S-adenosylmethionine blocks tumorigenesis and with immune checkpoint inhibitor enhances anti-cancer efficacy against BRAF mutant and wildtype melanomas.

Despite marked success in treatment with immune checkpoint inhibitor (CPI), only a third of patients are responsive. Thus, melanoma still has one of the highest prevalence and mortality rates; which has led to a search for novel combination therapies that might complement CPI. Aberrant methylomes are one of the mechanisms of resistance to CPI therapy. S-adenosylmethionine (SAM), methyl donor of important epigenetic processes, has significant anti-cancer effects in several malignancies; however, SAM's effect has never been extensively investigated in melanoma. We demonstrate that SAM modulates phenotype switching of melanoma cells and directs the cells towards differentiation indicated by increased melanogenesis (melanin and melanosome synthesis), melanocyte-like morphology, elevated Mitf and Mitf activators' expression, increased antigen expression, reduced proliferation, and reduced stemness genes' expression. Consistently, providing SAM orally, reduced tumor growth and progression, and metastasis of syngeneic BRAF mutant and wild-type (WT) melanoma mouse models. Of note, SAM and anti-PD-1 antibody combination treatment had enhanced anti-cancer efficacy compared to monotherapies, showed significant reduction in tumor growth and progression, and increased survival. Furthermore, SAM and anti-PD-1 antibody combination triggered significantly higher immune cell infiltration, higher CD8+ T cells infiltration and effector functions, and polyfunctionality of CD8+ T cells in YUMMER1.7 tumors. Therefore, SAM combined with CPI provides a novel therapeutic strategy against BRAF mutant and WT melanomas and provides potential to be translated into clinic.

Building knowledge, optimising physical and mental health and setting up healthier life trajectories in South African women (Bukhali): a preconception randomised control trial part of the Healthy Life Trajectories Initiative (HeLTI).

INTRODUCTION: South Africa's evolving burden of disease is challenging due to a persistent infectious disease, burgeoning obesity, most notably among women and rising rates of non-communicable diseases (NCDs). With two thirds of women presenting at their first antenatal visit either overweight or obese in urban South Africa (SA), the preconception period is an opportunity to optimise health and offset transgenerational risk of both obesity and NCDs. METHODS AND ANALYSIS: Bukhali is the first individual randomised controlled trial in Africa to test the efficacy of a complex continuum of care intervention and forms part of the Healthy Life Trajectories Initiative (HeLTI) consortium implementing harmonised trials in Canada, China, India and SA. Starting preconception and continuing through pregnancy, infancy and childhood, the intervention is designed to improve nutrition, physical and mental health and health behaviours of South African women to offset obesity-risk (adiposity) in their offspring. Women aged 18-28 years (n=6800) will be recruited from Soweto, an urban-poor area of Johannesburg. The primary outcome is dual-energy X-ray absorptiometry derived fat mass index (fat mass divided by height2) in the offspring at age 5 years. Community health workers will deliver the intervention randomly to half the cohort by providing health literacy material, dispensing a multimicronutrient supplement, providing health services and feedback, and facilitating behaviour change support sessions to optimise: (1) nutrition, (2) physical and mental health and (3) lay the foundations for healthier pregnancies and early child development. ETHICS AND DISSEMINATION: Ethical approval has been obtained from the Human Ethics Research Committee University of the Witwatersrand, Johannesburg, South Africa (M1811111), the University of Toronto, Canada (19-0066-E) and the WHO Ethics Committee (ERC.0003328). Data and biological sample sharing policies are consistent with the governance policy of the HeLTI Consortium (https://helti.org) and South African government legislation (POPIA). The recruitment and research team will obtain informed consent. TRIAL REGISTRATION: This trial is registered with the Pan African Clinical Trials Registry (https://pactr.samrc.ac.za) on 25 March 2019 (identifier: PACTR201903750173871). PROTOCOL VERSION: 20 March 2022 (version #4). Any protocol amendments will be communicated to investigators, Institutional Review Board (IRB)s, trial participants and trial registries.

Prenatal stress and epigenetics.

In utero exposure to environmental stress in both animals and humans could result in long-term epigenome alterations which further lead to consequences for adaptation and development in the offspring. Epigenetics, especially DNA methylation, is considered one of the most widely studied and well-characterized mechanisms involved in the long-lasting effects of in utero stress exposure. In this review, we outlined evidence from animal and human prenatal research supporting the view that prenatal stress could lead to lasting, broad and functionally organized signatures in DNA methylation which, in turn, could mediate exposure-phenotype associations. We also emphasized the advantage of using stressor from quasi-randomly assigned experiments. Furthermore, we discuss challenges that still need to be addressed in this field in the future.

Associations among oxytocin receptor gene (OXTR) DNA methylation in adulthood, exposure to early life adversity, and childhood trajectories of anxiousness.

Recent models propose deoxyribonucleic acid methylation of key neuro-regulatory genes as a molecular mechanism underlying the increased risk of mental disorder associated with early life adversity (ELA). The goal of this study was to examine the association of ELA with oxytocin receptor gene (OXTR) methylation among young adults. Drawing from a 21-year longitudinal cohort, we compared adulthood OXTR methylation frequency of 46 adults (23 males and 23 females) selected for high or low ELA exposure based on childhood socioeconomic status and exposure to physical and sexual abuse during childhood and adolescence. Associations between OXTR methylation and teacher-rated childhood trajectories of anxiousness were also assessed. ELA exposure was associated with one significant CpG site in the first intron among females, but not among males. Similarly, childhood trajectories of anxiousness were related to one significant CpG site within the promoter region among females, but not among males. This study suggests that females might be more sensitive to the impact of ELA on OXTR methylation than males.

PARP-1 is required for retrieval of cocaine-associated memory by binding to the promoter of a novel gene encoding a putative transposase inhibitor.

Reward-related memory is an important factor in cocaine seeking. One necessary signaling mechanism for long-term memory formation is the activation of poly(ADP-ribose) polymerase-1 (PARP-1), via poly(ADP-ribosyl)ation. We demonstrate herein that auto-poly(ADP-ribosyl)ation of activated PARP-1 was significantly pronounced during retrieval of cocaine-associated contextual memory, in the central amygdala (CeA) of rats expressing cocaine-conditioned place preference (CPP). Intra-CeA pharmacological and short hairpin RNA depletion of PARP-1 activity during cocaine-associated memory retrieval abolished CPP. In contrast, PARP-1 inhibition after memory retrieval did not affect CPP reconsolidation process and subsequent retrievals. Chromatin immunoprecipitation sequencing revealed that PARP-1 binding in the CeA is highly enriched in genes involved in neuronal signaling. We identified among PARP targets in CeA a single gene, yet uncharacterized and encoding a putative transposase inhibitor, at which PARP-1 enrichment markedly increases during cocaine-associated memory retrieval and positively correlates with CPP. Our findings have important implications for understanding drug-related behaviors, and suggest possible future therapeutic targets for drug abuse.

Ankyrin-3 as a molecular marker of early-life stress and vulnerability to psychiatric disorders.

Exposure to early-life stress (ELS) may heighten the risk for psychopathology at adulthood. Here, in order to identify common genes that may keep the memory of ELS through changes in their methylation status, we intersected methylome analyses performed in different tissues and time points in rats, non-human primates and humans, all characterized by ELS. We identified Ankyrin-3 (Ank3), a scaffolding protein with a strong genetic association for psychiatric disorders, as a gene persistently affected by stress exposure. In rats, Ank3 methylation and mRNA changes displayed a specific temporal profile during the postnatal development. Moreover, exposure to prenatal stress altered the interaction of ankyrin-G, the protein encoded by Ank3 enriched in the post-synaptic compartment, with PSD95. Notably, to model in humans a gene by early stress interplay on brain phenotypes during cognitive performance, we demonstrated an interaction between functional variation in Ank3 gene and obstetric complications on working memory in healthy adult subjects. Our data suggest that alterations of Ank3 expression and function may contribute to the effects of ELS on the development of psychiatric disorders.

Maternal depression is associated with DNA methylation changes in cord blood T lymphocytes and adult hippocampi.

Depression affects 10-15% of pregnant women and has been associated with preterm delivery and later developmental, behavioural and learning disabilities. We tested the hypothesis that maternal depression is associated with DNA methylation alterations in maternal T lymphocytes, neonatal cord blood T lymphocytes and adult offspring hippocampi. Genome-wide DNA methylation of CD3+ T lymphocytes isolated from 38 antepartum maternal and 44 neonatal cord blood samples were analyzed using Illumina Methylation 450 K microarrays. Previously obtained methylation data sets using methylated DNA immunoprecipitation and array-hybridization of 62 postmortem hippocampal samples of adult males were re-analyzed to test associations with history of maternal depression. We found 145 (false discovery rate (FDR) q<0.05) and 2520 (FDR q<0.1) differentially methylated CG-sites in cord blood T lymphocytes of neonates from the maternal depression group as compared with the control group. However, no significant DNA methylation differences were detected in the antepartum maternal T lymphocytes of our preliminary data set. We also detected 294 differentially methylated probes (FDR q<0.1) in hippocampal samples associated with history of maternal depression. We observed a significant overlap (P=0.002) of 33 genes with changes in DNA methylation in T lymphocytes of neonates and brains of adult offspring. Many of these genes are involved in immune system functions. Our results show that DNA methylation changes in offspring associated with maternal depression are detectable at birth in the immune system and persist to adulthood in the brain. This is consistent with the hypothesis that system-wide epigenetic changes are involved in life-long responses to maternal depression in the offspring.

Pregnant women's cognitive appraisal of a natural disaster affects DNA methylation in their children 13 years later: Project Ice Storm.

Prenatal maternal stress (PNMS) can impact a variety of outcomes in the offspring throughout childhood and persisting into adulthood as shown in human and animal studies. Many of the effects of PNMS on offspring outcomes likely reflect the effects of epigenetic changes, such as DNA methylation, to the fetal genome. However, no animal or human research can determine the extent to which the effects of PNMS on DNA methylation in human offspring is the result of the objective severity of the stressor to the pregnant mother, or her negative appraisal of the stressor or her resulting degree of negative stress. We examined the genome-wide DNA methylation profile in T cells from 34 adolescents whose mothers had rated the 1998 Québec ice storm's consequences as positive or negative (that is, cognitive appraisal). The methylation levels of 2872 CGs differed significantly between adolescents in the positive and negative maternal cognitive appraisal groups. These CGs are affiliated with 1564 different genes and with 408 different biological pathways, which are prominently featured in immune function. Importantly, there was a significant overlap in the differentially methylated CGs or genes and biological pathways that are associated with cognitive appraisal and those associated with objective PNMS as we reported previously. Our study suggests that pregnant women's cognitive appraisals of an independent stressor may have widespread effects on DNA methylation across the entire genome of their unborn children, detectable during adolescence. Therefore, cognitive appraisals could be an important predictor variable to explore in PNMS research.

Astrocytic abnormalities and global DNA methylation patterns in depression and suicide.

Astrocytes are glial cells specific to the central nervous system and involved in numerous brain functions, including regulation of synaptic transmission and of immune reactions. There is mounting evidence suggesting astrocytic dysfunction in psychopathologies such as major depression, however, little is known about the underlying etiological mechanisms. Here we report a two-stage study investigating genome-wide DNA methylation associated with astrocytic markers in depressive psychopathology. We first characterized prefrontal cortex samples from 121 individuals (76 who died during a depressive episode and 45 healthy controls) for the astrocytic markers GFAP, ALDH1L1, SOX9, GLUL, SCL1A3, GJA1 and GJB6. A subset of 22 cases with consistently downregulated astrocytic markers was then compared with 17 matched controls using methylation binding domain-2 (MBD2) sequencing followed by validation with high-resolution melting and bisulfite Sanger sequencing. With these data, we generated a genome-wide methylation map unique to altered astrocyte-associated depressive psychopathology. The map revealed differentially methylated regions (DMRs) between cases and controls, the majority of which displayed reduced methylation levels in cases. Among intragenic DMRs, those found in GRIK2 (glutamate receptor, ionotropic kainate 2) and BEGAIN (brain-enriched guanylate kinase-associated protein) were most significant and also showed significant correlations with gene expression. Cell-sorted fractions were investigated and demonstrated an important non-neuronal contribution of methylation status in BEGAIN. Functional cell assays revealed promoter and enhancer-like properties in this region that were markedly decreased by methylation. Furthermore, a large number of our DMRs overlapped known Encyclopedia of DNA elements (ENCODE)-identified regulatory elements. Taken together, our data indicate significant differences in the methylation patterns specific to astrocytic dysfunction associated with depressive psychopathology, providing a potential framework for better understanding this disease phenotype.

MORC1 exhibits cross-species differential methylation in association with early life stress as well as genome-wide association with MDD.

Early life stress (ELS) is associated with increased vulnerability for diseases in later life, including psychiatric disorders. Animal models and human studies suggest that this effect is mediated by epigenetic mechanisms. In humans, epigenetic studies to investigate the influence of ELS on psychiatric phenotypes are limited by the inaccessibility of living brain tissue. Due to the tissue-specific nature of epigenetic signatures, it is impossible to determine whether ELS induced epigenetic changes in accessible peripheral cells, for example, blood lymphocytes, reflect epigenetic changes in the brain. To overcome these limitations, we applied a cross-species approach involving: (i) the analysis of CD34+ cells from human cord blood; (ii) the examination of blood-derived CD3+ T cells of newborn and adolescent nonhuman primates (Macaca mulatta); and (iii) the investigation of the prefrontal cortex of adult rats. Several regions in MORC1 (MORC family CW-type zinc finger 1; previously known as: microrchidia (mouse) homolog) were differentially methylated in response to ELS in CD34+ cells and CD3+ T cells derived from the blood of human and monkey neonates, as well as in CD3+ T cells derived from the blood of adolescent monkeys and in the prefrontal cortex of adult rats. MORC1 is thus the first identified epigenetic marker of ELS to be present in blood cell progenitors at birth and in the brain in adulthood. Interestingly, a gene-set-based analysis of data from a genome-wide association study of major depressive disorder (MDD) revealed an association of MORC1 with MDD.

The consequences of early-life adversity: neurobiological, behavioural and epigenetic adaptations.

During the perinatal period, the brain is particularly sensitive to remodelling by environmental factors. Adverse early-life experiences, such as stress exposure or suboptimal maternal care, can have long-lasting detrimental consequences for an individual. This phenomenon is often referred to as 'early-life programming' and is associated with an increased risk of disease. Typically, rodents exposed to prenatal stress or postnatal maternal deprivation display enhanced neuroendocrine responses to stress, increased levels of anxiety and depressive-like behaviours, and cognitive impairments. Some of the phenotypes observed in these models of early-life adversity are likely to share common neurobiological mechanisms. For example, there is evidence for impaired glucocorticoid negative-feedback control of the hypothalamic-pituitary-adrenal axis, altered glutamate neurotransmission and reduced hippocampal neurogenesis in both prenatally stressed rats and rats that experienced deficient maternal care. The possible mechanisms through which maternal stress during pregnancy may be transmitted to the offspring are reviewed, with special consideration given to altered maternal behaviour postpartum. We also discuss what is known about the neurobiological and epigenetic mechanisms that underpin early-life programming of the neonatal brain in the first generation and subsequent generations, with a view to abrogating programming effects and potentially identifying new therapeutic targets for the treatment of stress-related disorders and cognitive impairment.

Hydroxymethylation and DNA methylation profiles in the prefrontal cortex of the non-human primate rhesus macaque and the impact of maternal deprivation on hydroxymethylation.

5-Hydroxymethylcytosine (5hmC) is abundant in the brain, suggesting an important role in epigenetic control of neuronal functions. In this paper, we show that 5hmC and 5-methylcytosine (5mC) levels are coordinately distributed in gene promoters of the rhesus macaque prefrontal cortex. Although promoter hydroxymethylation and methylation are overall negatively correlated with expression, a subset of highly expressed genes involved in specific cerebral functions is associated with high levels of 5mC and 5hmC. These relationships were also observed in the mouse cortex. Furthermore, we found that early-life maternal deprivation is associated, in the adult monkey cortex, with DNA hydroxymethylation changes of promoters of genes related to neurological functions and psychological disorders. These results reveal that early social adversity triggers variations in brain DNA hydroxymethylation that could be detected in adulthood.

The genome-wide landscape of DNA methylation and hydroxymethylation in response to sleep deprivation impacts on synaptic plasticity genes.

Sleep is critical for normal brain function and mental health. However, the molecular mechanisms mediating the impact of sleep loss on both cognition and the sleep electroencephalogram remain mostly unknown. Acute sleep loss impacts brain gene expression broadly. These data contributed to current hypotheses regarding the role for sleep in metabolism, synaptic plasticity and neuroprotection. These changes in gene expression likely underlie increased sleep intensity following sleep deprivation (SD). Here we tested the hypothesis that epigenetic mechanisms coordinate the gene expression response driven by SD. We found that SD altered the cortical genome-wide distribution of two major epigenetic marks: DNA methylation and hydroxymethylation. DNA methylation differences were enriched in gene pathways involved in neuritogenesis and synaptic plasticity, whereas large changes (>4000 sites) in hydroxymethylation where observed in genes linked to cytoskeleton, signaling and neurotransmission, which closely matches SD-dependent changes in the transcriptome. Moreover, this epigenetic remodeling applied to elements previously linked to sleep need (for example, Arc and Egr1) and synaptic partners of Neuroligin-1 (Nlgn1; for example, Dlg4, Nrxn1 and Nlgn3), which we recently identified as a regulator of sleep intensity following SD. We show here that Nlgn1 mutant mice display an enhanced slow-wave slope during non-rapid eye movement sleep following SD but this mutation does not affect SD-dependent changes in gene expression, suggesting that the Nlgn pathway acts downstream to mechanisms triggering gene expression changes in SD. These data reveal that acute SD reprograms the epigenetic landscape, providing a unique molecular route by which sleep can impact brain function and health.

The early-life social environment and DNA methylation.

DNA methylation is a chemical modification of DNA that confers, upon identical sequences, different identities that are reflected in different gene expression programming. DNA methylation has a well-established role in cellular differentiation by providing a mechanism for one genome to express multiple phenotypes in a multicellular organism. Recent data point however to the possibility that in addition to the innate process of cellular differentiation, DNA methylation can serve as a genome adaptation mechanism, adapting genome function to changing environmental contexts including social environments. A critical time point for this process is early life when cues from the social and physical environments define lifelong trajectories of physical and mental health. DNA methylation and additional epigenetic modifications could therefore serve as molecular links between 'nurture' and 'nature'. Data that are consistent with this new role for DNA methylation as a mechanism for conferring an 'environment' specific identity to DNA will be discussed.

Transgenic mice as a model of pre-clinical Alzheimer's disease.

At diagnosis, Alzheimer's disease (AD) brains are extensively burdened with plaques and tangles and display a degree of synaptic failure most likely beyond therapeutic treatment. It is therefore crucial to identify early pathological events in the progression of the disease. While it is not currently feasible to identify and study early, pre-clinical stages of AD, transgenic (Tg) models offer a valuable tool in this regard. Here we investigated cognitive, structural and biochemical CNS alterations occurring in our newly developed McGill-Thyl-APP Tg mice (over-expressing the human amyloid precursor protein with the Swedish and Indiana mutations) prior to extracellular plaque deposition. Pre-plaque, 3-month old Tg mice already displayed cognitive deficits concomitant with reorganization of cortical cholinergic pre-synaptic terminals. Conformational specific antibodies revealed the early appearance of intracellular amyloid ß (Aß)-oligomers and fibrillar oligomers in pyramidal neurons of cerebral cortex and hippocampus. At the same age, the cortical levels of insulin degrading enzyme -a well established Aß-peptidase, were found to be significantly down-regulated. Our results suggest that, in the McGill-Thy1-APP Tg model, functional, structural and biochemical alterations are already present in the CNS at early, pre-plaque stages of the pathology. Accumulation of intraneuronal neurotoxic Aß-oligomers (possibly caused by a failure in the clearance machinery) is likely to be the culprit of such early, pre-plaque pathology. Similar neuronal alterations might occur prior to clinical diagnosis in AD, during a yet undefined 'latent' stage. A better understanding of such pre-clinical AD might yield novel therapeutic targets and or diagnostic tools.

Child health, developmental plasticity, and epigenetic programming.

Plasticity in developmental programming has evolved in order to provide the best chances of survival and reproductive success to the organism under changing environments. Environmental conditions that are experienced in early life can profoundly influence human biology and long-term health. Developmental origins of health and disease and life-history transitions are purported to use placental, nutritional, and endocrine cues for setting long-term biological, mental, and behavioral strategies in response to local ecological and/or social conditions. The window of developmental plasticity extends from preconception to early childhood and involves epigenetic responses to environmental changes, which exert their effects during life-history phase transitions. These epigenetic responses influence development, cell- and tissue-specific gene expression, and sexual dimorphism, and, in exceptional cases, could be transmitted transgenerationally. Translational epigenetic research in child health is a reiterative process that ranges from research in the basic sciences, preclinical research, and pediatric clinical research. Identifying the epigenetic consequences of fetal programming creates potential applications in clinical practice: the development of epigenetic biomarkers for early diagnosis of disease, the ability to identify susceptible individuals at risk for adult diseases, and the development of novel preventive and curative measures that are based on diet and/or novel epigenetic drugs.

Organ and gestational age effects of maternal nutrient restriction on global methylation in fetal baboons.

BACKGROUND: A sub-optimal intrauterine environment alters the trajectory of fetal development with profound effects on life-time health. Altered methylation, a proposed epigenetic mechanism responsible for these changes, has been studied in non-primate species but not nonhuman primates. We tested the hypotheses that global methylation in fetal baboon demonstrates organ specificity, gestational age specificity, and changes with maternal nutritional status. METHODS: We measured global DNA methylation in fetuses of control fed (CTR) and nutrient restricted mothers fed 70% of controls (MNR) for brain, kidney, liver and heart at 0.5 and 0.9 gestation (G). RESULTS: We observed organ and gestation specific changes that were modified by maternal diet. Methylation in CTR fetuses was highest in frontal cortex and lowest in liver. MNR decreased methylation in 0.5G kidney and increased methylation in 0.9G kidney and frontal cortex. CONCLUSION: These results demonstrate a potential epigenetic mechanism whereby reduced maternal nutrition has long-term programming effects on fetal organ development.

Epigenetic programming of phenotypic variations in reproductive strategies in the rat through maternal care.

Studies across multiple organisms reveal considerable phenotypic variation in reproductive tactics. In some species, this variation is associated with maternal effects in which variation in maternal investment results in stable individual differences in reproductive function. Recent studies with the rat suggest that maternal effects can alter the function of neuroendocrine systems associated with female sexual behaviour as well as maternal behaviour. These maternal effects appear to be mediated by epigenetic modifications at the promoter for oestrogen receptor alpha (ERalpha) and subsequent effects on gene expression. The tissue-specific nature of such effects may underlie the co-ordinated variation in multiple forms of reproductive function, resulting in distinct reproductive strategies.

High intracellular concentrations of amyloid-beta block nuclear translocation of phosphorylated CREB.

The beta-amyloid peptide (Abeta) is considered responsible for the pathogenesis of Alzheimer's disease. Despite the magnitude of reports describing a neurotoxic role of extracellular Abeta, the role for intracellular Abeta (iAbeta) has not been elucidated. We previously demonstrated that in rat pheochromocytoma cells expression of moderate levels of Abeta results in the up-regulation of phospho-extracellular signal-regulated kinases (ERK1)/2 along with an elevation of cyclic AMP-response element (CRE)-regulated gene expression; however, the effect of high intracellular levels of Abeta were not examined. Towards this goal we generated constructs that endogenously produce different expression levels of iAbeta in a human cell line. We show a bimodal response to Abeta in a neural human cell line. A moderate increase of endogenous Abeta up-regulates certain cyclic AMP-response element-binding protein (CREB) responsive genes such as presenilin 1, presenilin 2, brain-derived neurotrophic factor, and mRNA and protein levels by CREB activation and Synapsin 1 nuclear translocation. On the other hand, high-loads of iAbeta resulted in sustained hyper-phosphorylation of CREB that did not translocate to the nucleus and did not stimulate activation of CRE-regulated gene expression. Our study suggests that variations in levels of iAbeta could influence signaling mechanisms that lead to phosphorylation of CREB, its nuclear translocation and CRE-regulated genes involved in production of Abeta and synaptic plasticity in opposite directions.