing and trafficking of the precursor, as suggested by reduced axonal transport of AVP from the hypothalamic PVN, finally contributing to cDI symptoms and low trait anxiety. In an F2 panel, this SNP co‐segregated with fluid intake and showed a partial contribution to low anxiety‐related behaviour, indicated by its co‐segregation with time spent on the open arms of the elevated plus‐maze in a subset of F2 mice. Thus, the SNP‐associated deficit in plasma and central AVP contributes to signs of cDI and, at least partially, to low trait anxiety, both features being typical of LAB animals.

Profiling Trait Anxiety: Transcriptome Analysis Reveals Cathepsin B (Ctsb) as a Novel Candidate Gene for Emotionality in Mice

Behavioral endophenotypes are determined by a multitude of counteracting but precisely balanced molecular and physiological mechanisms. In this study, we aim to identify potential novel molecular targets that contribute to the multigenic trait “anxiety”. We used microarrays to investigate the gene expression profiles of different brain regions within the limbic system of mice which were selectively bred for either high (HAB) or low (LAB) anxiety-related behavior, and also show signs of comorbid depression-like behavior. We identified and confirmed sex-independent differences in the basal expression of 13 candidate genes, using tissue from the entire brain, including coronin 7 (Coro7), cathepsin B (Ctsb), muscleblind-like 1 (Mbnl1), metallothionein 1 (Mt1), solute carrier family 25 member 17 (Slc25a17), tribbles homolog 2 (Trib2), zinc finger protein 672 (Zfp672), syntaxin 3 (Stx3), ATP-binding cassette, sub-family A member 2 (Abca2), ectonucleotide pyrophosphatase/phosphodiesterase 5 (Enpp5), high mobility group nucleosomal binding domain 3 (Hmgn3) and pyruvate dehydrogenase beta (Pdhb). Additionally, we confirmed brain region-specific differences in the expression of synaptotagmin 4 (Syt4). Our identification of about 90 polymorphisms in Ctsb suggested that this gene might play a critical role in shaping our mouse models behavioral endophenotypes. Indeed, the assessment of anxiety-related and depression-like behaviors of Ctsb knock-out mice revealed an increase in depression-like behavior in females. Altogether, our results suggest that Ctsb has significant effects on emotionality, irrespective of the tested mouse strain, making it a promising target for future pharmacotherapy.

Bidirectional rescue of extreme genetic predispositions to anxiety: impact of CRH receptor 1 as epigenetic plasticity gene in the amygdala.

The continuum of physiological anxiety up to psychopathology is not merely dependent on genes, but is orchestrated by the interplay of genetic predisposition, gene x environment and epigenetic interactions. Accordingly, inborn anxiety is considered a polygenic, multifactorial trait, likely to be shaped by environmentally driven plasticity at the genomic level. We here took advantage of the extreme genetic predisposition of the selectively bred high (HAB) and low anxiety (LAB) mouse model exhibiting high vs low anxiety-related behavior and tested whether and how beneficial (enriched environment) vs detrimental (chronic mild stress) environmental manipulations are capable of rescuing phenotypes from both ends of the anxiety continuum. We provide evidence that (i) even inborn and seemingly rigid behavioral and neuroendocrine phenotypes can bidirectionally be rescued by appropriate environmental stimuli, (ii) corticotropin-releasing hormone receptor 1 (Crhr1), critically involved in trait anxiety, shows bidirectional alterations in its expression in the basolateral amygdala (BLA) upon environmental stimulation, (iii) these alterations are linked to an increased methylation status of its promoter and, finally, (iv) binding of the transcription factor Yin Yang 1 (YY1) to the Crhr1 promoter contributes to its gene expression in a methylation-sensitive manner. Thus, Crhr1 in the BLA is critically involved as plasticity gene in the bidirectional epigenetic rescue of extremes in trait anxiety.

Selective Breeding for High Anxiety Introduces a Synonymous SNP That Increases Neuropeptide S Receptor Activity

Neuropeptide S (NPS) has generated substantial interest due to its anxiolytic and fear-attenuating effects in rodents, while a corresponding receptor polymorphism associated with increased NPS receptor (NPSR1) surface expression and efficacy has been implicated in an increased risk of panic disorder in humans. To gain insight into this paradox, we examined the NPS system in rats and mice bred for high anxiety-related behavior (HAB) versus low anxiety-related behavior, and, thereafter, determined the effect of central NPS administration on anxiety- and fear-related behavior. The HAB phenotype was accompanied by lower basal NPS receptor (Npsr1) expression, which we could confirm via in vitro dual luciferase promoter assays. Assessment of shorter Npsr1 promoter constructs containing a sequence mutation that introduces a glucocorticoid receptor transcription factor binding site, confirmed via oligonucleotide pull-down assays, revealed increased HAB promoter activity—an effect that was prevented by dexamethasone. Analogous to the human NPSR1 risk isoform, functional analysis of a synonymous single nucleotide polymorphism in the coding region of HAB rodents revealed that it caused a higher cAMP response to NPS stimulation. Assessment of the behavioral consequence of these differences revealed that intracerebroventricular NPS reversed the hyperanxiety of HAB rodents as well as the impaired cued-fear extinction in HAB rats and the enhanced fear expression in HAB mice, respectively. These results suggest that alterations in the NPS system, conserved across rodents and humans, contribute to innate anxiety and fear, and that HAB rodents are particularly suited to resolve the apparent discrepancy between the preclinical and clinical findings to date.

Blunted HPA axis reactivity reveals glucocorticoid system dysbalance in a mouse model of high anxiety-related behavior

Depression and anxiety disorders are often characterized by altered hypothalamic-pituitary-adrenal (HPA) axis re-/activity. However, the presence of a molecular link between dysbalanced neuroendocrine regulation and psychopathologies is not yet fully established. Earlier, we reported that high (HAB), normal (NAB) and low (LAB) anxiety-related behavior mice express divergent anxiety-related and passive/active coping phenotypes. Here, we studied mechanisms that might contribute to the different HPA axis reactivity observed in HAB, NAB and LAB mice and their involvement in the regulation of anxiety-related behavior and passive/active coping style. We found that HAB mice respond with significantly reduced corticosterone (CORT) secretion to an acute stressful stimulus and a blunted response in the Dex/CRH test compared to NAB and LAB mice. At the molecular level, higher expression of the glucocorticoid receptor (GR/Nr3c1) and decreased corticotropin-releasing hormone receptor 1 (CRHR1) expression were observed in the pituitary of HAB mice. We further analyzed whether these stress mediators differed between the HAB, NAB and LAB lines in limbic system-associated brain regions and whether their interplay contributes to the phenotype. Interestingly, not only in the pituitary but also in almost all brain regions investigated, GR expression was significantly higher in HAB mice. In contrast, the amount of CORT in the brain structures analyzed was significantly lower in these animals. The expression of CRHR1 varied in the prefrontal cortex only. Since glucocorticoids regulate both GR and CRHR1, we treated HAB and NAB mice chronically with CORT. After 6 weeks of administration, reduced anxiety- and depression-like behaviors were observed in HAB mice, whereas increased anxiety was found in NABs. In both groups, GR, but not CRHR1, were significantly reduced. Taken together, our study proposes HAB mice as an animal model of simultaneous features of increased anxiety-related and depression-like behaviors with blunted HPA axis reactivity suggesting a dysregulated GR/CORT system as one key mechanism behind their phenotype.