Integrative DNA Methylation and Gene Expression Analysis in the Prefrontal Cortex of Mexicans Who Died by Suicide

BackgroundSuicide 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.MethodsIn 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.ResultsWe 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.ConclusionOur 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.     

Increased temporal cortex ER stress proteins in depressed subjects who died by suicide.

Regulation of ER stress proteins, such as the 78-kilodalton glucose regulated protein (GRP78) by chronic treatment with mood stabilizing drugs suggests that this family of proteins may be involved in the pathophysiology of mood disorders. Indeed, increased levels of GRP78, GRP94, and calreticulin, a third member of the ER stress protein family, were found in temporal cortex of subjects with major depressive disorder who died by suicide compared with controls and subjects who died by other means. No such differences were found in subjects with other psychiatric disorders such as bipolar disorder or schizophrenia. These data suggest a potential role for ER stress proteins in severe depression that merits further study.     

Neuropeptide Y Y(1) and Y(2) receptor mRNA expression in the prefrontal cortex of psychiatric subjects. Relationship of Y(2) subtype to suicidal behavior.

It has been hypothesized that the neuropeptide Y (NPY) system is involved in the pathogenesis of mood disorder. In this study, Y(1) and Y(2) receptor mRNA expression levels were analyzed in the dorsolateral prefrontal cortex of subjects affected with major depression, bipolar disorder, or schizophrenia and compared to normal controls. No significant alterations in Y(1) or Y(2) mRNA expression levels were observed between the groups. However, the Y(2) mRNA expression was elevated in layer IV in subjects with suicide as a cause of death. For the Y(1) mRNA expression, there was a negative correlation with increasing subject age in the prefrontal cortex. Analysis of covariance revealed a significant elevation of the Y(1) mRNA expression levels in individuals with a current history of marijuana use but no other drug. In summary, the current results suggest distinct alterations of the prefrontal Y(1) and Y(2) neuronal populations in aging and suicide.     

Investigation of m1/m4 muscarinic receptors in the anterior cingulate cortex in schizophrenia, bipolar disorder, and major depression disorder.

Abnormal cholinergic neurotransmission has been suggested to occur in psychiatric illness. Therefore, this study investigated cholinergic muscarinic receptors in the anterior cingulate cortex (ACC) of schizophrenia, bipolar disorder and major depression disorder (n=15 per group). We used quantitative autoradiography to measure [(3)H]pirenzepine binding to M1 and M4 receptors. Brain tissue was obtained from the Stanley Foundation Neuropathology Consortium. [(3)H]pirenzepine binding was higher in superficial laminae (I-II) than in deep laminae (III-VI) of the ACC. There was a significant 24% reduction in the density of [(3)H]pirenzepine in the deep laminae and a significant 19% reduction in the upper laminae of the ACC in the schizophrenia group compared to the control group. There were no differences in [(3)H]pirenzepine binding in any laminae of the ACC in the bipolar or major depression groups compared with the control group, except for a trend towards decreased [(3)H]pirenzepine binding in subjects with major depression relative to control subjects. We also detected a significant effect of suicide on [(3)H]pirenzepine binding in the ACC in subjects who died as a result of suicide relative to those who did not, which was more evident in patients with schizophrenia. A significant effect of the onset of the disease was also observed that was more evident in patients with bipolar disorder. The study provides evidence of decreased muscarinic receptor density in the ACC in schizophrenia but no evidence for significant changes in these receptors in the bipolar and major depression groups. The changes observed in schizophrenia may contribute to dysfunctional ACC neural circuits.     

Role of the endocannabinoid system in depression and suicide

Depression is one of the most prevalent forms of neuropsychiatric disorder and is a major cause of suicide worldwide. The prefrontal cortex is a crucial brain region that is thought to be involved in the regulation of mood, aggression and/or impulsivity and decision making, which are altered in suicidality. Evidence of the role of the endocannabinoid (EC) system in the neurobiology of neuropsychiatric disorders is beginning to emerge. The behavioral effects of ECs are believed to be mediated through the central cannabinoid CB1 receptor. Alterations in the levels of ECs, and in the density and coupling efficacy of CB1 receptors, have been reported in the prefrontal cortex of depressed and alcoholic suicide victims. These findings support our hypothesis that altered EC function contributes to the pathophysiological aspects of suicidal behavior. Here, we provide a brief overview of the role of the EC system in alcoholism, depression and suicide, and discuss possible therapeutic interventions and directions for future research.     

Brain noradrenergic receptors in major depression and schizophrenia.

The binding of [125I]p-iodoclonidine to alpha-2, and/or [125I]iodopindolol to beta-1 and beta-2 adrenoceptors was measured in right prefrontal cortex (Brodmann's area 10) and right hippocampus from subjects with DSM-III-R diagnoses of major depression (n = 15) or schizophrenia (n = 8) as well as from control subjects (n = 20). No significant differences between study groups were observed in binding to alpha-2 adrenoceptors in any of the six layers of prefrontal cortex or in any of the hippocampal fields. Likewise, there were no significant differences in beta-1 or beta-2 adrenoceptor binding in any of the hippocampal fields between control and major depressive subjects. In contrast, binding to beta-1 adrenoceptors, but not beta-2 adrenoceptors, was significantly lower (-13 to -27%) in most hippocampal fields of schizophrenic subjects as compared to control subjects or to major depressives. Alterations in beta-1 adrenoceptor binding in the hippocampus of schizophrenics provide further evidence for a role of central noradrenergic neurons in the neurochemical pathology of schizophrenia.     

Neurotransmitter receptors and monoamine metabolites in the brains of patients with Alzheimer-type dementia and depression,and suicides

In patients with Alzheimer-type dementia, in addition to the well-known losses of cholinergic neurones, there is evidence of degeneration of the noradrenergic and serotonergic innervation of the cerebral cortex. While noradrenergic and cholinergic receptors are preserved there is a loss of serotonin S1 and S2 receptors, particularly in the temporal lobe. The loss of serotonin S2 receptors may occur at an early stage of the disease and, in temporal and frontal cortex, is correlated with the loss of somatostatin immunoreactivity. In patients dying in hospital with depression, and in individuals committing suicide, there are no consistent changes in monoamine metabolites. Noradrenergic, serotonergic, and other neurotransmitter receptors were found to be unchanged, although there was a moderate decrease in imipramine binding in a small group (n = 6) of subjects with a history of depression, who had committed suicide.     

Brain-derived neurotrophic factor and tyrosine kinase B receptor signalling in post-mortem brain of teenage suicide victims

Teenage suicide is a major public health concern, but its neurobiology is not very well understood. Stress and major mental disorders are major risk factors for suicidal behaviour, and it has been shown that brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (TrkB) are not only regulated by stress but are also altered in these illnesses. We therefore examined if BDNF/TrkB signalling is altered in the post-mortem brain of teenage suicide victims. Protein and mRNA expression of BDNF and of TrkB receptors were determined in the prefrontal cortex (PFC), Brodmann's Area 9 (BA 9), and hippocampus obtained from 29 teenage suicide victims and 25 matched normal control subjects. Protein expression was determined using the Western blot technique; mRNA levels by a quantitative RT-PCR technique. The protein expression of BDNF was significantly decreased in the PFC of teenage suicide victims compared with normal control subjects, whereas no change was observed in the hippocampus. Protein expression of TrkB full-length receptors was significantly decreased in both PFC and hippocampus of teenage suicide victims without any significant changes in the truncated form of TrkB receptors. mRNA expression of both BDNF and TrkB was significantly decreased in the PFC and hippocampus of teenage suicide victims compared with normal control subjects. These studies indicate a down-regulation of both BDNF and its receptor TrkB in the PFC and hippocampus of teenage suicide victims, which suggests that stress and altered BDNF may represent a major vulnerability factor in teenage suicidal behaviour.     

Relationship of psychopathology to the human serotonin1B genotype and receptor binding kinetics in postmortem brain tissue.

Knockout of the 5-HT1B gene in mice results in increased aggression, as well as alcohol and cocaine consumption. Given the clinical association of aggression, suicide, alcoholism, and substance abuse, we studied relationship of psychopathology to the human 5-HT1B receptor gene (N = 178) and postmortem human 5-HT1B receptor binding (N = 96) in the brain. The sample comprised: 71 suicide victims, 107 nonsuicides, 45 with a history of major depression and 79 without, 64 with a history of a alcoholism or substance abuse and 60 without, as well as 36 with a history of pathological aggression and 42 without. Single-strand conformational polymorphism (SSCP) analysis and DNA sequencing techniques were used to screen the coding region of the human 5-HT1B receptor gene in genomic DNA isolated from postmortem human brain tissue. Two common polymorphisms were identified in the 5-HT1B receptor gene, involving a silent C to T substitution at nucleotide 129 and a silent G to C substitution at nucleotide 861 of the coding region. These polymorphisms were found with the same frequency in the suicide and the nonsuicide groups and in those with and without a history of major depression, alcoholism, or pathological aggression. The binding indices (Bmax and KD of the 5-HT1B receptor in prefrontal cortex also did not differ in suicides and controls, major depression, alcoholism, and cases with a history of pathological aggression. The C129 or G861 allele had 20% fewer 5-HT1B receptor compared to the 129T or 861C allele. We did not identify a relationship between suicide, major depression, alcoholism, or pathological aggression with 5-HT1B receptor binding indices or genotype.     

Volumetric analysis of the prefrontal cortex, amygdala, and hippocampus in major depression.

Magnetic resonance imaging (MRI) studies in depressed subjects report smaller volumes of amygdala, hippocampus, inferior anterior cingulate, and the orbital prefrontal cortex (OPFC), components of the limbic-cortico-thalamic circuit. Major depression occurs more commonly in women, raising the possibility of an additional psychopathological process affecting women and not men. We sought to determine whether volumetric differences related to mood disorders are dependent on sex. Eight male and 10 female depressed subjects, meeting DSM III R criteria for a major depressive episode, and eight male and 10 female healthy volunteers had MRI scans on a 1.5 T GE Signa Advantage scanner. The regions of interest included amygdala, hippocampus, inferior anterior cingulate, and OPFC. In all analyses, regional volumes were normalized for total cerebral volume. Volumetric changes in the ROIs showed a significant sex by diagnosis interaction, indicating a different pattern of volumetric changes in depressed males compared with females relative to controls. Relative to sex-matched controls, the left inferior anterior cingulate was smaller in depressed males (23%) compared with depressed females (11%). Depressed females but not depressed males had smaller amygdala compared with controls (F-value = 4.946, p = 0.033). No significant volumetric differences were noted in the hippocampus or OPFC. No volumetric correlations were noted with clinical variables, depression subtypes, or a reported history of sexual or physical abuse. Sex may affect volumetric deficits in amygdala and anterior cingulate cortex in mood disorders, but no effects were found in the hippocampus or OPFC. The biology of mood disorders in females may differ in some aspects from males, and may contribute to the higher rate of depression in women.     

Brain region specific alterations in the protein and mRNA levels of protein kinase A subunits in the post-mortem brain of teenage suicide victims.

Protein kinase A (PKA), a critical component of the adenylyl cyclase signaling system, phosphorylates crucial proteins and has been implicated in the pathophysiology of depression and suicide. The objective of the study was to examine if changes in PKA activity or in the protein and messenger RNA (mRNA) expression of any of its subunits are related to the pathophysiology of teenage suicide. We determined PKA activity and the protein and mRNA expression of different subunits of PKA in cytosol and membrane fractions obtained from the prefrontal cortex, (PFC) hippocampus, and nucleus accumbens (NA) of post-mortem brain from 17 teenage suicide victims and 17 nonpsychiatric control subjects. PKA activity was significantly decreased in the PFC but not the hippocampus of teenage suicide victims as compared with controls. However, the protein and mRNA expression of only two PKA subunits, that is, PKA RIalpha and PKA RIbeta, but not any other subunits were significantly decreased in both membrane and cytosol fractions of the PFC and protein expression of RIalpha and RIbeta in the NA of teenage suicide victims as compared to controls. A decrease in protein and mRNA expression of two specific PKA subunits may be associated with the pathogenesis of teenage suicide, and this decrease may be brain region specific, which may be related to the specific behavioral functions associated with these brain areas. Whether these changes in PKA subunits are related to suicidal behavior or are a result of suicide or are specific to suicide is not clear at this point.     

Differential gene expression in a rat model of depression based on persistent differences in exploratory activity

Affective disorders are often accompanied by changes in motivation and anxiety. We investigated the genome-wide gene expression patterns in an animal model of depression that separates Wistar rats belonging into clusters of persistently high anxiety/low motivation to explore and low anxiety/high motivation to explore (low explorers and high explorers, LE and HE, respectively), in three brain regions previously implicated in mood disorders (raphe, hippocampus and the frontal cortex). Several serotonin-, GABA-, and glutamatergic genes were differentially expressed in LE- and HE-rats. The analysis of Gene Ontology biological process terms associated with the differentially regulated genes identified a significant overrepresentation of genes involved in the neuron development, morphogenesis, and differentiation; the most enriched pathways from the Kyoto Encyclopedia of Genes and Genomes were the Wnt signalling, MAPK signalling, long-term potentiation, and long-term depression pathways. These findings corroborate some expression data from other models of depression, and suggest additional targets.     

Low GABA concentrations in occipital cortex and anterior cingulate cortex in medication-free, recovered depressed patients

Studies using proton magnetic resonance spectroscopy (1H-MRS) indicate that unmedicated, acutely depressed patients have decreased levels of gamma-aminobutyric acid (GABA) in the occipital cortex. The aim of this study was to use 1H-MRS to determine if changes in occipital and frontal cortical GABA levels were present in patients with a history of depression who had recovered and were no longer taking medication. We used 1H-MRS to measure levels of GABA in both occipital cortex and anterior cingulate cortex/prefrontal cortex in medication-free, fully recovered subjects with a history of recurrent unipolar depression. Levels of GABA in both occipital and anterior cingulate cortex were significantly lower in recovered depressed subjects than healthy controls. Our data provide preliminary evidence that a history of recurrent depression is associated with decreased GABA levels in anterior cingulate cortex and occipital cortex. These changes could represent part of the neurobiological vulnerability to recurrent depressive episodes.     

GABAergic neurons immunoreactive for calcium binding proteins are reduced in the prefrontal cortex in major depression.

Post-mortem morphometric studies report reductions in the average density and size of cortical neurons in the dorsolateral prefrontal cortex (dlPFC) and orbitofrontal cortex (ORB) in major depressive disorder (MDD). The contribution of specific neuronal phenotypes to this general pathology in depression is still unclear. Post-mortem sections from the dlPFC and ORB regions of 14 subjects with MDD and 11 controls were immunostained to visualize calbindin-immunoreactive (CB-IR) and parvalbumin-immunoreactive (PV-IR) presumptive GABAergic neurons. A three-dimensional cell counting probe was used to assess the cell packing density and size of CB-IR neurons in layers II+IIIa and PV-IR neurons in layers III-VI. The density of CB-IR neurons was significantly reduced by 50% in depression in the dlPFC and there was a trend toward reduction in the ORB. The size of CB-IR somata was significantly decreased (18%) in depression in the dlPFC with a trend toward reduction in the ORB. In contrast, there was no difference in the density of PV-IR neurons between the depressed and control groups in the dlPFC. The size of PV-IR neuronal soma was unchanged in depressed compared to control subjects in either dlPFC or ORB. In depression, subpopulations of GABAergic neurons may be affected differently in dlPFC and ORB. A significant reduction in the density and size of GABAergic interneurons immunoreactive for calcium binding proteins was found predominantly in the dlPFC region. These cellular changes are consistent with recent neuroimaging studies revealing a reduction in the cortical levels of GABA in depression.     

Altered histamine H3 receptor radioligand binding in post-mortem brain samples from subjects with psychiatric diseases

Background and purpose:

Histamine is a modulatory neurotransmitter in the brain. Auto- and hetero-histamine H₃ receptors are present in human brain and are potential targets of antipsychotics. These receptors may also display disease-related abnormalities in psychiatric disorders. Here we have assessed how histamine H₃ receptors in human brain may be affected in schizophrenia, bipolar disorder, major depression.

Experimental approach:

Histamine H₃ receptor radioligand binding assays were applied to frozen post-mortem prefrontal and temporal cortical sections and anterior hippocampal sections from subjects with schizophrenia, bipolar disorder, major depression and matched controls.

Key results:

Compared with the controls, increased H₃ receptor radioligand binding was found in dorsolateral prefrontal cortex of schizophrenic subjects (especially the ones who were treated with atypical antipsychotics), and bipolar subjects with psychotic symptoms. No differences in H₃ receptor radioligand binding were found in the temporal cortex. In hippocampal formation of control subjects, H₃ receptor radioligand binding was prominent in dentate gyrus, subiculum, entorhinal cortex and parasubiculum. Decreased H₃ binding was found in the CA4 area of bipolar subjects. Decreased H₃ binding in CA2 and presubiculum of medication-free bipolar subjects was also seen.

Conclusions and implications:

The results suggest that histamine H₃ receptors in the prefrontal cortex take part in the modulation of cognition, which is impaired in schizophrenic subjects and bipolar subjects with psychotic symptoms. Histamine H₃ receptors probably regulate connections between hippocampus and various cortical and subcortical regions and could also be involved in the neuropathology of schizophrenia and bipolar disorder.     

Open questions in current models of antidepressant action

Research on depression and antidepressant drugs is necessary, as many patients display poor response to therapy. Different symptomatic and pathophysiological features have been proposed as end points of the depressive phenotype and of the antidepressant action, including anhedonia, depressed mood, alterations in morphology and activity of some brain areas (amygdala, nucleus accumbens, hippocampus, prefrontal cortex and cingulate cortex), modifications in the connectivity between brain structures, changes in neurotransmitters (serotonin, noradrenaline, glutamate and neuropeptides), brain plasticity (neurogenesis, neurotrophins) and abnormal function of the hypothalamic-pituitary adrenal axis. However, few models have been proposed to describe how these end points could induce the depressive phenotype and are involved in the mechanism of action of antidepressants. Here we propose a connectionist-inspired network of depression and antidepressant action, in which the different aetiological factors participating in the release of a depressive episode are represented by input nodes, the different symptomatic as well as pathophysiological end points are represented by an intermediate layer, and the onset of depression or of comorbid disease is represented by the output node. The occurrence of depression and the mechanism of the antidepressant action thus depend upon the weight of the interactions between the different end points, none of them being per se crucial to the onset of a depressive phenotype or to the antidepressant action. This model is heuristic to draw future lines of research concerning new antidepressant therapies, designing new animal models of depression and for a better understanding of the depressive pathology and of its comorbid pathology such as anxiety disorders.