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.

Prenatal S-Adenosine Methionine (SAMe) Induces Changes in Gene Expression in the Brain of Newborn Mice That Are Prevented by Co-Administration of Valproic Acid (VPA).

In previous studies, we produced changes in gene expression in the brain of mice by early postnatal administration of valproic acid (VPA), with distinct differences between genders. The addition of S-adenosine methionine (SAMe) normalized the expression of most genes in both genders, while SAMe alone induced no changes. We treated pregnant dams with a single injection of VPA on day 12.5 of gestation, or with SAMe during gestational days 12-14, or by a combination of VPA and SAMe. In the frontal half of the brain, we studied the expression of 770 genes of the pathways involved in neurophysiology and neuropathology using the NanoString nCounter method. SAMe, but not VPA, induced statistically significant changes in the expression of many genes, with differences between genders. The expression of 112 genes was changed in both sexes, and another 170 genes were changed only in females and 31 only in males. About 30% of the genes were changed by more than 50%. One of the most important pathways changed by SAMe in both sexes was the VEGF (vascular endothelial growth factor) pathway. Pretreatment with VPA prevented almost all the changes in gene expression induced by SAMe. We conclude that large doses of SAMe, if administered prenatally, may induce significant epigenetic changes in the offspring. Hence, SAMe and possibly other methyl donors may be epigenetic teratogens.

Personalized Cardio-Metabolic Responses to an Anti-Inflammatory Nutrition Intervention in Obese Adolescents: A Randomized Controlled Crossover Trial.

SCOPE: Chronic inflammation and hypoadiponectinemia are characteristics of obesity-induced insulin resistance (IR). The effect of an anti-inflammatory nutrition supplement (AINS) on IR and adiponectin biology in overweight adolescents was investigated. The secondary objective was to examine the extent to which individuals' biomarker profiles, derived from baseline phenotypes, predicted response or not to the AINS. Additionally, the impact of DNA methylation on intervention efficacy was assessed. METHODS AND RESULTS: Seventy overweight adolescents (13-18 years) were recruited to this randomized controlled crossover trial. Participants received an AINS (long chain n-3 PUFA, vitamin C, α-tocopherol, green tea extract, and lycopene) and placebo for 8 weeks each. Homeostatic model assessment (HOMA)-IR, adiponectin, inflammatory profiles, and DNA methylation were assessed. HOMA-IR was unchanged in the total cohort. High-molecular-weight (HMW) adiponectin was maintained following the AINS while it decreased over time following the placebo intervention. HOMA-IR decreased in 40% of subjects (responders) following the AINS. Responders' pretreatment phenotype was characterized by higher HOMA-IR, total and LDL cholesterol, but similar BMI in comparison to nonresponders. HMW adiponectin response to the AINS was associated with bidirectional modulation of adipogenic gene methylation. CONCLUSION: The AINS modulated adiponectin biology, an early predictor of type 2 diabetes risk, was associated with bidirectional modulation of adipogenic gene methylation in weight-stable overweight adolescents. HOMA-IR decreased in a sub-cohort of adolescents with an adverse metabolic phenotype. Thus, suggesting that more stratified or personalized nutrition approaches may enhance efficacy of dietary interventions.

Methyl donor S-adenosylmethionine (SAM) supplementation attenuates breast cancer growth, invasion, and metastasis in vivo; therapeutic and chemopreventive applications.

DNA hypomethylation coordinately targets various signaling pathways involved in tumor growth and metastasis. At present, there are no approved therapeutic modalities that target hypomethylation. In this regard, we examined the therapeutic plausibility of using universal methyl group donor S-adenosylmethionine (SAM) to block breast cancer development, growth, and metastasis through a series of studies in vitro using two different human breast cancer cell lines (MDA-MB-231 and Hs578T) and in vivo using an MDA-MB-231 xenograft model of breast cancer. We found that SAM treatment caused a significant dose-dependent decrease in cell proliferation, invasion, migration, anchorage-independent growth and increased apoptosis in vitro. These results were recapitulated in vivo where oral administration of SAM reduced tumor volume and metastasis in green fluorescent protein (GFP)-tagged MDA-MB-231 xenograft model. Gene expression analyses validated the ability of SAM to decrease the expression of several key genes implicated in cancer progression and metastasis in both cell lines and breast tumor xenografts. SAM was found to be bioavailable in the serum of experimental animals as determined by enzyme-linked immunosorbent assay and no notable adverse side effects were seen including any change in animal behavior. The results of this study provide compelling evidence to evaluate the therapeutic potential of methylating agents like SAM in patients with breast cancer to reduce cancer-associated morbidity and mortality.

Therapeutic benefits of the methyl donor S-adenosylmethionine on nerve injury-induced mechanical hypersensitivity and cognitive impairment in mice.

Despite considerable advances in understanding mechanisms involved in chronic pain, effective treatment remains elusive. Comorbid conditions including anxiety, depression, and cognitive impairment further impact quality of life. Chronic pain is associated with reversible changes in brain anatomy and function and with long-term changes in gene expression. Epigenetic mechanisms, including DNA methylation, contribute to wide-spread and long-lasting reprogramming of gene expression. We previously reported decreases in global DNA methylation in the mouse frontal cortex 6 months after induction of neuropathic pain using the spared nerve injury (SNI) model. Here, we examined the therapeutic effect of increasing DNA methylation using the methyl donor S-adenosylmethionine (SAM). S-adenosylmethionine is marketed as a nutritional supplement for a range of conditions including liver disease, depression, osteoarthritis, fibromyalgia, and dementia. Three months after SNI or sham surgery, animals were treated with SAM (20 mg/kg, 3×/week) or saline orally for 4 months, and the impact on sensory, motor, motivational, and cognitive indices was measured. S-adenosylmethionine attenuated SNI-induced mechanical hypersensitivity and reduced active avoidance of mechanical stimuli but had no effect on cold sensitivity or motor capacity. S-adenosylmethionine completely blocked nerve injury-induced cognitive impairment and attenuated SNI-induced decreases in global DNA methylation in the frontal cortex. In summary, chronic oral administration of the methyl donor, SAM, attenuated sensory and cognitive symptoms associated with nerve injury in mice. These effects may be mediated, in part, through modulation of DNA methylation in the central nervous system by systemic administration of the methyl donor SAM.

Preclinical optimization of a broad-spectrum anti-bladder cancer tri-drug regimen via the Feedback System Control (FSC) platform.

Therapeutic outcomes of combination chemotherapy have not significantly advanced during the past decades. This has been attributed to the formidable challenges of optimizing drug combinations. Testing a matrix of all possible combinations of doses and agents in a single cell line is unfeasible due to the virtually infinite number of possibilities. We utilized the Feedback System Control (FSC) platform, a phenotype oriented approach to test 100 options among 15,625 possible combinations in four rounds of assaying to identify an optimal tri-drug combination in eight distinct chemoresistant bladder cancer cell lines. This combination killed between 82.86% and 99.52% of BCa cells, but only 47.47% of the immortalized benign bladder epithelial cells. Preclinical in vivo verification revealed its markedly enhanced anti-tumor efficacy as compared to its bi- or mono-drug components in cell line-derived tumor xenografts. The collective response of these pathways to component drugs was both cell type- and drug type specific. However, the entire spectrum of pathways triggered by the tri-drug regimen was similar in all four cancer cell lines, explaining its broad spectrum killing of BCa lines, which did not occur with its component drugs. Our findings here suggest that the FSC platform holds promise for optimization of anti-cancer combination chemotherapy.

Pharmacological methyl group donors block skeletal metastasis in vitro and in vivo.

BACKGROUND AND PURPOSE: DNA hypomethylation was previously implicated in metastasis. In the present study, we examined whether methyl supplementation with the universal methyl donor S-adenosylmethionine (SAM) inhibits prostate cancer associated skeletal metastasis. EXPERIMENTAL APPROACH: Highly invasive human prostate cancer cells PC-3 and DU-145 were treated with vehicle alone, S-adenosylhomocysteine (SAH) or SAM and their effects on tumour cell proliferation, invasion, migration and colony formation were monitored. For in vivo studies, control (SAH) and SAM-treated PC-3 cells were injected into the tibia of Fox chase SCID mice and skeletal lesions were determined by X-ray and µCT. To understand possible mechanisms involved, we delineated the effect of SAM on the genome-wide methylation profile of PC-3 cells. KEY RESULTS: Treatment with SAM resulted in a dose-dependent inhibition of tumour cell proliferation, invasion, cell migration, colony formation and cell cycle characteristics. Animals injected with 250 µM SAM-treated cells developed significantly smaller skeletal lesions, which were associated with increases in bone volume to tumour volume ratio and connectivity density as well as decreased trabecular spacing. Genome-wide methylation analysis showed differential methylation in several key signalling pathways implicated in prostate cancer including the signal transducer and activator of transcription 3 (STAT3) pathway. A selective STAT3 inhibitor decreased tumour cell invasion, effects which were less pronounced as compared with SAM. CONCLUSIONS AND IMPLICATIONS: These studies provide a possible mechanism for the role of DNA demethylation in the development of skeletal metastasis and a rationale for the use of hypermethylation pharmacological agents to impede the development and progression of skeletal metastasis.

Placental oxidative stress and decreased global DNA methylation are corrected by copper in the Cohen diabetic rat.

Fetal Growth Restriction (FGR) is a leading cause for long term morbidity. The Cohen diabetic sensitive rats (CDs), originating from Wistar, develop overt diabetes when fed high sucrose low copper diet (HSD) while the original outbred Sabra strain do not. HSD induced FGR and fetal oxidative stress, more prominent in the CDs, that was alleviated more effectively by copper than by the anti-oxidant vitamins C and E. Our aim was to evaluate the impact of copper or the anti-oxidant Tempol on placental size, protein content, oxidative stress, apoptosis and total DNA methylation. Animals were mated following one month of HSD or regular chow diet and supplemented throughout pregnancy with either 0, 1 or 2 ppm of copper sulfate or Tempol in their drinking water. Placental weight on the 21st day of pregnancy decreased in dams fed HSD and improved upon copper supplementation. Placental/fetal weight ratio increased among the CDs. Protein content decreased in Sabra but increased in CDs fed HSD. Oxidative stress biochemical markers improved upon copper supplementation; immunohistochemistry for oxidative stress markers was similar between strains and diets. Caspase 3 was positive in more placentae of dams fed HSD than those fed RD. Placental global DNA methylation was decreased only among the CDs dams fed HSD. We conclude that FGR in this model is associated with smaller placentae, reduced DNA placental methylation, and increased oxidative stress that normalized with copper supplementation. DNA hypomethylation makes our model a unique method for investigating genes associated with growth, oxidative stress, hypoxia and copper.

Maternal programming of defensive responses through sustained effects on gene expression.

There are profound maternal effects on individual differences in defensive responses and reproductive strategies in species ranging literally from plants to insects to birds. Maternal effects commonly reflect the quality of the environment and are most likely mediated by the quality of the maternal provision (egg, propagule, etc.), which in turn determines growth rates and adult phenotype. In this paper we review data from the rat that suggest comparable forms of maternal effects on defensive responses stress, which are mediated by the effects of variations in maternal behavior on gene expression. Under conditions of environmental adversity maternal effects enhance the capacity for defensive responses in the offspring. In mammals, these effects appear to 'program' emotional, cognitive and endocrine systems towards increased sensitivity to adversity. In environments with an increased level of adversity, such effects can be considered adaptive, enhancing the probability of offspring survival to sexual maturity; the cost is that of an increased risk for multiple forms of pathology in later life.

Reversal of maternal programming of stress responses in adult offspring through methyl supplementation: altering epigenetic marking later in life.

Stress responses in the adult rat are programmed early in life by maternal care and associated with epigenomic marking of the hippocampal exon 1(7) glucocorticoid receptor (GR) promoter. To examine whether such epigenetic programming is reversible in adult life, we centrally infused the adult offspring with the essential amino acid L-methionine, a precursor to S-adenosyl-methionine that serves as the donor of methyl groups for DNA methylation. Here we report that methionine infusion reverses the effect of maternal behavior on DNA methylation, nerve growth factor-inducible protein-A binding to the exon 1(7) promoter, GR expression, and hypothalamic-pituitary-adrenal and behavioral responses to stress, suggesting a causal relationship among epigenomic state, GR expression, and stress responses in the adult offspring. These results demonstrate that, despite the inherent stability of the epigenomic marks established early in life through behavioral programming, they are potentially reversible in the adult brain.