Reduction of DNMT3a and RORA in the nucleus accumbens plays a causal role in post-traumatic stress disorder-like behavior: reversal by combinatorial epigenetic therapy.

Post-traumatic stress disorder (PTSD) is an incapacitating trauma-related disorder, with no reliable therapy. Although PTSD has been associated with epigenetic alterations in peripheral white blood cells, it is unknown where such changes occur in the brain, and whether they play a causal role in PTSD. Using an animal PTSD model, we show distinct DNA methylation profiles of PTSD susceptibility in the nucleus accumbens (NAc). Data analysis revealed overall hypomethylation of different genomic CG sites in susceptible animals. This was correlated with the reduction in expression levels of the DNA methyltransferase, DNMT3a. Since epigenetic changes in diseases involve different gene pathways, rather than single candidate genes, we next searched for pathways that may be involved in PTSD. Analysis of differentially methylated sites identified enrichment in the RAR activation and LXR/RXR activation pathways that regulate Retinoic Acid Receptor (RAR) Related Orphan Receptor A (RORA) activation. Intra-NAc injection of a lentiviral vector expressing either RORA or DNMT3a reversed PTSD-like behaviors while knockdown of RORA and DNMT3a increased PTSD-like behaviors. To translate our results into a potential pharmacological therapeutic strategy, we tested the effect of systemic treatment with the global methyl donor S-adenosyl methionine (SAM), for supplementing DNA methylation, or retinoic acid, for activating RORA downstream pathways. We found that combined treatment with the methyl donor SAM and retinoic acid reversed PTSD-like behaviors. Thus, our data point to a novel approach to the treatment of PTSD, which is potentially translatable to humans.

Fetal glucocorticoid receptor (Nr3c1) deficiency alters the landscape of DNA methylation of murine placenta in a sex-dependent manner and is associated to anxiety-like behavior in adulthood.

Prenatal stress defines long-term phenotypes through epigenetic programming of the offspring. These effects are potentially mediated by glucocorticoid release and by sex. We hypothesized that the glucocorticoid receptor (Gr, Nr3c1) fashions the DNA methylation profile of offspring. Consistent with this hypothesis, fetal Nr3c1 heterozygosity leads to altered DNA methylation landscape in fetal placenta in a sex-specific manner. There was a significant overlap of differentially methylated genes in fetal placenta and adult frontal cortex in Nr3c1 heterozygotes. Phenotypically, Nr3c1 heterozygotes show significantly more anxiety-like behavior than wildtype. DNA methylation status of fetal placental tissue is significantly correlated with anxiety-like behavior of the same animals in adulthood. Thus, placental DNA methylation might predict behavioral phenotypes in adulthood. Our data supports the hypothesis that Nr3c1 influences DNA methylation at birth and that DNA methylation in placenta correlates with adult frontal cortex DNA methylation and anxiety-like phenotypes.

DNA methylation of the serotonin transporter gene (SLC6A4) is associated with brain function involved in processing emotional stimuli.

BACKGROUND: The aim of the present study was to investigate the association of fMRI blood oxygen-level dependent (BOLD) reactivity with the level of epigenetic methylation of SLC6A4 in blood DNA from a sample of healthy participants and patients with major depressive disorder (MDD). METHODS: We investigated patients with MDD and healthy controls using fMRI and an emotional attention-shifting task. We assessed site-specific DNA methylation of a previously characterized SLC6A4 region in peripheral blood DNA using pyrosequencing. RESULTS: Our study involved 25 patients with MDD and 35 healthy controls. Activation in the anterior insula elicited by negative emotional content was significantly positively associated with the degree of SLC6A4 methylation. Significantly negative associations were observed between activation in the posterior insula and the degree of SLC6A4 methylation when judging the geometry of pictures after seeing negative in contrast to positive emotional stimuli. Healthy controls with a high degree of SLC6A4 methylation depicted significantly more activity elicited by positive stimuli in limbic regions and more activity elicited by negative stimuli in limbic as well as cognitive control regions than those with a low degree of SLC6A4 methylation. LIMITATIONS: It is impossible to measure methylation directly in the brain and thus we assessed peripheral methylation of SLC6A4. Since the association was cross-sectional, no conclusion about cause and effect can be drawn. CONCLUSION: Our study provides further support to the hypothesis that particular DNA methylation states that are associated with brain function during emotion processing are detectable in the periphery.

DNA methylation of the serotonin transporter gene in peripheral cells and stress-related changes in hippocampal volume: a study in depressed patients and healthy controls.

Serotonin plays an important role in the etiology of depression. Serotonin is also crucial for brain development. For instance, animal studies have demonstrated that early disruptions in the serotonin system affect brain development and emotion regulation in later life. A plausible explanation is that environmental stressors reprogram the serotonin system through epigenetic processes by altering serotonin system gene expression. This in turn may affect brain development, including the hippocampus, a region with dense serotonergic innervations and important in stress-regulation. The aim of this study was to test whether greater DNA methylation in specific CpG sites at the serotonin transporter promoter in peripheral cells is associated with childhood trauma, depression, and smaller hippocampal volume. We were particularly interested in those CpG sites whose state of methylation in peripheral cells had previously been associated with in vivo measures of brain serotonin synthesis. Thirty-three adults with Major Depressive Disorder (MDD) (23 females) and 36 matched healthy controls (21 females) were included in the study. Depressive symptoms, childhood trauma, and high-resolution structural MRI for hippocampal volume were assessed. Site-specific serotonin transporter methylation was assessed using pyrosequencing. Childhood trauma, being male, and smaller hippocampal volume were independently associated with greater peripheral serotonin transporter methylation. Greater serotonin transporter methylation in the depressed group was observed only in SSRI-treated patients. These results suggest that serotonin transporter methylation may be involved in physiological gene-environment interaction in the development of stress-related brain alterations. The results provide some indications that site-specific serotonin transporter methylation may be a biomarker for serotonin-associated stress-related psychopathology.

Performance of a clonal-based HIV-1 tropism phenotypic assay.

Adequate determination of HIV-1 tropism is important in clinical and research settings. Genotypic and phenotypic approaches to evaluate tropism have been described. Phenotypic assays are widely used to determine HIV-1 tropism because of their sensitivity to detect minor CXCR4-using variants (X4). However they cannot differentiate mixed quasi-species of R5 and X4 viruses from dual-tropic viruses. We describe here a clonal-based HIV-1 tropism phenotypic assay. Env-pseudo-typed viruses were produced by co-transfection of the env expression plasmid pcDNA3.1/V5HisTOPO and a backbone vector pNL4-3.Luc.E-R- that expresses the entire HIV-1 genome except for env and vpr in 293T cell cultures. Co-receptor use was tested by infecting U87.CD4.CCR5+ and U87.CD4.CXCR4+ cells in the presence or absence of co-receptor inhibitors, using 10 clones from each sample. The ability of the assay to detect minor variants in a viral population was assessed by mixing X4 and R5 clones using different ratios. Both R5 and X4 minority variants were detected when present at greater than 0.4% in a mixture of envelope populations. This assay can be useful in both clinical and research laboratories.

Metalloestrogenic effects of quantum dots.

AIM: To investigate the metalloestrogenic effects of cadmium telluride quantum dots (QDs) in both human breast cancer cells and in vivo in mice. MATERIALS & METHODS: Human breast cancer cells (MCF-7 cells) were utilized to study QDs, cadmium and 17ß-estradiol induced estrogen-related genomic and nongenomic signaling. Female prepubescent and ovariectomized adult mice were treated with CdTe QDs to assess whether QD-induced estrogenicity would lead to uterine changes. RESULTS & DISCUSSION: Our findings demonstrate that in vitro cadmium-containing QDs induce cellular proliferation, estrogen receptor α activation, and biphasic phosphorylation of AKT and ERK1/2, comparable with 17ß-estradiol. Green QDs elicited a more robust estrogenic response than orange QDs. Addition of the selective estrogen receptor antagonist, ICI 182780, completely abolished all QD-induced estrogenic effects, suggesting that QD-induced estrogenic signaling is mediated via the estrogen receptor. In vivo, chronic treatment of mice with QDs led to a two- to three-fold increase in uterine weight, comparable or greater than 17ß-estradiol. CONCLUSION: These findings suggest that certain cadmium-containing nanocrystals are endocrine disruptors, whose effects can exceed those induced by ionic cadmium or 17ß-estradiol.

Reversibility of the effects of the chemotherapeutic regimen for non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone, on the male rat reproductive system and progeny outcome.

The chemotherapeutic agents used to treat non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), have adverse effects on male reproductive function and progeny outcome. To determine the reversibility of these effects, male rats received a CHOP treatment mimicking human exposure. CHOP reduced testicular and epididymal weights; these remained decreased after 9 weeks recovery. This treatment also decreased testicular sperm number and increased spermatozoal DNA damage. Although sperm production returned to control values after 9 weeks recovery, DNA damage persisted. Decreased litter size, and increased pre- and post-implantation losses were observed among litters sired by CHOP-exposed males. Litter size and pre-implantation loss returned to control within 3 weeks post-treatment and post-implantation loss by 6 weeks. Thus, effects of CHOP on progeny outcome were reversed 9 weeks post-treatment, although germ cell DNA breaks remained elevated. These data suggest that the ability to sire viable progeny may not be a sensitive measure of spermatozoal quality in rats.

Effects of the chemotherapeutic agents for non-Hodgkin lymphoma, cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP), on the male rat reproductive system and progeny outcome.

Chemotherapy of non-Hodgkin lymphoma (NHL) with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) is associated with significant gonadal damage. Our goal was to determine the impact of CHOP chemotherapy on the male reproductive system, fertility, and progeny outcome in the rat model. Adult male Sprague-Dawley rats received saline or CHOP, 4 cycles of 3 weeks each, at doses analogous to 1/3x, 2/3x, or 1x the human dose; males were mated to evaluate effects on progeny outcome. Reproductive organ weights were significantly decreased in the 1x CHOP-exposed group. The spermatozoal contents of the testes and epididymides were decreased in 1x CHOP-treated males; the 1/3x and 2/3x doses also affected testicular sperm contents. Seminiferous tubule diameters were decreased by 20% in 1x CHOP-treated males. Damage ranged from the presence of small vacuoles in the epithelium to tubules deprived of spermatocytes and spermatids and was accompanied by an increased incidence of germ cell apoptosis. The acridine orange assay revealed a significant increase in sperm with abnormal DNA integrity profiles in the 1x CHOP group. Despite effects on germ cell number and quality, CHOP-exposed rats remained fertile. However, a 50% decrease in live fetuses was observed in litters sired by 1x CHOP-treated males due to a significant increase in both pre-implantation and postimplantation losses; postimplantation loss was also elevated among litters sired by 2/3x CHOP-treated males. Thus, CHOP treatment affected both the quantity and quality of male germ cells; conceptal loss is a sensitive measure of the integrity of the male genome.