Cognitive Decline, Cerebral-Spleen Tryptophan Metabolism, Oxidative Stress, Cytokine Production, and Regulation of the Txnip Gene in a Triple Transgenic Mouse Model of Alzheimer Disease.

Pathologic inflammation in response to injury, infection, or oxidative stress is a proposed mechanism relating cognitive decline to dementia. The kynurenine pathway and thioredoxin-interacting protein (TXNIP) activity regulate inflammation and neurotoxicity in Alzheimer disease (AD). We examined cognitive deficits, kynurenine pathway mediators, TXNIP, and oxidative damage in the cerebrum and spleen, including inflammatory cytokine production by stimulated splenocytes, from female triple transgenic (3xTg-AD) mice in early and late stages of disease progression, and characterized tissue-specific epigenetic regulation of Txnip gene expression. We show that cognitive deficits in 7-month-old 3xTg-AD mice are associated with a stable increase in cerebrum and spleen tryptophan metabolites, with a concomitant increase in amyloid ß 40 (Aß40)/Aß42 and tau/hyperphosphorylated tau pathologies and a coordinated reduction in spleen proinflammatory cytokine production in 17-month-old mice. The enhanced cerebrum TXNIP expression is associated with increased histone acetylation, transcription factor [Aß42 or CCCTC-binding factor (CTCF)] binding, and Txnip promoter hypomethylation, whereas the attenuated spleen TXNIP expression is associated with increased histone methylation, reduced CTCF binding, and Txnip promoter hypermethylation. These results suggest a causal relationship among epigenomic state, TXNIP expression, cerebral-spleen tryptophan metabolism, inflammatory cytokine production, and cognitive decline; and they provide a potential mechanism for Txnip gene regulation in normal and pathologic conditions, suggesting TXNIP levels may be a useful predictive or diagnostic biomarker for Aß40/Aß42 targeted AD therapies.

Epigenetic Silencing of TAP1 in Aldefluor+ Breast Cancer Stem Cells Contributes to Their Enhanced Immune Evasion.

Avoiding detection and destruction by immune cells is key for tumor initiation and progression. The important role of cancer stem cells (CSCs) in tumor initiation has been well established, yet their ability to evade immune detection and targeting is only partly understood. To investigate the ability of breast CSCs to evade immune detection, we identified a highly tumorigenic population in a spontaneous murine mammary tumor based on increased aldehyde dehydrogenase activity. We performed tumor growth studies in immunocompetent and immunocompromised mice. In immunocompetent mice, growth of the spontaneous mammary tumor was restricted; however, the Aldefluor+ population was expanded, suggesting inherent resistance mechanisms. Gene expression analysis of the sorted tumor cells revealed that the Aldefluor+ tumor cells has decreased expression of transporter associated with antigen processing (TAP) genes and co-stimulatory molecule CD80, which would decrease susceptibility to T cells. Similarly, the Aldefluor+ population of patient tumors and 4T1 murine mammary cells had decreased expression of TAP and co-stimulatory molecule genes. In contrast, breast CSCs identified by CD44+ CD24- do not have decreased expression of these genes, but do have increased expression of C-X-C chemokine receptor type 4. Decitabine treatment and bisulfite pyrosequencing suggests that DNA hypermethylation contributes to decreased TAP gene expression in Aldefluor+ CSCs. TAP1 knockdown resulted in increased tumor growth of 4T1 cells in immunocompetent mice. Together, this suggests immune evasion mechanisms in breast CSCs are marker specific and epigenetic silencing of TAP1 in Aldefluor+ breast CSCs contributes to their enhanced survival under immune pressure. Stem Cells 2018;36:641-654.

Presymptomatic Alterations in Amino Acid Metabolism and DNA Methylation in the Cerebellum of a Murine Model of Niemann-Pick Type C Disease.

The fatal neurodegenerative disorder Niemann-Pick type C (NPC) is caused in most cases by mutations in NPC1, which encodes the late endosomal NPC1 protein. Loss of NPC1 disrupts cholesterol trafficking from late endosomes to the endoplasmic reticulum and plasma membrane, causing cholesterol accumulation in late endosomes/lysosomes. Neurons are particularly vulnerable to this cholesterol trafficking defect, but the pathogenic mechanisms through which NPC1 deficiency causes neuronal dysfunction remain largely unknown. Herein, we have investigated amino acid metabolism in cerebella of NPC1-deficient mice at different stages of NPC disease. Imbalances in amino acid metabolism were evident from increased branched chain amino acid and asparagine levels and altered expression of key enzymes of glutamine/glutamate metabolism in presymptomatic and early symptomatic NPC1-deficient cerebellum. Increased levels of several amino acid intermediates of one-carbon metabolism indicated disturbances in folate and methylation pathways. Alterations in DNA methylation were apparent in decreased expression of DNA methyltransferase 3a and methyl-5'-cytosine-phosphodiester-guanine-domain binding proteins, reduced 5-methylcytosine immunoreactivity in the molecular and Purkinje cell layers, demethylation of genome-wide repetitive LINE-1 elements, and hypermethylation in specific promoter regions of single-copy genes in NPC1-deficient cerebellum at early stages of the disease. Alterations in amino acid metabolism and epigenetic changes in the cerebellum at presymptomatic stages of NPC disease represent previously unrecognized mechanisms of NPC pathogenesis.

Epigenetic traces of childhood maltreatment in peripheral blood: a new strategy to explore gene-environment interactions.

Maltreatment in childhood affects mental health over the life course. New research shows that early life experiences alter the genome in a way that can be measured in peripheral blood samples decades later. These findings suggest a new strategy for exploring gene-environment interactions and open opportunities for translational epigenomic research.

Noncanonical regulation of imprinted gene Igf2 by amyloid-beta 1-42 in Alzheimer's disease.

Reduced insulin-like growth factor 2 (IGF2) levels in Alzheimer's disease (AD) may be the mechanism relating age-related metabolic disorders to dementia. Since Igf2 is an imprinted gene, we examined age and sex differences in the relationship between amyloid-beta 1-42 (Aß42) accumulation and epigenetic regulation of the Igf2/H19 gene cluster in cerebrum, liver, and plasma of young and old male and female 5xFAD mice, in frontal cortex of male and female AD and non-AD patients, and in HEK293 cell cultures. We show IGF2 levels, Igf2 expression, histone acetylation, and H19 ICR methylation are lower in females than males. However, elevated Aß42 levels are associated with Aß42 binding to Igf2 DMR2, increased DNA and histone methylation, and a reduction in Igf2 expression and IGF2 levels in 5xFAD mice and AD patients, independent of H19 ICR methylation. Cell culture results confirmed the binding of Aß42 to Igf2 DMR2 increased DNA and histone methylation, and reduced Igf2 expression. These results indicate an age- and sex-related causal relationship among Aß42 levels, epigenomic state, and Igf2 expression in AD and provide a potential mechanism for Igf2 regulation in normal and pathological conditions, suggesting IGF2 levels may be a useful diagnostic biomarker for Aß42 targeted AD therapies.

Effects of paternal high-fat diet and maternal rearing environment on the gut microbiota and behavior.

Exposing a male rat to an obesogenic high-fat diet (HFD) influences attractiveness to potential female mates, the subsequent interaction of female mates with infant offspring, and the development of stress-related behavioral and neural responses in offspring. To examine the stomach and fecal microbiome's potential roles, fecal samples from 44 offspring and stomach samples from offspring and their fathers were collected and bacterial community composition was studied by 16 small subunit ribosomal RNA (16S rRNA) gene sequencing. Paternal diet (control, high-fat), maternal housing conditions (standard or semi-naturalistic housing), and maternal care (quality of nursing and other maternal behaviors) affected the within-subjects alpha-diversity of the offspring stomach and fecal microbiomes. We provide evidence from beta-diversity analyses that paternal diet and maternal behavior induced community-wide shifts to the adult offspring gut microbiome. Additionally, we show that paternal HFD significantly altered the adult offspring Firmicutes to Bacteroidetes ratio, an indicator of obesogenic potential in the gut microbiome. Additional machine-learning analyses indicated that microbial species driving these differences converged on Bifidobacterium pseudolongum. These results suggest that differences in early-life care induced by paternal diet and maternal care significantly influence the microbiota composition of offspring through the microbiota-gut-brain axis, having implications for adult stress reactivity.

The effect of background strain on the behavioral phenotypes of the MDGA2+/- mouse model of autism spectrum disorder.

The membrane-associated mucin (MAM) domain containing glycosylphosphatidylinositol anchor 2 protein single knock-out mice (MDGA2+/- ) are models of ASD. We examined the behavioral phenotypes of male and female MDGA2+/- and wildtype mice on C57BL6/NJ and C57BL6/N backgrounds at 2 months of age and measured MDGA2, neuroligin 1 and neuroligin 2 levels at 7 months. Mice on the C57BL6/NJ background performed better than those on the C57BL6/N background in visual ability and in learning and memory performance in the Morris water maze and differed in measures of motor behavior and anxiety. Mice with the MDGA2+/- genotype differed from WT mice in motor, social and repetitive behavior and anxiety, but most of these effects involved interactions between MDGA2+/- genotype and background strain. The background strain also influenced MDGA2 levels and NLGN2 association in MDGA2+/- mice. Our findings emphasize the importance of the background strain used in studies of genetically modified mice.

Acetylation-induced transcription is required for active DNA demethylation in methylation-silenced genes.

A hallmark of vertebrate genes is that actively transcribed genes are hypomethylated in critical regulatory sequences. However, the mechanisms that link gene transcription and DNA hypomethylation are unclear. Using a trichostatin A (TSA)-induced replication-independent demethylation assay with HEK 293 cells, we show that RNA transcription is required for DNA demethylation. Histone acetylation precedes but is not sufficient to trigger DNA demethylation. Following histone acetylation, RNA polymerase II (RNAP II) interacts with the methylated promoter. Inhibition of RNAP II transcription with actinomycin D, alpha-amanitin, or CDK7-specific small interfering RNA inhibits DNA demethylation. H3 trimethyl lysine 4 methylation, a marker of actively transcribed genes, was associated with the cytomegalovirus promoter only after demethylation. TSA-induced demethylation of the endogenous cancer testis gene GAGE follows a similar sequence of events and is dependent on RNA transcription as well. These data suggest that DNA demethylation follows rather than precedes early transcription and point towards a novel function for DNA demethylation as a memory of actively transcribed genes.

Author Correction: A novel mechanism of plasminogen activation in epithelial and mesenchymal cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Decitabine Response in Breast Cancer Requires Efficient Drug Processing and Is Not Limited by Multidrug Resistance.

Dysregulation of DNA methylation is an established feature of breast cancers. DNA demethylating therapies like decitabine are proposed for the treatment of triple-negative breast cancers (TNBC) and indicators of response need to be identified. For this purpose, we characterized the effects of decitabine in a panel of 10 breast cancer cell lines and observed a range of sensitivity to decitabine that was not subtype specific. Knockdown of potential key effectors demonstrated the requirement of deoxycytidine kinase (DCK) for decitabine response in breast cancer cells. In treatment-naïve breast tumors, DCK was higher in TNBCs, and DCK levels were sustained or increased post chemotherapy treatment. This suggests that limited DCK levels will not be a barrier to response in patients with TNBC treated with decitabine as a second-line treatment or in a clinical trial. Methylome analysis revealed that genome-wide, region-specific, tumor suppressor gene-specific methylation, and decitabine-induced demethylation did not predict response to decitabine. Gene set enrichment analysis of transcriptome data demonstrated that decitabine induced genes within apoptosis, cell cycle, stress, and immune pathways. Induced genes included those characterized by the viral mimicry response; however, knockdown of key effectors of the pathway did not affect decitabine sensitivity suggesting that breast cancer growth suppression by decitabine is independent of viral mimicry. Finally, taxol-resistant breast cancer cells expressing high levels of multidrug resistance transporter ABCB1 remained sensitive to decitabine, suggesting that the drug could be used as second-line treatment for chemoresistant patients.

Shaping adult phenotypes through early life environments.

A major question in the biology of stress and environmental adaptation concerns the neurobiological basis of how neuroendocrine systems governing physiological regulatory mechanisms essential for life (metabolism, immune response, organ function) become harmful. The current view is that a switch from protection to damage occurs when vulnerable phenotypes are exposed to adverse environmental conditions. In accordance with this theory, sequelae of early life social and environmental stressors, such as childhood abuse, neglect, poverty, and poor nutrition, have been associated with the emergence of mental and physical illness (i.e., anxiety, mood disorders, poor impulse control, psychosis, and drug abuse) and an increased risk of common metabolic and cardiovascular diseases later in life. Evidence from animal and human studies investigating the associations between early life experiences (including parent-infant bonding), hypothalamus-pituitary-adrenal axis activity, brain development, and health outcome provide important clues into the neurobiological mechanisms that mediate the contribution of stressful experiences to personality development and the manifestation of illness. This review summarizes our current molecular understanding of how early environment influences brain development in a manner that persists through life and highlights recent evidence from rodent studies suggesting that maternal care in the first week of postnatal life establishes diverse and stable phenotypes in the offspring through epigenetic modification of genes expressed in the brain that shape neuroendocrine and behavioral stress responsivity throughout life.

DNA methyltransferase 1 knockdown activates a replication stress checkpoint.

DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), gammaH2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response.

Epigenetic effects of glucocorticoids.

The early nurturing environment has persistent influences on developmental programming of inter-individual differences in metabolic and endocrine function that contribute to emotional and cognitive performance through life. These effects are mediated, in part, through neonatal programming of hypothalamic-pituitary-adrenal (HPA) axis function. Animal models support this hypothesis. For example, in the rat natural variations in maternal care influence HPA axis stress reactivity in the offspring via long-term changes in tissue-specific gene expression. Studies in vivo and in vitro show that maternal licking and grooming increases glucocorticoid receptor expression in the offspring via increased hippocampal serotonergic tone accompanied by increased histone acetylase transferase activity, histone acetylation and DNA demethylation mediated by the transcription factor nerve growth factor-inducible protein-A. These effects are reversed by early postnatal cross-fostering and by pharmacological manipulations, including trichostatin A (TSA) and l-methionine administration in adulthood. These studies demonstrate that an epigenetic state of a gene can be established through early in life experience, and is potentially reversible in adult life. Accordingly, epigenetic modifications in target gene promoters in response to environmental demand may ensure stable yet dynamic regulation that mediates persistent changes in biological and behavioral phenotype over the lifespan.

Retinoic acid and arsenic trioxide induce lasting differentiation and demethylation of target genes in APL cells.

Acute promyelocytic leukemia (APL) is characterized by arrested differentiation of promyelocytes. Patients treated with all-trans retinoic acid (ATRA) alone experience relapse, while patients treated with ATRA and arsenic trioxide (ATO) are often relapse-free. This suggests sustained changes have been elicited by the combination therapy. To understand the lasting effects of the combination therapy, we compared the effects of ATRA and ATO on NB4 and ATRA-resistant NB4-MR2 APL cells during treatment versus post treatment termination. After treatment termination, NB4 cells treated with ATRA or ATO reverted to non-differentiated cells, while combination-treated cells remained terminally differentiated. This effect was diminished in NB4-MR2 cells. This suggests combination treatment induced more permanent changes. Combination treatment induced higher expression of target genes (e.g., transglutaminase 2 and retinoic acid receptor beta), which in NB4 cells was sustained post treatment termination. To determine whether sustained epigenetic changes were responsible, we quantified the enrichment of histone modifications by chromatin immunoprecipitation, and CpG methylation by bisulfite-pyrosequencing. While ATRA and combination treatment induced similar histone acetylation enrichment, combination treatment induced greater demethylation of target genes, which was sustained. Therefore, sustained demethylation of target genes by ATRA and ATO combination treatment is associated with lasting differentiation and gene expression changes.

The transcription factor nerve growth factor-inducible protein a mediates epigenetic programming: altering epigenetic marks by immediate-early genes.

Maternal care alters epigenetic programming of glucocorticoid receptor (GR) gene expression in the hippocampus, and increased postnatal maternal licking/grooming (LG) behavior enhances nerve growth factor-inducible protein A (NGFI-A) transcription factor binding to the exon 1(7) GR promoter within the hippocampus of the offspring. We tested the hypothesis that NGFI-A binding to the exon 1(7) GR promoter sequence marks this sequence for histone acetylation and DNA demethylation and that such epigenetic alterations subsequently influence NGFI-A binding and GR transcription. We report that (1) NGFI-A binding to its consensus sequence is inhibited by DNA methylation, (2) NGFI-A induces the activity of exon 1(7) GR promoter in a transient reporter assay, (3) DNA methylation inhibits exon 1(7) GR promoter activity, and (4) whereas NGFI-A interaction with the methylated exon 1(7) GR promoter is reduced, NGFI-A overexpression induces histone acetylation, DNA demethylation, and activation of the exon 1(7) GR promoter in transient transfection assays. Site-directed mutagenesis assays demonstrate that NGFI-A binding to the exon 1(7) GR promoter is required for such epigenetic reprogramming. In vivo, enhanced maternal LG is associated with increased NGFI-A binding to the exon 1(7) GR promoter in the hippocampus of pups, and NGFI-A-bound exon 1(7) GR promoter is unmethylated compared with unbound exon 1(7) GR promoter. Knockdown experiments of NGFI-A in hippocampal primary cell culture show that NGFI-A is required for serotonin-induced DNA demethylation and increased exon 1(7) GR promoter expression. The data are consistent with the hypothesis that NGFI-A participates in epigenetic programming of GR expression.

Regional-specific global cytosine methylation and DNA methyltransferase expression in the adult rat hippocampus.

Recent observations suggest that DNA methylation plays an important role in memory and long-term potentiation (LTP) in the hippocampus and is involved in programming the offspring epigenome in response to maternal care. Global DNA methylation is believed to be stable postnatally and to be similar across tissues in the adult mammal. It has also been a long held belief that DNA methyltransferases (DNMTs) play a very limited role in postmitotic tissues. Recent data suggests a more dynamic role for DNA methylation in the brain postnatally, therefore we examined the global state of methylation and the expression of the known DNMTs in the different regions of the hippocampus. We observed strikingly different levels of global methylation in the adult rat dentate gyrus (DG) and CA1 region in comparison with the CA2 and CA3 regions. mRNA levels of DNA methyltransferases exhibited similar regional specificity and were correlated with global DNA methylation levels. These regional differences in global methylation and expression of the DNA methylation machinery in the adult brain are consistent with the emerging hypothesis that DNA methylation may play a dynamic physiological role in the adult brain.

Epigenetic programming by maternal behavior.

Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of LG and ABN were found to have differences in DNA methylation, as compared to offspring of 'low-LG-ABN' mothers. These differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible.

Effects of paternal high-fat diet and rearing environment on maternal investment and development of defensive responses in the offspring.

Paternal preconception risk factors (e.g. stress, diet, drug use) correlate with metabolic dysfunction in offspring, which is often comorbid with depressive and anxiety-like phenotypes. Detection of these risk factors or deleterious phenotypes informs a female about prevailing ecological demands, in addition to potential adverse environment-induced phenotypes that may be disseminated to her offspring. We examined whether a F0 male rat's prior exposure to an obesogenic high-fat diet (HFD) influences a female's attraction towards a male, subsequent mother-infant interactions and the development of defensive (emotional) responses in the F1 offspring. Females displayed less interest in the HFD exposed F0 males relative to control diet-exposed F0 males. Dams that reared F1 offspring in larger, semi-naturalistic housing provided more licking and grooming and active arched-back-nursing behavior. However, some of these effects interacted with paternal experience. F0 HFD and maternal rearing environment revealed sex-dependent, between group differences in F1 offspring wean weight, juvenile social interactions and anxiety-like behavior in adolescence. Our results show for the first time in mammals that male exposure to HFD may contribute to stable behavioral variation among females in courtship, maternal care, even when the females are not directly exposed to a HFD, and anxiety-like behavior in F1 offspring. Furthermore, when offspring were exposed to a predatory threat, hypothalamic Crf gene regulation was influenced by early housing. These results, together with our previous findings, suggest that paternal experience and maternal rearing conditions can influence maternal behavior and development of defensive responses of offspring.

A novel mechanism of plasminogen activation in epithelial and mesenchymal cells.

Cancer dissemination is initiated by the movement of cells into the vasculature which has been reported to be triggered by EMT (epithelial to mesenchymal transition). Cellular dissemination also requires proteases that remodel the extracellular matrix. The protease, plasmin is a prominent player in matrix remodeling and invasion. Despite the contribution of both EMT and the plasminogen activation (PA) system to cell dissemination, these processes have never been functionally linked. We reveal that canonical Smad-dependent TGFß1 signaling and FOXC2-mediated PI3K signaling in cells undergoing EMT reciprocally modulate plasminogen activation partly by regulating the plasminogen receptor, S100A10 and the plasminogen activation inhibitor, PAI-1. Plasminogen activation and plasminogen-dependent invasion were more prominent in epithelial-like cells and were partly dictated by the expression of S100A10 and PAI-1.

S100A10, a novel biomarker in pancreatic ductal adenocarcinoma.

Pancreatic cancer is arguably the deadliest cancer type. The efficacy of current therapies is often hindered by the inability to predict patient outcome. As such, the development of tools for early detection and risk prediction is key for improving outcome and quality of life. Here, we introduce the plasminogen receptor S100A10 as a novel predictive biomarker and a driver of pancreatic tumor growth and invasion. We demonstrated that S100A10 mRNA and protein are overexpressed in human pancreatic tumors compared to normal ducts and nonductal stroma. S100A10 mRNA and methylation status were predictive of overall survival and recurrence-free survival across multiple patient cohorts. S100A10 expression was driven by promoter methylation and the oncogene KRAS. S100A10 knockdown reduced surface plasminogen activation, invasiveness, and in vivo growth of pancreatic cancer cell lines. These findings delineate the clinical and functional contribution of S100A10 as a biomarker in pancreatic cancer.