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Additional file 3: Figure S3. Principal component analysis (PCA) of BeneMin baseline proteomics data for male vs female participants. Principal component analysis (PCA) of BeneMin baseline proteomics data for (a) 60 male participants and (b) 23 female participants in all four weight change categories. Results were illustrated using the ggbiplot R package and are coloured according to the category of weight change from baseline measured at follow-up.

Additional file 4: Figure S4. Principal component analysis (PCA) of BeneMin baseline proteomics data for minocycline vs placebo treatment groups. Principal component analysis (PCA) of BeneMin baseline proteomics data for (a) 39 participants treated with minocycline and (b) 44 participants treated with placebo as adjunctive treatment, in all four weight change categories. Results were illustrated using the ggbiplot R package and are coloured according to the category of weight change from baseline measured at follow-up.

Additional file 1: Figure S1. Heatmap of BeneMin baseline proteomics data for male vs female participants. Heatmap of BeneMin baseline proteomics data for (a) 60 male participants and (b) 23 female participants in all four weight change categories. The dendrograms above and to the left side of the heatmap correspond to the results of hierarchical clustering analysis (HCA) for samples and proteins, respectively (the distance matrix of which was computed using the Euclidean method and clustering using the complete linkage method). The bars below the top dendrogram are coloured according to the type of treatment (minocycline vs placebo) and the category of weight change from baseline measured at follow-up. Heatmap was plotted using the pheatmap R package with row scaling.

Additional file 2: Figure S2. Heatmap of BeneMin baseline proteomics data for minocycline vs placebo treatment groups. Heatmap of BeneMin baseline proteomics data for (a) 39 participants treated with minocycline and (b) 44 participants treated with placebo as adjunctive treatment, in all four weight change categories. The dendrograms above and to the left side of the heatmap correspond to the results of hierarchical clustering analysis (HCA) for samples and proteins, respectively (the distance matrix of which was computed using the Euclidean method and clustering using the complete linkage method). The bars below the top dendrogram are coloured according to gender and the category of weight change from baseline measured at follow-up. Heatmap was plotted using the pheatmap R package with row scaling.

Background Early-life stress is a key risk for psychiatric disorders that may produce changes in the neurodevelopment. N-methyl-d-aspartate receptor (NMDAR) have been associated with the pathophysiology of schizophrenia and evidence supports that epigenetic changes in NMDAR imply deficiencies in excitatory neurotransmission suggest its role in the neurobiology of psychoses (Uno and Coyle, 2019; Fachim et al., 2019; Gulchina et al., 2017). Aims: Although previous studies have shown abnormalities in the glutamatergic system in animal model of schizophrenia, it is not known if there are equivalent mRNA/protein alterations and DNA methylation changes in the brains of rats reared in isolation. Thus, in order to improve the knowledge of glutamatergic system role in psychosis, we investigated the NR1 and NR2 mRNA/protein and the DNA methylation levels of Grin1, Grin2a and Grin2b promoter region in the prefrontal cortex (PFC) and hippocampus (HIPPO) of male Wistar rats after isolation rearing. Furthermore, because the Parvalbumin (PV) deficit is the most consistent finding across animal models and schizophrenia itself, we also evaluated the expression of PV and other related GABAergic genes (REL and GAD1) in the brain of rats undergoing social isolation rearing as a validation of this animal model. We hypothesized that isolation rearing reduces mRNA and protein expressions of NMDAR subunits and cause DNA methylation changes. Methods Wistar rats were kept isolated or grouped (n=10/group) from weaning (21 days after birth) to 10 weeks and then exposed to the Open Field Test to assess locomotion. Afterwards the behavioural tests, the tissues were dissected for RNA/DNA extraction and NMDAR subunits were analysed using qRT-PCR, ELISA and pyrosequencing. Data were analysed by parametric tests. Results Isolated-reared animals presented: (i) decreased mRNA levels of Grin1 (p=0.011), Grin2a (p=0.039) and Grin2b (p=0.037) in the PFC followed by reduction in the GABAergic markers; (ii) increased NR1 protein levels in the HIPPO (p=0.001); (iii) hypermethylation of Grin1 at CpG5 in the PFC (p=0.047) and Grin2b CpG4 in the HIPPO when compared to grouped (p=0.024). Moreover, isolated and grouped animals presented a negative correlation between Grin1 mRNA and Grin1 methylation levels at CpG5 in the PFC (r: -0.577; p=0.010) and isolated rats presented a negative correlation between Grin2b methylation at CpG4 and NR2 protein levels in the HIPPO (r: -0.753; p=0.012). Discussion This study supports the hypothesis that the NMDAR methylation changes found in the brain tissues may underlie the NMDAR mRNA/protein expression alterations caused by the isolation period. These results highlighted the importance of the environmental influence during the development that may lead to cognitive impairments in adulthood. Moreover, we demonstrated that the social isolation rearing during 10 weeks causes long-lasting behavioral changes that may be more associated with late stages of schizophrenia. Our study contributes to the identification of the epigenetic mechanisms involved in the neuropathophysiology of schizophrenia, which can bring new pharmacotherapeutic strategies and to identify biomarkers that can improve the early interventions in schizophrenia patients. Finally, our data thus reinforce the validity of rats reared in social isolation after weaning in modelling aspects of schizophrenia, highlighting the glutamatergic and GABAergic features involved principally in the cognitive impairments related to prefrontal cortex.

Aim: We investigated morning cortisol, stress, rs1006737 and childhood trauma relationship with CACNA1C methylation. Materials & methods: Morning cortisol release, childhood trauma and perceived stress were collected and genotyping for rs1006737 conducted in 103 adult males. Genomic DNA extracted from saliva was bisulphite converted and using pyrosequencing methylation determined at 11 CpG sites within intron 3 of CACNA1C. Results: A significant negative correlation between waking cortisol and overall mean methylation was found and a positive correlation between CpG5 methylation and perceived stress. Conclusion: CACNA1C methylation levels may be related to cortisol release and stress perception. Future work should evaluate the influence of altered CACNA1C methylation on stress reactivity to investigate this as a potential mechanism for mental health vulnerability.