High-fat diet during adulthood interacts with prenatal stress, affecting both brain inflammatory and neuroendocrine markers in male rats.

Prenatal stress (PNS) affects foetal programming and, through an interaction with subsequent challenges, can increase vulnerability to mood and metabolic disorders. We have previously shown that, following PNS, adult male rats are characterized by increased vulnerability to a metabolic stressor experienced at adulthood (8-week-high-fat diet-HFD). In this study, we specifically assessed whether PNS might interact with an adult metabolic challenge to induce an inflammatory phenotype. Changes in the expression levels of inflammatory (Il-1ß, Tnf-α, Il-6) and of stress response mediators (Nr3c1, Fkbp5) as well as of mood and metabolic regulators (Bdnf, Ghs-R) were investigated in the hippocampus, prefrontal cortex and hypothalamus, brain regions involved in the pathogenesis of depression and prone to inflammation in response to stress. Overall, PNS reduced the expression of Bdnf and Tnf-α, while HFD administered at adulthood counteracted this effect suggesting that PNS impinges upon the same pathways regulating responses to a metabolic challenge at adulthood. Furthermore, HFD and PNS affected the expression of both Nr3c1 and Fkbp5, two neuroendocrine mediators involved in the response to stress, metabolic challenges and in the modulation of the emotional profile (as shown by the correlation between Fkbp5 and the time spent in the open arms of the elevated plus-maze). Overall, these results indicate that the same metabolic and neuroendocrine effectors engaged by PNS are affected by metabolic challenges at adulthood, providing some mechanistic insight into the well-known comorbidity between mood and metabolic disorders.

A peripheral signature of Alzheimer's disease featuring microbiota-gut-brain axis markers.

BACKGROUND: Increasing evidence links the gut microbiota (GM) to Alzheimer's disease (AD) but the mechanisms through which gut bacteria influence the brain are still unclear. This study tests the hypothesis that GM and mediators of the microbiota-gut-brain axis (MGBA) are associated with the amyloid cascade in sporadic AD. METHODS: We included 34 patients with cognitive impairment due to AD (CI-AD), 37 patients with cognitive impairment not due to AD (CI-NAD), and 13 cognitively unimpaired persons (CU). We studied the following systems: (1) fecal GM, with 16S rRNA sequencing; (2) a panel of putative MGBA mediators in the blood including immune and endothelial markers as bacterial products (i.e., lipopolysaccharide, LPS), cell adhesion molecules (CAMs) indicative of endothelial dysfunction (VCAM-1, PECAM-1), vascular changes (P-, E-Selectin), and upregulated after infections (NCAM, ICAM-1), as well as pro- (IL1ß, IL6, TNFα, IL18) and anti- (IL10) inflammatory cytokines; (3) the amyloid cascade with amyloid PET, plasma phosphorylated tau (pTau-181, for tau pathology), neurofilament light chain (NfL, for neurodegeneration), and global cognition measured using MMSE and ADAScog. We performed 3-group comparisons of markers in the 3 systems and calculated correlation matrices for the pooled group of CI-AD and CU as well as CI-NAD and CU. Patterns of associations based on Spearman's rho were used to validate the study hypothesis. RESULTS: CI-AD were characterized by (1) higher abundance of Clostridia_UCG-014 and decreased abundance of Moryella and Blautia (p < .04); (2) elevated levels of LPS (p < .03), upregulation of CAMs, Il1ß, IL6, and TNFα, and downregulation of IL10 (p < .05); (3) increased brain amyloid, plasma pTau-181, and NfL (p < 0.004) compared with the other groups. CI-NAD showed (1) higher abundance of [Eubacterium] coprostanoligenes group and Collinsella and decreased abundance of Lachnospiraceae_ND3007_group, [Ruminococcus]_gnavus_group and Oscillibacter (p < .03); (2) upregulation of PECAM-1 and TNFα (p < .03); (4) increased plasma levels of NfL (p < .02) compared with CU. Different GM genera were associated with immune and endothelial markers in both CI-NAD and CI-AD but these mediators were widely related to amyloid cascade markers only in CI-AD. CONCLUSIONS: Specific bacterial genera are associated with immune and endothelial MGBA mediators, and these are associated with amyloid cascade markers in sporadic AD. The physiological mechanisms linking the GM to the amyloid cascade should be further investigated to elucidate their potential therapeutic implications.

Higher immune-related gene expression in major depression is independent of CRP levels: results from the BIODEP study.

Compelling evidence demonstrates that some individuals suffering from major depressive disorder (MDD) exhibit increased levels of inflammation. Most studies focus on inflammation-related proteins, such as serum or plasma C-reactive protein (CRP). However, the immune-related modifications associated with MDD may be not entirely captured by CRP alone. Analysing mRNA gene expression levels, we aimed to identify broader molecular immune-related phenotypes of MDD. We examined 168 individuals from the non-interventional, case-control, BIODEP study, 128 with a diagnosis of MDD and 40 healthy controls. Individuals with MDD were further divided according to serum high-sensitivity (hs)CRP levels (n = 59 with CRP <1, n = 33 with CRP 1-3 and n = 36 with CRP >3 mg/L). We isolated RNA from whole blood and performed gene expression analyses using RT-qPCR. We measured the expression of 16 immune-related candidate genes: A2M, AQP4, CCL2, CXCL12, CRP, FKBP5, IL-1-beta, IL-6, ISG15, MIF, GR, P2RX7, SGK1, STAT1, TNF-alpha and USP18. Nine of the 16 candidate genes were differentially expressed in MDD cases vs. controls, with no differences between CRP-based groups. Only CRP mRNA was clearly associated with serum CRP. In contrast, plasma (proteins) IL-6, IL-7, IL-8, IL-10, IL-12/IL-23p40, IL-16, IL-17A, IFN-gamma and TNF-alpha, and neutrophils counts, were all differentially regulated between CRP-based groups (higher in CRP >3 vs. CRP <1 and/or controls), reflecting the gradient of CRP values. Secondary analyses on MDD individuals and controls with CRP values <1 mg/L (usually interpreted as 'no inflammation') confirmed MDD cases still had significantly different mRNA expression of immune-related genes compared with controls. These findings corroborate an immune-related molecular activation in MDD, which appears to be independent of serum CRP levels. Additional biological mechanisms may then be required to translate this mRNA signature into inflammation at protein and cellular levels. Understanding these mechanisms will help to uncover the true immune abnormalities in depression, opening new paths for diagnosis and treatment.

Alterations in 'inflammatory' pathways in the rat prefrontal cortex as early biological predictors of the long-term negative consequences of exposure to stress early in life.

Early life stress, especially when experienced during the first period of life, affects the brain developmental trajectories leading to an enhanced vulnerability for stress-related psychiatric disorders later in life. Although both clinical and preclinical studies clearly support this association, the biological pathways deregulated by such exposure, and the effects in shaping the neurodevelopmental trajectories, have so far been poorly investigated. By using the prenatal stress (PNS) model, a well-established rat model of early life stress, we performed transcriptomic analyses in the prefrontal cortex of rats exposed or not to PNS and sacrificed at different postnatal days (PNDs 21, 40, 62). We first investigated the long-lasting mechanisms and pathways affected in the PFC. We have decided to focus on the prefrontal cortex because we have previously shown that this brain region is highly sensitive to PNS exposure. We found that adult animals exposed to PNS show alterations in 389 genes, mainly involved in stress and inflammatory signalling. We then wanted to establish whether PNS exposure could also affect the neurodevelopmental trajectories in order to identify the most critical temporal window. We found that PNS rats show the most significant changes during adolescence (between PND 40 versus PND 21), with alterations of several pathways related to stress, inflammation and metabolism, which were maintained until adulthood.

Social isolation in adolescence and long-term changes in the gut microbiota composition and in the hippocampal inflammation: Implications for psychiatric disorders - Dirk Hellhammer Award Paper 2021.

Exposure to early adverse experiences induces persistent changes in physiological, emotional and behavioural functions predisposing the individual to an enhanced vulnerability to develop different disorders during lifespan. The adverse outcomes depend upon the timing of the stressful experiences, and in this contest, adolescence represents a key sensitive period for brain development. Among the biological systems involved, gut microbiota has recently been proposed to act on the interplay between the stress response, brain functions and immune system, through the gut-brain axis communication. In the current study we aimed to evaluate, in a preclinical model, changes over time in the microbiota community structure in physiological condition and in response to stress during adolescence. We also aimed to correlate the microbiota composition to the inflammatory status in brain. We used the preclinical model of social deprivation in rats during adolescence, based on the lack of all social contacts, for four weeks after weaning, followed by re-socialization until adulthood. We collected fecal samples at different post-natal days to investigate the short- and long-lasting effects of social isolation on gut microbiota composition and we collected brain areas (dorsal and ventral hippocampus) samples at killing to measure a panel of inflammatory and microglia activation markers. 16 S metataxonomic sequencing analysis revealed that microbial changes were influenced by age in both isolated and controls rats, regardless of sex, whereas social isolation impacted the microbial composition in a sex-dependent manner. A multivariate analysis showed that social isolation induced short-term gut microbiota alterations in females but not in males. We also identified several stress-related genera associated with social isolation condition. In brain areas we found a specific inflammatory pattern, in dorsal and ventral hippocampus, that significantly correlated with gut microbiota composition. Overall, in this study we reported a novel sex-specific association between gut microbiota composition and inflammatory response related to social isolation paradigm during adolescence, suggesting that stressful experiences during this sensitive period could have a long-lasting impact on the development of different biological systems that could in turn influence the vulnerability to develop mental disorders later in life.

Corrigendum: The Long-Term Effects of Early Life Stress on the Modulation of miR-19 Levels.

[This corrects the article DOI: 10.3389/fpsyt.2020.00389.].

The Long-Term Effects of Early Life Stress on the Modulation of miR-19 Levels.

MicroRNAs (miRNAs), one of the major small non-coding RNA classes, have been proposed as regulatory molecules in neurodevelopment and stress response. Although alterations in miRNAs profiles have been implicated in several psychiatric and neurodevelopmental disorders, the contribution of individual miRNAs in brain development and function is still unknown. Recent studies have identified miR-19 as a key regulator of brain trajectories, since it drives the differentiation of neural stem cells into mature neurons. However, no findings are available on how vulnerability factors for these disorders, such as early life stress (ELS), can modulate the expression of miR-19 and its target genes. To reach our aim, we investigated miR-19 modulation in human hippocampal progenitor stem cells (HPCs) treated with cortisol during 3 days of proliferation and harvested immediately after the end of the treatment or after 20 days of differentiation into mature neurons. We also analyzed the long-term expression changes of miR-19 and of its validated target genes, involved in neurodevelopment and inflammation, in the hippocampus of adult rats exposed or not to prenatal stress (PNS). Interestingly, we observed a significant downregulation of miR-19 levels both in proliferating (FC = -1.59, p-value = 0.022 for miR-19a; FC = -1.79, p-value = 0.016 for miR-19b) as well as differentiated HPCs (FC = -1.28, p-value = 0.065 for miR-19a; FC = -1.75, p-value = 0.047 for miR-19b) treated with cortisol. Similarly, we found a long-term decrease of miR-19 levels in the hippocampus of adult PNS rats (FC = -1.35, p-value = 0.025 for miR-19a; FC = -1.43, p-value = 0.032 for miR-19b). Among all the validated target genes, we observed a significant increase of NRCAM (FC = 1.20, p-value = 0.027), IL4R (FC = 1.26, p-value = 0.046), and RAPGEF2 (FC = 1.23, p-value = 0.020).We suggest that ELS can cause a long-term downregulation of miR-19 levels, which may be responsible of alterations in neurodevelopmental pathways and in immune/inflammatory processes, leading to an enhanced risk for mental disorders later in life. Intervention strategies targeting miR-19 may prevent alterations in these pathways, reducing the ELS-related effects.

Short-Chain Fatty Acids and Lipopolysaccharide as Mediators Between Gut Dysbiosis and Amyloid Pathology in Alzheimer's Disease.

BACKGROUND: Metagenomic data support an association between certain bacterial strains and Alzheimer's disease (AD), but their functional dynamics remain elusive. OBJECTIVE: To investigate the association between amyloid pathology, bacterial products such as lipopolysaccharide (LPS) and short chain fatty acids (SCFAs: acetate, valerate, butyrate), inflammatory mediators, and markers of endothelial dysfunction in AD. METHODS: Eighty-nine older persons with cognitive performance from normal to dementia underwent florbetapir amyloid PET and blood collection. Brain amyloidosis was measured with standardized uptake value ratio versus cerebellum. Blood levels of LPS were measured by ELISA, SCFAs by mass spectrometry, cytokines by using real-time PCR, and biomarkers of endothelial dysfunction by flow cytometry. We investigated the association between the variables listed above with Spearman's rank test. RESULTS: Amyloid SUVR uptake was positively associated with blood LPS (rho≥0.32, p≤0.006), acetate and valerate (rho≥0.45, p < 0.001), pro-inflammatory cytokines (rho≥0.25, p≤0.012), and biomarkers of endothelial dysfunction (rho≥0.25, p≤0.042). In contrast, it was negatively correlated with butyrate (rho≤-0.42, p≤0.020) and the anti-inflammatory cytokine IL10 (rho≤-0.26, p≤0.009). Endothelial dysfunction was positively associated with pro-inflammatory cytokines, acetate and valerate (rho≥0.25, p≤0.045) and negatively with butyrate and IL10 levels (rho≤-0.25, p≤0.038). CONCLUSION: We report a novel association between gut microbiota-related products and systemic inflammation with brain amyloidosis via endothelial dysfunction, suggesting that SCFAs and LPS represent candidate pathophysiologic links between the gut microbiota and AD pathology.

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 < 0.013) and for the majority of the most abundant genera (p < 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 < 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.

Long-term effects of stress early in life on microRNA-30a and its network: Preventive effects of lurasidone and potential implications for depression vulnerability.

Exposure to early life stress can interfere with neurodevelopmental trajectories to increase the vulnerability for psychiatric disorders later in life. With this respect, epigenetic mechanisms play a key role for the long-lasting changes in brain functions that may elicit and sustain psychopathologic outcomes. Here, we investigated DNA methylation changes as possible epigenetic mechanism mediating the effect of prenatal stress (PNS), an experimental paradigm associated with behavioral and molecular alterations relevant for psychiatric disorders. We identified 138 genes as being differentially methylated in the prefrontal cortex (PFC) and in the hippocampus (HIP) of male and female adult rats exposed to PNS. Among these genes, miR-30a and Neurod1 emerged as potential players for the negative outcomes associated with PNS exposure. Indeed, in addition to showing consistent methylation differences in both brain regions and in both sexes, and interacting with each other, they are both involved in Axon guidance and Neurotrophin signaling, which are important to neurodevelopmental disorders. We also found a significant reduction in the expression of a panel of genes (CAMK2A, c-JUN, LIMK1, MAP2K1, MAP2K2, PIK3CA and PLCG1) that belong to these two biological pathways and are also validated targets of miR-30a, pointing to a down-regulation of these pathways as a consequence of PNS exposure. Interestingly, we also found that miR-30a levels were significantly upregulated in depressed patients exposed to childhood trauma, as compared to control individuals. Importantly, we also found that a sub-chronic treatment with the atypical antipsychotic drug, lurasidone, during adolescence was able to prevent the up-regulation of miR-30a and normalized the expression of its target genes in response to PNS exposure. Our results demonstrate that miR-30a undergoes epigenetic changes following early life stress exposure and suggest that this miRNA could play a key role in producing broad and long-lasting alterations in neuroplasticity-related pathways, contributing to the etiology of psychiatric disorders.

Correction: Whole-blood expression of inflammasome- and glucocorticoid-related mRNAs correctly separates treatment-resistant depressed patients from drug-free and responsive patients in the BIODEP study.

We have corrected this Article post-publication, because Dr. Cattaneo's affiliation details were originally incorrect (she was affiliated with three institutions but is in fact only linked to one: Biological Psychiatry Unit, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia). These changes reflect in both the PDF and HTML versions of this Article.

Whole-blood expression of inflammasome- and glucocorticoid-related mRNAs correctly separates treatment-resistant depressed patients from drug-free and responsive patients in the BIODEP study.

The mRNA expression signatures associated with the 'pro-inflammatory' phenotype of depression, and the differential signatures associated with depression subtypes and the effects of antidepressants, are still unknown. We examined 130 depressed patients (58 treatment-resistant, 36 antidepressant-responsive and 36 currently untreated) and 40 healthy controls from the BIODEP study, and used whole-blood mRNA qPCR to measure the expression of 16 candidate mRNAs, some never measured before: interleukin (IL)-1-beta, IL-6, TNF-alpha, macrophage inhibiting factor (MIF), glucocorticoid receptor (GR), SGK1, FKBP5, the purinergic receptor P2RX7, CCL2, CXCL12, c-reactive protein (CRP), alpha-2-macroglobulin (A2M), acquaporin-4 (AQP4), ISG15, STAT1 and USP-18. All genes but AQP4, ISG15 and USP-18 were differentially regulated. Treatment-resistant and drug-free depressed patients had both increased inflammasome activation (higher P2RX7 and proinflammatory cytokines/chemokines mRNAs expression) and glucocorticoid resistance (lower GR and higher FKBP5 mRNAs expression), while responsive patients had an intermediate phenotype with, additionally, lower CXCL12. Most interestingly, using binomial logistics models we found that a signature of six mRNAs (P2RX7, IL-1-beta, IL-6, TNF-alpha, CXCL12 and GR) distinguished treatment-resistant from responsive patients, even after adjusting for other variables that were different between groups, such as a trait- and state-anxiety, history of childhood maltreatment and serum CRP. Future studies should replicate these findings in larger, longitudinal cohorts, and test whether this mRNA signature can identify patients that are more likely to respond to adjuvant strategies for treatment-resistant depression, including combinations with anti-inflammatory medications.

Prospective cohort study of early biosignatures of response to lithium in bipolar-I-disorders: overview of the H2020-funded R-LiNK initiative.

BACKGROUND: Lithium is recommended as a first line treatment for bipolar disorders. However, only 30% of patients show an optimal outcome and variability in lithium response and tolerability is poorly understood. It remains difficult for clinicians to reliably predict which patients will benefit without recourse to a lengthy treatment trial. Greater precision in the early identification of individuals who are likely to respond to lithium is a significant unmet clinical need. STRUCTURE: The H2020-funded Response to Lithium Network (R-LiNK; http://www.r-link.eu.com/ ) will undertake a prospective cohort study of over 300 individuals with bipolar-I-disorder who have agreed to commence a trial of lithium treatment following a recommendation by their treating clinician. The study aims to examine the early prediction of lithium response, non-response and tolerability by combining systematic clinical syndrome subtyping with examination of multi-modal biomarkers (or biosignatures), including omics, neuroimaging, and actigraphy, etc. Individuals will be followed up for 24 months and an independent panel will assess and classify each participants' response to lithium according to predefined criteria that consider evidence of relapse, recurrence, remission, changes in illness activity or treatment failure (e.g. stopping lithium; new prescriptions of other mood stabilizers) and exposure to lithium. Novel elements of this study include the recruitment of a large, multinational, clinically representative sample specifically for the purpose of studying candidate biomarkers and biosignatures; the application of lithium-7 magnetic resonance imaging to explore the distribution of lithium in the brain; development of a digital phenotype (using actigraphy and ecological momentary assessment) to monitor daily variability in symptoms; and economic modelling of the cost-effectiveness of introducing biomarker tests for the customisation of lithium treatment into clinical practice. Also, study participants with sub-optimal medication adherence will be offered brief interventions (which can be delivered via a clinician or smartphone app) to enhance treatment engagement and to minimize confounding of lithium non-response with non-adherence. CONCLUSIONS: The paper outlines the rationale, design and methodology of the first study being undertaken by the newly established R-LiNK collaboration and describes how the project may help to refine the clinical response phenotype and could translate into the personalization of lithium treatment.