Identification of a miRNAs signature associated with exposure to stress early in life and enhanced vulnerability for schizophrenia: New insights for the key role of miR-125b-1-3p in neurodevelopmental processes

Epidemiological and clinical studies have provided evidence for a role of both genetic and environmental factors, such as stressful experiences early in life, in the pathogenesis of Schizophrenia (SZ) and microRNAs (miRNAs) have been suggested to play a key role in the interplay between the environment and our genome. In this study, we conducted a miRNOme analysis in different samples (blood of adult subjects exposed to childhood trauma, brain (hippocampus) of rats exposed to prenatal stress and human hippocampal progenitor cells treated with cortisol) and we identified miR-125b-1-3p as a down-regulated miRNA in all the three datasets. Interestingly, a significant down-regulation was observed also in SZ patients exposed to childhood trauma. To investigate the biological systems targeted by miR-125b-1-3p and also involved in the effects of stress, we combined the list of biological pathways modulated by predicted and validated target genes of miR-125b-1-3p, with the biological systems significantly regulated by cortisol in the in vitro model. We found, as common pathways, the CXCR4 signaling, the G-alpha signaling, and the P2Y Purigenic Receptor Signaling Pathway, which are all involved in neurodevelopmental processes. Our data, obtained from the combining of miRNAs datasets across different tissues and species, identified miR-125b-1-3p as a key marker associated with the long-term effects of stress early in life and also with the enhanced vulnerability of developing SZ. The identification of such a miRNA biomarker could allow the early detection of vulnerable subjects for SZ and could provide the basis for the development of preventive therapeutic strategies

Glucocorticoids prime the inflammatory response of human hippocampal cells through up-regulation of inflammatory pathways

Increased pro-inflammatory cytokines and an overactive hypothalamic-pituitary-adrenal (HPA) axis have both been implicated in the pathogenesis of depression. However, these explanations appear contradictory because glucocorticoids are well recognised for their anti-inflammatory effects. Two hypotheses exist to resolve this paradox: the mediating presence of glucocorticoid receptor resistance, or the possibility that glucocorticoids can potentiate inflammatory processes in some circumstances. We sought to investigate these hypotheses in a cell model with significant relevance to depression: human hippocampal progenitor cells. We demonstrated that dexamethasone in vitro given for 24 hours and followed by a 24 hours rest interval before an immune challenge potentiates inflammatory effects in these neural cells, that is, increases the IL-6 protein secretion induced by stimulation with IL-1\u3b2 (10 ng/mL for 24 hours) by + 49% (P < 0.05) at a concentration of 100 nM and by + 70% (P < 0.01) for 1 \u3bcM. These effects are time- and dose-dependent and require activation of the glucocorticoid receptor. Gene expression microarray assays using Human Gene 2.1st Array Strips demonstrated that glucocorticoid treatment up-regulated several innate immune genes, including chemokines and Nod-like receptor, NLRP6; using transcription factor binding motifs we found limited evidence that glucocorticoid resistance was induced in the cells. Our data suggests a mechanism by which stress may prime the immune system for increased inflammation and suggests that stress and inflammation may be synergistic in the pathogenesis of depression

Transcriptomic analyses and leukocyte telomere length measurement in subjects exposed to severe recent stressful life events

Stressful life events occurring in adulthood have been found able to affect mood and behavior, thus increasing the vulnerability for several stress-related psychiatric disorders. However, although there is plenty of clinical data supporting an association between stressful life events in adulthood and an enhanced vulnerability for psychopathology, the underlying molecular mechanisms are still poorly investigated. Thus, in this study we performed peripheral/whole-genome transcriptomic analyses in blood samples obtained from 53 adult subjects characterized for recent stressful life events occurred within the previous 6 months. Transcriptomic data were analyzed using Partek Genomics Suite; pathway and network analyses were performed using Ingenuity Pathway Analysis and GeneMANIA Software. We found 207 genes significantly differentially expressed in adult subjects who reported recent stressful life experiences (n=21) compared with those without such experiences (n=32). Moreover, the same subjects exposed to such stressful experiences showed a reduction in leukocyte telomere length. A correlation analyses between telomere length and transcriptomic data indicated an association between the exposures to recent stressful life events and the modulation of several pathways, mainly involved in immune-inflammatory-related processes and oxidative stress, such as natural killer cell signaling, interleukin-1 (IL-1) signaling, MIF regulation of innate immunity and IL-6 signaling. Our data suggest an association between exposures to recent stressful life events in adulthood and alterations in the immune, inflammatory and oxidative stress pathways, which could be also involved in the negative effect of stressful life events on leukocyte telomere length. The modulation of these mechanisms may underlie the clinical association between the exposure to recent Stressful life events in adulthood and an enhanced vulnerability to develop psychiatric diseases in adulthood

Transcriptomics in Interferon-α-Treated Patients Identifies Inflammation-, Neuroplasticity- and Oxidative Stress-Related Signatures as Predictors and Correlates of Depression

Owing to the unique opportunity to assess individuals before and after they develop depression within a short timeframe, interferon-α (IFN-α) treatment for chronic hepatitis C virus (HCV) infection is an ideal model to identify molecular mechanisms relevant to major depression, especially in the context of enhanced inflammation. Fifty-eight patients were assessed prospectively, at baseline and monthly over 24 weeks of IFN-α treatment. New-onset cases of depression were determined using the Mini International Neuropsychiatric Interview (MINI). Whole-blood transcriptomic analyses were conducted to investigate the following: (1) baseline gene expression differences associated with future development of IFN-α-induced depression, before IFN-α, and (2) longitudinal gene expression changes from baseline to weeks 4 or 24 of IFN-α treatment, separately in those who did and did not develop depression. Transcriptomics data were analyzed using Partek Genomics Suite (1.4-fold, FDR adjusted pless than or equal to0.05) and Ingenuity Pathway Analysis Software. Twenty patients (34%) developed IFN-α-induced depression. At baseline, 73 genes were differentially expressed in patients who later developed depression compared with those who did not. After 4 weeks of IFN-α treatment, 592 genes were modulated in the whole sample, representing primarily IFN-α-responsive genes. Substantially more genes were modulated only in patients who developed depression (n=506, compared with n=70 in patients who did not), with enrichment in inflammation-, neuroplasticity- and oxidative stress-related pathways. A similar picture was observed at week 24. Our data indicate that patients who develop IFN-α-induced depression have an increased biological sensitivity to IFN-α, as shown by larger gene expression changes, and specific signatures both as predictors and as correlates

BDNF alterations as consequences of childhood trauma experiences

Indeed, exposure to childhood trauma experiences is associated with increased vulnerability for several kinds of disorders including mental illnesses (van Winkel et al., 2013). This increased vulnerability is due to the fact that childhood is a critical period for the brain development, where the brain is not completely developed yet and thus high vulnerable to adverse events. The Brain-Derived Neutrotrophic factor (BDNF) is a major neuronal growth factor in the brain, regulating neurogenesis, neuronal maturation and survival, and synaptic plasticity (Tsankova et al., 2007). It has been proposed that BDNF could be the link between early life stress and the vulnerability to psychiatric disorders (Schroeder et al., 2010). The focus of this work was to investigate BDNF levels in the peripheral blood of control subjects characterized for exposures to stressful experiences during childhood. By using Real Time PCR we measured total BDNF mRNA levels and the results indicated a strong reduction of total BDNF mRNA levels in individuals exposed to stress early in life versus not exposed individuals (-32%, p<0.01 vs not exposed subjects). Then, we assessed the contribution of the different BDNF transcripts on BDNF total modulation, and we found that transcripts IV and IX were significantly reduced (-71%, p<0.05; -35%, p<0.05, respectively). In order to investigate the molecular mechanisms underlying these long lasting changes in BDNF expression, we evaluated the role of DNA methylation and miRNAs as possible epigenetic mechanisms. We assessed the DNA methylation levels of specific CpGs in both the transcripts IV and IX, however, no significant changes in association to stress exposures were observed. Then, we focused on miRNAs targeting BDNF and we identified, by using biostatistical tools, the main predicted and validated miRNAs targeting BDNF 5\u2019 UTR region, CDS region and 3\u2019 UTR region (total number of miRNAs = 805). Due to the high number of miRNAs targeting BDNF that we identified, we used a whole genome approach and we run a miRNome analysis. We found that 26 of the 805 miRNAs targeting BDNF are significantly up regulated in response to stress early in life; 18 targeting the 5\u2019 UTR region, 4 targeting the CDS region and 4 targeting the 3\u2019 UTR region (Fold Change>1.1; p<0,05 for all the 26 miRNAs). Bioinformatic analysis, using the DIANA miRPath program (v.2.0) identified the signal transduction, the neurotrophin signalling and the long-term potentiation pathways as the most significant pathways modulated by the significant miRNAs. In conclusion we found that total BDNF mRNA levels are reduced in the peripheral blood of subjects exposed to stressful experiences during childhood and that miRNAs could be in part responsible for these long lasting changes in BDNF expression. In the future, a better characterization of miRNAs modulation may identify novel targets for preventative therapies in subjects with a history of childhood trauma, which are at higher risk to develop psychiatric disorders in the adulthood

Emotional Regulation in Teens and Improvement of Constructive Skills (EmoTIConS): study protocol for a randomized controlled trial

Background: Emotional dysregulation (ED) constitutes a relevant factor involved in the onset and maintenance of many mental disorders. Targeting ED during adolescence could be a determinant both to identify high-risk individuals and to promote preventive interventions. This study will aim to evaluate the impact of a brief Dialectical Behavioral Therapy (DBT)-based intervention for adolescent students by measuring changes in emotional regulation skills and impulsive behaviors. Moreover, alterations in biological features related to stress response and inflammation will be assessed as potential biological variables associated with ED. Methods: This is a randomized trial. A total of 20 classes of adolescent students will be recruited among high schools in Brescia, a city in northern Italy. They will be randomized to the psychoeducational intervention (experimental group) or to a control condition (control group). The intervention will be based on DBT Skills Training for Emotional Problem Solving for Adolescents, and will consist of four monthly, 2-h sessions (for a total of 8 h) scheduled during regular school time. Participants will be assessed at baseline, post-intervention, and at 3 and 6 months of follow-up. The primary outcome measures will be represented by changes in the use of emotional regulation skills and by changes in the frequency of impulsive behaviors. Salivary samples will be collected at baseline and post-intervention to explore possible biological features underlying ED. Discussion: Data from the present project will offer the opportunity to better understand the complex phenomenon of ED. Repeated assessment will cover several domains (emotional, behavioral, social, biological) as potential factors associated with ED. Moreover, it will be possible to establish the effect of the proposed intervention, thus helping to improve knowledge on the impact of school-based universal preventive programs. Finally, the current trial will propose an integrated screening and intervention-based model. Ultimately, this could reduce barriers to youths’ mental health care by fostering collaboration between schools and mental health services. Trial registration: ClinicalTrials.gov NCT04349709. Registered on April 16, 2020

Comparison of Bioinformatics Pipelines and Operating Systems for the Analyses of 16S rRNA Gene Amplicon Sequences in Human Fecal Samples

Amplicon high-throughput sequencing of 16S ribosomal RNA (rRNA) gene is currently the most widely used technique to investigate complex gut microbial communities. Microbial identification might be influenced by several factors, including the choice of bioinformatic pipelines, making comparisons across studies difficult. Here, we compared four commonly used pipelines (QIIME2, Bioconductor, UPARSE and mothur) run on two operating systems (OS) (Linux and Mac), to evaluate the impact of bioinformatic pipeline and OS on the taxonomic classification of 40 human stool samples. We applied the SILVA 132 reference database for all the pipelines. We compared phyla and genera identification and relative abundances across the four pipelines using the Friedman rank sum test. QIIME2 and Bioconductor provided identical outputs on Linux and Mac OS, while UPARSE and mothur reported only minimal differences between OS. Taxa assignments were consistent at both phylum and genus level across all the pipelines. However, a difference in terms of relative abundance was identified for all phyla (p &lt; 0.013) and for the majority of the most abundant genera (p &lt; 0.028), such as Bacteroides (QIIME2: 24.5%, Bioconductor: 24.6%, UPARSE-linux: 23.6%, UPARSE-mac: 20.6%, mothur-linux: 22.2%, mothur-mac: 21.6%, p &lt; 0.001). The use of different bioinformatic pipelines affects the estimation of the relative abundance of gut microbial community, indicating that studies using different pipelines cannot be directly compared. A harmonization procedure is needed to move the field forward