S22 Molecular neuropsychopharmacology

were analyzed by mass spectrometry, functional proteinassociation networks were analysed and candidate proteinswere identified and prioritized. The resulting proteinlists were curated using statistical and functional criteriato prioritize for the most significant Cntn6 interactor,which we name Interacting protein of Cntn6 (IPC6).To confirm the interaction of Cntn6 and IPC6, thesestudies were complemented with in vitro and in vivo co-immunoprecipitation experiments. Trans-membrane inter-action of these proteins in the post-synapse was confirmedby subcellular fractionation experiments and cell-bindingand -aggregation assays. Neuro-anatomical studies inmouse by immunohistochemistry and in situ hybridizationconfirmed co-expression of Cntn6 and IPC6 in the cortexand thalamus. Furthermore, the function of this interactionwas demonstrated by morphological analysis of transfectedmurine neural progenitor cells. Based on these data wepropose that Cntn6 participates in a synaptic protein-interaction network that engages several other establishedASD genes. These studies provide a starting point fordevelopment and testing of novel pharmaceuticals that areable to restore appropriate synaptic function in ASD.

Reference(s)

[1] Pinto, D., Pagnamenta, A.T., Klei, L., Anney, R.,Merico, D., Regan, R., & Conroy, J., 2010. Functionalimpact of global rare copy number variation in autismspectrum disorders. Nature, 466, 368–372.

[2] Pinto, D., Pagnamenta, A.T., Klei, L., Anney, R.,Merico, D., Regan, R., & Conroy, J., 2010. Functionalimpact of global rare copy number variation in autismspectrum disorders. Nature, 466, 368–372.

[3] Takeda, Y., Akasaka, K., Lee, S., Kobayashi, S.,Kawano, H., Murayama, S., 2003. Impaired motorcoordination in mice lacking neural recognitionmolecule NB-3 of the contactin/F3 subgroup. Journalof Neurobiology, 56, 252–265.

P.1.023 Understanding epigenetic changes in

CNS disorders: histone modification

regulates LPS-induced inflammatory

transcriptional response

G. Rigillo1 °, M. Zoli2, F. Tascedda1, N. Brunello1,J.M.C. Blom3. 1University of Modena and ReggioEmilia, Life Sciences, Modena, Italy; 2University ofModena and Reggio Emilia, Biomedical Metabolic andNeural Sciences, Modena, Italy; 3University of Modenaand Reggio Emilia, Medical and Surgical Sciences forChildren and Adults, Modena, Italy

Background and Purpose of the study: Epigeneticmodifications of both DNA and histone proteins are now

emerging as fundamental mechanisms by which neuronsadapt their transcriptional response to environmentalcues, such as, immune stimuli or stress [1]. Geneticpolymorphisms in a large number of genes quantitativelyand qualitatively affect immune and inflammatoryresponses altering the susceptibility to or course of alarge number of CNS diseases with an inflammatorycomponent. A peripheral infection induces innate immunecells to produce pro-inflammatory cytokines acting on thebrain to cause sickness behavior. Persistent activation ofthe peripheral immune system, such as during systemicinfections, cancer or autoimmune diseases, causes immunesignaling to the brain that can lead to an exacerbation ofsickness and the development of symptoms of depressionin vulnerable individuals [2]. Furthermore, modificationof histone status is known to regulate the transcription ofspecific genes involved in inflammatory conditions. Giventhis, the purpose of our study was to better understand themolecular mechanisms at the chromatin level underlyingneural-immune interaction in sickness behavior in twospecific brain areas: hippocampus and hypothalamus.More specifically we studied: (1) the transcriptionalactivity induced by LPS immune stimulus, in particular theexpression of pro-inflammatory cytokines and the imme-diate early gene c-Fos; (2) histone H3 phosphorylation atserine10 and acetylation at lysine14 (H3S10p-K14ac) as achromatin remodeling mechanism that mediated the LPStranscriptional activity [3]; (3) how the H3S10p-K14acselectively turned on the transcription of specific genepromoters.

Methods: Adult male rats were injected with LPS(0.830mg/ml/kg i.p.) a potent activator of the immunesystem which causes short-term changes indicative ofsickness behavior and sacrificed 2 hours after theinjection. Hippocampus and hypothalamus were dissectedimmediately and used in all analyses; western blot ofnuclear fractions was used to measure histone H3 phos-phoacetylation (S10K14). Pro-inflammatory cytokines, theimmediate early gene cFos and inducible nitric oxidesynthase (iNOS) gene expression were analyzed byreal time PCR. Chromatin immuno-precipitation analysis(ChIP) was performed to evaluate the effect of LPS onH3S10p-K14ac at cFos, iNos, Interleukin-1b (IL-1b) andInterleukin-6 (IL-6) promoters.

Results: The mRNA levels of IL-1b, IL-6, iNOS andcFos were markedly enhanced 2 hours after an LPSinflammatory challenge (p< 0.05). H3 phosphoacetyla-tion was significantly enhanced in hippocampus andhypothalamus of rats treated with LPS with respect tocontrols (p< 0.01). Moreover, the activation of promotertranscription, mediated by H3S10p-K14ac, was specific inour target genes and in the two examined areas.

Molecular neuropsychopharmacology S23

Conclusions: Our data suggest a possible role forhistone H3 phosphoacetylation in LPS induced sicknessbehavior as well as in the regulation of transcriptionalmechanisms involved in the immune response. In this way,histone modification may represent the first of a cascadeof genetic events that determine the behavioral alterationsand sensitivity to stress observed after activation of theimmune system.

Reference(s)

[1] Toyokawa S, Uddin M, Koenen KC, Galea S, 2012.How does the social environment ‘get into the mind’?Epigenetics at the intersection of social and psychiatricepidemiology. Social Science & Medicine 74, 67−74.

[2] Dantzer R, O’Connor JC, Freund GG, Johnson RW,Kelley KW, 2008. From inflammation to sickness anddepression: when the immune system subjugates thebrain. Nat Rev Neurosci. Jan; 9(1): 46−56.

[3] Ottaviani E, Accorsi A, Rigillo G, Malagoli D,Blom JM, Tascedda F, 2013. Epigenetic modificationin neurons of the mollusc Pomacea canaliculata afterimmune challenge. Brain Res. I 537, 18−26.

P.1.024 Epigenetic regulation of A2A gene

transcription in a model of binge eating

in the amygdala complex of female rats

M.V. Micioni Di Bonaventura1 °, C. Cifani1, M. Pucci2,C. Lambertucci3, R. Volpini3, M. Maccarrone4,C. D’Addario2. 1University of Camerino, Schoolof Pharmacy Pharmacology Unit, Camerino (MC),Italy; 2University of Teramo, Faculty of Bioscienceand Technology for Food Agriculture and Environment,Teramo, Italy; 3University of Camerino, School ofPharmacy Medicinal Chemistry Unit, Camerino (MC),Italy; 4University of Rome, Campus Bio-Medico, Rome,Italy

Purpose of the study: Eating disorders, especiallybinge eating disorder (BED) and bulimia nervosa, aremental illness triggered by dieting, stress, and negativeaffective states [1]. Despite a growing recognition of theconsequences of bulimia nervosa and of BED on publichealth, satisfactory treatments are not available at present.In search of new effective pharmacological for the controlof Binge Eating (BE), we here used a well-characterizedanimal model of BE [2] to study the epigenetic regulationof the Adenosine Receptor A2A gene, receptor alreadyknown to have an effect on food intake [3].

Methods: We used an animal model of BE whichincluded four groups (rats fed normally and not stressedor stressed, rats exposed to cycles of restriction/refeeding

and then stressed, or not stressed). Biological material(e.g., total RNA and DNA) were isolated from selectedrat brain region, amygdala. To assess mRNA abundancesand to quantify gene promoters DNA methylation, we usedReal-Time RT-PCR and Bisulfite pyrosequencing.

Results: Gene expression analysis carried out onamygdala of stressed rats (S) revealed a significantincrease of A2AR mRNA when compared to not stressed(NS) in rats exposed to restriction (R+NS: 1.43±0.32,R+S: 2.68±0.39, p< 0.05). The administration of A2ARagonist (VT 7) induced in stressed and restricted rats(R+S) a significant increase of A2AR mRNA level whencompared to control group (Veh) (Veh: 2.68±0.39, VT7 6.63±1.28, p< 0.001), whereas a significant decreasein rats treated with the A2AR antagonist (ANR 94) wasobserved (1.68±0.60, p< 0.001 vs VT 7 rats).Pyrosequencing analysis revealed a significant reduction

of % DNA methylation at A2AR promoter region in foodrestricted and exposed to acute stress rats respect to R+NSgroup (R+NS: 4.54±0.30, R+S: 3.77±0.20, p< 0.05).Also, significant changes in the DNA methylation status ofA2AR promoter were observed in restricted and exposedto stress rats after administration of VT 7 and ANR 94.We observed a decrease of DNA methylation in VT 7rats (VT 7: 3.25±0.25, Veh 3.77±0.20 p< 0.05) and ahypermethylation in ANR 94 rats (5.55±0.34, p< 0.001vs both Veh and VT 7) respect to control group.

Conclusions: The increase of A2A mRNA elicited bystress in restricted animals could indicate a compensatorymechanism, given the already proposed use of pharma-cological agents acting via A2A in controlling BE [3]and thus suggesting that A2A activation strongly inducingreceptor gene up-regulation could be of relevance inreducing food consumption. Moreover, this result is furthersupported by the epigenetic regulation of this receptor genetranscription, evident by the reduction in DNA methylationat gene promoter which might be responsible of theincrease in gene expression.

Reference(s)

[1] Freeman LM, Gil KM. Daily stress, coping, anddietary restraint in binge eating. Int J Eat Disord.2004; 36: 204−12.

[2] Cifani C, Polidori C, Melotto S, Ciccocioppo R,Massi M. Preclinical model of binge-eating elicited byyo−yo dieting and stressful exposure to food: effectof sibutramine, fluoxetine, topiramate and midazolam.Psychopharmacology. 2009; 204: 113–125.

[3] Micioni Di Bonaventura MV, Cifani C, Lambertucci C,Volpini R, Cristalli G, Massi M. A(2A) adenosinereceptor agonists reduce both high-palatability andlow-palatability food intake in female rats. BehavPharmacol. 2012; 23: 567–574.