El Chérif Ibrahim

Responder and nonresponder patients exhibit different peripheral transcriptional signatures during major depressive episode.

To date, it remains impossible to guarantee that short-term treatment given to a patient suffering from a major depressive episode (MDE) will improve long-term efficacy. Objective biological measurements and biomarkers that could help in predicting the clinical evolution of MDE are still warranted. To better understand the reason nearly half of MDE patients respond poorly to current antidepressive treatments, we examined the gene expression profile of peripheral blood samples collected from 16 severe MDE patients and 13 matched controls. Using a naturalistic and longitudinal design, we ascertained mRNA and microRNA (miRNA) expression at baseline, 2 and 8 weeks later. On a genome-wide scale, we detected transcripts with roles in various biological processes as significantly dysregulated between MDE patients and controls, notably those involved in nucleotide binding and chromatin assembly. We also established putative interactions between dysregulated mRNAs and miRNAs that may contribute to MDE physiopathology. We selected a set of mRNA candidates for quantitative reverse transcriptase PCR (RT-qPCR) to validate that the transcriptional signatures observed in responders is different from nonresponders. Furthermore, we identified a combination of four mRNAs (PPT1, TNF, IL1B and HIST1H1E) that could be predictive of treatment response. Altogether, these results highlight the importance of studies investigating the tight relationship between peripheral transcriptional changes and the dynamic clinical progression of MDE patients to provide biomarkers of MDE evolution and prognosis.

Severity of experimental autoimmune encephalomyelitis is unexpectedly reduced in mice born to vitamin D-deficient mothers

Accumulating data indicate that vitamin D, a sun-induced hormone, plays a key role in multiple sclerosis (MS) etiology. Notably, it has been shown that there is a remarkable season of birth effect in MS. We surmised that gestational vitamin D deficiency is a risk factor for MS. To test this hypothesis, a vitamin D deficiency was induced in C57BL/6 female mice 6 weeks prior to conception and prolonged until offspring birth. Contrary to our prediction, we show here that adult offspring exposed to developmental vitamin D deficiency (DVD) developed a striking milder and delayed experimental autoimmune encephalomyelitis (EAE), when compared to control offspring. Using reverse transcription and quantitative real-time PCR, we measured the expression level of 22 candidate transcripts in the spleen, the cerebrum and the spinal cord, at Day0 and Day30 post-immunization. We report here that, at Day30 post-immunization, TNF, osteopontin, H2-Eb were over-expressed and IFN was under-expressed in the spinal cord of control mice and not in DVD mice. Another discrepancy between nervous and immune systems was observed: expression of IL4 was dysregulated exclusively in the spleen. Reduced symptom severity in DVD mice can partially be explained by a nervous system-restricted over-expression of vitamin D receptor (VDR), two heat shock proteins (HSP90, HSPa8) and FK506 binding protein 1a (FKBP1a), at Day0. Our clinical test and molecular findings converge to indicate that maternal hypovitaminosis D imprints the foetus and alters the susceptibility of the offspring to EAE. We propose a new hypothesis to explain our unexpected observations.

Dissociating Bottom-Up and Top-Down Mechanisms in the Cortico-Limbic System during Emotion Processing

The cortico-limbic system is critically involved in emotional responses and resulting adaptive behaviors. Within this circuit, complementary regions are believed to be involved in either the appraisal or the regulation of affective state. However, the respective contribution of these bottom-up and top-down mechanisms during emotion processing remains to be clarified. We used a new functional magnetic resonance imaging (fMRI) paradigm varying 3 parameters: emotional valence, emotional congruency, and allocation of attention, to distinguish the functional variation in activity and connectivity between amygdala, anterior cingulate cortex (ACC), and dorsolateral prefrontal cortex (DLPFC). Bottom-up appraisal of negative compared with positive stimuli led to a greater amygdala response and stronger functional interaction between amygdala and both dorsal ACC and DLPFC. Top-down resolution of emotional conflict was associated with increased activity within ACC and higher functional connectivity between this structure, and both the amygdala and DLPFC. Finally, increased top-down attentional control caused greater engagement of the DLPFC, accompanied by increased connectivity between DLPFC and dorsal ACC. This novel task provides an efficient tool for exploring bottom-up and top-down processes underlying emotion and may be particularly helpful for investigating the neurofunctional underpinnings of psychiatric disorders.

Genome-wide analysis of familial dysautonomia and kinetin target genes with patient olfactory ecto-mesenchymal stem cells.

Familial dysautonomia (FD) is a rare inherited neurodegenerative disorder. The most common mutation is a c.2204+6T>C transition in the 5′ splice site (5′ss) of IKBKAP intron 20, which causes a tissue‐specific skipping of exon 20, resulting in lower synthesis of IKAP/hELP1 protein. To better understand the specificity of neuron loss in FD, we modeled the molecular mechanisms of IKBKAP mRNA splicing by studying human olfactory ecto‐mesenchymal stem cells (hOE‐MSCs) derived from FD patient nasal biopsies. We explored how the modulation of IKBKAP mRNA alternative splicing impacts the transcriptome at the genome‐wide level. We found that the FD transcriptional signature was highly associated with biological functions related to the development of the nervous system. In addition, we identified target genes of kinetin, a plant cytokinin that corrects IKBKAP mRNA splicing and increases the expression of IKAP/hELP1. We identified this compound as a putative regulator of splicing factors and added new evidence for a sequence‐specific correction of splicing. In conclusion, hOE‐MSCs isolated from FD patients represent a promising avenue for modeling the altered genetic expression of FD, demonstrating a methodology that can be applied to a host of other genetic disorders to test the therapeutic potential of candidate molecules. Hum Mutat 33:530–540, 2012.

CX3CR1 is dysregulated in blood and brain from schizophrenia patients

The molecular mechanisms underlying schizophrenia remain largely unknown. Although schizophrenia is a mental disorder, there is increasing evidence to indicate that inflammatory processes driven by diverse environmental factors play a significant role in its development. With gene expression studies having been conducted across a variety of sample types, e.g., blood and postmortem brain, it is possible to investigate convergent signatures that may reveal interactions between the immune and nervous systems in schizophrenia pathophysiology. We conducted two meta-analyses of schizophrenia microarray gene expression data (N=474) and non-psychiatric control (N=485) data from postmortem brain and blood. Then, we assessed whether significantly dysregulated genes in schizophrenia could be shared between blood and brain. To validate our findings, we selected a top gene candidate and analyzed its expression by RT-qPCR in a cohort of schizophrenia subjects stabilized by atypical antipsychotic monotherapy (N=29) and matched controls (N=31). Meta-analyses highlighted inflammation as the major biological process associated with schizophrenia and that the chemokine receptor CX3CR1 was significantly down-regulated in schizophrenia. This differential expression was also confirmed in our validation cohort. Given both the recent data demonstrating selective CX3CR1 expression in subsets of neuroimmune cells, as well as behavioral and neuropathological observations of CX3CR1 deficiency in mouse models, our results of reduced CX3CR1 expression adds further support for a role played by monocyte/microglia in the neurodevelopment of schizophrenia.

Mood disorders are associated with a more severe hypovitaminosis D than schizophrenia

Patients with psychiatric disorders display high levels of hypovitaminosis D (<50nmol/L). It remains unclear whether it is associated with specific diagnoses. To further explore vitamin D status in psychiatric inpatients, 82 individuals with mood disorders or schizophrenia/schizoaffective disorders were included. Hypovitaminosis D was significantly lower in patients with mood disorders than patients with schizophrenia (standardized β coefficient=0.385, p=0.007). Further studies are warranted to determine specific causes of hypovitaminosis D and the interest of supplementation.

Predisposition to treatment response in major depressive episode: A peripheral blood gene coexpression network analysis

Antidepressant efficacy is insufficient, unpredictable and poorly understood in major depressive episode (MDE). Gene expression studies allow for the identification of significantly dysregulated genes but can limit the exploration of biological pathways. In the present study, we proposed a gene coexpression analysis to investigate biological pathways associated with treatment response predisposition and their regulation by microRNAs (miRNAs) in peripheral blood samples of MDE and healthy control subjects. We used a discovery cohort that included 34 MDE patients that were given 12-week treatment with citalopram and 33 healthy controls. Two replication cohorts with similar design were also analyzed. Expression-based gene network was built to define clusters of highly correlated sets of genes, called modules. Association between each modules first principal component of the expression data and clinical improvement was tested in the three cohorts. We conducted gene ontology analysis and miRNA prediction based on the module gene list. Nine of the 59 modules from the gene coexpression network were associated with clinical improvement. The association was partially replicated in other cohorts. Gene ontology analysis demonstrated that 4 modules were associated with cytokine production, acute inflammatory response or IL-8 functions. Finally, we found 414 miRNAs that may regulate one or several modules associated with clinical improvement. By contrast, only 12 miRNAs were predicted to specifically regulate modules unrelated to clinical improvement. Our gene coexpression analysis underlines the importance of inflammation-related pathways and the involvement of a large miRNA program as biological processes predisposing associated with antidepressant response.

How to: Measuring blood cytokines in biological psychiatry using commercially available multiplex immunoassays

Cytokines produced by both immune and non-immune cells are likely to play roles in the development and/or progression of psychiatric disorders. Indeed, many investigators have compared the blood cytokine levels in psychiatric patients with those of healthy controls or monitored their levels in patients during disease progression to identify biomarkers. Nevertheless, very few studies have confirmed that such cytokines remain stable in healthy individuals through periods of weeks and months. This is an important issue to consider before using blood cytokine levels as biomarkers of disease traits, disease state, or treatment response. Although multiplex assay technology represents an advance in identifying biomarkers because it allows simultaneous examination of large panels of analytes from a small volume of sample, it is necessary to verify whether these assays yield enough sensitivity and reproducibility when applied to the blood from neuropsychiatric patients. Therefore, we compared two multiplex immunoassays, the bead-based Luminex® (Bio-Rad) and the electro-chemiluminescence-based V-plex® (MesoScaleDiscovery), for the detection and quantification of 31 cytokines, chemokines and growth factors in both the sera and plasma of patients with major depressive episodes (MDE) and age- and sex-matched healthy control subjects during a 30-week period. Although both platforms exhibited low coefficients of variability (CV) between the duplicates in the calibration curves, the linearity was better in general for the V-PLEX® platform. However, neither platform was able to detect the absolute values for all of the tested analytes. Among the 16 analytes that were detected by both assays, the intra-assay reproducibility was in general better with the V-PLEX® platform. Although it is not a general rule that the results from sera and plasma will be correlated, consistent results were more frequent with the V-PLEX® platform. Furthermore, the V-PLEX® results were more consistent with the gold standard ELISA simplex assay for IL-6 in both sera and plasma. The intra-individual variability of the measurements, among the sera and plasma for the 4 samples harvested from each healthy individual, was low for Eotaxin, G-CSF, IL-4, IL-7, IL-9, IL-12p40, IL-12p70, IL-15, MIP-1β, PDGF-BB, TNF, TNF-β and VEGF, but intermediate or high for IFN-γ, IL-6, IL-8, IL-10, and IP10. Together, these data suggest that extreme caution is needed in translating the results of multiplex cytokine profiling into biomarker discovery in psychiatry.

MicroRNA screening identifies a link between NOVA1 expression and a low level of IKAP in familial dysautonomia

ABSTRACT Familial dysautonomia (FD) is a rare neurodegenerative disease caused by a mutation in intron 20 of the IKBKAP gene (c.2204+6T>C), leading to tissue-specific skipping of exon 20 and a decrease in the synthesis of the encoded protein IKAP (also known as ELP1). Small non-coding RNAs known as microRNAs (miRNAs) are important post-transcriptional regulators of gene expression and play an essential role in the nervous system development and function. To better understand the neuronal specificity of IKAP loss, we examined expression of miRNAs in human olfactory ecto-mesenchymal stem cells (hOE-MSCs) from five control individuals and five FD patients. We profiled the expression of 373 miRNAs using microfluidics and reverse transcription coupled to quantitative PCR (RT-qPCR) on two biological replicate series of hOE-MSC cultures from healthy controls and FD patients. This led to the total identification of 26 dysregulated miRNAs in FD, validating the existence of a miRNA signature in FD. We then selected the nine most discriminant miRNAs for further analysis. The signaling pathways affected by these dysregulated miRNAs were largely within the nervous system. In addition, many targets of these dysregulated miRNAs had been previously demonstrated to be affected in FD models. Moreover, we found that four of our nine candidate miRNAs target the neuron-specific splicing factor NOVA1. We demonstrated that overexpression of miR-203a-3p leads to a decrease of NOVA1, counter-balanced by an increase of IKAP, supporting a potential interaction between NOVA1 and IKAP. Taken together, these results reinforce the choice of miRNAs as potential therapeutic targets and suggest that NOVA1 could be a regulator of FD pathophysiology. Summary: A miRNA screening conducted in olfactory stem cells from patients links the neuron-specific splicing factor NOVA1 to neurodegeneration in familial dysautonomia.

Translational Identification of Transcriptional Signatures of Major Depression and Antidepressant Response

Major depressive disorder (MDD) is a highly prevalent mental illness whose therapy management remains uncertain, with more than 20% of patients who do not achieve response to antidepressants. Therefore, identification of reliable biomarkers to predict response to treatment will greatly improve MDD patient medical care. Due to the inaccessibility and lack of brain tissues from living MDD patients to study depression, researches using animal models have been useful in improving sensitivity and specificity of identifying biomarkers. In the current study, we used the unpredictable chronic mild stress (UCMS) model and correlated stress-induced depressive-like behavior (n = 8 unstressed vs. 8 stressed mice) as well as the fluoxetine-induced recovery (n = 8 stressed and fluoxetine-treated mice vs. 8 unstressed and fluoxetine-treated mice) with transcriptional signatures obtained by genome-wide microarray profiling from whole blood, dentate gyrus (DG), and the anterior cingulate cortex (ACC). Hierarchical clustering and rank-rank hypergeometric overlap (RRHO) procedures allowed us to identify gene transcripts with variations that correlate with behavioral profiles. As a translational validation, some of those transcripts were assayed by RT-qPCR with blood samples from 10 severe major depressive episode (MDE) patients and 10 healthy controls over the course of 30 weeks and four visits. Repeated-measures ANOVAs revealed candidate trait biomarkers (ARHGEF1, CMAS, IGHMBP2, PABPN1 and TBC1D10C), whereas univariate linear regression analyses uncovered candidates state biomarkers (CENPO, FUS and NUBP1), as well as prediction biomarkers predictive of antidepressant response (CENPO, NUBP1). These data suggest that such a translational approach may offer new leads for clinically valid panels of biomarkers for MDD.