Exploring the use of immunomethylomics in the characterization of depressed patients: A proof-of-concept study.

Alterations in DNA methylation and inflammation could represent valid biomarkers for the stratification of patients with major depressive disorder (MDD). This study explored the use of DNA-methylation based immunological cell-type profiles in the context of MDD and symptom severity over time. In 119 individuals with MDD, DNA-methylation was assessed on whole blood using the Illumina Infinium MethylationEPIC 850 k BeadChip. Quality control and data processing, as well as cell type estimation was conducted using the RnBeads package. The cell type composition was estimated using epigenome-wide DNA methylation signatures, applying the Houseman method, considering six cell types (neutrophils, natural killer cells (NK), B cells, CD4 + T cells, CD8 + T cells and monocytes). Two cytokines (IL-6 and IL-1ß) and hsCRP were quantified in serum. We performed a hierarchical cluster analysis on the six estimated cell-types and tested the differences between these clusters in relation to the two cytokines and hsCRP, depression severity at baseline, and after 6 weeks of treatment (celecoxib/placebo + vortioxetine). We performed a second cluster analysis with cell-types and cytokines combined. ANCOVA was used to test for differences across clusters. We applied the Bonferroni correction. After quality control, we included 113 participants. Two clusters were identified, cluster 1 was high in CD4 + cells and NK, cluster 2 was high in CD8 + T-cells and B-cells, with similar fractions of neutrophils and monocytes. The clusters were not associated with either of the two cytokines and hsCRP, or depression severity at baseline, but cluster 1 showed higher depression severity after 6 weeks, corrected for baseline (p = 0.0060). The second cluster analysis found similar results: cluster 1 was low in CD8 + T-cells, B-cells, and IL-1ß. Cluster 2 was low in CD4 + cells and natural killer cells. Neutrophils, monocytes, IL-6 and hsCRP were not different between the clusters. Participants in cluster 1 showed higher depression severity at baseline than cluster 2 (p = 0.034), but no difference in depression severity after 6 weeks. DNA-methylation based cell-type profiles may be valuable in the immunological characterization and stratification of patients with MDD. Future models should consider the inclusion of more cell-types and cytokines for better a prediction of treatment outcomes.

Long-term characterisation of the relationship between change in depression severity and change in inflammatory markers following inflammation-stratified treatment with vortioxetine augmented with celecoxib or placebo.

BACKGROUND: Major depressive disorder (MDD) is a highly prevalent condition with a substantial incidence of relapse or treatment resistance. A subset of patients show evidence of low-grade inflammation, with these patients having a higher likelihood of more severe or difficult to treat courses of illness. Anti-inflammatory treatment of MDD has been investigated with mixed results, and no known studies have included assessments beyond cessation of the anti-inflammatory agent, meaning it remains unknown if any benefit from treatment persists. The objective of the present study was to investigate treatment outcomes up to 29 weeks post-cessation of celecoxib or placebo augmentation of an antidepressant, and how concentrations of selected inflammatory markers change over the same period. METHODS: The PREDDICT parallel-group, randomised, double-blind, placebo-controlled trial (University of Adelaide, Australia) ran from December 2017 to April 2020. Participants with MDD were stratified into normal range or elevated inflammation strata according to screening concentrations of high sensitivity C-reactive protein (hsCRP). Participants were randomised to treatment with vortioxetine and celecoxib or vortioxetine and placebo for six weeks, and vortioxetine alone for an additional 29 weeks (35 total weeks). Following a previous publication of results from the six-week RCT phase, exploratory analyses were performed on Montgomery-Åsberg Depression Rating Scale (MADRS) scores, response and remission outcomes, and selected peripheral inflammatory markers across the entire study duration up to week 35. RESULTS: Participants retained at each observation were baseline N=119, week 2 N=115, week 4 N=103, week 6 N=104, week 8 N=98, week 22 N=81, and week 35 N=60. Those in the elevated hsCRP celecoxib-augmented group had a statistically significantly greater reduction in MADRS score from baseline to week 35 compared to all other groups, demonstrating the greatest clinical improvement long-term, despite no group or strata differences at preceding time points. Response and remission outcomes did not differ by treatment group or hsCRP strata at any time point. Changes in hsCRP between baseline and week 35 and Tumour Necrosis Factor-α (TNF-α) concentrations between baseline and week 6 and baseline and week 35 were statistically significantly associated with MADRS scores observed at week 6 and week 35 respectively, with reducing TNF-α concentrations associated with reducing MADRS scores and vice versa in each case. A post-hoc stratification of the participant cohort by baseline TNF-α concentrations led to significant prediction by the derived strata on clinical response at weeks 6, 8 and 35, with participants with elevated baseline TNF-α less likely to achieve clinical response. INTERPRETATION: The present analysis suggests for the first time a possible longer-term clinical benefit of celecoxib augmentation of vortioxetine in inflammation-associated MDD treatment. However, further research is needed to confirm the finding and to ascertain the reason for such a delayed effect. Furthermore, the trial suggests that TNF-α may have a stronger relationship with anti-inflammatory MDD treatment outcomes than hsCRP, and should be investigated further for potential predictive utility. CLINICAL TRIALS REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12617000527369p. Registered on 11 April 2017, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12617000527369p.

Long-Term Immunomodulatory Impact of VNS on Peripheral Cytokine Profiles and Its Relationship with Clinical Response in Difficult-to-Treat Depression (DTD).

Vagus nerve stimulation (VNS) represents a long-term adjunctive treatment option in patients with difficult-to-treat depression (DTD). Anti-inflammatory effects have been discussed as a key mechanism of action of VNS. However, long-term investigations in real-world patients are sparse. In this naturalistic observational study, we collected data on cytokines in peripheral blood in n = 6 patients (mean age 47.8) with DTD and VNS treatment at baseline and at 6 months follow-up. We have identified clusters of peripheral cytokines with a similar dynamic over the course of these 6 months using hierarchical clustering. We have investigated cytokine changes from baseline to 6 months as well as the relationship between the cytokine profile at 6 months and long-term response at 12 months. After 6 months of VNS, we observed significant correlations between cytokines (p < 0.05) within the identified three cytokine-pairs which were not present at baseline: IL(interleukin)-6 and IL-8; IL-1ß and TNF-α; IFN-α2 and IL-33. At 6 months, the levels of all the cytokines of interest had decreased (increased in non-responders) and were lower (5-534 fold) in responders to VNS than in non-responders: however, these results were not statistically significant. VNS-associated immunomodulation might play a role in long-term clinical response to VNS.

Inflammation-stratified augmentation of vortioxetine with celecoxib: Results from a double-blind, randomized, placebo-controlled trial in major depressive disorder.

Low-grade inflammation is considered as a pathophysiological mechanism in a subtype of patients with major depressive disorder (MDD). Anti-inflammatory drugs have shown efficacy in treating MDD. However, it remains unclear how to identify suitable patients for anti-inflammatory treatment of depression. This study investigates the predictive value of pre-treatment high-sensitivity C-Reactive Protein (hsCRP) stratification on the outcome of celecoxib augmentation of vortioxetine. The PREDDICT study was conducted as a randomized, double-blind, placebo-controlled 6-week trial on augmentation of vortioxetine with celecoxib between December 2017 and April 2020 at the University of Adelaide (Australia). The present analysis focusses on the question of whether the pre-treatment hsCRP measurement and stratification of patients to depression with inflammation (hsCRP >3 mg/L) or without inflammation (hsCRP ≤3 mg/L) has an impact on the outcome of anti-inflammatory treatment with celecoxib. A total of n = 119 mostly treatment-resistant MDD patients with moderate to severe symptomatology were recruited in the trial. There was no effect of treatment group (celecoxib or placebo), pre-treatment hsCRP strata (with/without inflammation), or interaction between the two terms on treatment outcome. The results of the current analysis do not support the hypothesis that pre-treatment hsCRP level is predictive for response to anti-inflammatory treatment with celecoxib in MDD patients. Further research is needed to identify appropriate biomarkers for the prediction of anti-inflammatory treatment outcome in depression. CLINICAL TRIALS REGISTRATION: Australian New Zealand Clinical Trials Registry (ANZCTR), ACTRN12617000527369p. Registered on 11 April 2017, http://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?ACTRN=12617000527369p.

DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels.

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7-/- mice. Although palmitoylation of barttin in kidneys of Zdhhc7-/- animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7-/- mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

Brain microstructural changes in mice persist in adulthood and are modulated by the palmitoyl acyltransferase ZDHHC7.

For a long time, mice have been classified as adults with completely mature brains at 8 weeks of age, but recent research suggests that developmental brain changes occur for up to 6 months. In particular, adolescence coincides with dramatic changes of neuronal structure and function in the brain that influence the connectivity between areas like hippocampus and medial prefrontal cortex (mPFC). Neuronal development and plasticity are regulated in part by the palmitoyl acyltransferase ZDHHC7, which modulates structural connectivity between hippocampus and mPFC. The aim of the current study was to investigate whether developmental changes take place in hippocampus and mPFC microstructure even after 8 weeks of age and whether deficiency of ZDHHC7 impacts such age-dependent alterations. Altogether, 46 mice at 11, 14 or 17 weeks of age with a genetic Zdhhc7 knockout (KO) or wild type (WT) were analysed with neuroimaging and diffusion tensor-based fibre tractography. The hippocampus and mPFC regions were compared regarding fibre metrics, supplemented by volumetric and immunohistological analyses of the hippocampus. In WT animals, we identified age-dependent changes in hippocampal fibre lengths that followed a U-shaped pattern, whereas in mPFC, changes were linear. In Zdhhc7-deficient animals, the fibre statistics were reduced in both regions, whereas the hippocampus volume and the intensities of myelin and neurofilament were higher in 11-week-old KO mice compared to WTs. Our results confirmed ongoing changes of microstructure in mice up to 17 weeks old and demonstrate that deleting the Zdhhc7 gene impairs fibre development, suggesting that palmitoylation is important in this process.

The role of BDNF methylation and Val66 Met in amygdala reactivity during emotion processing.

Epigenetic alterations of the brain-derived neurotrophic factor (BDNF) gene have been associated with psychiatric disorders in humans and with differences in amygdala BDNF mRNA levels in rodents. This human study aimed to investigate the relationship between the functional BDNF-Val66 Met polymorphism, its surrounding DNA methylation in BDNF exon IX, amygdala reactivity to emotional faces, and personality traits. Healthy controls (HC, n = 189) underwent functional MRI during an emotional face-matching task. Harm avoidance, novelty seeking and reward dependence were measured using the Tridimensional Personality Questionnaire (TPQ). Individual BDNF methylation profiles were ascertained and associated with several BDNF single nucleotide polymorphisms surrounding the BDNF-Val66 Met, amygdala reactivity, novelty seeking and harm avoidance. Higher BDNF methylation was associated with higher amygdala reactivity (x = 34, y = 0, z = -26, t(166) = 3.00, TFCE = 42.39, p(FWE) = .045), whereby the BDNF-Val66 Met genotype per se did not show any significant association with brain function. Furthermore, novelty seeking was negatively associated with BDNF methylation (r = -.19, p = .015) and amygdala reactivity (r = -.17, p = .028), while harm avoidance showed a trend for a positive association with BDNF methylation (r = .14, p = .066). The study provides first insights into the relationship among BDNF methylation, BDNF genotype, amygdala reactivity and personality traits in humans, highlighting the multidimensional relations among genetics, epigenetics, and neuronal functions. The present study suggests a possible involvement of epigenetic BDNF modifications in psychiatric disorders and related brain functions, whereby high BDNF methylation might reduce BDNF mRNA expression and upregulate amygdala reactivity.

RGS2 ggenetic variation: association analysis with panic disorder and dimensional as well as intermediate phenotypes of anxiety.

Accumulating evidence from mouse models points to the G protein-coupled receptor RGS2 (regulator of G-protein signaling 2) as a promising candidate gene for anxiety in humans. Recently, RGS2 polymorphisms were found to be associated with various anxiety disorders, e.g., rs4606 with panic disorder (PD), but other findings have been negative or inconsistent concerning the respective risk allele. To further examine the role of RGS2 polymorphisms in the pathogenesis of PD, we genotyped rs4606 and five additional RGS2 tag single nucleotide polymorphisms (SNPs; rs16834831, rs10801153, rs16829458, rs1342809, rs1890397) in two independent PD samples, comprising 531 matched case/control pairs. The functional SNP rs4606 was nominally associated with PD when both samples were combined. The upstream SNP rs10801153 displayed a Bonferroni-resistant significant association with PD in the second and the combined sample (P = 0.006 and P = 0.017). We furthermore investigated the effect of rs10801153 on dimensional anxiety traits, a behavioral avoidance test (BAT), and an index for emotional processing in the respective subsets of the total sample. In line with categorical results, homozygous risk (G) allele carriers displayed higher scores on the Agoraphobic Cognitions Questionnaire (ACQ; P = 0.015) and showed significantly more defensive behavior during fear provoking situations (P = 0.001). Furthermore, significant effects on brain activation in response to angry (P = 0.013), happy (P = 0.042) and neutral faces (P = 0.032) were detected. Taken together, these findings provide further evidence for the potential role of RGS2 as a candidate gene for PD.

Serotonin transporter gene methylation is associated with hippocampal gray matter volume.

BACKGROUND: The serotonin transporter (5-HTT) and the 5-HTTLPR/rs25531 polymorphisms in its gene (SLC6A4) have been associated with depression, increased stress-response, and brain structural alterations such as reduced hippocampal volumes. Recently, epigenetic processes including SLC6A4 promoter methylation were shown to be affected by stress, trauma, or maltreatment and are regarded to be involved in the etiology of affective disorders. However, neurobiological correlates of SLC6A4 promoter methylation have never been studied or compared to genotype effects by means of human neuroimaging hitherto METHODS: Healthy subjects were recruited in two independent samples (N = 94, N = 95) to obtain structural gray matter images processed by voxel-based morphometry (VBM8), focusing on hippocampal, amygdala, and anterior cingulate gyrus gray matter structure. SLC6A4 promoter methylation within an AluJb element and 5-HTTLPR/rs25531 genotypes were analyzed in view of a possible impact on local gray matter volume RESULTS: Strong associations of AluJb methylation and hippocampal gray matter volumes emerged within each sample separately, which in the combined sample withstood most conservative alpha-corrections for the entire brain. The amygdala, insula, and caudate nucleus showed similar associations. The 5-HTTLPR/rs25531 showed no main effect on gray matter, and the effect of methylation rates on hippocampal structure was comparable among the genotype groups CONCLUSIONS: Methylation within the AluJb appears to have strong effects on hippocampal gray matter volumes, indicating that epigenetic processes can alter brain structures crucially involved in stress-related disorders. Different ways of regulating SLC6A4 expression might involve exonization or transcription factor binding as potentially underlying mechanisms, which, however, is speculative and warrants further investigation.

Unraveling epigenomic signatures and effectiveness of electroconvulsive therapy in treatment-resistant depression patients: a prospective longitudinal study.

BACKGROUND: Electroconvulsive therapy (ECT) benefits patients with treatment-resistant depression (TRD), but the underlying biological processes are unclear. We conducted an epigenome-wide association study in 32 TRD patients undergoing ECT to depict ECT-associated methylation changes. Illness severity and ECT outcomes were assessed with the Montgomery-Åsberg Depression Rating Scale at baseline (T0) and 1 month after its end (T1). Methylation was profiled at T0 and T1 with the Illumina Infinium Methylation EPIC BeadChip array. RESULTS: Longitudinal T0-T1 analyses showed 3 differentially methylated probes (DMPs) with nominal p values ≤ 10-5, with 2 annotated in the genes CYB5B and PVRL4. Including covariates, we found 4 DMPs for symptoms variation, annotated in FAM20C, EPB41, OTUB1 and ADARB1, and 3 DMPs for response status, with 2 annotated in IQCE and FAM20C. Regional analysis revealed 54 differentially methylated regions (DMRs) with nominal p value area ≤ 0.05, with 9 presenting adjusted p-value area ≤ 0.10, annotated in MCF2L, SLC25A24, RUNX3, MIR637, FOXK2, FAM180B, POU6F1, ALS2CL and CCRL2. Considering covariates, we found 21 DMRs for symptoms variation and 26 DMRs for response (nominal p value area ≤ 0.05), with 4 presenting adjusted p-value area ≤ 0.10 for response, annotated in SNORD34, NLRP6, GALNT2 and SFT2D3. None remained significant after false discovery rate correction. Notably, ADARB1 variants are associated with suicide attempt in patients with psychiatric disorders, and SLC25A24 relates to conduct disorder. Several DMPs and DMRs are annotated in genes associated with inflammatory/immune processes. Longitudinal analyses on females (n = 22) revealed statistically significant DMRs (adjusted p value area ≤ 0.05) and trend-significant DMRs (adjusted p value area ≤ 0.07) for symptoms variation and response status, annotated in genes related to psychiatric disorders (ZFP57, POLD4, TRIM10, GAS7, ADORA2A, TOLLIP), trauma exposure (RIPOR2) and inflammatory/immune responses (LAT, DLX4, POLD4, FAM30A, H19). Pathway analysis on females revealed enrichment for transcriptional activity, growth factors, DNA maintenance, and immune pathways including IRF7 and IRF2. CONCLUSION: Although no significant results were found for the whole cohort, the study provides insights into ECT-associated methylation changes, highlighting DMPs and DMRs related to ECT outcomes. Analyses on females revealed significant DMRs and pathways related to psychiatric disorders and inflammatory/immune processes.

DNA methylation changes in association with trauma-focused psychotherapy efficacy in treatment-resistant depression patients: a prospective longitudinal study.

Background: Stressful events increase the risk for treatment-resistant depression (TRD), and trauma-focused psychotherapy can be useful for TRD patients exposed to early life stress (ELS). Epigenetic processes are known to be related to depression and ELS, but there is no evidence of the effects of trauma-focused psychotherapy on methylation alterations.Objective: We performed the first epigenome-wide association study to investigate methylation changes related to trauma-focused psychotherapies effects in TRD patients.Method: Thirty TRD patients assessed for ELS underwent trauma-focused psychotherapy, of those, 12 received trauma-focused cognitive behavioural therapy, and 18 Eye Movement Desensitization and Reprocessing (EMDR). DNA methylation was profiled with Illumina Infinium EPIC array at T0 (baseline), after 8 weeks (T8, end of psychotherapy) and after 12 weeks (T12 - follow-up). We examined differentially methylated CpG sites and regions, as well as pathways analysis in association with the treatment.Results: Main results obtained have shown 110 differentially methylated regions (DMRs) with a significant adjusted p-value area associated with the effects of trauma-focused psychotherapies in the entire cohort. Several annotated genes are related to inflammatory processes and psychiatric disorders, such as LTA, GFI1, ARID5B, TNFSF13, and LST1. Gene enrichment analyses revealed statistically significant processes related to tumour necrosis factor (TNF) receptor and TNF signalling pathway. Stratified analyses by type of trauma-focused psychotherapy showed statistically significant adjusted p-value area in 141 DMRs only for the group of patients receiving EMDR, with annotated genes related to inflammation and psychiatric disorders, including LTA, GFI1, and S100A8. Gene set enrichment analyses in the EMDR group indicated biological processes related to inflammatory response, particularly the TNF signalling pathway.Conclusion: We provide preliminary valuable insights into global DNA methylation changes associated with trauma-focused psychotherapies effects, in particular with EMDR treatment.

Stressful events increase treatment-resistant depression, and trauma-focused psychotherapy can be useful for these patients.Epigenome-wide data shows changes associated with trauma-focused psychotherapies, especially eye movement desensitization and reprocessing therapy, in treatment-resistant depression patients.Genes and biological pathways related to inflammatory and immune systems are among the most statistically significant results.

Epigenetic modification related to cognitive changes during a cognitive training intervention in depression.

BACKGROUND: DNA methylation as a biomarker is well suited to investigate dynamic processes, such as symptom improvement. For this study we focus on epigenomic state or trait markers as early signatures of cognitive improvement in individuals receiving a cognitive intervention. We performed a first epigenome-wide association study (EWAS) on patients with cognitive dysfunction in depression comparing those with vs without cognitive dysfunction and those cognitively improving vs non-improving following a cognitive intervention. METHOD: Data from a randomized controlled trial (RCT) were used for this analysis, where cognitive function of 112 patients randomly assigned to a personalized cognitive intervention was compared to standard cognitive treatment. Cognition was measured for this study using the four cognitive tasks from the THINC-it battery. We compared individuals with cognitive impairment with individuals without cognitive impairment at baseline and after a cognitive intervention of 8 weeks. Blood for DNA methylation analysis (Illumina Infinium MethylationEPIC 850 k BeadChip) was collected at baseline and 8 weeks into the treatment. For the baseline analysis, after quality control, the final sample comprised 90 individuals, and analyses at week 8 were performed on 84 individuals. Data cleaning, quality control, and differential methylation analysis of DNA methylation data was performed using the RnBeads package (R). Analyses were corrected for gender, age, depression score (MADRS), reported years of education, height and weight, as well as surrogate variables estimated by the pipeline used. The within-individual paired longitudinal analysis was performed using Welch's t-test. RESULTS: Analyses at baseline and at week 8 did not show any genome-wide significant CpGs (p < 5 × 10-8) comparing patients with and without cognitive impairment. The most significant result in the baseline analysis comparing the groups with and without cognitive impairment at baseline is located in an open Sea region with predominantly regulatory qualities (cg10962945; 6.61 × 10-7). The most significant CpG at 8 weeks was also located in open sea, though in exon 13 of the NTRK2-gene, linked to the BDNF pathway (cg13620631, 5.56 × 10-7). Finally, a within-individual paired longitudinal analysis with only patients that show improved cognitive function over time was performed, showing 65 CpGs that overlapped between the 1% most significant of this analysis and the 1% most significant CpGs from the cross-sectional analysis at 8 weeks. CONCLUSION: Our result suggest that DNA methylation can be suitable to capture early signs of treatment response of a cognitive intervention in depression. In our layered approach we could capture dynamics that can help differentiate between biological trait and state markers of cognitive function in depression. Despite not being genome-wide significant, the CpG locations returned by our analysis comparing patients with and without cognitive impairment, are in line with prior knowledge on pathways and genes relevant for depression treatment and cognition.

Longitudinal early epigenomic signatures inform molecular paths of therapy response and remission in depressed patients.

Introduction: The etiology of major depressive disorder (MDD) involves the interaction between genes and environment, including treatment. Early molecular signatures for treatment response and remission are relevant in a context of personalized medicine and stratification and reduce the time-to-decision. Therefore, we focused the analyses on patients that responded or remitted following a cognitive intervention of 8 weeks. Methods: We used data from a randomized controlled trial (RCT) with MDD patients (N = 112) receiving a cognitive intervention. At baseline and 8 weeks, blood for DNA methylation (Illumina Infinium MethylationEPIC 850k BeadChip) was collected, as well as MADRS. First, responders (N = 24; MADRS-reduction of at least 50%) were compared with non-responders (N = 60). Then, we performed longitudinal within-individual analyses, for response (N = 21) and for remission (N = 18; MADRS smaller or equal to 9 and higher than 9 at baseline), respectively, as well as patients with no change in MADRS over time. At 8 weeks the sample comprised 84 individuals; 73 patients had DNA methylation for both time-points. The RnBeads package (R) was used for data cleaning, quality control, and differential DNA-methylation (limma). The within-individual paired longitudinal analysis was performed using Welch's t-test. Subsequently gene-ontology (GO) pathway analyses were performed. Results: No CpG was genome-wide significant CpG (p < 5 × 10-8). The most significant CpG in the differential methylation analysis comparing response versus non-response was in the IQSEC1 gene (cg01601845; p = 1.53 × 10-6), linked to neurotransmission. The most significant GO-terms were linked to telomeres. The longitudinal response analysis returned 67 GO pathways with a p < 0.05. Two of the three most significant pathways were linked to sodium transport. The analysis for remission returned 46 GO terms with a p-value smaller than 0.05 with pathways linked to phosphatase regulation and synaptic functioning. The analysis with stable patients returned mainly GO-terms linked to basic cellular processes. Discussion: Our result suggest that DNA methylation can be suitable to capture early signs of treatment response and remission following a cognitive intervention in depression. Despite not being genome-wide significant, the CpG locations and GO-terms returned by our analysis comparing patients with and without cognitive impairment, are in line with prior knowledge on pathways and genes relevant for depression treatment and cognition. Our analysis provides new hypotheses for the understanding of how treatment for depression can act through DNA methylation and induce response and remission.

Exploratory Analysis of the Effects of Celecoxib on Cognitive Function in Vortioxetine-Treated Patients With Major Depressive Disorder in the PREDDICT Study: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

Objective: Major depressive disorder (MDD) remains difficult to treat, with many patients resistant to existing treatments or experiencing relapse. Cognitive dysfunction is associated with more severe clinical outcomes. Vortioxetine has shown efficacy in remediating depression-associated cognitive impairment. Anti-inflammatory augmentation of antidepressants is a new strategy in treating depression and has not previously been assessed for effects on cognition in depression.Methods: Exploratory analyses were performed on secondary outcome cognitive data from the PREDDICT parallel-group, randomized, double-blind, placebo-controlled trial at the University of Adelaide (Australia). Participants (N = 119) with MDD (validated with Mini-International Neuropsychiatric Interview for DSM-IV) were treated with vortioxetine and celecoxib or vortioxetine and placebo for 6 weeks between December 2017 and April 2020. Measures included objective cognition composite scores (Choice Reaction Time, N-Back, Digit Symbol Substitution Test, Trail Making Task Part B), subjective cognition scores (Perceived Deficits Questionnaire), and global cognition composite scores (combined objective and subjective scores) derived from the THINC integrated tool (THINC-it). High-sensitivity C-reactive protein (hsCRP) measured at baseline and week 6 was tested for a predictive relationship with cognitive outcomes.Results: Cognition composite scores demonstrated improvement by week 6 in both treatment groups. However, there was no significant interaction between change over time and treatment group. HsCRP did not have a significant relationship with any tested cognition measures.Conclusions: Both treatment groups showed a reduction in depression-associated cognitive impairment. No superior clinical effect was reported for the add-on celecoxib group. HsCRP was modulated by neither vortioxetine nor add-on celecoxib.Trial Registration: ANZCTR identifier: ACTRN12617000527369.

Immune gene co-expression signatures implicated in occurence and persistence of cognitive dysfunction in depression.

Cognitive dysfunction contributes significantly to the burden caused by Major Depressive Disorder (MDD). Yet, while compelling evidence suggests that different biological processes play a part in both MDD aetiology and the development of cognitive decline more generally, we only begin to understand the molecular underpinnings of depression-related cognitive impairment. Developments in psychometric assessments, molecular high-throughput methods and systems biology derived analysis strategies advance this endeavour. Here, we aim to identify gene expression signatures associated with cognitive dysfunction and cognitive improvement following therapy using RNA sequencing to analyze the whole blood-derived transcriptome of altogether 101 MDD patients who enrolled in the CERT-D study. The mRNA(Nova)Seq based transcriptome was analyzed from whole blood taken at baseline assessment, and patients' cognitive performance was measured twice at baseline and following eight weeks of therapy by means of the THINC integrated tool. Thirty-six patients showed comparatively low cognitive performance at baseline assessment, and 32 patients showed comparatively strong cognitive improvement following therapy. Differential gene expression analysis was performed using limma to a significance threshold of 0.05 and a logFC cutoff of |1.2|. Although we observed some indications for expression differences related to low cognitive performance and cognitive therapy response, signals did not withstand adjustment for multiple testing. Applying WGCNA, we retrieved altogether 25 modules of co-expressed genes and we used a combination of correlational and linear analyses to identify modules related to baseline cognitive performance and cognitive improvement following therapy. Three immune modules reflected distinct but interrelated immune processes (the yellow module: neutrophil-mediated immunity, the darkorange module: interferon signaling, the tan module: platelet activation), and higher expression of the yellow (r = -0.21, p < .05), the dark orange (r = 0.2, p < .05), and the tan (r = -0.23, p < .05) module correlated significantly negatively with patients' cognitive baseline performance. Patients' cognitive baseline performance was a significant predictor of the darkorange module (b = -0.039, p < .05) and the tan module's expression (b = 0.02, p < .05) and was close to becoming a significant predictor of the yellow module's expression (b = -0.02, p = .05). Furthermore, patients characterized by comparatively low cognitive performance at baseline showed significantly higher expression of the tan module when compared to all other patients F(1,97) = 4.32, p < .05, η= 0.04. Following eight weeks of treatment, we observed altogether significant improvement in patients' cognitive performance (b = 0.30, p < .001), and patients with comparatively high cognitive gain showed noticeably lower, but not significantly lower F(1,98) = 3.76, p = .058, expression of a dark turquoise module, which reflects complement and B-cell-associated immune processes. Noteworthy, the relation between cognitive performance and module expression remained observable after controlling for symptom severity and BMI, which partly accounted for variance in module expression. As such, our findings provide further evidence for the involvement of immune processes in MDD related cognitive dysfunction and they suggest that different immune processes contribute to the development and long-term persistence of cognitive dysfunction in the context of depression.

Immunological changes following electroconvulsive therapy in multiple sclerosis.

INTRODUCTION: Electroconvulsive therapy (ECT) is a well-established treatment option in case of treatment-resistant depression (TRD). Only a few cases of ECT in depressed patients with multiple sclerosis (MS) were reported so far suggesting efficacy for the treatment of severe depression in MS, while data on possible neurological deterioration remained unclear. METHODS: In this case study we report on a case of a middle-aged man with MS. He was on dimethyl fumarate for relapse prevention since 2019 and without signs of active disease in a recent cerebral MRI. He suffered from treatment-resistant severe bipolar depression and thus received a total of 14 ECT sessions. We changed from right-unilateral to bilateral stimulation technique after the 7th session. We rated depression severity and measured biomarkers of neurodegeneration and inflammation before and after the ECT series to determine the impact of ECT on tolerance, response and neurobiology. RESULTS: The ECT series was tolerated well without neurological deterioration and any new neurological symptoms. The seizure quality was sufficient on average. We saw partial response corresponding to an improvement of about 35% in BDI-II and MADRS. The concentration of inflammation and neurodegeneration biomarkers was low both pre-treatment and post-treatment with increases from pre- to post ECT mainly in the CCL-2 pathway. CONCLUSION: In our patient with TRD and MS ECT was safe and feasible. We did not see any neurobiological signs of disease activation of MS or neurodegeneration during the course of ECT, which may even be beneficial as it led to increase in the neuroprotective CCL-2 pathway in the presented patient.

Functional assessment of a promoter polymorphism in S100B, a putative risk variant for bipolar disorder.

Calcium-binding protein S100B has been implicated in the pathology of bipolar affective disorder (BPAD) and schizophrenia (SZ). S100B protein levels are elevated in serum of patients with both disorders compared to controls. We previously reported genetic association of a SNP in the promoter of S100B, rs3788266, with a psychotic form of BPAD. To test for genotypic effects of rs3788266 in vivo, S100B serum protein levels were measured in 350 Irish and German subjects of known S100B genotype. The functional effect of rs3788266 on S100B promoter activity was studied using the luciferase reporter system in U373MG glioblastoma and SH-SY5Y neuroblastoma cell lines. Allelic effects of rs3788266 on protein complex formation at the S100B promoter were investigated by an electrophoretic mobility shift assay. Higher mean serum S100B levels were associated with the risk G allele of rs3788266 in BPAD cases (P = 0.0001), unaffected relatives of BPAD cases (P < 0.0001) and unrelated controls (P < 0.0001). Consistent with the in vivo findings, luciferase gene expression was significantly increased in the presence of the G allele compared to the A allele in SH-SY5Y (P = <0.0001), and in U373MG (P = <0.0008) cell lines. The binding affinity of both SH-SY5Y and U373MG protein complexes for the S100B promoter was significantly stronger in the presence of G allele compared to the A allele promoter fragments. These data support rs3788266 as a functional promoter variant in the S100B gene where the presence of the G allele promotes increased gene expression and is associated with increased serum levels of the protein.

Acute stress reveals different impacts in male and female Zdhhc7-deficient mice.

Numerous processes of neuronal development and synaptic plasticity in the brain rely on the palmitoyl acyltransferase ZDHHC7, as it palmitoylates various synaptic and extrasynaptic proteins such as neural cell adhesion molecule (NCAM) or gamma-aminobutyric acid (GABAA) receptors. In addition, ZDHHC7 palmitoylates sex steroid hormone receptors and is, therefore, indirectly linked to mental disorders that often occur because of or in conjunction with stress. In this work, we investigated how ZDHHC7 affects stress responses in mice. For this purpose, genetically modified mice with a knockout of the Zdhhc7 gene (KO) and wild-type (WT) littermates of both sexes were exposed to acute stressors or control conditions and examined with regard to their behavior, brain microstructure, gene expression, and synaptic plasticity. While no behavioral effects of acute stress were found, we did find that acute stress caused reduced mRNA levels of Esr1 and Esr2 coding for estrogen receptor α and ß in the medial prefrontal cortex of male WT and KO mice. Strikingly, after acute stress only male KO mice showed reduced mean fiber lengths of the medioventral hippocampus. Furthermore, Zdhhc7-deficiency impaired synaptic plasticity in mice of both sexes, while acute stress improved it in females, but not in male mice. Taken together, our findings suggest that ZDHHC7 plays a modulatory role in the brain that leads to sex-specific stress responses, possibly due to estrogen receptor-mediated signaling pathways.

Emotion specific modulation of automatic amygdala responses by 5-HTTLPR genotype.

A functional polymorphism in the serotonin transporter gene (5-HTTLPR) has been reported to modulate amygdala responsiveness to negative environmental cues. However, it remains unclear whether 5-HTTLPR modulates amygdala responses specifically to negative stimuli or rather to emotionally salient stimuli in general. In 44 healthy subjects, amygdala responses to subliminally presented happy and sad facial expressions were assessed by means of fMRI at 3 Tesla. All subjects were genotyped for 5-HTTLPR and the recently discovered 5-HTT rs25531. We observed a robust emotion by genotype group interaction in the right amygdala. Risk allele carriers (S or L(G)) showed similar amygdala responses to happy faces compared to homozygous L(A)L(A) carriers but increased amygdala responses to sad faces. The right amygdala was the only anatomical region across the whole brain demonstrating this interaction at a reasonable threshold. It appears that whereas 5-HTT gene variation modulates automatic amygdala responsiveness to sad faces, no such association was found for happy faces. We conclude that 5-HTTLPR genotype predominantly impacts the central processing predominantly of negative environmental cues but not of emotionally salient stimuli in general.

Norepinephrine and serotonin transporter genes: impact on treatment response in depression.

BACKGROUND/AIMS: The norepinephrine transporter (NET) and serotonin transporter (5-HTT) genes constitute promising candidate genes in major depression. Seven polymorphisms in the promoter, intronic and exonic region of the NET gene, as well as serotonin-transporter-linked promoter region (5-HTTLPR) and 5-HTT rs25531 polymorphisms were analyzed with respect to antidepressant treatment response with particular attention to gender effects and subtypes of melancholic or anxious depression. METHODS: 252 unrelated Caucasian patients (f = 142; m = 110) with major depression were genotyped for NET and 5-HTT polymorphisms. Genotype effects on Hamilton Depression Rating Scale score changes over 6 weeks of antidepressant treatment were analyzed using analysis of covariance with repeated measures. RESULTS: There was no effect of any of the 7 investigated NET, or the two 5-HTT polymorphisms, on the overall treatment response. An additional -/CT insertion/deletion (ins/del) polymorphism (rs58532686), however, was significantly associated with melancholic depression, with a better response in 12 patients carrying the deletion. Stratification for anxious versus nonanxious depression revealed a significantly detrimental effect of the less active 5-HTTLPR S allele (p = 0.007) and 5-HTTLPR/5-HTT rs25531 haplotypes on treatment response in patients with anxious depression. CONCLUSION: The present findings do not support a major impact of the NET and 5-HTT genes on antidepressant treatment response in major depression per se. However, there might be an impact of a -/CT ins/del polymorphism in the enhancer domain of the NET gene on treatment response in melancholic depression, which remains to be functionally investigated in future studies. The observed significant influence of the 5-HTT gene variation on antidepressant treatment in anxious depression points to anxious depression as a potential diagnostic entity of its own, requiring specific diagnostic and therapeutic attention.