Elke Binder

Functional polymorphisms in the interleukin-6 and serotonin transporter genes, and depression and fatigue induced by interferon-α and ribavirin treatment

Depression and fatigue are frequent side effects of interferon-α (IFN-α) treatment, and there is compelling evidence that the inflammatory response system (including interleukin-6, IL-6) and the serotonergic system is important in the pathophysiology of such symptoms. Functional polymorphisms in the promoter region of the IL-6 gene (rs1800795) and serotonin transporter gene (5-HTTLPR) have been identified as regulating these systems. The present study aimed to determine if these polymorphisms were associated with the development of depression and fatigue during IFN-α and ribavirin treatment. Ninety-eight Caucasian patients receiving pegylated IFN-α and ribavirin treatment for chronic hepatitis C virus at Kings College Hospital, London, and Emory University Hospital, Atlanta, participated in this prospective cohort study. Symptoms of depression and fatigue were measured before treatment and at weeks 4, 8, 12 and 24 during treatment. The ‘low IL-6’ synthesizing genotype (CC) was associated with significantly fewer symptoms of depression (effect size=0.7 at week 24; F=9.4, d.f.=436, P=0.002). The ‘high transcription’ serotonin transporter (5-HTT) genotype (LL) was also associated with significantly fewer symptoms of depression, but with a much smaller effect (effect size=0.2 at week 24; F=4.5, d.f.=436, P=0.03). Neither polymorphisms were associated with symptoms of fatigue (IL-6: F=1.2, d.f.=430, P=0.2; 5-HTT: F=0.5, d.f.=430, P=0.5). The smaller effects of the 5-HTT polymorphism on depression may be explained by an interaction between the genes (F=5.0, d.f.=434, P=0.02): the ‘protective’ effect of the 5-HTTLPR polymorphism was evident only in the presence of the ‘low IL-6’ genotype (F=5.4, d.f.=64, P=0.02), not in the presence of the ‘high IL-6’ genotype (F=2.2, d.f.=369, P=0.1). The association between the IL-6 polymorphism and reduced risk of depressive symptoms confirms the role of the inflammatory response system in the pathophysiology of IFN-α-induced depression; in contrast, the effect of the 5-HTT gene was small and perhaps dependent on the status of the inflammatory response.

Regular voluntary exercise reduces anxiety-related behaviour and impulsiveness in mice.

We embarked on a study to delineate the behavioural changes in mice after 4 weeks of voluntary exercise. As an initial behavioural characterization, we exposed the control and exercising mice to a modified hole board and an open field test. As compared to control mice, exercising animals showed clear signs of increased behavioural inhibition (e.g. a longer latency to enter unprotected areas), suggesting increased anxiety in these animals. In addition, the exercising mice were reluctant to spend time in the open fields centre during the beginning of the 30-min open field test, but compensated for this at later times. Paradoxically, the exercising animals showed more rearings on the board of the modified hole board, indicating decreased anxiety. Thus, the behavioural inhibition seen in exercising mice is likely to represent decreased stress responsiveness at the behavioural level which can also be interpreted as reduced impulsiveness. To clarify whether voluntary exercise evolves in more or less anxiety-related behaviour, we exposed animals to the elevated plus-maze and the dark-light box, two selective tests for unconditioned anxiety. Clearly, compared to the control animals, exercising mice spent significantly more time on the open arm of the plus-maze and spent double the amount of time in the light compartment of the dark-light box. Taken together, we conclude that long-term voluntary exercise appears to result in decreased anxiety-related behaviour and impulsiveness. Thus, our observations fit into the concept that regular exercise strengthens endogenous stress coping mechanisms, thereby protecting the organism against the deleterious effects of stress.

Impact of high and low anxiety on cognitive performance in a modified hole board test in C57BL/6 and DBA/2 mice.

We investigated the interaction between behavioural dimensions and cognitive performance in the inbred mouse strains C57BL/6 and DBA/2, which have previously been found to differ in cognitive performance and emotionality. Because it has never been evaluated whether cognitive performance and emotional behaviour are interrelated in these strains, we analysed various behavioural dimensions and cognitive functions in parallel using the modified hole board test. We could show that naive BL6 and DBA mice distinctly differed in terms of anxiety‐related behaviour. Principal component analysis on the phenotyping data showed that anxiety‐related behaviour was described by identical parameters and was not correlated to locomotion in the two strains. During cognitive testing, DBA mice habituated faster and performed better than BL6 mice. Principal component analysis indicated a close correlation between anxiety‐related behaviour and cognitive performance in DBA mice, being associated with a highly successful cognitive performance. In BL6 mice, cognition was correlated to general exploration. This correlation turned out to be less successful in performing the modified hole board test. Our findings support the idea that high anxiety may interact with specific cognitive processing, thus offering a promising animal model for future preclinical research on the interaction of anxiety and cognition.

Differential analysis of behavior and diazepam-induced alterations in C57BL/6N and BALB/c mice using the modified hole board test.

A variety of test procedures are used in preclinical research on behavioral pharmacology and to dissociate behavioral differences or pharmacologically induced behavioral alterations several independent tests are usually performed. In the present study we introduce a modified hole board procedure for mice which allows us to investigate a variety of behavioral parameters such as anxiety, risk assessment, exploration, locomotion, food-intake inhibition, novelty seeking, and arousal by using only one test. The modified hole board was established by investigating the behavior of two inbred mouse strains, C57BL/6 and BALB. Significant differences in terms of locomotor activity, general exploration, and other parameters were found. Moreover, strain-specific exploration strategies could be detected in the modified hole board. Further, the test was validated by investigating the effects of diazepam as standard anxiolytic on the behavior in both mouse strains. Acute administration of diazepam (1 and 3 mg/kg) induced strong sedative effects in a dose-dependent manner in C57BL/6 mice. In BALB mice, the lower dosage of diazepam showed an activating and anxiolytic action while the 3 mg dosage revealed a slight sedative but still anxiolytic effect in these animals. Taken together, the results demonstrate that the modified hole board enables to differentially investigate behavioral phenotypes and also pharmacologically-induced behavioral alterations in mice. Therefore, this new strategy allows to reduce the number of experimental animals and the time needed, thus, representing an effective screening-tool for behavioral investigations.

Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB₁ receptor blockade.

Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of β-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral β-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors.

A DNA methylation biomarker of alcohol consumption

The lack of reliable measures of alcohol intake is a major obstacle to the diagnosis and treatment of alcohol-related diseases. Epigenetic modifications such as DNA methylation may provide novel biomarkers of alcohol use. To examine this possibility, we performed an epigenome-wide association study of methylation of cytosine-phosphate-guanine dinucleotide (CpG) sites in relation to alcohol intake in 13 population-based cohorts (ntotal=13 317; 54% women; mean age across cohorts 42–76 years) using whole blood (9643 European and 2423 African ancestries) or monocyte-derived DNA (588 European, 263 African and 400 Hispanic ancestry) samples. We performed meta-analysis and variable selection in whole-blood samples of people of European ancestry (n=6926) and identified 144 CpGs that provided substantial discrimination (area under the curve=0.90–0.99) for current heavy alcohol intake (⩾42 g per day in men and ⩾28 g per day in women) in four replication cohorts. The ancestry-stratified meta-analysis in whole blood identified 328 (9643 European ancestry samples) and 165 (2423 African ancestry samples) alcohol-related CpGs at Bonferroni-adjusted P<1 × 10−7. Analysis of the monocyte-derived DNA (n=1251) identified 62 alcohol-related CpGs at P<1 × 10-7. In whole-blood samples of people of European ancestry, we detected differential methylation in two neurotransmitter receptor genes, the γ-Aminobutyric acid-A receptor delta and γ-aminobutyric acid B receptor subunit 1; their differential methylation was associated with expression levels of a number of genes involved in immune function. In conclusion, we have identified a robust alcohol-related DNA methylation signature and shown the potential utility of DNA methylation as a clinically useful diagnostic test to detect current heavy alcohol consumption.

Leucine supplementation protects from insulin resistance by regulating adiposity levels.

Background Leucine supplementation might have therapeutic potential in preventing diet-induced obesity and improving insulin sensitivity. However, the underlying mechanisms are at present unclear. Additionally, it is unclear whether leucine supplementation might be equally efficacious once obesity has developed. Methodology/Principal Findings Male C57BL/6J mice were fed chow or a high-fat diet (HFD), supplemented or not with leucine for 17 weeks. Another group of HFD-fed mice (HFD-pairfat group) was food restricted in order to reach an adiposity level comparable to that of HFD-Leu mice. Finally, a third group of mice was exposed to HFD for 12 weeks before being chronically supplemented with leucine. Leucine supplementation in HFD-fed mice decreased body weight and fat mass by increasing energy expenditure, fatty acid oxidation and locomotor activity in vivo. The decreased adiposity in HFD-Leu mice was associated with increased expression of uncoupling protein 3 (UCP-3) in the brown adipose tissue, better insulin sensitivity, increased intestinal gluconeogenesis and preservation of islets of Langerhans histomorphology and function. HFD-pairfat mice had a comparable improvement in insulin sensitivity, without changes in islets physiology or intestinal gluconeogenesis. Remarkably, both HFD-Leu and HFD-pairfat mice had decreased hepatic lipid content, which likely helped improve insulin sensitivity. In contrast, when leucine was supplemented to already obese animals, no changes in body weight, body composition or glucose metabolism were observed. Conclusions/Significance These findings suggest that leucine improves insulin sensitivity in HFD-fed mice by primarily decreasing adiposity, rather than directly acting on peripheral target organs. However, beneficial effects of leucine on intestinal gluconeogenesis and islets of Langerhanss physiology might help prevent type 2 diabetes development. Differently, metabolic benefit of leucine supplementation is lacking in already obese animals, a phenomenon possibly related to the extent of the obesity before starting the supplementation.

Convergent animal and human evidence suggests a role of PPM1A gene in response to antidepressants.

BACKGROUND Antidepressant drugs are used as first-line treatment in depression, but response has been shown to be highly heterogeneous, with drugs often failing to have the desired therapeutic effect. We report on an integrative analysis from the Genome-Based Therapeutic Drugs for Depression (GENDEP) study using gene expression from mice to inform prioritization in a human pharmacogenetic study. METHODS The same two antidepressants were used in mice and humans: escitalopram (a serotonin reuptake inhibitor) and nortriptyline (a norepinephrine reuptake inhibitor). The animal study used four inbred strains of mice (129S1/SvlmJ, C57LB/6J, DBA/2J, and FVB/NJ). Hippocampus mRNA levels were measured in 144 animals using the Affymetrix MOE 430 v2 chip. RESULTS Based on gene-expression analysis of strain-by-drug interactions, 17 genes differentially expressed with nortriptyline or escitalopram versus saline were prioritized in the human pharmacogenetic analysis. Single nucleotide polymorphisms tagging common sequence variation in human orthologs of these genes were tested for association with response to antidepressants in 706 participants of the GENDEP human pharmacogenetic study, treated with escitalopram or nortriptyline for 12 weeks, with available high-quality Illumina 610 quad array genotyping. Several polymorphisms in the protein phosphatase 1A gene (PPM1A) remained significantly associated with response to nortriptyline in humans after correction for multiple comparisons within the gene. PPM1A encodes a phosphatase involved in mitogen-activated protein kinase signaling and cell stress response. CONCLUSIONS Convergent evidence from mice and humans suggests a role of the PPM1A in response to noradrenergic but not serotonergic antidepressants.

Antidepressants and the resilience to early-life stress in inbred mouse strains

Rationale Selecting an effective treatment for patients with major depressive disorder is a perpetual problem for psychiatrists. It is of particular interest to explore the interaction between genetic predisposition and environmental factors. Objectives Mouse inbred strains vary in baseline performance in depression-related behaviour tests, which were originally validated as tests of antidepressant response. Therefore, we investigated interactions between environmental stress, genotype, and drug response in a multifactorial behaviour study. Method Our study design included four inbred mouse strains (129S1/SvlmJ, C57LB/6J, DBA/2J and FVB/NJ) of both sexes, two subjected to environmental manipulations (maternal separation and unpredictable chronic mild stress) and two representative of treatment with antidepressants (escitalopram and nortryptiline vs. vehicle). The mice treated with antidepressants were further divided into those administered acute (1 day) and subchronic (14 days) regimes, giving 144 experimental groups in all, each with at least seven animals. All animals were tested using the Porsolt forced-swim test (FST) and the hole-board test. Results Despite a 24-h maternal separation (MS) or a 14-day unpredictable chronic mild stress protocol, most animals seemed to be resilient to the stress induced. One compelling finding is the long-lasting, strain-specific effect of MS resulting in an increased depression-like behaviour in the Porsolt FST and elevated anxiety-related behaviour in the hole-board test seen in 129S1/SvImJ mice. Nortriptyline was effective in reversing the effect of MS in the FST in 129S1/SvlmJ male mice. Conclusion A single 24-h maternal separation of pups from their mother on postnatal day 9 is a sufficient insult to result in a depression-like phenotype in adult 129S1/SvImJ mice but not in C57LB/6 J, DBA/2 J, and FVB/NJ mice.

Antidepressant-dependent mRNA changes in mouse associated with hippocampal neurogenesis in a mouse model of depression

Rationale Monoaminergic imbalances play a role in the pathogenesis of depression and most common antidepressant drugs act on monoamine neurotransmitters. However, the lag time between restoring neurochemical balance and symptom improvement suggests that the response to drugs involves complex biological events downstream of primary targets that have not yet been fully characterized. Here, we report a mouse mRNA expression study to evaluate the effect of escitalopram (a serotonergic antidepressant) and nortriptyline (a noradrenergic antidepressant) on genes that are involved in the pathogenesis of depression and to assess the similarities and differences between two drugs on gene expression levels. Methods Genome-wide RNA expression data from the hippocampal tissues of four inbred mouse strains (129S1/SvlmJ, C57LB/6J, DBA/2J and FVB/NJ) were treated with varying doses of either nortriptyline (NRI) or escitalopram (SSRI) and subjected to two different depressogenic protocols. Following robust multichip average normalization, we applied the nonparametric RankProd approach to identify differentially expressed genes in response to drugs across the four strains. Pathway analysis was subsequently carried out on top-ranking genes to gain further biological insights. Results A total of 371 genes were significantly differentially expressed in response to nortriptyline, whereas 383 were altered by escitalopram. Genes involved in the pathways of integrin signalling (Fnlb, Mapk1, Mapk8), synaptic transmission (Cacnb1, Dnajc5, Kcnma1, Slc1a2) or Huntington disease (Crebbp, Dlg4, Ncor1) were altered by both nortriptyline and escitalopram. Several biological processes and pathways were identified, which could explain the divergence between the molecular mechanisms of nortriptyline and escitalopram. Conclusion From a large-scale animal study, we obtain gene sets comprised of commonly and differentially expressed genes in response to different antidepressant drug treatments. The results may help to characterize the response to antidepressant treatment, shed further light on the neurobiology of depressive disorders and inform future animal and human studies. Finally, the top-ranking pathways from Ingenuity provide further evidence for the hippocampal neurogenesis hypothesis of major depressive disorders.