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Background: Major depressive disorder (MDD) is a leading cause of disability with significant mortality risk. Despite progress in our understanding of the etiology of MDD, the underlying molecular changes in the brain remain poorly understood. Extracellular vesicles (EVs) are lipid-bound particles that can reflect the molecular signatures of the tissue of origin. We aimed to optimize a streamlined EV isolation protocol from postmortem brain tissue and determine whether EV RNA cargo, particularly microRNAs (miRNAs), have an MDD-specific profile., Methods: EVs were isolated from postmortem human brain tissue. Quality was assessed using western blots, transmission electron microscopy, and microfluidic resistive pulse sensing. EV RNA was extracted and sequenced on Illumina platforms. Functional follow-up was performed in silico., Results: Quality assessment showed an enrichment of EV markers, as well as a size distribution of 30 to 200 nm in diameter, and no contamination with cellular debris. Small RNA profiling indicated the presence of several RNA biotypes, with miRNAs and transfer RNAs being the most prominent. Exploring miRNA levels between groups revealed decreased expression of miR-92a-3p and miR-129-5p, which was validated by qPCR and was specific to EVs and not seen in bulk tissue. Finally, in silico functional analyses indicate potential roles for these 2 miRNAs in neurotransmission and synaptic plasticity., Conclusion: We provide a streamlined isolation protocol that yields EVs of high quality that are suitable for molecular follow-up. Our findings warrant future investigations into brain EV miRNA dysregulation in MDD., (© The Author(s) 2024. Published by Oxford University Press on behalf of CINP.)

Major depressive disorder (MDD) is a common psychiatric illness that manifests in sex-influenced ways. Men and women may experience depression differently and also respond to various antidepressant treatments in sex-influenced ways. Ketamine, which is now being used as a rapid-acting antidepressant, is likely the same. To date, the majority of studies investigating treatment outcomes in MDD do not disaggregate the findings in males and females, and this is also true for ketamine. This review aims to highlight that gap by exploring pre-clinical data-at a behavioral, molecular, and structural level-and recent clinical trials. Sex hormones, particularly estrogen and progesterone, influence the response at all levels examined, and sex is therefore a critical factor to examine when looking at ketamine response. Taken together, the data show females are more sensitive to ketamine than males, and it might be possible to monitor the phase of the menstrual cycle to mitigate some risks associated with the use of ketamine for females with MDD. Based on the studies reviewed in this article, we suggest that ketamine should be administered adhering to sex-specific considerations., (© The Author(s) 2021. Published by Oxford University Press on behalf of CINP.)

Background: Suicide represents a major health concern, especially in developing countries. While many demographic risk factors have been proposed, the underlying molecular pathology of suicide remains poorly understood. A body of evidence suggests that aberrant DNA methylation and expression is involved. In this study, we examined DNA methylation profiles and concordant gene expression changes in the prefrontal cortex of Mexicans who died by suicide., Methods: In collaboration with the coroner's office in Mexico City, brain samples of males who died by suicide (n = 35) and age-matched sudden death controls (n = 13) were collected. DNA and RNA were extracted from prefrontal cortex tissue and analyzed with the Infinium Methylation480k and the HumanHT-12 v4 Expression Beadchips, respectively., Results: We report evidence of altered DNA methylation profiles at 4430 genomic regions together with 622 genes characterized by differential expression in cases vs controls. Seventy genes were found to have concordant methylation and expression changes. Metacore-enriched analysis identified 10 genes with biological relevance to psychiatric phenotypes and suicide (ADCY9, CRH, NFATC4, ABCC8, HMGA1, KAT2A, EPHA2, TRRAP, CD22, and CBLN1) and highlighted the association that ADCY9 has with various pathways, including signal transduction regulated by the cAMP-responsive element modulator, neurophysiological process regulated by the corticotrophin-releasing hormone, and synaptic plasticity. We therefore went on to validate the observed hypomethylation of ADCY9 in cases vs control through targeted bisulfite sequencing., Conclusion: Our study represents the first, to our knowledge, analysis of DNA methylation and gene expression associated with suicide in a Mexican population using postmortem brain, providing novel insights for convergent molecular alterations associated with suicide., (© The Author(s) 2021. Published by Oxford University Press on behalf of CINP.)

Background: There is a lack of reliable biomarkers for major depressive disorder (MDD) in clinical practice. However, several studies have shown an association between alterations in microRNA levels and MDD, albeit none of them has taken advantage of machine learning (ML)., Method: Supervised and unsupervised ML were applied to blood microRNA expression profiles from a MDD case-control dataset (n = 168) to distinguish between (1) case vs control status, (2) MDD severity levels defined based on the Montgomery-Asberg Depression Rating Scale, and (3) antidepressant responders vs nonresponders., Results: MDD cases were distinguishable from healthy controls with an area-under-the receiver-operating characteristic curve (AUC) of 0.97 on testing data. High- vs low-severity cases were distinguishable with an AUC of 0.63. Unsupervised clustering of patients, before supervised ML analysis of each cluster for MDD severity, improved the performance of the classifiers (AUC of 0.70 for cluster 1 and 0.76 for cluster 2). Antidepressant responders could not be successfully separated from nonresponders, even after patient stratification by unsupervised clustering. However, permutation testing of the top microRNA, identified by the ML model trained to distinguish responders vs nonresponders in each of the 2 clusters, showed an association with antidepressant response. Each of these microRNA markers was only significant when comparing responders vs nonresponders of the corresponding cluster, but not using the heterogeneous unclustered patient set., Conclusions: Supervised and unsupervised ML analysis of microRNA may lead to robust biomarkers for monitoring clinical evolution and for more timely assessment of treatment in MDD patients., (© The Author(s) 2020. Published by Oxford University Press on behalf of CINP.)

Introduction: Antidepressant drugs are effective therapies for major depressive disorder; however, they are frequently associated with side effects. Although there is some evidence for a relationship between genetic variation and side effects, little is known regarding the role of dynamic molecular factors as moderators of side effects. The aim of this study was to assess microRNA (miRNA) changes associated with side effects during escitalopram treatment and their downstream effects on target gene expression., Methods: A total 160 patients with major depressive disorder from the CAN-BIND-1 cohort were included. Side effects were assessed with the Toronto Side Effect Scale after 2 weeks of treatment with escitalopram. We assessed the relationship between side effects and changes in peripheral expression of miRNAs between baseline and week 2. For miRNA whose expression changed, we used target prediction algorithms to identify putative messenger RNA (mRNA) targets and assessed their expression., Results: Nausea was experienced by 42.5% of patients. We identified 45 miRNAs whose expression changed on initiation of escitalopram treatment, of which 10 displayed a negative association with intensity of nausea (miR15b-5p, miR17-5p, miR20a-5p, miR20b-5p, miR103a-3p, miR103b, miR106a-5p, miR182-5p, miR185-5p, and miR660-5p). Additionally, we found negative associations between 4 microRNAs (miR20a-5p, miR106a-5p, miR185-5p, miR660-5p) and mRNA targets. The expression of the miR185-5p target, CAMK2δ was significantly decreased [log 2 mean = -0.048 (0.233)] between weeks 0 and 2 (P = .01)]., Conclusions: We identified an overexpression of miR185-5p during escitalopram treatment of major depressive disorder, which was negatively associated with intensity of nausea, and identified a potential mRNA target that may mediate this effect., (© The Author(s) 2019. Published by Oxford University Press on behalf of CINP.)

Background: The polyamines are a group of ubiquitous low-molecular-weight aliphatic molecules that play an essential role in various physiological functions of the mammalian CNS. Previous literature has indicated alterations in the expression of polyamine-related genes in the brains of individuals who died by suicide, including downregulation of spermidine/spermine N1-acetyltransferase, a key enzyme involved in polyamine catabolism. One such polyamine, agmatine, has been shown to act as an antidepressant in animal models of depressive-like behavior. However, agmatine concentrations have not been explored in postmortem human brain of individuals who died by suicide., Methods: To measure agmatine in postmortem human brain tissue, we employed our previously published high-resolution capillary gas chromatography in combination with mass spectrometry method. Using this method, we analyzed agmatine levels in a total of 120 tissue samples from Brodmann areas 4, 11, and 44 of 40 male subjects comprising controls (n=13), individuals who died by suicide and met criteria for major depressive disorder (n=14), and subjects who died by suicide and did not meet criteria for major depressive disorder (n=13)., Results: Agmatine fell within the expected nanomolar range and was significantly reduced in the cortex of suicides, irrespective of meeting criteria for major depressive disorder compared with controls., Conclusions: This is the first gas chromatography-mass spectrometry study to analyze agmatine concentrations in human postmortem brain of individuals who died by suicide. These results add to our mechanistic understanding of the role that the polyamine stress response pathway may play in the neurobiology of major depression and/or suicide.

Background: Major depressive disorder is a debilitating illness, which is most commonly treated with antidepressant drugs. As the majority of patients do not respond on their first trial, there is great interest in identifying biological factors that indicate the most appropriate treatment for each patient. Studies suggest that microRNA represent excellent biomarkers to predict antidepressant response., Methods: We investigated the expression of miR-1202, miR-135a, and miR-16 in peripheral blood from 2 cohorts of depressed patients who received 8 weeks of antidepressant therapy. Expression was quantified at baseline and after treatment, and its relationship to treatment response and depressive symptoms was assessed., Results: In both cohorts, responders displayed lower baseline miR-1202 levels compared with nonresponders, which increased following treatment., Conclusions: Ultimately, our results support the involvement of microRNA in antidepressant response and suggest that quantification of their levels in peripheral samples represents a valid approach to informing treatment decisions., (© The Author 2017. Published by Oxford University Press on behalf of CINP.)

Background: Major depressive disorder has been associated with dysfunctional astrocytic networks. The underlying causes, extent, and consequences of such dysfunctions remain to be characterized. Astrocyte-astrocyte communication occurs principally through gap junction channels primarily formed by connexin 30 and 43 (CX30 and CX43). We previously reported decreased connexin expression in the prefrontal cortex of depressed suicides. In the present study, we investigated whether these changes are mediated by epigenetic regulation, and expanded gene expression quantifications to other cortical and subcortical regions to assess the regional distribution of connexion disruptions in depressed suicides., Methods: The expression of CX30 and CX43 was measured by real-time PCR in samples of neocortex (Brodmann areas 4 and 17), cerebellar cortex, mediodorsal thalamus, and caudate nucleus of 22 depressed suicides and 22 matched sudden-death controls. Chromatin immunoprecipitation was used to measure enrichment levels of the repressive chromatin mark H3K9me3 in the prefrontal cortex., Results: We found a consistent downregulation of connexin genes in all regions examined, except in the cerebellum where an increase in the expression of CX30 was measured and using chromatin immunoprecipitation we observed an enrichment of H3K9me3 for both Cx30 and Cx43 in the prefrontal cortex., Conclusions: Our study shows widespread astrocytic CX gene repression in depressed suicides that is mediated, at least in part, through epigenetic mechanisms. Taken together, these findings support the notion of widespread cerebral astrocytic dysfunction in major depressive disorder., (© The Author 2016. Published by Oxford University Press on behalf of CINP.)

Glutamate receptors are promising drug targets for the treatment of urgent suicide ideation and chronic major depressive disorder (MDD) that may lead to suicide completion. Antagonists of glutamatergic NMDA receptors reduce depressive symptoms faster than traditional antidepressants, with beneficial effects occurring within hours. Glutamate is the prominent excitatory input to the noradrenergic locus coeruleus (LC). The LC is activated by stress in part through this glutamatergic input. Evidence has accrued demonstrating that the LC may be overactive in MDD, while treatment with traditional antidepressants reduces LC activity. Pathological alterations of both glutamatergic and noradrenergic systems have been observed in depressive disorders, raising the prospect that disrupted glutamate-norepinephrine interactions may be a central component to depression and suicide pathobiology. This study examined the gene expression levels of glutamate receptors in post-mortem noradrenergic LC neurons from subjects with MDD (most died by suicide) and matched psychiatrically normal controls. Gene expression levels of glutamate receptors or receptor subunits were measured in LC neurons collected by laser capture microdissection. MDD subjects exhibited significantly higher expression levels of the NMDA receptor subunit genes, GRIN2B and GRIN2C, and the metabotropic receptor genes, GRM4 and GRM5, in LC neurons. Gene expression levels of these receptors in pyramidal neurons from prefrontal cortex (BA10) did not reveal abnormalities in MDD. These findings implicate disrupted glutamatergic-noradrenergic interactions at the level of the stress-sensitive LC in MDD and suicide, and provide a theoretical mechanism by which glutamate antagonists may exert rapid antidepressant effects.

Mitochondrial dysfunction, oxidative stress, and alterations in DNA methylation, are all associated with the pathophysiology of bipolar disorder (BD). We therefore studied the relationship between oxidative stress and DNA methylation in patients with BD with an excellent response to lithium treatment, their affected and unaffected relatives and healthy controls. Transformed lymphoblasts were cultured in the presence or absence of lithium chloride (0.75 mM). DNA and proteins were extracted from the cells to determine levels of 8-hydroxy-2-deoxyguanosine (8-OHdG), 5-methylcytosine (5-mc), mitochondrial complex I and glutathione peroxidase (GPx) activities. Methylation was decreased in BD subjects and their relatives compared to controls and remained so after lithium treatment in BD subjects but not in their relatives. 8-OHdG levels and complex I activity did not differ between groups before and after lithium treatment. Finally, relatives of patients showed increased GPx activity before and after lithium treatment, which negatively correlated with 5-mc levels. Changes in global methylation may be specific for BD and lithium may be involved in glutathione regulation. The present study supports the importance of DNA methylation to the pathophysiology of BD and the therapeutic potential of antioxidants in this illness.

MicroRNAs (miRNAs) are small, non-coding RNA molecules that play an important role in the post-transcriptional regulation of mRNA. These molecules have been the subject of growing interest as they are believed to control the regulation of a large number of genes, including those expressed in the brain. Evidence suggests that miRNAs could be involved in the pathogenesis of neuropsychiatric disorders. Alterations in metabolic enzymes of the polyamine system have been reported to play a role in predisposition to suicidal behaviour. We have previously shown the expression of the polyamine genes SAT1 and SMOX to be down-regulated in the brains of suicide completers. In this study, we hypothesized that the dysregulation of these genes in depressed suicide completers could be influenced by miRNA post-transcriptional regulation. Using a stringent target prediction analysis, we identified several miRNAs that target the 3'UTR of SAT1 and SMOX. We profiled the expression of 10 miRNAs in the prefrontal cortex (BA44) of suicide completers (N = 15) and controls (N = 16) using qRT-PCR. We found that several miRNAs showed significant up-regulation in the prefrontal cortex of suicide completers compared to psychiatric healthy controls. Furthermore, we demonstrated a significant correlation between these miRNAs and the expression levels of both SAT1 and SMOX. Our results suggest a relationship between miRNAs and polyamine gene expression in the suicide brain, and postulate a mechanism for SAT1 and SMOX down-regulation by post-transcriptional activity of miRNAs.

An altered polyamine system has been suggested to play a key role in mood disorders and suicide, a hypothesis corroborated by the evidence that lithium inhibits the polyamine mediated stress response in the rat brain. Recent post-mortem studies have shown that spermidine/spermine N1-acetyltransferase (SAT1), the key regulator of cellular polyamine content, is under-expressed in brains from suicide victims compared to controls. In our study we tested the effect of in vitro lithium treatment on SAT1 gene and protein expression in B lymphoblastoid cell lines (BLCLs) from bipolar disorder (BD) patients who committed suicide (and for which BLCLs were collected prior to their death), BD patients with high and low risk of suicide and a sample of non-psychiatric controls. Baseline mRNA levels were similar in the four groups of subjects (p > 0.05). Lithium had no effect in suicide completers (p > 0.05) while it significantly increased SAT1 expression in the high risk (p < 0.001) and low risk (p < 0.01) groups as well as in controls (p < 0.001). Protein and mRNA levels were not correlated; lithium significantly reduced protein levels only in the control sample (p < 0.05). Our findings suggest that SAT1 transcription is influenced by lithium and that this effect is altered in BD patients who completed suicide, further supporting a role for polyamines in suicide.

Brain oxysterol levels, which are enzymatic oxidation products of cholesterol (Chl), have been proposed to reflect the dynamic process of physiological synapse maintenance and repair of nerve terminals within the central nervous system (CNS), due to the turnover of membrane Chl. Modifications of oxysterols have important implications in neurological conditions, especially in neurodegenerative and psychiatric disorders in which alterations of synaptic plasticity or cell signalling are implicated, such as depression. Oxysterols can diffuse across the blood-brain barrier and have been hypothesized to provide a mechanism by which the brain can eliminate excess Chl to maintain a steady state. Relations of 24-hydroxycholesterol (24OH) and 27-hydroxycholesterol (27OH) specifically may provide a depiction of CNS Chl homeostasis. Thus, the objective of this study was to integrate oxysterol measures and gene expression measures in an effort to identify how they may relate to depression and suicide. Using post-mortem human prefrontal cortex tissue, quantification of metabolites by GC-MS and gene expression by qRT-PCR were performed with the aim to provide a characterization of enzymatic oxidative Chl homeostasis. Results show a significant increase in 24OH, which suggests a higher turnover of Chl to 24OH in the prefrontal cortex of suicide cases. An increase in 24OH may, in combination with liver-X receptor activation, explain the observed reduction of low central and peripheral Chl in suicide and would have implications for synapse maintenance and loss in the neuropathology of depression and suicide.

The synapsin family of neuronal phosphoproteins is composed of three genes (SYN1, SYN2 and SYN3) with alternative splicing resulting in a number of variants with various levels of homology. These genes have been postulated to play significant roles in several neuropsychiatric disorders, including bipolar disorder, schizophrenia and epilepsy. Epigenetic regulatory mechanisms, such as histone modifications in gene regulatory regions, have also been proposed to play a role in a number of psychiatric disorders, including bipolar disorder and major depressive disorder. One of the best characterized histone modifications is histone 3 lysine 4 tri-methylation (H3K4me3), an epigenetic mark shown to be highly enriched at transcriptional start sites and associated with active transcription. In the present study we have quantified the expression of transcript variants of the three synapsin genes and investigated their relationship to H3K4me3 promoter enrichment in post-mortem brain samples. We found that histone modification marks were significantly increased in bipolar disorder and major depression and this effect was correlated with significant increases in gene expression. Our findings suggest that synapsin dysregulation in mood disorders is mediated in part by epigenetic regulatory mechanisms.

Altered polyamine metabolism has been consistently observed as underlying the suicide process. We recently performed a global analysis of polyamine gene expression across the brains of suicide completers, and identified up-regulation of four genes, arginase II (ARG2), S-adenosylmethionine decarboxylase (AMD1), and antizymes 1 and 2 (OAZ1 and OAZ2), which play essential roles in polyamine biosynthesis. To determine if a shared epigenetic mechanism is involved in their overexpression in the prefrontal cortex, we measured promoter levels of tri-methyl modified histone-3-lysine-4 (H3K4me3), a marker of open chromatin, and assessed its association with suicide and gene expression. We identified increased H3K4me3 in the promoter region of OAZ1 in suicide, and found that H3K4me3 was correlated with the expression of OAZ1 and ARG2. Overall, our findings indicate that the H3K4me3 modification plays an important role in the regulation of polyamine biosynthesis, and that this mechanism may be involved in the neurobiology of suicide.

The noradrenergic locus coeruleus (LC) is the principal source of brain norepinephrine, a neurotransmitter thought to play a major role in the pathology of major depressive disorder (MDD) and in the therapeutic action of many antidepressant drugs. The goal of this study was to identify potential mediators of brain noradrenergic dysfunction in MDD. Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor-β superfamily, is a critical mediator of noradrenergic neuron differentiation during development and has neurotrophic and neuroprotective effects on mature catecholaminergic neurons. Real-time PCR of reversed transcribed RNA isolated from homogenates of LC tissue from 12 matched pairs of MDD subjects and psychiatrically normal control subjects revealed low levels of BMP7 gene expression in MDD. No differences in gene expression levels of other members of the BMP family were observed in the LC, and BMP7 gene expression was normal in the prefrontal cortex and amygdala in MDD subjects. Laser capture microdissection of noradrenergic neurons, astrocytes, and oligodendrocytes from the LC revealed that BMP7 gene expression was highest in LC astrocytes relative to the other cell types, and that the MDD-associated reduction in BMP7 gene expression was limited to astrocytes. Rats exposed to chronic social defeat exhibited a similar reduction in BMP7 gene expression in the LC. BMP7 has unique developmental and trophic actions on catecholamine neurons and these findings suggest that reduced astrocyte support for pontine LC neurons may contribute to pathology of brain noradrenergic neurons in MDD.

In recent years, gene expression, genetic association, and metabolic studies have implicated the polyamine system in psychiatric conditions, including suicide. Given the extensive regulation of genes involved in polyamine metabolism, as well as their interconnections with the metabolism of other amino acids, we were interested in further investigating the expression of polyamine-related genes across the brain in order to obtain a more comprehensive view of the dysregulation of this system in suicide. To this end, we examined the expression of genes related to polyamine metabolism across 22 brain regions in a sample of 29 mood-disordered suicide completers and 16 controls, and identified 14 genes displaying differential expression. Among these, altered expression of spermidine/spermine N1-acetyltransferase, spermine oxidase, and spermine synthase, has previously been observed in brains of suicide completers, while the remainder of the genes represent novel findings. In addition to genes with direct involvement in polyamine metabolism, including S-adenosylmethionine decarboxylase, ornithine decarboxylase antizymes 1 and 2, and arginase II, we identified altered expression of several more distally related genes, including aldehyde dehydrogenase 3 family, member A2, brain creatine kinase, mitochondrial creatine kinase 1, glycine amidinotransferase, glutamic-oxaloacetic transaminase 1, and arginyl-tRNA synthetase-like. Many of these genes displayed altered expression across several brain regions, strongly implying that dysregulated polyamine metabolism is a widespread phenomenon in the brains of suicide completers. This study provides a broader view of the nature and extent of the dysregulation of the polyamine system in suicide, and highlights the importance of this system in the neurobiology of suicide.

Several chromosomal regions have been linked to bipolar disorder (BD). However, the search for specific genes has been hampered by inconsistent findings, partly due to genetic and phenotypic heterogeneity. We focused on lithium-responsive bipolar patients, a subgroup thought to be more homogeneous and conducted a multistage study including an initial linkage study followed up by fine mapping and gene expression. Our sample consisted of 36 families (275 genotyped individuals, 132 affected) recruited through probands who were responders to long-term lithium treatment. We conducted a genome-wide scan with 811 microsatellite markers followed by fine mapping. Gene expression studies of candidate regions were conducted on six post-mortem prefrontal brain regions of 20 individuals (8 BD and 12 controls). We identified regions 3p25, 3p14 and 14q11 as showing the highest genome-wide linkage signal (LOD 2.53, 2.04 and 3.19, respectively). Fine mapping provided further support for 3p25, while only modest support was found in the other two regions. We identified a group of synaptic, mitochondrial and apoptotic genes with altered expression patterns in BD. Analysis of an independent microarray dataset supported the implication of synapse-related and mitochondrial genes in BD. In conclusion, using two complementary strategies, we found evidence of linkage to lithium-responsive BD on 3p25, 3p14 and 14q11 as well as significantly dysregulated genes on these regions suggesting altered synaptic and mitochondrial function in BD. Further studies are warranted to demonstrate the functional role of these genes in BD.

Alterations in the levels of spermine synthase (SMS) and spermine oxidase (SMOX), two enzymes involved in polyamine metabolism, have previously been observed in brains of suicide completers. To characterize the roles played by genetic and epigenetic factors in determining expression levels of these genes, as well as to identify potential mechanisms by which to explain our findings in suicide completers, we (1) assessed the role of promoter polymorphisms in determining expression in the brain and in vitro, and (2) examined CpG methylation and levels of methylated histone H3 lysine-27 in the promoter regions of these genes in the prefrontal cortex of suicide completers and healthy controls. We identified several promoter haplotypes in SMS and SMOX, but found no consistent effects of haplotype on expression levels in either the brain or in reporter gene assays performed in three different cell lines. We also found no overall effects of epigenetic factors in determining expression, with the exception of a relationship between CpG methylation at one site in the promoter of SMOX and its expression in Brodmann area 8/9. In conclusion, the genetic and epigenetic factors examined in this study show little influence on the expression levels of SMS and SMOX, and do not appear to be responsible for the dysregulated expression of these genes in suicide completers.

An association between low levels of serum cholesterol and violent or suicidal behaviour has frequently been reported, but it remains unclear how cholesterol in the peripheral system might be related to the brain functions involved in mediating suicidal behaviour. To our knowledge, there have been no previous studies aimed at answering the important question of whether there are differences in cholesterol within the brains of suicide completers. In the present study, cholesterol content was measured in cortical and subcortical tissue of brains from 41 male suicide completers and 21 male controls that died of sudden causes with no direct influence on brain tissue. No significant differences in cholesterol content were found between suicides and controls in the frontal cortex, amygdala or hippocampus. However, when the suicide completers were stratified into violent (n=31) or non-violent (n=10) groups based on the method of death, violent suicides were found to have lower grey-matter cholesterol content overall compared to non-violent suicides [F(1,111)=4.75, p=0.03], specifically in the frontal cortex (t=-4.16, d.f.=37, p<0.0005). Further exploration of the frontal cortex revealed that violent suicides had lower cholesterol content compared to non-violent suicides in the orbitofrontal cortex (t=-2.01, d.f.=36, p=0.05) and the ventral prefrontal cortex (t=-2.49, d.f.=37, p=0.02). This study represents the first direct examination of cholesterol content in brain tissue from suicide completers, and the present findings provide added support for the relationship between low cholesterol and violent or suicidal behaviour.