Abstract

   Suicidal behaviors are frequent in mood disorders patients but only a
   subset of them ever complete suicide. Understanding predisposing
   factors for suicidal behaviors in high risk populations is of major
   importance for the prevention and treatment of suicidal behaviors. The
   objective of this project was to investigate gene expression changes
   associated with suicide in brains of mood disorder patients by
   microarrays (Affymetrix HG-U133 Plus2.0) in the dorsolateral prefrontal
   cortex (DLPFC: 6 Non-suicides, 15 suicides), the anterior cingulate
   cortex (ACC: 6NS, 9S) and the nucleus accumbens (NAcc: 8NS, 13S).
   ANCOVA was used to control for age, gender, pH and RNA degradation,
   with P≤0.01 and fold change±1.25 as criteria for significance. Pathway
   analysis revealed serotonergic signaling alterations in the DLPFC and
   glucocorticoid signaling alterations in the ACC and NAcc. The gene with
   the lowest p-value in the DLPFC was the 5-HT2A gene, previously
   associated both with suicide and mood disorders. In the ACC 6
   metallothionein genes were down-regulated in suicide (MT1E, MT1F, MT1G,
   MT1H, MT1X, MT2A) and three were down-regulated in the NAcc (MT1F,
   MT1G, MT1H). Differential expression of selected genes was confirmed by
   qPCR, we confirmed the 5-HT2A alterations and the global
   down-regulation of members of the metallothionein subfamilies MT 1 and
   2 in suicide completers. MTs 1 and 2 are neuro-protective following
   stress and glucocorticoid stimulations, suggesting that in suicide
   victims neuroprotective response to stress and cortisol may be
   diminished. Our results thus suggest that suicide-specific expression
   changes in mood disorders involve both glucocorticoids regulated
   metallothioneins and serotonergic signaling in different regions of the
   brain.

Introduction

   Suicide is one of the leading causes of death in the world particularly
   for young males and projections of global mortality indicate that
   suicide rates are rising [47][1] and will remain a leading cause of
   injury and death worldwide [48][2]. The number of suicides is
   approximately 1 million per year in the world [49][3]. In the United
   States suicide is the eleventh leading cause of death for all ages and
   the third leading cause among adolescents [50][4]. One third of those
   suicides visited a health care professional within a week before
   committing suicide [51][4]. This suggests that better methods are
   needed in assessment of suicidal intent in at risk populations.

   Mood disorders (major depressive disorder (MDD) and bipolar disorder
   (BD)) constitute prominent risk factors for suicide [52][5]–[53][7].
   Accordingly, lifetime risk for suicide in mood disorders patients is
   particularly high when compared to the general population
   [54][8]–[55][12]. In BD patients suicide attempts are specifically
   associated with depressive episodes rather than manic episodes
   [56][13]. Despite current advances in the understanding of suicide
   predisposing factors, existing diagnostic algorithms are inadequate and
   the identification of individuals at high risk for suicide is
   frequently missed [57][14].

   Suicide and suicidal behaviors have been consistently linked to
   monoaminergic (serotonin, norepinephrine and dopamine)
   neurotransmission alterations [58][15]–[59][17] and to alterations in
   the hypothalamic-pituitary-adrenal (HPA) axis [60][18]–[61][20].
   Postmortem receptor/transporter binding studies have also confirmed
   alterations in the number and affinity of monoaminergic receptors in
   the brains of suicides [62][17], [63][21]. Interestingly, both
   serotonergic neurotransmission imbalance and HPA axis over-activity are
   related to stressful events and were proposed as risk factors for
   suicide [64][22]. Recent evidence has shown that childhood abuse alters
   HPA stress responses and increases the risk of suicide through
   epigenetic regulation of glucocorticoid receptor expression underlining
   the role played by the HPA-stress response in the susceptibility to
   suicide [65][23].

   Recently, microarray technology has been used to investigate in
   post-mortem brain global gene expression alterations associated with
   mood disorders and suicide compared to psychiatrically normal controls
   [66][24]. A number of studies specifically explored gene expression
   alterations in post-mortem brains of suicides versus control subjects
   in prefrontal regions [67][25]–[68][30] and in the limbic system
   [69][31], [70][32] with very few replicated results due probably to
   methodological and demographic differences in the samples. These
   studies focused mainly on the investigation of genes differentially
   expressed between psychiatric cases that committed suicide and
   controls, but did not compare the psychiatric patients who commit
   suicide from those who do not. To our knowledge, only two recent
   articles investigated suicide specific changes in the brain by
   comparing suicide versus non-suicide psychiatric patients. Garbett et
   al. observed a prefrontal cortical down-regulation of the HTR2A
   receptor in subjects with schizophrenia who committed suicide using
   Affymetrix arrays [71][33]. Also, Kim et al. (2007) reanalyzed a large
   microarray data set to investigate gene expression in major depression
   and schizophrenic patients who did and did not commit suicide, in the
   prefrontal cortex (BA10/46) [72][30]. They found alterations on 2 genes
   across diagnoses, PLSCR4 (phospholipid scramblase 4) and EMX2 (empty
   spiracles homolog 2 (Drosophila)) and several members of the
   metallothionein subfamily 1 (MT1E, MT1X, MT1M, MT1G, MT1H, MT1F) as
   being specifically down-regulated among the schizophrenics who
   committed suicide [73][30].

   Mental disorders and particularly mood disorders contribute
   significantly but not exclusively to the risk of suicide. Determining
   the expression changes in the brain that differentiate suicide from
   non-suicide mood disorder brains should help identify the genes
   specifically involved in suicide. In this study, we investigated a
   cohort of mood disorder subjects (BD and MDD) for gene expression
   changes specifically associated with suicide in the DLPFC, ACC, and
   NAcc, three regions commonly associated with suicidal behavior
   alterations, mood, control of impulses, motivation, reward and
   pleasure, behaviors know to be altered in mood disorders.

Methods

Subjects

   Brain samples were obtained at the University of California,
   Irvine/Davis Brain Repository through a uniform process approved by the
   Institutional Review Board. Written informed consent was obtained from
   the next-of-kin of the deceased. All subjects went through an extensive
   review of multiple sources of information including the medical
   examiner’s conclusions, coroner’s investigation, medical and
   psychiatric records, toxicology results, and interviews of the
   decedents’ next-of-kin, in order to rule out as much as possible any
   neurological disorder. Additionally, a neuropathological examination of
   each brain was conducted in order to exclude cases with cerebrovascular
   disease (infarcts or hemorrhages), subdural hematoma, or other
   significant pathological features. Our psychological autopsy protocol
   is based largely on procedures validated by Kelly and Mann [74][34].
   Details of death are evaluated in order to assign an Agonal Factor
   Score to each subject in accordance with previous guidelines hardy
   [75][35], [76][36]. Previous work consistently showed an association
   between agonal factors and tissue quality [77][37]–[78][40]. Brains are
   initially screened for agonal factors at the time of consent in order
   to exclude brains with factors potentially affecting nucleic acids and
   proteins. Subjects with history of drugs or alcohol dependence were
   also excluded from the present sample. Non-suicide subjects, including
   mood disorder subjects and normal controls, died rapidly from either
   cardiac related events or motor-vehicle accidents.

   The human brain dissection and freezing protocol is described in detail
   elsewhere [79][41]. Briefly, the whole brain is first inspected and
   photographed before the brainstem and cerebellum are separated from the
   cerebrum. The cerebrum is coronally sliced from anterior to posterior
   and slices are photographed, labeled, and bagged before being frozen.
   Small 5 cm squares are dissected for both pH and RNA quality
   measurements. Freezing is done by placing the bagged slices between
   super-cooled aluminum plates, then in a pre-cooled freezing rack before
   putting the whole rack in a −140°C freezer for a minimum of 10 minutes.
   The brains are stored in −80°C freezers until dissected and processed
   for extractions. The brain regions were dissected on dry ice from the
   left hemisphere of frozen tissue according to visible landmarks near
   the regions of interest under the direction of Dr E. G. Jones. Grey
   matter punches were taken from the cortical regions and from the middle
   of NAcc region.

   Grey matter brain samples (25 mg) were used to extract total RNA with
   the Trizol method (Invitrogen, Carlsbad, CA), followed by a cleaning up
   using a silica based mini-spin column (Qiagen RNeasy Mini Kit,
   Valencia, CA). RNA integrity was evaluated using 18S and 28S ratios and
   RNA integrity numbers (RIN) using the 2100 Agilent Bioanalyzer (Santa
   Clara, CA). All subjects were clinically characterized with the
   psychological autopsy method and died suddenly without prolonged agonal
   state.

Microarray Analysis

   Cel files from Affymetrix HG-U133 Plus2.0 chips were processed using
   the RMA algorithm. Statistical analysis was performed using Partek (St.
   Louis, MO). An ANOVA model was used to identify the differentially
   expressed genes between the non suicides and the suicides in a sample
   of mood disorder patients controlling for age, gender, RIN, and pH.
   Additionally, the AffyRNAdeg function was used to generate 3′/5′ slopes
   indicating RNA degradation and used as covariate in the ANOVA model
   [80][42]. Genes were considered as differentially expressed if they had
   a P-value of less than 0.01 and a fold change of ±1.25. Pathway
   analysis was performed using Ingenuity (Redwood City, CA) and the
   differentially expressed probe sets in each region.

   Outlier detection was conducted in order to exclude problematic arrays
   using a combination of principal component analysis (PCA), and RNAdeg
   Slopes. In the DLPFC, 21 subjects were analyzed by microarrays, 15
   suicides (3 females/12 males) and 6 non-suicides (2 females/4 males).
   In the ACC, a total of 15 subjects were included in the microarray
   analysis, 9 suicides (3 females/6 males) and 6 non-suicides (3
   females/3 males). In the NAcc a total of 21 subjects were analyzed by
   microarrays, 13 suicides (3 females/10 males) and 8 non-suicides (2
   females/6 males). For the microarray experiment sample (cohort 1),
   demographic and physiological variables used for the ANOVA model and to
   assess the RNA quality (age, pH, PMI or RIN numbers) can be found in
   [81]Table 1 for the group averages and standard deviation and [82]Table
   S1 for the detailed information. The entire dataset has been deposited
   to the Gene Expression Omnibus (GEO) with the Accession Number
   [83]GSE6306 ([84]Table S6).

Table 1. Demographic and physiological details for the subjects included in
the microarray gene expression study sample (cohort 1).

   DLPFC ACC NAcc
   Suicide (n = 15) NS (n = 6) Suicide (n = 9) NS (n = 6) Suicide (n = 13)
   NS (n = 8)
   Age (Mean±SD) 44±14 42±17 43±9 53±10 43±9 54±8
   pH (Mean±SD) 6.8±0.3 6.9±0.4 6.9±0.3 6.7±0.3 6.9±0.3 6.8±0.2
   PMI (Mean±SD) 25±4.2 23±8.3 24±5.0 25±5.0 24±4.7 25±6.6
   Slope (Mean±SD) 3.3±0.6 2.9±0.3 3.6±0.3 4.0±0.5 3.0±0.2 2.9±0.4
   RIN (Mean±SD) 7.9±0.5 7.9±0.6 8.0±0.4 8.0±0.3 8.1±0.4 8.0±0.5
   [85]Open in a new tab

   Mean±standard deviation values (SD) are presented. The slope was
   calculated using the AffyRNAdeg function. DLPFC: dorsolateral
   prefrontal cortex, ACC: anterior cingulate cortex, NAcc: nucleus
   accumbens.

Confirmation of Differential Expression

   Confirmation of a subset of genes was done was done in an extended
   cohort including also normal controls by quantitative real-time
   polymerase chain reaction (qPCR) using the SYBR Green chemistry.
   Normalization of the qPCR results was performed using the geNorm method
   developed by Vandesompele et al. [86][43], this method consists on a
   factor calculation normalization based on the geometric mean of
   multiple endogenous control genes, in this case we used CAMK2A and
   SDHA. Samples were run in triplicate and confirmation of the results
   was determined by ANOVA controlling for age, pH, and diagnosis. Primers
   were designed to amplify regions close to the corresponding target
   sequences in the microarray experiments and spanning two exons in order
   to only amplify mRNA (primers are available on request). Experiments
   were performed in a 384 well plate in a total volume of 10 µl on a 7900
   HT real time sequence detector (Applied Biosystems, Foster City, CA).
   The qPCR confirmation cohort (cohort 2) also included normal controls
   and showed no differences in terms of age, pH, gender or PMI between
   the groups ([87] Table 2 and [88]Table S2).

Table 2. Demographic and physiological details for the subjects included in
the qPCR validation sample (cohort 2).

   DLPFC ACC NAcc
   Suicide (n = 20) NS (n = 11) Control (n = 27) Suicide (n = 10) NS
   (n = 7) Controls (n = 18) Suicide (n = 13) NS (n = 7) Control (n = 10)
   Age (Mean±SD) 44±12 52±11 50±15 43±11 54±9 49±14 46±9 49±9 41±11
   pH (Mean±SD) 6.9±0.2 6.9±0.2 6.9±0.3 7.0±0.2 6.9±0.2 7.0±0.2 6.9±0.2
   6.9±0.2 6.9±0.2
   PMI (Mean± SD) 26±4 24±6 20±7 24±5 26±5 20±5 25±5 24±6 21±5
   RIN (Mean ± SD) 7.9±0.5 7.8±0.7 7.8±0.6 8.2±1 8.3±1 8.0±1 NA NA NA
   [89]Open in a new tab

   Mean±standard deviation values (SD) are presented. DLPFC: dorsolateral
   prefrontal cortex, ACC: anterior cingulate cortex, NAcc: nucleus
   accumbens, NA: not available.

Results

   No significant differences between the suicide and non-suicide groups
   were observed for the major demographic variables such as pH, age, RIN,
   PMI and RNAdeg slope ([90] Table 1 and [91]Table S1). In order to
   control for possible confounding effects, demographic variables, such
   as diagnosis and age, and RNA quality measures (RIN and slope) were
   used as covariates.

   A total of 161 genes were found to be differentially expressed in the
   DLPFC ([92]Table S3). The most statistically significant gene in this
   region was the 5-HT2A gene (P = 0.00022, FC = −1.28), previously
   identified as being implicated in both mood disorders and in suicide
   consistently. The decrease of 5-HT2A expression in the suicide group
   was confirmed by qPCR (data not shown). Enrichment of canonical
   pathways analysis using Ingenuity confirmed that the serotonin receptor
   signaling pathway was altered in suicide brain tissue ([93] Fig. 1 a).
   Another gene of interest previously shown to be involved in suicide
   [94][44] and that was altered in the DLPFC was the adenylate cyclase 1
   (brain) (ADCY1) gene that codes for a brain specific form of adenylate
   cyclase responsible of the production of the second messenger cyclic
   AMP, ADCY was significantly down-regulated in suicide versus
   non-suicide patients. Finally, period homolog 1 (PER1), a major
   component of the circadian rhythm system, was also down-regulated in
   suicides compared to non-suicides. Clock genes have been in the past
   associated with mood disorders [95][45] but not specifically with
   suicide. In summary, the DLPFC data shows it is possible to detect
   significant expression alterations in human brain areas between
   suicides and non-suicides brains and confirms the involvement of
   serotonergic and second messenger system alterations in suicide.

Figure 1. Canonical pathway enrichment analysis using Ingenuity.

   [96]Figure 1
   [97]Open in a new tab

   A In the DLPFC. B In the ACC. C In the NAcc.

   In the ACC, 139 annotated probe sets were differentially expressed
   between the suicides and non-suicides cases ([98]Table S4). Of these,
   eight probe sets corresponded to seven metallothionein family members
   (MT1E, MT1M, MT1F, MT1G, MT1H, MT1X, MT2A), located as a cluster on
   chromosome 16q13 ([99]Table 3). All these probe sets were significantly
   down-regulated in suicide victims with fold changes varying from −1.46
   to −1.83. Pathway analysis conducted using Ingenuity on the 139
   differentially expressed probe sets revealed that the most over
   represented pathway was glucocorticoid receptor signaling ([100] Fig. 1
   b). Further exploration of the probe sets altered in the
   glucocorticoids receptor signaling pathway revealed a global
   down-regulation of metallothionein subfamilies 1 and 2 genes. In fact,
   a closer look at the expression levels of all the metallothionein genes
   revealed that most of the genes in subfamilies 1 and 2 where
   down-regulated in the ACC, including pseudogenes co-localized in 16q13
   ([101] Fig. 2 ). Interestingly, the brain specific MT3 gene was highly
   expressed, but showed no differences in gene expression like
   subfamilies 1 and 2.

Table 3. qPCR Confirmation of decreased expression of metallothionein
subfamilies 1 and 2.

        NACC         ACC
        P-value T    P-value T
   MT1E 0.09    3.25 0.02    6.36
   MT1F 0.02    7.23 0.05    4.14
   MT1G 0.01    7.63 0.01    7.67
   MT1H 0.01    8.96 0.00    14.19
   MT1X 0.03    5.65 0.02    5.99
   MT2A 0.03    5.83 0.04    4.72
   [102]Open in a new tab

   Significant differences are in bold for one tailed t-test confirming
   the direction of the change.

Figure 2. Expression levels of 11 metallothionein probe sets corresponding to
9 gene as measured by microarrays (HG-U133Plus 2.0) in the ACC and NAcc of
non-suicide (grey bars) and suicide (black bars) mood disorder subjects.

   [103]Figure 2
   [104]Open in a new tab

   Significant genes/probe sets are labeled by * (P<0.01) and # (P<0.05)
   signs.

   In the NAcc, 146 probe sets were differentially expressed between
   suicides and non-suicides ([105]Table S5). Three metallothionein genes
   were also down-regulated in the NACC (MT1F, MT1G, MT1H) among the
   suicide victims and like in the ACC several others were also
   down-regulated although at a P<0.05 level. IPA did not reveal any
   canonical pathway as being over-represented, as opposed to the ACC
   ([106] Fig. 1 c), only three MT genes passed the significance criteria.
   However, 7 probe sets in total were differentially expressed at the
   0.05 level confirming the overall dysregulation in this family of genes
   ([107] Fig. 2 ). The Wolfram syndrome gene (WFS1), previously
   associated with psychiatric disorders and suicide [108][46]–[109][48],
   was also down-regulated among the suicide victims in the NACC.

Confirmation of Results by qPCR

   Group averages for demographic and physiological variables for the
   subjects (cohort 2) included in the confirmation experiments per region
   can be seen in [110]Table 2 and for subjects in cohort 2 used in this
   analysis can be seen in [111]Table 2 and detailed demographic data in
   table S2. All the subjects used the validation cohort had high pH
   (mean = 6.9) and RIN values (mean = 8), and an average the PMI of 23
   hours (8.5 to 37 hours). Metallotionein gene expression down-regulation
   in both the ACC and NACC was confirmed by qPCR for all the functional
   variants in sub-families 1 and 2 known to be expressed in the brain
   (MT1E, MT1F, MT1G, MT1H, MT1X, MT2A). In summary, the pattern of
   metallothionein expression was confirmed to be lower in the suicides
   compared to the non-suicides in both the ACC and NACC for all the genes
   examined ([112] Figs. 3 and [113]4 ). When compared to normal controls,
   metallothionein levels tended to be elevated in the non-suicide mood
   disorder group, especially in the NAcc, while lower specifically in the
   suicide group.

Figure 3. Expression levels in the NACC of six metallothionein genes in as
measured by qPCR in psychiatrically normal controls and mood disorder
patients that did (Mood-S) or did not commit suicide (Mood-NS).

   [114]Figure 3
   [115]Open in a new tab

   * denotes significance at the P<0.05 versus the Mood-S group.

Figure 4. qPCR confirmation in the ACC.

   [116]Figure 4
   [117]Open in a new tab

   Expression levels in the ACC of six metallothionein genes measured by
   qPCR and the GE-Norm method between psychiatrically normal controls and
   mood disorder patients that did (Mood-S) or did not commit suicide
   (Mood-NS). * denotes significance at the P<0.05 versus the Mood-S
   group.

Discussion

   A gene expression survey by microarrays in three brain regions (DLPFC,
   NAcc and ACC) revealed alterations in the serotonergic and second
   messenger systems in the DLPFC and a consistent down-regulation of
   several members of the metallothionein subfamilies 1 and 2 genes in the
   NAcc and ACC in mood disorder suicides. Our strategy consisted on using
   high quality brains (short PMI, high pH, high RIN) with no history of
   drug dependence, as well as the development of a general linear model
   in which factors known to influence RNA quality and expression were
   taken into account when exploring the suicide specific expression
   changes. This is to our knowledge the first study investigating suicide
   specific changes in mood disorders in three highly relevant areas as
   they are involved in the control of impulses and in mood disorders
   (DLPFC, ACC) and in motivation-reward mechanisms (NAcc).

   We studied gene expression changes in the DLPFC, an area previously
   associated with both mood disorders, control of impulses and suicide.
   Interestingly, the most statistically significant gene differentially
   expressed in suicide victims was the 5-HT2A serotonergic receptor, with
   a1.28 fold-change reduction in mood disorder subjects that committed
   suicide versus non-suicide mood disorder subjects. Genetic association
   studies have often shown that mutations in the 5-HT2A receptor are
   associated with mood disorders and suicide [118][49], as well as with
   the response to antidepressants [119][50]. Additionally, gene
   expression post-mortem studies have revealed alterations 5-HT2Areceptor
   levels in mood disorder subject suicides when compared to controls.
   However the direction of the alterations in 5-HT2A is controversial,
   with some reports showing elevations in the 5-HT2A binding and
   expression [120][51]–[121][55] while others showed reduced levels or no
   differences [122][56]–[123][58]. Recently, a well designed study
   including matched suicides and controls also confirmed the absence of
   changes in 5-HT2 binding but reported a decrease in neuronal densities
   in the prefrontal cortex in a sample of predominantly major depressed
   suicides [124][59]. In another recent study designed to investigate
   5-HT2A levels in major depression, Shelton et al. reported an increase
   in 5-HT2A expression in major depression subjects, but reported no
   differences when comparing suicides versus non-suicide subjects
   [125][55]. In this study, we aimed to investigate suicide specific
   changes in mood disorder patients and observed a significant decrease
   in 5-HT2A levels in suicide which can at least partially explain
   previous discordant findings when comparing mood disorder to controls
   or suicides to controls. The reduction in 5-HT2A levels is also in
   concordance with another study comparing suicide versus non-suicide
   schizophrenia subjects using also Affymetrix microarrays [126][33],
   suggesting that this reduction in 5-HT2A levels might be involved in
   suicide irrespective of the diagnosis. We also observed decreased
   expression in suicide group of the brain specific ADCY1, a brain
   specific form of adenylate cyclase, suggesting altered
   neurotransmission and cyclic AMP signaling in suicide and confirms
   previous observations pointing to cAMP signaling alterations in suicide
   and mood disorders [127][60]–[128][63] and in animal models of
   depression [129][64]. Both serotonergic alterations and second
   messenger systems underline global neurotransmission alterations in the
   DLPFC that seem to play a role in the predisposition to commit suicide.

   In both the ACC and NAcc decreased expression was observed in gene
   members of two subfamilies (1 and 2) of the metallothionein super
   family in the suicides versus the non-suicides mood disorder subjects.
   These changes were confirmed successfully Metallothioneins are small
   single chain proteins with a high content of cysteine residues
   organized in specific sequences enabling the formation of thiolate
   cluster and the binding of certain metals react to oxidative stress,
   glucocorticoids and inflammatory mediators [130][65]. There are four
   subfamilies of metallothioneins proteins in mammals corresponding to
   types 1 to 4, all metallothionein genes are closely located in
   chromosome 16q13. MT1 and MT2 are expressed ubiquitously and highly
   co-expressed, whereas MT4 is not expressed in the brain and MT3 is
   brain specific and expressed mainly in hippocampus amygdala and cortex
   in zinc enriched neurons and astrocytes [131][66]. Consistent with
   this, MT3 was also observed to be expressed in the three brain areas
   investigated but was not differentially expressed like MT1 and 2
   subfamilies. MT3, unlike MT 1 and 2 is not regulated by glucocorticoids
   and stress, which can explain this difference [132][67]–[133][72].

   MT1 and MT2 are not only known to be responsive to several forms of
   stress and to glucocorticoids [134][73], they also play an important
   role in neuroprotection [135][66]. In animal models of stress, both MT1
   and MT2 mRNA were elevated as much as 30-fold following just 12 h (one
   cycle) of restraint stress whereas MT3 gene expression was not altered
   by stress [136][74]. The induction occurred at the transcriptional
   level and was mediated by the activation of glucocorticoid receptor
   [137][74]. In a stress mouse model, treatment with a glucocorticoid
   receptor antagonist (RU 486) prior to restraint stress inhibited MT
   induction by at least 50% indication that at least in part MT induction
   is under direct control of the glucocorticoid receptor at the
   transcriptional levels [138][75]. Glucocorticoids induction of
   metallothioneins expression is done through the direct binding of the
   ligand activated glucocorticoid receptor to the glucocorticoid response
   elements (GRE) in the promoter of MTs [139][76], [140][77].
   Dexamethasone has been shown for instance to increase significantly the
   levels of MT and immobilization stress has also been shown to increase
   selectively MT1 and MT2 of rat brains [141][70]. MT1 and 2 play a
   neuroprotective role against stress and glucocorticoids elevation by a
   mechanism of induced expression of metallothioneins in astrocytes
   followed by an extracellular release and internalization by neurons
   after binding to the receptor megalin that mediates the transport of
   MTs into neurons [142][78]. Thus, MT1 and 2 sub-families, that play an
   important role in neuroprotection from the effects of behavioral stress
   and in response to cortisol, are significantly reduced in mood disorder
   suicides compared to non-suicides. Interestingly, in another recent
   microarray study using Affymetrix Exon 1.0 ST arrays, Shelton et al.
   observed and confirmed by qPCR a decreased expression of MT1M in the
   BA10 of depressed subjects compared to controls [143][79].

   Alterations in the hypothalamic-pituitary-adrenal (HPA) axis in
   depressive states and suicide have been consistently observed for more
   than 40 years [144][19], [145][80]. It has also been extensively shown
   that depressed states are specifically linked to both chronic stress
   and elevated cortisol levels relevant of HPA axis hyperactivity in
   depression [146][81]. Many studies have observed higher baseline
   cortisol values in young depressed patients [147][82] and cortisol
   hypersecretion in depression [148][83]. We propose in this study that
   decreased levels of MT1 and 2 can result in a higher sensitivity in
   suicide completers to the effects of depression-associated cortisol
   levels in the brain. If these results are confirmed in a larger
   independent cohort, pharmacological agents that increase the expression
   of MT 1 and 2 metallothionein sub-families or their transport to
   neurons by the megalin receptor without necessarily altering HPA axis
   activity can be envisioned for the treatment and prevention of suicide
   and suicidal behaviors in mood disorder patients.

   The effect of known confounding variables in post-mortem brain gene
   expression studies (pH, PMI, age, gender, diagnosis and RNA quality)
   was controlled by conducting an ANCOVA using significant variable as
   covariates. One of the weaknesses of this study is the relatively small
   size of the microarray sample. However, we were able to confirm the
   observed results in a larger sample and to explore MT expression in
   control subjects also by qPCR in a larger sample in both the ACC and
   NAcc ([149] Figs. 3 and [150]4 ). Another problem observed was the
   difference in age between the non-suicides and the suicides in the NAcc
   and ACC. This was expected as suicide is particularly prevalent in
   younger populations [151][2], [152][2], [153][7,84] with about 200,000
   young people that die every year in the world by suicide [154][85]. For
   that reason, we included age into the statistical model in order to
   exclude genes altered by age only. Ultimately however, MT subfamilies 1
   and 2 down-regulation in suicide mood disorder subjects should be
   tested in peripheral tissue such as blood lymphocytes, where they have
   been shown to be expressed [155][86], in order to confirm these results
   in a clinical population and to investigate the validity of these
   changes as a biomarker for suicide.

   In conclusion, while expression profiling confirmed and further
   extended the implication of serotonergic genes in suicide in the DLPFC,
   the present study also reveals a significant decrease in expression of
   metallothioneins subfamilies1 and 2, a family of genes involved in
   neuroprotection and in cortisol/stress response, in suicide victims
   compared to non-suicide mood disorder patients. Because cortisol levels
   are known to be elevated in depressive states, the observed decreased
   in MT expression in the suicide group might point to a failed
   neuroprotective mechanism against cortisol in this group and point to a
   new marker and possible pharmacological target for suicide and suicidal
   behavior.

Supporting Information

   Table S1

   Complete demographic variables for the subjects included in the
   microarray analysis per brain region. (RIN: Agilent 2100 RNA Integrity
   Number; MDD: major depressive disorder; BD: bipolar disorder; NS: non
   suicide; MOD: method of death).

   (DOC)
   [156]Click here for additional data file.^ (101.5KB, doc)
   Table S2

   Complete demographic variables for the subjects included in the
   confirmation qPCR analysis of the DLPFC, ACC and Nacc. (MDD: major
   depressive disorder; BD: bipolar disorder; NS: non suicide; PMI:
   post-mortem interval; MOD: method of death).

   (DOC)
   [157]Click here for additional data file.^ (186.5KB, doc)
   Table S3

   Differentially expressed probe sets between suicide and non-suicide
   mood disorder subjects in the DLPFC.

   (DOC)
   [158]Click here for additional data file.^ (75KB, doc)
   Table S4

   Differentially expressed probe sets between suicide and non-suicide
   mood disorder subjects in the ACC (139).

   (DOC)
   [159]Click here for additional data file.^ (185KB, doc)
   Table S5

   Differentially expressed probe sets between suicide and non-suicide
   mood disorder subjects in the NAcc (146).

   (DOC)
   [160]Click here for additional data file.^ (189.5KB, doc)
   Table S6

   Accession numbers for the chips included in the study.

   (DOC)
   [161]Click here for additional data file.^ (91KB, doc)

Footnotes

   Competing Interests: The authors have declared that no competing
   interests exist.

   Funding: The authors are members of the Pritzker Neuropsychiatric
   Disorders Research Consortium, which is supported by the Pritzker
   Neuropsychiatric Disorders Research Fund L.L.C. A shared intellectual
   property agreement exists between this philanthropic fund and the
   University of Michigan, Stanford University, the Weill Medical College
   of Cornell University, HudsonAlpha Institute of Biotechnology, the
   Universities of California at Davis (UCD), and at Irvine (UCI), to
   encourage the development of appropriate findings for research and
   clinical applications. The research was further supported by the
   William Lion Penzner Foundation (Dept of Psychiatry, UCI), Della Martin
   Foundation (to WEB, AS) and the National Alliance for Research on
   Schizophrenia and Depression (NARSAD) Young Investigator Award (to AS).
   The funders had no role in study design, data collection and analysis,
   decision to publish, or preparation of the manuscript.

References