David Stacey

RASGRF2 regulates alcohol-induced reinforcement by influencing mesolimbic dopamine neuron activity and dopamine release

The firing of mesolimbic dopamine neurons is important for drug-induced reinforcement, although underlying genetic factors remain poorly understood. In a recent genome-wide association metaanalysis of alcohol intake, we identified a suggestive association of SNP rs26907 in the ras-specific guanine-nucleotide releasing factor 2 (RASGRF2) gene, encoding a protein that mediates Ca2+-dependent activation of the ERK pathway. We performed functional characterization of this gene in relation to alcohol-related phenotypes and mesolimbic dopamine function in both mice and adolescent humans. Ethanol intake and preference were decreased in Rasgrf2−/− mice relative to WT controls. Accordingly, ethanol-induced dopamine release in the ventral striatum was blunted in Rasgrf2−/− mice. Recording of dopamine neurons in the ventral tegmental area revealed reduced excitability in the absence of Ras-GRF2, likely because of lack of inhibition of the IA potassium current by ERK. This deficit provided an explanation for the altered dopamine release, presumably linked to impaired activation of dopamine neurons firing. Functional neuroimaging analysis of a monetary incentive–delay task in 663 adolescent boys revealed significant association of ventral striatal activity during reward anticipation with a RASGRF2 haplotype containing rs26907, the SNP associated with alcohol intake in our previous metaanalysis. This finding suggests a link between the RASGRF2 haplotype and reward sensitivity, a known risk factor for alcohol and drug addiction. Indeed, follow-up of these same boys at age 16 y revealed an association between this haplotype and number of drinking episodes. Together, these combined animal and human data indicate a role for RASGRF2 in the regulation of mesolimbic dopamine neuron activity, reward response, and alcohol use and abuse.

Cytokine levels in major depression are related to childhood trauma but not to recent stressors

BACKGROUND Major depressive disorder (MDD) is a stress-related psychiatric disorder. A subgroup of MDD patients is characterized by increased inflammatory activation. We aimed to investigate whether increased inflammation particularly occurs in MDD patients with a history of stressful early or later life experiences. METHODS Serum levels of tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 were determined in N=214 MDD patients and N=180 healthy controls (HC). Childhood trauma (Childhood Trauma Questionnaire - CTQ), adverse life events of the past 12 months (List of Threatening Experiences Questionnaire - LTE-Q), and perceived stress in the past month (Perceived Stress Scale - PSS) were analyzed with regard to cytokine levels. RESULTS Pro-inflammatory cytokine levels were not related to global scores of adverse events or perceived stress covering different time points ranging from childhood to the past month. However, in the subgroup of traumatized MDD patients, higher severity of childhood sexual abuse was associated with higher levels of both IL-6 and TNF-α in a linear fashion. CONCLUSIONS Our data suggest a linear relationship between childhood sexual abuse and increased pro-inflammatory cytokine levels in MDD patients, while more recent stressful life events were not related to these inflammatory markers.

Evidence of an IFN-γ by early life stress interaction in the regulation of amygdala reactivity to emotional stimuli

INTRODUCTION Since numerous studies have found that exposure to early life stress leads to increased peripheral inflammation and psychiatric disease, it is thought that peripheral immune activation precedes and possibly mediates the onset of stress-associated psychiatric disease. Despite early studies, IFNγ has received little attention relative to other inflammatory cytokines in the context of the pathophysiology of affective disorders. Neuroimaging endophenotypes have emerged recently as a promising means of elucidating these types of complex relationships including the modeling of the interaction between environmental factors and genetic predisposition. Here we investigate the GxE relationship between early-life stress and genetic variants of IFNγ on emotion processing. METHODS To investigate the impact of the relationship between genetic variants of IFNγ (rs1861494, rs2069718, rs2430561) and early life stress on emotion processing, a sample of healthy adults (n=409) undergoing an emotional faces paradigm in an fMRI study were genotyped and analysed. Information on early life stress was obtained via Childhood Trauma Questionnaire (CTQ). RESULTS A positive association between early life stress and amygdala reactivity was found. Specifically, the main effect of genotype of rs1861494 on amygdala reactivity indicates a higher neural response in C allele carriers compared to T homozygotes, while we did not find main effects of rs2069718 and rs2430561. Importantly, interaction analyses revealed a specific interaction between IFNγ genotype (rs1861494) and early life stress affecting amygdala reactivity to emotional faces, resulting from a positive association between CTQ scores and amygdala reactivity in C allele carriers while this association was absent in T homozygotes. CONCLUSIONS Our findings indicate that firstly the genetic variant of IFNγ (rs1861494) is involved with the regulation of amygdala reactivity to emotional stimuli and secondly, that this genetic variant moderates effects of early life stress on emotion processing. These findings reiterate the importance that inflammatory genes play in the interaction with early life stress and the regulation of emotion processing.

TNF receptors (TNFR) 1 and 2 exert distinct region‐specific effects on striatal and hippocampal grey matter volumes (VBM) in healthy adults – Revision

Tumour necrosis factor alpha (TNFα) has been implicated in the pathophysiology of neurodegenerative and neuropsychiatric disease, with research highlighting a role for TNFα in hippocampal and striatal regulation. TNFα signals are primarily transduced by TNF receptors 1 and 2 (TNFR1 and TNFR2), encoded by TNFRSF1A and TNFRSF1B, which exert opposing effects on cell survival (TNFR1, neurodegenerative; TNFR2, neuroprotective). We therefore sought to explore the respective roles of TNFR1 and TNFR2 in the regulation of hippocampal and striatal morphology in an imaging genetics study. Voxel‐based morphometry was used to analyse the associations between TNFRSF1A (rs4149576 and rs4149577) and TNFRSF1B (rs1061624) genotypes and grey matter structure. The final samples comprised a total of 505 subjects (mean age = 33.29, SD = 11.55 years; 285 females and 220 males) for morphometric analyses of rs1061624 and rs4149576, and 493 subjects for rs4149577 (mean age = 33.20, SD = 11.56 years; 281 females and 212 males). Analyses of TNFRSF1A single nucleotide polymorphisms (SNPs) rs4149576 and rs4149577 showed highly significant genotypic associations with striatal volume but not the hippocampus. Specifically, for rs4149576, G homozygotes were associated with reduced caudate nucleus volumes relative to A homozygotes and heterozygotes, whereas for rs4149577, reduced caudate volumes were observed in C homozygotes relative to T homozygotes and heterozygotes. Analysis of the TNFRSF1B SNP rs1061624 yielded a significant association with hippocampal but not with striatal volume, whereby G homozygotes were associated with increased volumes relative to A homozygotes and heterozygotes. Our findings indicate a role for TNFR1 in regulating striatal but not hippocampal morphology, as well as a complementary role for TNFR2 in hippocampal but not in striatal morphology.

No evidence of DISC1-associated morphological changes in the hippocampus, anterior cingulate cortex, or striatum in major depressive disorder cases and healthy controls

BACKGROUND DISC1 imaging genetics studies in healthy controls, schizophrenia, and bipolar disorder cases have revealed morphological changes in brain regions involved in the pathophysiology of psychiatric disease including the hippocampus, anterior cingulate cortex (ACC), and the striatum. However, many of these studies have yielded discordant findings so there is a need for replication. Furthermore, despite evidence from human genetic studies and animal models implicating DISC1 in major depressive disorder (MDD), a DISC1 imaging genetics study in MDD cases has yet to be published. Thus, using neuroimaging data from MDD cases and a large sample of healthy controls we aimed to identify morphological changes representing neurobiological mechanisms underlying the association between DISC1 and MDD. METHODS We utilized structural magnetic resonance imaging (sMRI) data from 512 healthy controls and 171 current MDD (SCID interview) cases, each with genotype data for non-synonymous DISC1 SNPs rs3738401, rs6675281, and rs821616. RESULTS Region of interest analyses failed to reveal DISC1-associated morphological changes in the hippocampus, ACC, or striatum in MDD patients and healthy controls. Whole brain exploratory analyses identified a nominally significant cluster mapping to the border of the precentral and postcentral gyri associated with rs821616 in healthy controls only (p(uncorrected)<0.001). LIMITATIONS We focused our analyses exclusively on three, but previously heavily studied, SNPs in DISC1. CONCLUSIONS Our findings suggest that morphological changes in the hippocampus, ACC, and/or striatum of MDD patients do not represent neurobiological mechanisms underlying the association between DISC1 and MDD. However, we urge replication in independent samples of MDD cases.

Peripheral blood gene expression analysis implicates B lymphocyte proliferation and ribosomal S26 transcripts in cognitive dysfunction in people with remitted Major Depression

Residual impairments in cognitive domains including attention, memory, executive function, and social perception occur in about 40% of patients with Major Depressive Disorder (MDD) following remission from acute episodes (Conradi et al., 2011), and are associated with reductions in pre-morbid functioning and higher unemployment rates (Baune et al., 2010; Evans et al., 2014). Cognitive impairment in MDD may represent a trait marker, trait-byillness interaction marker, or endophenotype of MDD that is independent of the acute depressive state and particularly active in a subgroup of MDD patients (Hasler et al., 2004; Iverson et al., 2011). We explored the potential molecular underpinnings of such a ‘cognitive’ MDD phenotype in a well-matched pilot sample of remitted MDD patients with poorer versus better performance on cognitive testing. Because the phenotype is likely highly complex, polygenic, and heterogeneous, we performed weighted gene coexpression network analysis (WGCNA), a hypothesis-free systems biology approach that identifies ‘modules’ of co-regulated – and therefore functionally related – genes in transcriptomic datasets. After constructing a gene co-expression network from samples of all patients, we determined whether modules were correlated with poorer versus better cognitive performance. We additionally explored whether modules were also correlated with a continuous measure of cognitive performance in both groups. We utilized whole blood transcriptomic data from remitted MDD patients from the Adelaide Cognitive Function and Mood Study (CoFaMS, HREC RAH protocol no. 111230g) matched for race, gender, age, Hamilton Depression Scale score, presence of psychotic features during any depressive episode, educational status, annual household income, current drugand alcohol use, and

Evidence of increased risk for major depressive disorder in individuals homozygous for the high-expressing 5-HTTLPR/rs25531 (LA) allele of the serotonin transporter promoter

Stacey, David; Cohen-Woods, Sarah; Toben, Catherine; Arolt, Volker; Dannlowski, Udo; Baune, Bernhard T.

Co-expression network analysis of peripheral blood transcriptome identifies dysregulated protein processing in endoplasmic reticulum and immune response in recurrent MDD in older adults

The molecular factors involved in the pathophysiology of major depressive disorder (MDD) remain poorly understood. One approach to examine the molecular basis of MDD is co-expression network analysis, which facilitates the examination of complex interactions between expression levels of individual genes and how they influence biological pathways affected in MDD. Here, we applied an unsupervised gene-network based approach to a prospective experimental design using microarray genome-wide gene expression from the peripheral whole blood of older adults. We utilised the Sydney Memory and Ageing Study (sMAS, N = 521) and the Older Australian Twins Study (OATS, N = 186) as discovery and replication cohorts, respectively. We constructed networks using Weighted Gene Co-expression Network Analysis (WGCNA), and correlated identified modules with four subtypes of depression: single episode, current, recurrent, and lifetime MDD. Four modules of highly co-expressed genes were associated with recurrent MDD (N = 27) in our discovery cohort (FDR<0.2), with no significant findings for a single episode, current or lifetime MDD. Functional characterisation of these modules revealed a complex interplay between dysregulated protein processing in the endoplasmic reticulum (ER), and innate and adaptive immune response signalling, with possible involvement of pathogen-related pathways. We were underpowered to replicate findings at the network level in an independent cohort (OATS), however; we found a significant overlap for 9 individual genes with similar co-expression and dysregulation patterns associated with recurrent MDD in both cohorts. Overall, our findings support other reports on dysregulated immune response and protein processing in the ER in MDD and provide novel insights into the pathophysiology of depression.