Lasting Changes In Gene Expression Research Articles

Astrocytic transcriptional and epigenetic mechanisms of drug addiction.

Addiction is a leading cause of disease burden worldwide and remains a challenge in current neuroscience research. Drug-induced lasting changes in gene expression are mediated by transcriptional and epigenetic regulation in the brain and are thought to underlie behavioral adaptations. Emerging evidence implicates astrocytes in regulating drug-seeking behaviors and demonstrates robust transcriptional response to several substances of abuse. This review focuses on the astrocytic transcriptional and epigenetic mechanisms of drug action.

Breastfeeding lifespan control of growth, maintenance, and metabolism of small intestinal epithelium.

Gastrointestinal epithelial cells respond to milk-born molecules throughout breastfeeding, influencing growth, and development. The rapid renewal of the small intestine depends on the proliferation in the crypt that drives cell fates. We used early weaning model to investigate immediate and late effects of breastfeeding on proliferation, differentiation of jejunal epithelial cells. Wistar rats were either allowed to suckle (S) until 21 postnatal days or submitted to early weaning (EW) at 15 days. By comparing ages (18, 60, and 120 days), we found that EW decreased Ki67 indices and villi height at 18 and 60 days (p < 0.05), and at 120 days they were similar between diets. Proliferative reduction and augmented expression of Cdkn1b (p27 gene) were parallel. In the stem cell niche, EW increased the number and activity (Defa24) of Paneth cells at 18 and 60 days (p < 0.05), and Lgr5 and Ascl2 genes showed inverted responses between ages. Among target cells, EW decreased goblet cell number at 18 and 60 days (p < 0.05) and increased it at 120 days (p < 0.05), whereas enteroendocrine marker genes were differentially altered. EW reduced enterocytes density at 18 days (p < 0.05), and at 120 days this population was decreased (vs. 60 days). Among cell fate crypt-controlling genes, Notch and Atoh1 were the main targets of EW. Metabolically, intraperitoneal glucose tolerance was immediately reduced (18 days), being reverted until 120 days (p < 0.05). Currently, we showed that breastfeeding has a lifespan influence on intestinal mucosa and on its stem cell compartment. We suggest that, although jejunum absorptive function is granted after early weaning, the long lasting changes in gene expression might prime the mucosa with a different sensitivity to gut disorders that still have to be further explored.

Exploring genetic variation that influences brain methylation in attention-deficit/hyperactivity disorder

Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder caused by an interplay of genetic and environmental factors. Epigenetics is crucial to lasting changes in gene expression in the brain. Recent studies suggest a role for DNA methylation in ADHD. We explored the contribution to ADHD of allele-specific methylation (ASM), an epigenetic mechanism that involves SNPs correlating with differential levels of DNA methylation at CpG sites. We selected 3896 tagSNPs reported to influence methylation in human brain regions and performed a case-control association study using the summary statistics from the largest GWAS meta-analysis of ADHD, comprising 20,183 cases and 35,191 controls. We observed that genetic risk variants for ADHD are enriched in ASM SNPs and identified associations with eight tagSNPs that were significant at a 5% false discovery rate (FDR). These SNPs correlated with methylation of CpG sites lying in the promoter regions of six genes. Since methylation may affect gene expression, we inspected these ASM SNPs together with 52 ASM SNPs in high LD with them for eQTLs in brain tissues and observed that the expression of three of those genes was affected by them. ADHD risk alleles correlated with increased expression (and decreased methylation) of ARTN and PIDD1 and with a decreased expression (and increased methylation) of C2orf82. Furthermore, these three genes were predicted to have altered expression in ADHD, and genetic variants in C2orf82 correlated with brain volumes. In summary, we followed a systematic approach to identify risk variants for ADHD that correlated with differential cis-methylation, identifying three novel genes contributing to the disorder.

SU1 - EXPLORING GENETIC VARIATION THAT INFLUENCES BRAIN METHYLATION IN ATTENTION-DEFICIT/HYPERACTIVITY DISORDER (ADHD)

Background Allele-Specific Methylation (ASM) is a common epigenetic mechanism that involves SNPs correlating with differential levels of methylation at CpG sites. Epigenetic changes play a crucial role in driving lasting changes in gene expression in several tissues, including the brain. As alteration of DNA methylation levels has recently been linked to ADHD symptoms, our aim is to explore the contribution of ASM to ADHD through a case-control association study using different GWAS datasets. Methods We performed a primary SNP selection based on two previous studies that identified ASM variants in different brain regions of post-mortem human samples. Subsequently, from those variants we selected a total of 3,896 SNPs following these criteria: associations in cis between the SNP and methylation at a CpG site, correlation of methylation levels with gene expression (R2) ≥ 0.2, tagSNPs for each CpG site (LD; r2≥0.85). These candidates were inspected in summary statistics data from the first GWAS meta-analysis of adult and childhood ADHD, performed by iPSYCH and the Psychiatric Genomics Consortium (PGC) in 20,183 cases and 35,191 controls. We applied False Discovery Rate (FDR) correction for multiple testing (5% FDR, corrected P≤ 6.78e-05) to determine the top hits. These signals were followed-up to retrieve (i) SNPs located within the same CpG site and thereafter (ii) SNPs in high linkage disequilibrium with the associated SNPs (using the same FDR threshold). Impact of the variants on gene expression levels were further explored in the GTEx database. Results The case-control association study identified eight tagSNPs surviving FDR, which correlate with differential methylation of six CpG sites. Top hits were followed-up to retrieve other SNPs that correlate with methylation of the same CpG sites, making a total of 39 candidate SNPs. The six CpG sites are located in the promoter regions of five genes, not previously related to ADHD. Notably, the risk SNPs associated with three of the genes are eQTLs for those genes in various brain tissues, including the cerebellar hemisphere and the cerebellum. Discussion We have directed existing knowledge of ASM to evaluate its possible contribution to ADHD both at genetic and epigenetic levels. We followed a systematic approach by employing cis-acting ASM variants to pinpoint candidate genes for ADHD. Interestingly, several of the identified variants are eQTLs for genes that are expressed in brain. Our aim is now to further investigate the role of these genes in the development of the ADHD phenotype.

The Toll-Like Receptor 3 Agonist Poly(I:C) Induces Rapid and Lasting Changes in Gene Expression Related to Glutamatergic Function and Increases Ethanol Self-Administration in Rats.

Growing evidence suggests that neuroimmune signaling via Toll-like receptors (TLRs) alters brain circuitry related to alcohol use disorders. Both ethanol (EtOH) exposure and the TLR3 agonist, poly(I:C), increase brain TLR3 expression in neurons and glia. Furthermore, previous studies have shown that cortical TLR3 expression is correlated with lifetime EtOH intake in humans. The current experiments investigated the consequences of poly(I:C) treatment on gene expression in 2 brain regions contributing to alcohol reinforcement, the insular cortex (IC) and nucleus accumbens (Acb) and on operant EtOH self-administration, in Long Evans rats. TLR3 activation increased mRNA levels of neuroimmune genes (TLR3, COX2), glutamatergic genes (mGluR2, mGluR3, GLT1), and the trophic factor BDNF in Acb and IC. Furthermore, increases in each of these genes were correlated with increases in TLR3 mRNA, suggesting that TLR3 induction of these genes may impact excitatory transmission in IC and Acb. TLR3 activation also increased EtOH self-administration 18days postinjection and enhanced the effects of the mGluR2/3 agonist LY379268 to reduce EtOH self-administration following poly(I:C). Together, these findings suggest lasting consequences of TLR3 activation on gene expression including increases in Group II mGluRs in the Acb. Furthermore, we show an important role for TLR3 signaling in EtOH intake, and a functional involvement of Group II mGluRs.

Multiple neurons encode CrebB dependent appetitive long-term memory in the mushroom body circuit.

Lasting changes in gene expression are critical for the formation of long-term memories (LTMs), depending on the conserved CrebB transcriptional activator. While requirement of distinct neurons in defined circuits for different learning and memory phases have been studied in detail, only little is known regarding the gene regulatory changes that occur within these neurons. We here use the fruit fly as powerful model system to study the neural circuits of CrebB-dependent appetitive olfactory LTM. We edited the CrebB locus to create a GFP-tagged CrebB conditional knockout allele, allowing us to generate mutant, post-mitotic neurons with high spatial and temporal precision. Investigating CrebB-dependence within the mushroom body (MB) circuit we show that MB α/β and α'/β' neurons as well as MBON α3, but not in dopaminergic neurons require CrebB for LTM. Thus, transcriptional memory traces occur in different neurons within the same neural circuit.

A paternal methyl donor depleted diet leads to increased anxiety- and depression-like behavior in adult rat offspring.

Epigenetic mechanisms such as DNA methylation elicit lasting changes in gene expression and likely mediate gene–environment interactions that shape brain development, behavior, and emotional health. Myriad environmental factors influence DNA methylation, including methyl donor content in the paternal diet, could influence methylation in offspring via changes in the paternal germ line. The present study examines the effects of paternal methyl donor dietary deficiency on offspring’s emotional behaviors, including anxiety, social interaction, and depression-like behavior. We previously found that rats bred to display high levels of anxiety- and depression-like behavior exhibit diminished DNA methylation in the amygdala. We also observed that depleting dietary methyl donor content exacerbated the rats’ already high levels of anxiety- and depression-like behavior. Here we sought to determine whether paternal dietary methyl donor depletion elicits intergenerational effects on first generation (F1) offspring’s behavior (potentially triggering a similar increase in anxiety- and/or depression-like behavior). Thus, adult male rats prone to high anxiety/depression-like behavior, were fed either a methyl donor depleted (DEP) or control (CON) diet for 5 weeks prior to mating. They were paired with females and resultant F1 male offspring were subjected to a behavioral test battery in adulthood. F1-DEP offspring showed a similar behavioral profile to the F0 males, including greater depression-like behavior in the forced swim test (FST) and increased anxiety-like behavior in the open field test (OFT). Future work will interrogate molecular changes in the brains of F1 offspring that mediate these intergenerational effects of paternal methyl donor dietary content on offspring emotional behavior.

Assimiliating an Associative Trait: from Eco-Physiology to Epigenetics

The possible evolutionary significance of epigenetic memory and codes is a key problem for extended evolutionary synthesis and biosemiotics. In this paper, some less known original works are reviewed which highlight theoretical parallels between current evolutionary epigenetics, on the one hand, and its predecessors in the eco-physiology of higher nervous activity, on the other. Recently, these areas have begun to converge, with first evidence now indicating the possibility of transgenerational epigenetic inheritance of conditional associations in the mammalian nervous system, and related findings in other taxa. This can serve as an interesting example of evolutionary code-making, where the molecular mechanisms underlying arbitrary associations between stimuli involve lasting changes in gene expression that may be transmitted epigenetically across generations, and which in some cases could be further assimilated into the genome over subsequent evolution. Although preliminary, such epigenetic scenarios would also offer an interesting, if so far overlooked parallel to earlier research carried out by one of I.P. Pavlov’s leading students, acad. P.K. Anokhin, and his colleagues, but also by eminent eco-physiologists of the time, several of whom offered arguments for the possibility of unconditional reflexes representing evolutionarily later, specialized, and reduced forms of associative reflexes, from which they may be derived. Although discarded under the growing dominance of modern synthesis, these early epigenetic investigations may deserve renewed attention in the modern context, and if further confirmed, could open essentially new perspectives on the morphofunctional evolution of the nervous system.

P.2.037 - Differential effects of perturbations of the microbiome during the adolescent period induces long lasting behavioural effects in adult mice

It has become clear that the gut microbiota plays a key role in all aspects of health including mental health. The concept of the microbiota-gut-brain axis focuses on the ability of the microbiota to influence CNS function and modulate brain development and behaviour [1]. Microbiota composition can be affected by many different environmental factors such as stress, diet, exercise, illness and antibiotic treatment. Recent hypothesis suggests that neurodevelopment runs in parallel with establishment of an adult microbial profile and that disturbance or the lack thereof influences behaviour and brain function in adulthood [2]. Many different strategies can be utilized to investigate the role of the intestinal microbiota in host brain and behaviour. Here, we aimed to investigate whether exposure to antibiotic treatment or suboptimal diet both of which significantly affect microbiota composition resulted in long lasting effects on behaviour. Methods Male C57BL/6 mice were either exposed to high fat, a cafeteria diet or antibiotic treatment [3] for 21 days during the adolescence. We next assessed whether exposure to these known microbial interventions resulted in long lasting changes in gene expression and a variety of behaviours, in adulthood. Mice were tested for -like behaviour in the elevated plus maze and open field, memory in the novel object recognition task and fear in the fear conditioning paradigm. A T-test was used to compare antibiotic treatment with controls and ANOVA to compare controls and duet intervention, repeated measures were used when applicable. Results Both, diet and antibiotic treatment resulted in specific behavioural alterations. Antibiotic treatment alone resulted in increased anxiety-like behaviour in the elevated plus maze for the % time spend in the open arm (unpaired t-test, control M=17.64 SEM=±2.78 , antibiotic M=7.90 SEM=±1.99; t(17)=2.79 p=0.013), the % of entries into the open arm (unpaired t-test, control M=30.61 SEM=±2.95, antibiotic M=20.17 SEM=±2.86; t(17)=2.53 p=0.022) and head dips (unpaired t-test with Welch’s correction, control M=10.30 SEM=±1.76, antibiotic M=4.67 SEM=±0.80; t(12)=2.91 p=0.013), high fat diet induced increased contextual fear memory (two-way repeated measure ANOVA F(4, 54)=1.23, followed by Newman-Keuls multiple comparison test p Conclusion We demonstrate that short term exposure to known microbial altering factors during the adolescent period, a critical window of neurodevelopment, can induce long lasting changes on brain and behaviour. Further research needs to be done to investigate the mechanisms underlying the effects of altered microbiota on behaviour and brain circuits during this critical period.

Histone Posttranslational Modifications in Schizophrenia.

Schizophrenia is a complex neuropsychiatric disorder with high heritability; however, family and twin studies have indicated that environmental factors also play important roles in the etiology of disease. Environmental triggers exert their influence on behavior via epigenetic mechanisms. Epigenetic modifications, such as histone acetylation and methylation, as well as DNA methylation, can induce lasting changes in gene expression and have therefore been implicated in promoting the behavioral and neuronal behaviors that characterize this disorder. Importantly, because epigenetic processes are potentially reversible, they might serve as targets in the design of novel therapies in psychiatry. This chapter will review the current information regarding histone modifications in schizophrenia and the potential therapeutic relevance of such marks.

Insight into Cocaine-Induced Neural and Behavioral Adaptations Using Genome-Wide Expression Profiling and Epigenomic Analysis

Cocaine, like most other drugs of abuse, exerts long lasting changes in gene expression in the reward circuitry of the brain. This resultant remodelled reward circuitry is considered to underlie drug addiction and relapse. In recent years, genome-wide expression studies and epigenomic analysis of the affected regions in the reward circuitry have identified molecular changes associated with the structural remodeling of the circuitry. In particular, application of gene expression microarray and chromatin immunoprecipitation (ChIP) coupled to microarray (ChIP-chip) or sequencing (ChIP-seq) has provided unprecedented insights into the neural and behavioral adaptations in nucleus accumbens (NAc), the critical substrate of the reward circuitry, in cocaine addiction. Genome level expression profiling and epigenomic analysis leading to the recent advances in our understanding of cocaine-induced neural and behavioral adaptations are reviewed here. We examine the convergence of transcriptomic and epigenomic evidence, and discuss about emerging drug target candidates in cocaine-addiction.

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4. DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides. CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample. Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

Association of neonatal hypoxia with lasting changes in left ventricular gene expression: An animal model

Innovations in pediatric cardiovascular surgery have resulted in significant improvements in survival for children with congenital heart disease. In adults with such disease, however, surgical morbidity and mortality remain significant. We hypothesized that hypoxemia in early life causes lasting changes in gene expression in the developing heart and that such changes may persist into later life, affecting the physiology of the adult myocardium. Microarray expression analyses were performed with left ventricular tissue from 10- and 90-day-old rats exposed to hypoxia (inspired oxygen fraction 0.12) for the first 10 days after birth then subsequently reared in ambient air and with tissue from age-matched rats reared entirely in ambient air. Changes in expression of selected genes were confirmed with real-time reverse transcriptase polymerase chain reaction. Left ventricular cardiomyocytes were isolated from adult animals in both groups, and cellular morphology and viability were compared. Microarray analyses revealed significant changes in 1945 and 422 genes in neonates and adults, respectively. Changes in genes associated with adaptive vascular remodeling and energy homeostasis, as well as regulation of apoptosis, were confirmed by real-time reverse transcriptase polymerase chain reaction. The viability of cardiomyocytes isolated from hypoxic animals was significantly lower than in those from control animals (36.7% +/- 13.3% vs 85.0% +/- 2.9%, P = .024). Neonatal hypoxia is associated with significant changes in left ventricular gene expression in both neonatal and adult rats. This may have physiologic implications for the adult myocardium.

Intrinsic androgen-dependent gene expression patterns revealed by comparison of genital fibroblasts from normal males and individuals with complete and partial androgen insensitivity syndrome

BackgroundTo better understand the molecular programs of normal and abnormal genital development, clear-cut definition of androgen-dependent gene expression patterns, without the influence of genotype (46, XX vs. 46, XY), is warranted. Previously, we have identified global gene expression profiles in genital-derived fibroblasts that differ between 46, XY males and 46, XY females with complete androgen insensitivity syndrome (CAIS) due to inactivating mutations of the androgen receptor (AR). While these differences could be due to cell autonomous changes in gene expression induced by androgen programming, recent work suggests they could also be influenced by the location from which the fibroblasts were harvested (topology). To minimize the influence of topology, we compared gene expression patterns of fibroblasts derived from identical urogenital anlagen: the scrotum in normally virilized 46, XY males and the labia majora from completely feminized 46, XY individuals with CAIS.Results612 transcripts representing 440 unique genes differed significantly in expression levels between scrotum and CAIS labia majora, suggesting the effects of androgen programming. While some genes coincided with those we had identified previously (TBX3, IGFBP5, EGFR, CSPG2), a significant number did not, implying that topology had influenced gene expression in our previous experiments. Supervised clustering of gene expression data derived from a large set of fibroblast cultures from individuals with partial AIS revealed that the new, topology controlled data set better classified the specimens.ConclusionInactivating mutations of the AR, in themselves, appear to induce lasting changes in gene expression in cultured fibroblasts, independent of topology and genotype. Genes identified are likely to be relevant candidates to decipher androgen-dependent normal and abnormal genital development.

Novel transcription factors are induced by chronic cocaine treatment.

There are many other transcription factors that are regulated by cocaine, and there are several nontranscriptional mechanisms of regulating protein levels as well. However, the induction of the chronic AP-1 complex and the chronic Fras provides a mechanism capable of underlying long-lasting alterations in gene expression following chronic cocaine treatment. We hypothesize that while the well-known acute effects of cocaine are occurring, the acutely induced cAMP pathway, possibly in conjunction with other second messenger pathways, is also inducing two general types of alterations in gene expression. 1. Acutely induced significant alterations in gene expression are rapidly induced and relatively short-lived. These alterations in gene expression would allow the cell to rapidly adapt to cocaine in its environment without committing to long-term changes. 2. Additional alterations in gene expression are induced by each acute administration during chronic cocaine exposure. These latter alterations are gradually induced and long-lasting. With each acute activation, the level of induction of these latter longer-lasting alterations in gene expression would be relatively small and insignificant compared to the rapidly induced acute alterations in gene expression. Therefore, with each acute administration, the cell does not commit itself to long-term alterations. However, these small but longer lasting changes in gene expression would accumulate with each acute administration during chronic cocaine treatment, similar to the accumulation of the chronic AP-I complex (FIG. 4). The cumulative alterations which occur during repeated administrations eventually reach a level where they produce significant and long-lasting alterations in gene expression different from those induced acutely. This incremental induction of long-lasting alterations in gene expression would allow the cell to gradually commit to long-term alterations for the purpose of adapting to repeated long-term exposure to cocaine in its environment. These long-lasting alterations in gene expression may underlie the gradual induction of persistent changes in protein levels, neuronal signaling, and related behaviors. This hypothesis allows for the different adaptations observed following acute versus chronic cocaine administration at the biochemical, physiological, and behavioral levels. We hope these investigations provide ideas for specific pharmacological blockade or reversal of the long-term, versus short-term, alterations in gene expression that lead to addiction, without interfering with the animal's or human's ability to experience reward. This would be important for ensuring both efficacy and compliance.