The Orphan GPCR Receptor, GPR88, Interacts with Nuclear Protein Partners in the Cerebral Cortex.

GPR88 is an orphan G-protein-coupled receptor (GPCR) highly expressed in striatal medium spiny neurons (MSN), also found in cortical neurons at low level. In MSN, GPR88 has a canonical GPCR plasma membrane/cytoplasmic expression, whereas in cortical neurons, we previously reported an atypical intranuclear localization. Molecular size analysis suggests that GPR88, expressed in plasma membrane of MSN or in nuclear compartment of cortical neurons, corresponds to the full-length protein. By transfection of cortical neurons, we showed that GPR88 fluorescent chimeras exhibit a nuclear localization. This localization is contingent on the third intracytoplasmic loop and C-terminus domains, even though these domains do not contain any known nuclear localization signals (NLS). Using yeast two-hybrid screening with these domains, we identified the nuclear proteins ATRX, TOP2B, and BAZ2B, all involved in chromatin remodeling, as potential protein partners of GPR88. We also validated the interaction of GPR88 with these nuclear proteins by proximity ligation assay on cortical neurons in culture and coimmunoprecipitation experiments on cortical extracts from GPR88 wild-type (WT) and knockout (KO) mice. The identification of GPR88 subcellular partners may provide novel functional insights for nonclassical modes of GPCR action that could be relevant in the maturating process of neocortical neurons.

Cell therapy in Huntington's disease: Taking stock of past studies to move the field forward.

Huntington's disease (HD) is a rare inherited neurodegenerative disease that manifests mostly in adulthood with progressive cognitive, behavioral, and motor dysfunction. Neuronal loss occurs predominantly in the striatum but also extends to other brain regions, notably the cortex. Most patients die around 20 years after motor onset, although there is variability in the rate of progression and some phenotypic heterogeneity. The most advanced experimental therapies currently are huntingtin-lowering strategies, some of which are in stage 3 clinical trials. However, even if these approaches are successful, it is unlikely that they will be applicable to all patients or will completely halt continued loss of neural cells in all cases. On the other hand, cellular therapies have the potential to restore atrophied tissues and may therefore provide an important complementary therapeutic avenue. Pilot studies of fetal cell grafts in the 2000s reported the most dramatic clinical improvements yet achieved for this disease, but subsequent studies have so far failed to identify methodology to reliably reproduce these results. Moving forward, a major challenge will be to generate suitable donor cells from (nonfetal) cell sources, but in parallel there are a host of procedural and trial design issues that will be important for improving reliability of transplants and so urgently need attention. Here, we consider findings that have emerged from clinical transplant studies in HD to date, in particular new findings emerging from the recent multicenter intracerebral transplant HD study, and consider how these data may be used to inform future cell therapy trials.

Reduction of DNMT3a and RORA in the nucleus accumbens plays a causal role in post-traumatic stress disorder-like behavior: reversal by combinatorial epigenetic therapy.

Post-traumatic stress disorder (PTSD) is an incapacitating trauma-related disorder, with no reliable therapy. Although PTSD has been associated with epigenetic alterations in peripheral white blood cells, it is unknown where such changes occur in the brain, and whether they play a causal role in PTSD. Using an animal PTSD model, we show distinct DNA methylation profiles of PTSD susceptibility in the nucleus accumbens (NAc). Data analysis revealed overall hypomethylation of different genomic CG sites in susceptible animals. This was correlated with the reduction in expression levels of the DNA methyltransferase, DNMT3a. Since epigenetic changes in diseases involve different gene pathways, rather than single candidate genes, we next searched for pathways that may be involved in PTSD. Analysis of differentially methylated sites identified enrichment in the RAR activation and LXR/RXR activation pathways that regulate Retinoic Acid Receptor (RAR) Related Orphan Receptor A (RORA) activation. Intra-NAc injection of a lentiviral vector expressing either RORA or DNMT3a reversed PTSD-like behaviors while knockdown of RORA and DNMT3a increased PTSD-like behaviors. To translate our results into a potential pharmacological therapeutic strategy, we tested the effect of systemic treatment with the global methyl donor S-adenosyl methionine (SAM), for supplementing DNA methylation, or retinoic acid, for activating RORA downstream pathways. We found that combined treatment with the methyl donor SAM and retinoic acid reversed PTSD-like behaviors. Thus, our data point to a novel approach to the treatment of PTSD, which is potentially translatable to humans.

Epigenetic mechanisms underlie the crosstalk between growth factors and a steroid hormone.

Crosstalk between growth factors (GFs) and steroid hormones recurs in embryogenesis and is co-opted in pathology, but underlying mechanisms remain elusive. Our data from mammary cells imply that the crosstalk between the epidermal GF and glucocorticoids (GCs) involves transcription factors like p53 and NF-κB, along with reduced pausing and traveling of RNA polymerase II (RNAPII) at both promoters and bodies of GF-inducible genes. Essentially, GCs inhibit positive feedback loops activated by GFs and stimulate the reciprocal inhibitory loops. As expected, no alterations in DNA methylation accompany the transcriptional events instigated by either stimulus, but forced demethylation of regulatory regions broadened the repertoire of GF-inducible genes. We report that enhancers, like some promoters, are poised for activation by GFs and GCs. In addition, within the cooperative interface of the crosstalk, GFs enhance binding of the GC receptor to DNA and, in synergy with GCs, promote productive RNAPII elongation. Reciprocally, within the antagonistic interface GFs hyper-acetylate chromatin at unmethylated promoters and enhancers of genes involved in motility, but GCs hypoacetylate the corresponding regions. In conclusion, unmethylated genomic regions that encode feedback regulatory modules and differentially recruit RNAPII and acetylases/deacetylases underlie the crosstalk between GFs and a steroid hormone.

The Signature of Maternal Social Rank in Placenta Deoxyribonucleic Acid Methylation Profiles in Rhesus Monkeys.

The effects of social status on human health can be modeled in captive cohorts of nonhuman primates. This study shows that maternal social rank is associated with broad changes in DNA methylation in placentae of rhesus monkeys (N = 10). Differentially methylated genes between social ranks are enriched in signaling pathways playing major roles in placenta physiology. Moreover, the authors found significant overlaps with genes whose expression was previously associated with social rank in adult rhesus monkeys (Tung et al., 2012) and whose methylation was associated with perinatal stress in newborn humans and rhesus monkeys (Nieratschker et al., 2014). These results are consistent with the hypothesis that system-wide epigenetic changes in multiple tissues are involved in long-term adaptations to the social environment.

Role of DNA methylation in the nucleus accumbens in incubation of cocaine craving.

One of the major challenges of cocaine addiction is the high rate of relapse to drug use after periods of withdrawal. During the first few weeks of withdrawal, cue-induced cocaine craving intensifies, or "incubates," and persists over extended periods of time. Although several brain regions and molecular mechanisms were found to be involved in this process, the underlying epigenetic mechanisms are still unknown. Herein, we used a rat model of incubation of cocaine craving, in which rats were trained to self-administer cocaine (0.75 mg/kg, 6 h/d, 10 d), and cue-induced cocaine-seeking was examined in an extinction test after 1 or 30 d of withdrawal. We show that the withdrawal periods, as well as cue-induced cocaine seeking, are associated with broad, time-dependent enhancement of DNA methylation alterations in the nucleus accumbens (NAc). These gene methylation alterations were partly negatively correlated with gene expression changes. Furthermore, intra-NAc injections of a DNA methyltransferase inhibitor (RG108, 100 µm) abolished cue-induced cocaine seeking on day 30, an effect that persisted 1 month, whereas the methyl donor S-adenosylmethionine (500 µm) had an opposite effect on cocaine seeking. We then targeted two proteins whose genes were demethylated by RG108-estrogen receptor 1 (ESR1) and cyclin-dependent kinase 5 (CDK5). Treatment with an intra-NAc injection of the ESR1 agonist propyl pyrazole triol (10 nm) or the CDK5 inhibitor roscovitine (28 µm) on day 30 of withdrawal significantly decreased cue-induced cocaine seeking. These results demonstrate a role for NAc DNA methylation, and downstream targets of DNA demethylation, in incubation of cocaine craving.

Early life adversity alters normal sex-dependent developmental dynamics of DNA methylation.

Studies in rodents, nonhuman primates, and humans suggest that epigenetic processes mediate between early life experiences and adult phenotype. However, the normal evolution of epigenetic programs during child development, the effect of sex, and the impact of early life adversity on these trajectories are not well understood. This study mapped the genome-wide DNA methylation changes in CD3+ T lymphocytes from rhesus monkeys from postnatal day 14 through 2 years of age in both males and females and determined the impact of maternal deprivation on the DNA methylation profile. We show here that DNA methylation profiles evolve from birth to adolescence and are sex dependent. DNA methylation changes accompany imposed weaning, attenuating the difference between males and females. Maternal separation at birth alters the normal evolution of DNA methylation profiles and targets genes that are also affected by a later stage maternal separation, that is, weaning. Our results suggest that early life events dynamically interfere with the normal developmental evolution of the DNA methylation profile and that these changes are highly effected by sex.

Impact of traumatic brain injury on sleep structure, electrocorticographic activity and transcriptome in mice.

Traumatic brain injury (TBI), including mild TBI (mTBI), is importantly associated with vigilance and sleep complaints. Because sleep is required for learning, plasticity and recovery, we here evaluated the bidirectional relationship between mTBI and sleep with two specific objectives: (1) Test that mTBI rapidly impairs sleep-wake architecture and the dynamics of the electrophysiological marker of sleep homeostasis (i.e., non-rapid eye movement sleep delta (1-4Hz) activity); (2) evaluate the impact of sleep loss following mTBI on the expression of plasticity markers that have been linked to sleep homeostasis and on genome-wide gene expression. A closed-head injury model was used to perform a 48h electrocorticographic (ECoG) recording in mice submitted to mTBI or Sham surgery. mTBI was found to immediately decrease the capacity to sustain long bouts of wakefulness as well as the amplitude of the time course of ECoG delta activity during wakefulness. Significant changes in ECoG spectral activity during wakefulness, non-rapid eye movement and rapid eye movement sleep were observed mainly on the second recorded day. A second experiment was performed to measure gene expression in the cerebral cortex and hippocampus after a mTBI followed either by two consecutive days of 6h sleep deprivation (SD) or of undisturbed behavior (quantitative PCR and next-generation sequencing). mTBI modified the expression of genes involved in immunity, inflammation and glial function (e.g., chemokines, glial markers) and SD changed that of genes linked to circadian rhythms, synaptic activity/neuronal plasticity, neuroprotection and cell death and survival. SD appeared to affect gene expression in the cerebral cortex more importantly after mTBI than Sham surgery including that of the astrocytic marker Gfap, which was proposed as a marker of clinical outcome after TBI. Interestingly, SD impacted the hippocampal expression of the plasticity elements Arc and EfnA3 only after mTBI. Overall, our findings reveal alterations in spectral signature across all vigilance states in the first days after mTBI, and show that sleep loss post-mTBI reprograms the transcriptome in a brain area-specific manner and in a way that could be deleterious to brain recovery.

Modifiable factors associated with Huntington's disease progression in presymptomatic participants.

OBJECTIVE: Huntington's disease (HD) is a neurodegenerative disorder characterized by progressive motor, cognitive, and psychiatric symptoms. Our aim here was to identify factors that can be modified to slow disease progression even before the first symptoms appear. METHODS: We included 2636 presymptomatic individuals (comparison with family controls) drawn from the prospective observational cohort Enroll-HD, with more than 35 CAG repeats and at least two assessments of disease progression measured with the composite Huntington's disease rating Scale (cUHDRS). The association between sociodemographic factors, health behaviors, health history, and cUHDRS trajectory was assessed with a mixed-effects random forest using partial dependence plots and Shapley additive explanation method. RESULTS: Participants were followed by an average of 3.4 (SD = 1.97) years. We confirmed the negative impact of age and a high number of CAG repeats. We found that a high level of education, a body mass index (BMI) <23 kg/m2 before the age of 40 and >23 kg/m2 thereafter, alcohol consumption of <15 units per week, current coffee consumption and no smoking were linked to slow disease progression, as did no previous exposure to antidepressants or anxiolytic, no psychiatric history or comorbidities, and being female. Other comorbidities or marital status showed no major association with HD evolution. INTERPRETATION: Reducing modifiable risk factors for HD is one way to support the presymptomatic population. A high level of education, low-to-moderate alcohol consumption, no smoking, and BMI control are likely to slow disease progression in this population.

The signature of maternal rearing in the methylome in rhesus macaque prefrontal cortex and T cells.

Early-life adversity is associated with a broad scope of life-long health and behavioral disorders. Particularly critical is the role of the mother. A possible mechanism is that these effects are mediated by "epigenetic" mechanisms. Studies in rodents suggest a causal relationship between early-life adversity and changes in DNA methylation in several "candidate genes" in the brain. This study examines whether randomized differential rearing (maternal vs surrogate-peer rearing) of rhesus macaques is associated with differential methylation in early adulthood. The data presented here show that differential rearing leads to differential DNA methylation in both prefrontal cortex and T cells. These differentially methylated promoters tend to cluster by both chromosomal region and gene function. The broad impact of maternal rearing on DNA methylation in both the brain and T cells supports the hypothesis that the response to early-life adversity is system-wide and genome-wide and persists to adulthood. Our data also point to the feasibility of studying the impact of the social environment in peripheral T-cell DNA methylation.

A new approach to digitized cognitive monitoring: validity of the SelfCog in Huntington's disease.

Cognitive deficits represent a hallmark of neurodegenerative diseases, but evaluating their progression is complex. Most current evaluations involve lengthy paper-and-pencil tasks which are subject to learning effects dependent on the mode of response (motor or verbal), the countries' language or the examiners. To address these limitations, we hypothesized that applying neuroscience principles may offer a fruitful alternative. We thus developed the SelfCog, a digitized battery that tests motor, executive, visuospatial, language and memory functions in 15 min. All cognitive functions are tested according to the same paradigm, and a randomization algorithm provides a new test at each assessment with a constant level of difficulty. Here, we assessed its validity, reliability and sensitivity to detect decline in early-stage Huntington's disease in a prospective and international multilingual study (France, the UK and Germany). Fifty-one out of 85 participants with Huntington's disease and 40 of 52 healthy controls included at baseline were followed up for 1 year. Assessments included a comprehensive clinical assessment battery including currently standard cognitive assessments alongside the SelfCog. We estimated associations between each of the clinical assessments and SelfCog using Spearman's correlation and proneness to retest effects and sensitivity to decline through linear mixed models. Longitudinal effect sizes were estimated for each cognitive score. Voxel-based morphometry and tract-based spatial statistics analyses were conducted to assess the consistency between performance on the SelfCog and MRI 3D-T1 and diffusion-weighted imaging in a subgroup that underwent MRI at baseline and after 12 months. The SelfCog detected the decline of patients with Huntington's disease in a 1-year follow-up period with satisfactory psychometric properties. Huntington's disease patients are correctly differentiated from controls. The SelfCog showed larger effect sizes than the classical cognitive assessments. Its scores were associated with grey and white matter damage at baseline and over 1 year. Given its good performance in longitudinal analyses of the Huntington's disease cohort, it should likely become a very useful tool for measuring cognition in Huntington's disease in the future. It highlights the value of moving the field along the neuroscience principles and eventually applying them to the evaluation of all neurodegenerative diseases.

Self-Reported Social Relationship Capacities Predict Motor, Functional and Cognitive Decline in Huntington's Disease.

Huntington's Disease (HD) is an inherited neurodegenerative disease characterized by a combination of motor, cognitive, and behavioral disorders. The social and behavioral symptoms observed in HD patients impact their quality of life and probably explain their relational difficulties, conflicts, and social withdrawal. In this study, we described the development of the Social Relationship Self-Questionnaire (SRSQ), a self-reporting questionnaire that assesses how HD patients perceived their social relationships. The scale was proposed for 66 HD patients at an early stage of the disease, 32 PreHD patients (individuals carrying the mutant gene without motor symptoms), and 66 controls. The HD patients were included in a prospective longitudinal follow-up for an average of 1.07 years with motor, functional, cognitive, and behavioral assessments. Based on the HD patients' answers at baseline, we identified two domains in the SRSQ. The first domain was related to social motivation and correlated with cognitive performance. The second domain was related to emotional insight and correlated with behavioral symptoms such as apathy, anxiety, and irritability. We discovered that both SRSQ domain scores at baseline predicted future motor, functional, and cognitive decline in HD.

Cognitive decline in Huntington's disease in the Digitalized Arithmetic Task (DAT).

BACKGROUND: Efficient cognitive tasks sensitive to longitudinal deterioration in small cohorts of Huntington's disease (HD) patients are lacking in HD research. We thus developed and assessed the digitized arithmetic task (DAT), which combines inner language and executive functions in approximately 4 minutes. METHODS: We assessed the psychometric properties of DAT in three languages, across four European sites, in 77 early-stage HD patients (age: 52 ± 11 years; 27 females), and 57 controls (age: 50 ± 10, 31 females). Forty-eight HD patients and 34 controls were followed up to one year with 96 participants who underwent MRI brain imaging (HD patients = 46) at baseline and 50 participants (HD patients = 22) at one year. Linear mixed models and Pearson correlations were used to assess associations with clinical assessment. RESULTS: At baseline, HD patients were less accurate (p = 0.0002) with increased response time (p<0.0001) when compared to DAT in controls. Test-retest reliability in HD patients ranged from good to excellent for response time (range: 0.63-0.79) and from questionable to acceptable for accuracy (range: r = 0.52-0.69). Only DAT, the Mattis Dementia Rating Scale, the Symbol Digit Modalities Test, and Total Functional Capacity scores were able to detect a decline within a one-year follow-up in HD patients (all p< 0.05). In contrast with all the other cognitive tasks, DAT correlated with striatal atrophy over time (p = 0.037) but not with motor impairment. CONCLUSIONS: DAT is fast, reliable, motor-free, applicable in several languages, and able to unmask cognitive decline correlated with striatal atrophy in small cohorts of HD patients. This likely makes it a useful endpoint in future trials for HD and other neurodegenerative diseases.

Objectively characterizing Huntington's disease using a novel upper limb dexterity test.

BACKGROUND: The Clinch Token Transfer Test (C3t) is a bi-manual coin transfer task that incorporates cognitive tasks to add complexity. This study explored the concurrent and convergent validity of the C3t as a simple, objective assessment of impairment that is reflective of disease severity in Huntington's, that is not reliant on clinical expertise for administration. METHODS: One-hundred-and-five participants presenting with pre-manifest (n = 16) or manifest (TFC-Stage-1 n = 39; TFC-Stage-2 n = 43; TFC-Stage-3 n = 7) Huntington's disease completed the Unified Huntington's Disease Rating Scale and the C3t at baseline. Of these, thirty-three were followed up after 12 months. Regression was used to estimate baseline individual and composite clinical scores (including cognitive, motor, and functional ability) using baseline C3t scores. Correlations between C3t and clinical scores were assessed using Spearman's R and visually inspected in relation to disease severity using scatterplots. Effect size over 12 months provided an indication of longitudinal behaviour of the C3t in relation to clinical measures. RESULTS: Baseline C3t scores predicted baseline clinical scores to within 9-13% accuracy, being associated with individual and composite clinical scores. Changes in C3t scores over 12 months were small ([Formula: see text] ≤ 0.15) and mirrored the change in clinical scores. CONCLUSION: The C3t demonstrates promise as a simple, easy to administer, objective outcome measure capable of predicting impairment that is reflective of Huntington's disease severity and offers a viable solution to support remote clinical monitoring. It may also offer utility as a screening tool for recruitment to clinical trials given preliminary indications of association with the prognostic index normed for Huntington's disease.

Striatal GPR88 expression is confined to the whole projection neuron population and is regulated by dopaminergic and glutamatergic afferents.

GPR88, an orphan G protein-coupled receptor, was designated Strg/GPR88 for striatum-specific G protein-coupled receptor (K. Mizushima et al. (2000)Genomics, 69, 314-321). In this study, we focused on striatal GPR88 protein localization using a polyclonal antibody. We established that the distribution of immunoreactivity in rat brain matched that of GPR88 transcripts and provided evidence for its exclusive neuronal expression. GPR88 protein is abundant throughout the striatum of rat and primate, with expression limited to the two subsets of striatal projection medium spiny neurons (MSNs) expressing preprotachykinin-substance P or preproenkephalin mRNAs. Ultrastructural immunolabelling revealed the GPR88 concentration at post-synaptic sites along the somatodendritic compartments of MSNs, with pronounced preference for dendrites and dendritic spines. The GPR88-rich expression, in both striatal output pathways, designates this receptor as a potential therapeutic target for diseases involving dysfunction of the basal ganglia, such as Parkinson's disease. Hence, we investigated changes of GPR88 expression in a model of Parkinson's disease (unilateral 6-hydroxydopamine-lesioned rats) following repeated L-DOPA treatment. In dopamine-depleted striatum, GPR88 expression was differentially regulated, i.e. decreased in striatopallidal and increased in striatonigral MSNs. L-DOPA treatment led to a normalization of GPR88 levels through dopamine D1 and D2 receptor-mediated mechanisms in striatopallidal and striatonigral MSNs, respectively. Moreover, the removal of corticostriatal inputs, by ibotenate infusion, downregulated GPR88 in striatopallidal MSNs. These findings provide the first evidence that GPR88 is confined to striatal MSNs and indicate that L-DOPA-mediated behavioural effects in hemiparkinsonian rats may involve normalization of striatal GPR88 levels probably through dopamine receptor-mediated mechanisms and modulations of corticostriatal pathway activity.

International Guidelines for the Treatment of Huntington's Disease.

The European Huntington's Disease Network (EHDN) commissioned an international task force to provide global evidence-based recommendations for everyday clinical practice for treatment of Huntington's disease (HD). The objectives of such guidelines are to standardize pharmacological, surgical and non-pharmacological treatment regimen and improve care and quality of life of patients. A formalized consensus method, adapted from the French Health Authority recommendations was used. First, national committees (French and English Experts) reviewed all studies published between 1965 and 2015 included dealing with HD symptoms classified in motor, cognitive, psychiatric, and somatic categories. Quality grades were attributed to these studies based on levels of scientific evidence. Provisional recommendations were formulated based on the strength and the accumulation of scientific evidence available. When evidence was not available, recommendations were framed based on professional agreement. A European Steering committee supervised the writing of the final recommendations through a consensus process involving two rounds of online questionnaire completion with international multidisciplinary HD health professionals. Patients' associations were invited to review the guidelines including the HD symptoms. Two hundred and nineteen statements were retained in the final guidelines. We suggest to use this adapted method associating evidence base-medicine and expert consensus to other rare diseases.

Fetal glucocorticoid receptor (Nr3c1) deficiency alters the landscape of DNA methylation of murine placenta in a sex-dependent manner and is associated to anxiety-like behavior in adulthood.

Prenatal stress defines long-term phenotypes through epigenetic programming of the offspring. These effects are potentially mediated by glucocorticoid release and by sex. We hypothesized that the glucocorticoid receptor (Gr, Nr3c1) fashions the DNA methylation profile of offspring. Consistent with this hypothesis, fetal Nr3c1 heterozygosity leads to altered DNA methylation landscape in fetal placenta in a sex-specific manner. There was a significant overlap of differentially methylated genes in fetal placenta and adult frontal cortex in Nr3c1 heterozygotes. Phenotypically, Nr3c1 heterozygotes show significantly more anxiety-like behavior than wildtype. DNA methylation status of fetal placental tissue is significantly correlated with anxiety-like behavior of the same animals in adulthood. Thus, placental DNA methylation might predict behavioral phenotypes in adulthood. Our data supports the hypothesis that Nr3c1 influences DNA methylation at birth and that DNA methylation in placenta correlates with adult frontal cortex DNA methylation and anxiety-like phenotypes.

DNA methylation and transcription onset in the brain.

AIM: The goal of this study was to test the state of methylation of transcription start positions in DNA that are actively involved in transcription. MATERIALS & METHODS: We used sequential ChIP-bisulfite-sequencing with an antibody to RNpolII-PS5 to map the state of methylation of actively transcribing transcription start sites (TSS). RESULTS: TSS that RNApolII-PS5 physically bind to, are ubiquitously unmethylated. TSS that appear to be both heavily methylated and transcriptionally active are truly a mixture of unmethylated TSS with bound RNApolII-PS5 in some nuclei and unbound methylated TSS in other nuclei. CONCLUSION: TSS DNA methylation is universally inconsistent with transcription onset and could therefore serve as a digital count of the fraction of nuclei with methylation-silenced TSS.

Genome-wide DNA Methylation Changes in a Mouse Model of Infection-Mediated Neurodevelopmental Disorders.

BACKGROUND: Prenatal exposure to infectious or inflammatory insults increases the risk of neurodevelopmental disorders. Using a well-established mouse model of prenatal viral-like immune activation, we examined whether this pathological association involves genome-wide DNA methylation differences at single nucleotide resolution. METHODS: Prenatal immune activation was induced by maternal treatment with the viral mimetic polyriboinosinic-polyribocytidylic acid in middle or late gestation. Following behavioral and cognitive characterization of the adult offspring (n = 12 per group), unbiased capture array bisulfite sequencing was combined with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative real-time polymerase chain reaction analyses to quantify DNA methylation changes and transcriptional abnormalities in the medial prefrontal cortex of immune-challenged and control offspring. Gene ontology term enrichment analysis was used to explore shared functional pathways of genes with differential DNA methylation. RESULTS: Adult offspring of immune-challenged mothers displayed hyper- and hypomethylated CpGs at numerous loci and at distinct genomic regions, including genes relevant for gamma-aminobutyric acidergic differentiation and signaling (e.g., Dlx1, Lhx5, Lhx8), Wnt signaling (Wnt3, Wnt8a, Wnt7b), and neural development (e.g., Efnb3, Mid1, Nlgn1, Nrxn2). Altered DNA methylation was associated with transcriptional changes of the corresponding genes. The epigenetic and transcriptional effects were dependent on the offspring's age and were markedly influenced by the precise timing of prenatal immune activation. CONCLUSIONS: Prenatal viral-like immune activation is capable of inducing stable DNA methylation changes in the medial prefrontal cortex. These long-term epigenetic modifications are a plausible mechanism underlying the disruption of prefrontal gene transcription and behavioral functions in subjects with prenatal infectious histories.

Overlapping signatures of chronic pain in the DNA methylation landscape of prefrontal cortex and peripheral T cells.

We tested the hypothesis that epigenetic mechanisms in the brain and the immune system are associated with chronic pain. Genome-wide DNA methylation assessed in 9 months post nerve-injury (SNI) and Sham rats, in the prefrontal cortex (PFC) as well as in T cells revealed a vast difference in the DNA methylation landscape in the brain between the groups and a remarkable overlap (72%) between differentially methylated probes in T cells and prefrontal cortex. DNA methylation states in the PFC showed robust correlation with pain score of animals in several genes involved in pain. Finally, only 11 differentially methylated probes in T cells were sufficient to distinguish SNI or Sham individual rats. This study supports the plausibility of DNA methylation involvement in chronic pain and demonstrates the potential feasibility of DNA methylation markers in T cells as noninvasive biomarkers of chronic pain susceptibility.