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The localization, number, and function of postsynaptic AMPA-type glutamate receptors (AMPARs) are crucial for synaptic plasticity, a cellular correlate for learning and memory. The Hippo pathway member WWC1 is an important component of AMPAR-containing protein complexes. However, the availability of WWC1 is constrained by its interaction with the Hippo pathway kinases LATS1 and LATS2 (LATS1/2). Here, we explored the biochemical regulation of this interaction and found that it is pharmacologically targetable in vivo. In primary hippocampal neurons, phosphorylation of LATS1/2 by the upstream kinases MST1 and MST2 (MST1/2) enhanced the interaction between WWC1 and LATS1/2, which sequestered WWC1. Pharmacologically inhibiting MST1/2 in male mice and in human brain-derived organoids promoted the dissociation of WWC1 from LATS1/2, leading to an increase in WWC1 in AMPAR-containing complexes. MST1/2 inhibition enhanced synaptic transmission in mouse hippocampal brain slices and improved cognition in healthy male mice and in male mouse models of Alzheimer's disease and aging. Thus, compounds that disrupt the interaction between WWC1 and LATS1/2 might be explored for development as cognitive enhancers.

Cell-cell communication is mediated by membrane receptors and their ligands, such as the Eph/ephrin system, orchestrating cell migration during development and in diverse cancer types. Epigenetic mechanisms are key for integrating external "signals", e.g., from neighboring cells, into the transcriptome in health and disease. Previously, we reported ephrinA5 to trigger transcriptional changes of lncRNAs and protein-coding genes in cerebellar granule cells, a cell model for medulloblastoma. LncRNAs represent important adaptors for epigenetic writers through which they regulate gene expression. Here, we investigate a lncRNA-mediated targeting of DNMT1 to specific gene loci by the combined power of in silico modeling of RNA/DNA interactions and wet lab approaches, in the context of the clinically relevant use case of ephrinA5-dependent regulation of cellular motility of cerebellar granule cells. We provide evidence that Snhg15, a cancer-related lncRNA, recruits DNMT1 to the Ncam1 promoter through RNA/DNA triplex structure formation and the interaction with DNMT1. This mediates DNA methylation-dependent silencing of Ncam1, being abolished by ephrinA5 stimulation-triggered reduction of Snhg15 expression. Hence, we here propose a triple helix recognition mechanism, underlying cell motility regulation via lncRNA-targeted DNA methylation in a clinically relevant context., (© 2023. BioMed Central Ltd., part of Springer Nature.)

An adaptive stress response involves various mediators and circuits orchestrating a complex interplay of physiological, emotional, and behavioral adjustments. We identified a population of corticotropin-releasing hormone (CRH) neurons in the lateral part of the interstitial nucleus of the anterior commissure (IPACL), a subdivision of the extended amygdala, which exclusively innervate the substantia nigra (SN). Specific stimulation of this circuit elicits hyperactivation of the hypothalamic-pituitary-adrenal axis, locomotor activation, and avoidance behavior contingent on CRH receptor type 1 (CRHR1) located at axon terminals in the SN, which originate from external globus pallidus (GPe) neurons. The neuronal activity prompting the observed behavior is shaped by IPACL CRH and GPe CRHR1 neurons coalescing in the SN. These results delineate a previously unidentified tripartite CRH circuit functionally connecting extended amygdala and basal ganglia nuclei to drive locomotor activation and avoidance behavior.

Early life stress has profound effects on the development of the central nervous system. We exposed 9-day-old rat pups to a 24 h maternal deprivation (MD) and sacrificed them as young adults (60-day-old), with the aim to study the effects of early stress on forebrain circuitry. We estimated numbers of various immunohistochemically defined interneuron subpopulations in several neocortical regions and in the hippocampus. MD rats showed reduced numbers of parvalbumin-expressing interneurons in the CA1 region of the hippocampus and in the prefrontal cortex, compared with controls. Numbers of reelin-expressing and calretinin-expressing interneurons were also reduced in the CA1 and CA3 hippocampal areas, but unaltered in the neocortex of MD rats. The number of calbinin-expressing interneurons in the neocortex was similar in the MD rats compared with controls. We analyzed cell death in 15-day-old rats after MD and found no difference compared to control rats. Thus, our results more likely reflect the downregulation of markers than the actual loss of interneurons. To investigate synaptic activity in the hippocampus we immunostained for glutamatergic and inhibitory vesicular transporters. The number of inhibitory synapses was decreased in the CA1 and CA3 regions of the hippocampus in MD rats, with the normal number of excitatory synapses. Our results indicate complex, cell type-specific, and region-specific alterations in the inhibitory circuitry induced by maternal deprivation. Such alterations may underlie symptoms of MD at the behavioral level and possibly contribute to mechanisms by which early life stress causes neuropsychiatric disorders, such as schizophrenia., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Aksic, Poleksic, Aleksic, Petronijevic, Radonjic, Jakovcevski, Kapor, Divac, Filipovic and Jakovcevski.)

Several studies in humans and rodents suggest an association between impulsivity and activity of the stress response on the one hand and addiction vulnerability on the other. The neural cell adhesion molecule (NCAM) has been related to several neuropsychiatric disorders in humans. Constitutively NCAM-deficient (-/-) mice display enhanced novelty-induced behavior and hyperfunction of the hypothalamic-pituitary-adrenal axis. Here we hypothesize that NCAM deficiency causes an altered response to cocaine. Cocaine-induced behaviors of NCAM-/- mice and wild-type (+/+) littermates were analyzed in the conditioned place preference (CPP) test. c-fos mRNA levels were investigated by quantitative polymerase chain reaction (qPCR) to measure neural activation after exposure to the cocaine-associated context. NCAM-/- mice showed an elevated cocaine-induced sensitization, enhanced CPP, impaired extinction, and potentiated cocaine-induced hyperlocomotion and CPP after extinction. NCAM-/- showed no potentiated CPP as compared with NCAM+/+ littermates when a natural rewarding stimulus (ie, an unfamiliar female) was used, suggesting that the behavioral alterations of NCAM-/- mice observed in the CPP test are specific to the effects of cocaine. Activation of the prefrontal cortex and nucleus accumbens induced by the cocaine-associated context was enhanced in NCAM-/- compared with NCAM+/+ mice. Finally, cocaine-induced behavior correlated positively with novelty-induced behavior and plasma corticosterone levels in NCAM-/- mice and negatively with NCAM mRNA levels in the hippocampus and nucleus accumbens in wild-type mice. Our findings indicate that NCAM deficiency affects cocaine-induced CPP in mice and support the view that hyperfunction of the stress response system and reactivity to novelty predict the behavioral responses to cocaine., (© 2020 The Authors. Addiction Biology published by John Wiley & Sons Ltd on behalf of Society for the Study of Addiction.)

Background: The study aimed to examine the effect of early hyperglycemia on the morbidity/mortality of very low birth weight premature infants., Methods: This retrospective study included all premature infants with gestational age ≤32 gestational weeks, hospitalized at the Department of Intensive Neonatal Care, Clinical Center Kragujevac, during the period 2017-2019. Hyperglycemia was defined as glycemia of ≥12 mmol/l in one measurement, or > 10 mmol/l in two measurements, at repeated intervals of 2-4 hours. Glycemia was determined from capillary blood, using a gas analyzer of Gem Premier 3000, during the first 7 days of life. Continuous intravenous insulin infusion was administered after ineffective glucose restriction at glycemic values of > 14 mmol/l., Results: Patients with normoglycemia (41/72 (56.94%)) and hyperglycemia (31/72 (43.06%)) did not differ in gender, gestational age, mode of delivery and antenatal administration of steroids, while birth weight had a tendency to be lower in the hyperglycemic group (p=0.052). Hyperglycemia was significantly associated with a low APGAR score at the fifth minute (p=0.048), necrotizing enterocolitis (p=0.011), and shorter duration of mechanical ventilation (p=0.006). Hyperglycemia was associated with significantly more frequent fatal outcomes (35.5%) when compared with the normoglycemic group (4.9%). Accordingly, these patients required inotropic (r=0.036) and insulin therapy (r < 0.001) more often. Retinopathy of prematurity, bronchopulmonary dysplasia and sepsis did not correlate with hyperglycemia in our study. Intraventricular hemorrhage of the first degree was more often associated with normoglycemia in premature infants on prolonged mechanical ventilation while more severe intracranial hemorrhage was more common in the hyperglycemic group but did not result in statistical significance due to the small number of patients., Conclusions: Monitoring glucose levels in the blood of very low birth weight premature infants is clinically important because abnormalities in glucose homeostasis can have serious short-term and long-term consequences.

Neuropsychiatric disorders are a collective of cerebral conditions with a multifactorial and polygenetic etiology. Dysregulation of epigenetic profiles in the brain is considered to play a critical role in the development of neuropsychiatric disorders. SET domain, bifurcate 1 (SETDB1), functioning as a histone H3K9 specific methyltransferase, is not only critically involved in transcriptional silencing and local heterochromatin formation, but also affects genome-wide neuronal epigenetic profiles and is essential for 3D genome integrity. Here, we provide a review of recent advances towards understanding the role of SETDB1 in the central nervous system during early neurodevelopment as well as in the adult brain, with a particular focus on studies that link its functions to neuropsychiatric disorders and related behavioral changes, and the exploration of novel therapeutic strategies targeting SETDB1.

Chromatin immunoprecipitation (ChIP) in conjunction with qPCR or next generation sequencing (ChIP-seq) is used to detect protein-DNA interaction. Typically, DNA bound to a protein of interest is captured with an antibody against this protein, and DNA is then purified from DNA-protein complexes. Here, we describe a native Chromatin immunoprecipitation (N-ChIP) approach which is an efficient ChIP method with high resolution for histone modifications and a number of transcription factors. This protocol has been tailored for cultured primary rat astrocytes, and we included the preparation of astrocytic cell cultures in this protocol.

Objective: Vulnerability to eating disorders (EDs) is broadly assumed to be associated with early life stress. However, a careful examination of the literature shows that susceptibility to EDs may depend on the type, severity and timing of the stressor and the sex of the individual. We aimed at exploring the link between chronic prenatal stress and predisposition to EDs and metabolic disease., Methods: We used a chronic variable stress protocol during gestation to explore the metabolic response of male and female offspring to food restriction (FR), activity-based anorexia (ABA), binge eating (BE) and exposure to high fat (HF) diet., Results: Contrary to controls, prenatally stressed (PNS) female offspring showed resistance to ABA and BE and displayed a lower metabolic rate leading to hyperadiposity and obesity on HF diet. Male PNS offspring showed healthy responses to FR and ABA, increased propensity to binge and improved coping with HF compared to controls. We found that long-lasting abnormal responses to metabolic challenge are linked to fetal programming and adult hypothalamic dysregulation in PNS females, resulting from sexually dimorphic adaptations in placental methylation and gene expression., Conclusions: Our results show that maternal stress may have variable and even opposing effects on ED risk, depending on the ED and the sex of the offspring., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Objective: Patients with non-functioning pituitary adenomas exhibit high morbidity and mortality rates. Growth hormone deficiency and high doses of glucocorticoid substitution therapy have been identified as corresponding risk factors. Interestingly, high levels of endogenous cortisol in, e.g., patients with post-traumatic stress disorder or patients with Cushing's disease have been linked to shorter telomere length. Telomeres are noncoding DNA regions located at the end of chromosomes consisting of repetitive DNA sequences which shorten with ageing and hereby determine cell survival. Therefore, telomere length can serve as a predictor for the onset of disease and mortality in some endocrine disorders (e.g., Cushing's disease)., Design/methods: We examine telomere length from blood in patients (n = 115) with non-functioning pituitary adenomas (NFPA) in a cross-sectional case control (n = 106, age-, gender- matched) study using qPCR. Linear regression models were used to identify independent predictors of telomere length., Results: We show that patients with NFPA exhibited shorter telomeres than controls. No significant association of indices of growth hormone deficiency (IGF-1-level-SDS, years of unsubstituted growth hormone deficiency etc.) with telomere length was detected. Interestingly, linear regression analysis showed that hydrocortisone replacement dosage in patients with adrenal insufficiency (n = 52) was a significant predictor for shorter telomere length (β = 0.377; p = 0.018) independent of potential confounders. Median split analysis revealed that higher hydrocortisone intake (> 20 mg) was associated with significantly shorter telomeres., Conclusion: These observations strengthen the importance of adjusted glucocorticoid treatment in NFPA patients with respect to morbidity and mortality rates.

N 6 -methyladenosine (m 6 A) and N 6 ,2'-O-dimethyladenosine (m 6 Am) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed m 6 A/m, in the stress response in the adult brain in vivo is currently unknown. Here, we provide a detailed analysis of the stress epitranscriptome using m 6 A/m-seq, global and gene-specific m 6 A/m measurements. We show that stress exposure and glucocorticoids region and time specifically alter m 6 A/m and its regulatory network. We demonstrate that deletion of the methyltransferase Mettl3 or the demethylase Fto in adult neurons alters the m 6 A/m epitranscriptome, increases fear memory, and changes the transcriptome response to fear and synaptic plasticity. Moreover, we report that regulation of m 6 A/m is impaired in major depressive disorder patients following glucocorticoid stimulation. Our findings indicate that brain m 6 A/m represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the m 6 A/m response may contribute to the pathophysiology of stress-related psychiatric disorders., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

The interplay between corticotropin-releasing hormone (CRH) and the dopaminergic system has predominantly been studied in addiction and reward, while CRH-dopamine interactions in anxiety are scarcely understood. We describe a new population of CRH-expressing, GABAergic, long-range-projecting neurons in the extended amygdala that innervate the ventral tegmental area and alter anxiety following chronic CRH depletion. These neurons are part of a distinct CRH circuit that acts anxiolytically by positively modulating dopamine release.

Anorexia nervosa (AN) is a devastating eating disorder characterized by self-starvation that mainly affects women. Its etiology is unknown, which impedes successful treatment options leading to a limited chance of full recovery. Here, we show that gestation is a vulnerable window that can influence the predisposition to AN. By screening placental microRNA expression of naive and prenatally stressed (PNS) fetuses and assessing vulnerability to activity-based anorexia (ABA), we identify miR-340 as a sexually dimorphic regulator involved in prenatal programming of ABA. PNS caused gene-body hypermethylation of placental miR-340, which is associated with reduced miR-340 expression and increased protein levels of several target transcripts, GR, Cry2 and H3F3b. MiR-340 is linked to the expression of several nutrient transporters both in mice and human placentas. Using placenta-specific lentiviral transgenes and embryo transfer, we demonstrate the key role miR-340 plays in the mechanism involved in early life programming of ABA.

Excessive intake of high-caloric diets as well as subsequent development of obesity and diabetes mellitus may exert a wide range of unfavorable effects on the central nervous system (CNS) in the long-term. The potentially harmful effects of such diets were suggested to be mitigated by physical exercise. Here, we conducted a study investigating early effects of a cafeteria-diet on gray and white brain matter volume by means of voxel-based morphometry (VBM) and region-of-interest (ROI) analysis. Half of the mice performed voluntary wheel running to study if regular physical exercise prevents unfavorable effects of a cafeteria-diet. In addition, histological analyses for myelination and neurogenesis were performed. As expected, wheel running resulted in a significant increase of gray matter volume in the CA1-3 areas, the dentate gyrus and stratum granulosum of the hippocampus in the VBM analysis, while a positive effect of the cafeteria-diet was shown for the whole hippocampal CA1-3 area only in the ROI analysis, indicating a regional volume effect. It was earlier found that hippocampal neurogenesis may be related to volume increases after exercise. Interestingly, while running resulted in a significant increase in neurogenesis assessed by doublecortin (DCX)-labeling, this was not true for cafeteria diet. This indicates different underlying mechanisms for gray matter increase. Moreover, animals receiving cafeteria diet only showed mild deficits in long-term memory assessed by the puzzle-box paradigm, while executive functioning and short term memory were not affected. Our data therefore highlight that high caloric diet impacts on the brain and behavior. Physical exercise seems not to interact with these mechanisms.

Different types of sequencing biases have been described and subsequently improved for a variety of sequencing systems, mostly focusing on the widely used Illumina systems. Similar studies are missing for the SOLiD 5500xl system, a sequencer which produced many data sets available to researchers today. Describing and understanding the bias is important to accurately interpret and integrate these published data in various ongoing research projects. We report a particularly strong GC bias for this sequencing system when analyzing a defined gDNA mix of 5 microbes with a wide range of different GC contents (20-72%) when comparing to the expected distribution and Illumina MiSeq data from the same DNA pool. Since we observed this bias already under PCR-free conditions, changing the PCR conditions during library preparation - a common strategy to handle bias in the Illumina system - was not relevant. Source of the bias appeared to be an uneven heat distribution during the SOLiD emulsion PCR (ePCR) - for enrichment of libraries prior loading - since ePCR in either small pouches or in 96-well plates improved the GC bias. Sequencing of chromatin immunoprecipitated DNA (ChIP-seq) is a common approach in epigenetics. ChIP-seq of the mixed source histone mark H3K9ac (acetyl Histone H3 lysine 9), typically found on promoter regions and on gene bodies, including CpG islands, performed on a SOLiD 5500xl machine, resulted in major loss of reads at GC rich loci (GC content ≥ 62%), not explained by low sequencing depth. This was improved with adaptations of the ePCR.

Binge eating (BE) is a common aberrant form of eating behavior, characterized by overconsumption of food in a brief period of time. Recurrent episodes of BE constitute the BE disorder, which mostly affects females and is associated with early-life adversities. Here, we show that corticotropin releasing factor (CRF)-induced prenatal stress (PNS) in late gestation predisposes female offspring to BE-like behavior that coincides with hypomethylation of hypothalamic miR-1a and downstream dysregulation of the melanocortin system through Pax7/Pax3. Moreover, exposing the offspring to a methyl-balanced diet during adolescence prevents the dysregulation and predisposition from being triggered. We demonstrate that gestational programming, per se, will not lead to BE-like behavior, but pre-existing alterations due to prenatal programming are revealed only when challenged during adolescence. We provide experimental evidence for long-term epigenetic abnormalities stemming from PNS in predisposing female offspring to BE disorder as well as a potential non-invasive prevention strategy., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Major depressive disorder (MDD) is a multifactorial disease, weakly linked to multiple genetic risk factors. In contrast to that, environmental factors and "gene × environment" interaction between specific risk genes and environmental factors, such as severe or early stress exposure, have been strongly linked to MDD vulnerability. Stressors can act on the interface between an organism and the environment, the epigenome. The molecular foundation for the impact of stressors on the risk to develop MDD is based on the hormonal stress response itself: the glucocorticoid receptor (GR, encoded by NR3C1). NR3C1 can directly interact with the epigenome in the cell nucleus. Besides DNA methylation, histone modifications have been reported to be crucial targets for the interaction with the stress response system. Here, we review critical findings on the impact of the most relevant histone modifications, i.e. histone acetylation and methylation, in the context of MDD and related animal models. We discuss new treatment options which have been based on these findings, including histone deacetylase inhibitors (HDACis) and drugs targeting specific histone marks, closely linked to psychiatric disease. In this context we talk about contemporary and future approaches required to fully understand (1) the epigenetics of stress-related disease and (2) the mode of action of potential MDD drugs targeting histone modifications. This includes harnessing the unprecedented potentials of genome-wide analysis of the epigenome and transcriptome, in a cell type-specific manner, and the use of epigenome editing technologies to clearly link epigenetic marks on specific genomic loci to functional relevance.

Lysine (K) methyltransferase 2a (Kmt2a) and other regulators of H3 lysine 4 methylation, a histone modification enriched at promoters and enhancers, are widely expressed throughout the brain, but molecular and cellular phenotypes in subcortical areas remain poorly explored. We report that Kmt2a conditional deletion in postnatal forebrain is associated with excessive nocturnal activity and with absent or blunted responses to stimulant and dopaminergic agonist drugs, in conjunction with near-complete loss of spike-timing-dependent long-term potentiation in medium spiny neurons (MSNs). Selective ablation of Kmt2a, but not the ortholog Kmt2b, in adult ventral striatum/nucleus accumbens neurons markedly increased anxiety scores in multiple behavioral paradigms. Striatal transcriptome sequencing in adult mutants identified 262 Kmt2a-sensitive genes, mostly downregulated in Kmt2a-deficient mice. Transcriptional repression includes the 5-Htr2a serotonin receptor, strongly associated with anxiety- and depression-related disorders in human and animal models. Consistent with the role of Kmt2a in promoting gene expression, the transcriptional regulators Bahcc1, Isl1, and Sp9 were downregulated and affected by H3K4 promoter hypomethylation. Therefore, Kmt2a regulates synaptic plasticity in striatal neurons and provides an epigenetic drug target for anxiety and dopamine-mediated behaviors.

Background: Early childhood malnutrition affects 113 million children worldwide, impacting health and increasing vulnerability for cognitive and behavioral disorders later in life. Molecular signatures after childhood malnutrition, including the potential for intergenerational transmission, remain unexplored., Methods: We surveyed blood DNA methylomes (~483,000 individual CpG sites) in 168 subjects across two generations, including 50 generation 1 individuals hospitalized during the first year of life for moderate to severe protein-energy malnutrition, then followed up to 48 years in the Barbados Nutrition Study. Attention deficits and cognitive performance were evaluated with the Connors Adult Attention Rating Scale and Wechsler Abbreviated Scale of Intelligence. Expression of nutrition-sensitive genes was explored by quantitative reverse transcriptase polymerase chain reaction in rat prefrontal cortex., Results: We identified 134 nutrition-sensitive, differentially methylated genomic regions, with most (87%) specific for generation 1. Multiple neuropsychiatric risk genes, including COMT, IFNG, MIR200B, SYNGAP1, and VIPR2 showed associations of specific methyl-CpGs with attention and IQ. IFNG expression was decreased in prefrontal cortex of rats showing attention deficits after developmental malnutrition., Conclusions: Early childhood malnutrition entails long-lasting epigenetic signatures associated with liability for attention and cognition, and limited potential for intergenerational transmission., Competing Interests: Competing Financial Interests: The authors report no biomedical financial interests or potential conflicts of interest., (Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Astrocytes orchestrate arrangement and functions of neuronal circuits and of the blood-brain barrier. Dysfunctional astrocytes characterize mood disorders, here showcased by deregulation of the astrocyte end-feet protein Aquaporin-4 around blood vessels and, hypothetically, of the astrocyte-specific phagocytic protein MEGF10 to shape synapses. Development of mood disorders is often a result of 'gene × environment' interactions, regulated among others by histone modifications and related modulator enzymes, which rapidly promote adaptive responses. Thus, they represent ideal targets of drugs aimed at inducing stable effects with quick onsets. One of the prevalent features of histone modifications and their modulators is their cell-type specificity. Investigating cell type-specific epigenetic modulations upon drug administration might therefore help to implement therapeutic treatments., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Excessive intake of high-caloric diets as well as subsequent development of obesity and diabetes mellitus may exert a wide range of unfavorable effects on the central nervous system (CNS). It has been suggested that one mechanism in this context is the promotion of neuroinflammation. The potentially harmful effects of such diets were suggested to be mitigated by physical exercise. Here, we conducted a study investigating the effects of physical exercise in a cafeteria-diet mouse model on CNS metabolites by means of in vivo proton magnetic resonance spectroscopy ((1)HMRS). In addition postmortem histologic and real-time (RT)-PCR analyses for inflammatory markers were performed. Cafeteria diet induced obesity and hyperglycemia, which was only partially moderated by exercise. It also induced several changes in CNS metabolites such as reduced hippocampal glutamate (Glu), choline-containing compounds (tCho) and N-acetylaspartate (NAA)+N-acetyl-aspartyl-glutamic acid (NAAG) (tNAA) levels, whereas opposite effects were seen for running. No association of these effects with markers of central inflammation could be observed. These findings suggest that while voluntary wheel running alone is insufficient to prevent the unfavorable peripheral sequelae of the diet, it counteracted many changes in brain metabolites. The observed effects seem to be independent of neuroinflammation.

To investigate epigenetic contributions to Huntington's disease (HD) pathogenesis, we carried out genome-wide mapping of the transcriptional mark, trimethyl-histone H3-lysine 4 (H3K4me3) in neuronal nuclei extracted from prefrontal cortex of HD cases and controls using chromatin immunoprecipitation followed by deep-sequencing. Neuron-specific mapping of the genome-wide distribution of H3K4me3 revealed 136 differentially enriched loci associated with genes implicated in neuronal development and neurodegeneration, including GPR3, TMEM106B, PDIA6 and the Notch signaling genes hairy and enhancer of split 4 (HES4) and JAGGED2, supporting the view that the neuronal epigenome is affected in HD. Importantly, loss of H3K4me3 at CpG-rich sequences on the HES4 promoter was associated with excessive DNA methylation, reduced binding of nuclear proteins to the methylated region and altered expression of HES4 and HES4 targeted genes MASH1 and P21 involved in striatal development. Moreover, hypermethylation of HES4 promoter sequences was strikingly correlated with measures of striatal degeneration and age-of-onset in a cohort of 25 HD brains (r = 0.56, P = 0.006). Lastly, shRNA knockdown of HES4 in human neuroblastoma cells altered MASH1 and P21 mRNA expression and markedly increased mutated HTT-induced aggregates and cell death. These findings, taken together, suggest that epigenetic dysregulation of HES4 could play a critical role in modifying HD disease pathogenesis and severity., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Three-dimensional chromosomal conformations regulate transcription by moving enhancers and regulatory elements into spatial proximity with target genes. Here we describe activity-regulated long-range loopings bypassing up to 0.5 Mb of linear genome to modulate NMDA glutamate receptor GRIN2B expression in human and mouse prefrontal cortex. Distal intronic and 3' intergenic loop formations competed with repressor elements to access promoter-proximal sequences, and facilitated expression via a "cargo" of AP-1 and NRF-1 transcription factors and TALE-based transcriptional activators. Neuronal deletion or overexpression of Kmt2a/Mll1 H3K4- and Kmt1e/Setdb1 H3K9-methyltransferase was associated with higher-order chromatin changes at distal regulatory Grin2b sequences and impairments in working memory. Genetic polymorphisms and isogenic deletions of loop-bound sequences conferred liability for cognitive performance and decreased GRIN2B expression. Dynamic regulation of chromosomal conformations emerges as a novel layer for transcriptional mechanisms impacting neuronal signaling and cognition., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Neurodegenerative diseases, such as frontotemporal dementia (FTD), are often associated with behavioral deficits, but the underlying anatomical and molecular causes remain poorly understood. Here we show that forebrain-specific expression of FTD-associated mutant CHMP2B in mice causes several age-dependent neurodegenerative phenotypes, including social behavioral impairments. The social deficits were accompanied by a change in AMPA receptor (AMPAR) composition, leading to an imbalance between Ca(2+)-permeable and Ca(2+)-impermeable AMPARs. Expression of most AMPAR subunits was regulated by the brain-enriched microRNA miR-124, whose abundance was markedly decreased in the superficial layers of the cerebral cortex of mice expressing the mutant CHMP2B. We found similar changes in miR-124 and AMPAR levels in the frontal cortex and induced pluripotent stem cell-derived neurons from subjects with behavioral variant FTD. Moreover, ectopic miR-124 expression in the medial prefrontal cortex of mutant mice decreased AMPAR levels and partially rescued behavioral deficits. Knockdown of the AMPAR subunit Gria2 also alleviated social impairments. Our results identify a previously undescribed mechanism involving miR-124 and AMPARs in regulating social behavior in FTD and suggest a potential therapeutic avenue.

The neural cell adhesion molecule (NCAM) plays a crucial role in stress-related brain function, emotional behavior and memory formation. In this study, we investigated the functions of the glucocorticoid and serotonergic systems in mice constitutively deficient for NCAM (NCAM-/- mice). Our data provide evidence for a hyperfunction of the hypothalamic-pituitary-adrenal axis, with enlarged adrenal glands and increased stress-induced corticosterone release, but reduced hippocampal glucocorticoid receptor expression in NCAM-/- mice when compared to NCAM+/+ mice. We also obtained evidence for a hypofunction of 5-HT1A autoreceptors as indicated by increased 8-0H-DPAT-induced hypothermia. These findings suggest a disturbance of both humoral and neural stress systems in NCAM-/- mice. Accordingly, we not only confirmed previously observed hyperarousal of NCAM-/- mice in various anxiety tests, but also observed an increased response to novelty exposure in these animals. Spatial learning deficits of the NCAM-/- mice in a Morris Water maze persisted, even when mice were pretrained to prevent effects of novelty or stress. We suggest that NCAM-mediated processes are involved in both novelty/stress-related emotional behavior and in cognitive function during spatial learning.

Fragile X syndrome (FXS), the most common cause of inherited mental retardation and autism, is caused by transcriptional silencing of FMR1, which encodes the translational repressor fragile X mental retardation protein (FMRP). FMRP and cytoplasmic polyadenylation element-binding protein (CPEB), an activator of translation, are present in neuronal dendrites, are predicted to bind many of the same mRNAs and may mediate a translational homeostasis that, when imbalanced, results in FXS. Consistent with this possibility, Fmr1(-/y); Cpeb1(-/-) double-knockout mice displayed amelioration of biochemical, morphological, electrophysiological and behavioral phenotypes associated with FXS. Acute depletion of CPEB1 in the hippocampus of adult Fmr1(-/y) mice rescued working memory deficits, demonstrating reversal of this FXS phenotype. Finally, we find that FMRP and CPEB1 balance translation at the level of polypeptide elongation. Our results suggest that disruption of translational homeostasis is causal for FXS and that the maintenance of this homeostasis by FMRP and CPEB1 is necessary for normal neurologic function.

Background: Postmortem brain studies have shown that HDAC1-a lysine deacetylase with broad activity against histones and nonhistone proteins-is frequently expressed at increased levels in prefrontal cortex (PFC) of subjects diagnosed with schizophrenia and related disease. However, it remains unclear whether upregulated expression of Hdac1 in the PFC could affect cognition and behavior., Methods: Using adeno-associated virus, an Hdac1 transgene was expressed in young adult mouse PFC, followed by behavioral assays for working and long-term memory, repetitive activity, and response to novelty. Prefrontal cortex transcriptomes were profiled by microarray. Antipsychotic drug effects were explored in mice treated for 21 days with haloperidol or clozapine., Results: Hdac1 overexpression in PFC neurons and astrocytes resulted in robust impairments in working memory, increased repetitive behaviors, and abnormal locomotor response profiles in novel environments. Long-term memory remained intact. Over 300 transcripts showed subtle but significant changes in Hdac1-overexpressing PFC. Major histocompatibility complex class II (MHC II)-related transcripts, including HLA-DQA1/H2-Aa, HLA-DQB1/H2-Ab1, and HLA-DRB1/H2-Eb1, located in the chromosome 6p21.3-22.1 schizophrenia and bipolar disorder risk locus, were among the subset of genes with a more robust (>1.5-fold) downregulation in expression. Hdac1 levels declined during the course of normal PFC development. Antipsychotic drug treatment, including the atypical clozapine, did not affect Hdac1 levels in PFC but induced expression of multiple MHC II transcripts., Conclusions: Excessive HDAC1 activity, due to developmental defects or other factors, is associated with behavioral alterations and dysregulated expression of MHC II and other gene transcripts in the PFC., (© 2013 Society of Biological Psychiatry.)

Alzheimer's disease (AD) is a devastating neurodegenerative disorder and the most common cause of elderly dementia. In an effort to contribute to the potential of molecular approaches to reduce degenerative processes we have tested the possibility that the neural adhesion molecule L1 ameliorates some characteristic cellular and molecular parameters associated with the disease in a mouse model of AD. Three-month-old mice overexpressing mutated forms of amyloid precursor protein and presenilin-1 under the control of a neuron-specific promoter received an injection of adeno-associated virus encoding the neuronal isoform of full-length L1 (AAV-L1) or, as negative control, green fluorescent protein (AAV-GFP) into the hippocampus and occipital cortex. Four months after virus injection, the mice were analyzed for histological and biochemical parameters of AD. AAV-L1 injection decreased the Aβ plaque load, levels of Aβ42, Aβ42/40 ratio and astrogliosis compared with AAV-GFP controls. AAV-L1 injected mice also had increased densities of inhibitory synaptic terminals on pyramidal cells in the hippocampus when compared with AAV-GFP controls. Numbers of microglial cells/macrophages were similar in both groups, but numbers of microglial cells/macrophages per plaque were increased in AAV-L1 injected mice. To probe for a molecular mechanism that may underlie these effects, we analyzed whether L1 would directly and specifically interact with Aβ. In a label-free binding assay, concentration dependent binding of the extracellular domain of L1, but not of the close homolog of L1 to Aβ40 and Aβ42 was seen, with the fibronectin type III homologous repeats 1-3 of L1 mediating this effect. Aggregation of Aβ42 in vitro was reduced in the presence of the extracellular domain of L1. The combined observations indicate that L1, when overexpressed in neurons and glia, reduces several histopathological hallmarks of AD in mice, possibly by reduction of Aβ aggregation. L1 thus appears to be a candidate molecule to ameliorate the pathology of AD, when applied in therapeutically viable treatment schemes., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The exploration of brain epigenomes, which consist of various types of DNA methylation and covalent histone modifications, is providing new and unprecedented insights into the mechanisms of neural development, neurological disease and aging. Traditionally, chromatin defects in the brain were considered static lesions of early development that occurred in the context of rare genetic syndromes, but it is now clear that mutations and maladaptations of the epigenetic machinery cover a much wider continuum that includes adult-onset neurodegenerative disease. Here, we describe how recent advances in neuroepigenetics have contributed to an improved mechanistic understanding of developmental and degenerative brain disorders, and we discuss how they could influence the development of future therapies for these conditions.

The specificity of the response of an organism is an important variable influencing stress-related parameters and psychopathological states. We have shown that trait anxiety in C57BL/6 mice, determined by their emergence latencies in the free choice open field test, positively correlates with the long-term behavioral and neuroendocrinological changes induced by a stressor. Here, we show that this interindividual variability is caused by a different reactivity of the hypothalamus-pituitary-adrenal (HPA) axis upon exposure to a stressor. Mice with high trait anxiety (long emergence latency, LEL) display a more pronounced stress-induced activation of the HPA axis than mice with low trait anxiety (short emergence latency, SEL). Moreover, stress-induced activation of tyrosine hydroxylase and corticotropin-releasing hormone occurred in LEL but not SEL mice. In search of the molecular mechanisms underlying these differences, we found that under non-stressed conditions mRNA and protein levels of the glucocorticoid receptor in the hippocampus were higher in LEL mice compared to SEL mice. Also, systemic injection of the glucocorticoid receptor antagonist RU486 decreased the stress-induced activation of the HPA axis and the long-term anxiogenic effects of stress observed in LEL mice. Finally, the rewarding properties of cocaine were enhanced in LEL mice compared to SEL mice, suggesting a causal link between trait anxiety, stress activity and the behavioral responses to drugs of addiction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The growth arrest specific 5 (gas5) is a noncoding protein gene that hosts small nucleolar RNAs. Based on the observation that gas5 RNA level in the brain is highest in the hippocampus and remarkably enhanced in aged mice, we tested the hypothesis that gas5 is involved in functions controlled by the hippocampus and known to be affected by age, such as spatial learning and novelty-induced behaviors. We show that aged (22-month-old) C57BL/6 male mice have spatial-learning impairments, reduced novelty-induced exploration, and enhanced gas5 RNA levels in the hippocampus compared to young (3-month-old) mice. At both ages, levels of gas5 RNA in the hippocampus negatively correlated with novelty-induced exploration in the open field and elevated-plus maze tests. No correlations were found between gas5 RNA levels in the hippocampus and performance in the water maze test. The expression of gas5 RNA in the rest of the brain did not correlate with any behavioral parameter analyzed. Because variations in novelty-induced behaviors could be caused by stressfull experiences, we analyzed whether gas5 RNA levels in the hippocampus are regulated by acute stressors. We found that gas5 RNA levels in the hippocampus were upregulated by 50% 24 h after a psychogenic stressor (60-min olfactory contact with a rat) but were unchanged after exposure to an unfamiliar environment or after acquisition of new spatial information in a one-trial learning task. The present results suggest that strong psychogenic stressors upregulate gas5 RNA in the hippocampus, which in turn affects novelty-induced responses controlled by this region. We hypothesize that long-life exposure to stressors causes an age-dependent increase in hippocampal gas5 RNA levels, which could be responsible for age-related reduced novelty-induced behaviors, thus suggesting a new mechanism by which ageing and stress affect hippocampal function.

Antibodies differentiating between the mono-, di- and trimethylated forms of specific histone lysine residues are a critical tool in epigenome research, but show variable specificity, potentially limiting comparisons across studies and between samples. Using trimethyl histone H3 lysine 4 (H3K4me3)-a mark enriched at transcription start sites (TSS) of active genes-as an example, we describe how simple co-incubation with synthetic peptide of the K4me2 modification leads to increased specificity for K4me3 and a much sharper peak distribution proximal to TSS following chromatin immunoprecipitation and massively parallel sequencing (ChIP-Seq).

Histone methyltransferases specific for the histone H3-lysine 9 residue, including Setdb1 (Set domain, bifurcated 1)/Eset/Kmt1e are associated with repressive chromatin remodeling and expressed in adult brain, but potential effects on neuronal function and behavior remain unexplored. Here, we report that transgenic mice with increased Setdb1 expression in adult forebrain neurons show antidepressant-like phenotypes in behavioral paradigms for anhedonia, despair, and learned helplessness. Chromatin immunoprecipitation in conjunction with DNA tiling arrays (ChIP-chip) revealed that genomic occupancies of neuronal Setdb1 are limited to <1% of annotated genes, which include the NMDA receptor subunit NR2B/Grin2B and other ionotropic glutamate receptor genes. Chromatin conformation capture and Setdb1-ChIP revealed a loop formation tethering the NR2B/Grin2b promoter to the Setdb1 target site positioned 30 kb downstream of the transcription start site. In hippocampus and ventral striatum, two key structures in the neuronal circuitry regulating mood-related behaviors, Setdb1-mediated repressive histone methylation at NR2B/Grin2b was associated with decreased NR2B expression and EPSP insensitivity to pharmacological blockade of NR2B, and accelerated NMDA receptor desensitization consistent with a shift in NR2A/B subunit ratios. In wild-type mice, systemic treatment with the NR2B antagonist, Ro25-6981 [R-(R,S)-alpha-(4-hydroxyphenyl)-beta-methyl-4-(phenylmethyl)-1-piperidine propranol], and hippocampal small interfering RNA-mediated NR2B/Grin2b knockdown resulted in behavioral changes similar to those elicited by the Setdb1 transgene. Together, these findings point to a role for neuronal Setdb1 in the regulation of affective and motivational behaviors through repressive chromatin remodeling at a select set of target genes, resulting in altered NMDA receptor subunit composition and other molecular adaptations.

The rostro-medial cortex of the mouse and rat, considered the functional homolog to the primate prefrontal cortex (PFC), is of growing importance for preclinical models of schizophrenia and other neurodevelopmental diseases for which symptoms typically emerge in adolescence and early adulthood. Therefore, in order to explore molecular mechanisms operating during these critical stages of PFC development, it will be important to develop an efficient gene delivery system for the PFC of juvenile animals. To this end, adeno-associated virus (AAV)-based systems are increasingly used in mice for targeted gene delivery in specific brain regions such as the hippocampus. Strikingly, there is very little literature on vector-mediated gene expression in the rostro-medial cortex. In addition, multiple AAV serotypes exist based on differences in their envelope capsid proteins. However, to date, the large majority of studies in the central nervous system (CNS) have utilized the AAV2 serotype. This is typically limited to a very focal transduction pattern and therefore is not ideal for the murine PFC, which occupies several square millimeters in the rostral hemisphere. Here, we introduce a protocol for efficient, AAV9-serotype-mediated gene delivery in juvenile (postnatal day 21) and young adult PFC, resulting in long-lasting transgene expression.

Stress is an important physiological regulator of brain function in young and adult mammals. The mechanisms underlying regulation of the consequences of stress, and in particular severe chronic stress, are thus important to investigate. These consequences most likely involve changes in synaptic function of brain areas being part of neural networks that regulate responses to stress. Cell adhesion molecules have been shown to regulate synaptic function in the adult and we were thus interested to investigate a regulatory mechanism that could influence expression of three adhesion molecules of the immunoglobulin superfamily (NCAM, L1 and CHL1) after exposure of early postnatal and adult mice to repeated stress. We hypothesized that reduction of adhesion molecule expression after chronic stress, as observed previously in vivo, could be due to gene silencing of the three molecules by DNA methylation. Although adhesion molecule expression was reduced after exposure of C57BL/6 mice to stress, thus validating our stress paradigm as imposing changes in adhesion molecule expression, we did not observe differences in methylation of CpG islands in the promoter regions of NCAM, L1 and CHL1, nor in the promoter region of the glucocorticoid receptor in the hippocampus, the expression of which at the protein level was also reduced after stress. We must therefore infer that severe stress in mice of the C57BL/6 strain downregulates adhesion molecule levels by mechanisms that do not relate to DNA methylation.