Approaches and Findings from Gene Expression Profiling Studies of Suicide

Laura M. Fiori; Gustavo Turecki

NCBI Bookshelf. A service of the National Library of Medicine, National Institutes of Health.

Dwivedi Y, editor. The Neurobiological Basis of Suicide. Boca Raton (FL): CRC Press/Taylor & Francis; 2012.

The Neurobiological Basis of Suicide.

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Chapter 12Approaches and Findings from Gene Expression Profiling Studies of Suicide

Laura M. Fiori and Gustavo Turecki.

12.1. INTRODUCTION

Studies investigating neurological and biological factors associated with suicidal behaviors have been ongoing for decades. Initial molecular genetic studies largely focused on candidate genes and pathways, and while specific neurobiological alterations have been associated with suicide, we are far from reaching a comprehensive understanding of the underlying pathological processes associated with this complex phenotype. In order to expand its focus, suicide research moved toward high-throughput gene expression microarrays in an effort to identify novel biological pathways and molecular mechanisms associated with suicide. By analyzing tissues obtained from suicide completers and examining the expression of a vast number of genes in parallel, these technologies allow researchers to obtain a functional profile of gene expression, thus providing valuable insight into the overall biological processes underlying suicide. This chapter will first discuss the methodologies that have been used to profile gene expression alterations in suicide, and then will examine several of the neurobiological mechanisms that have been implicated. Following this, future uses of gene expression profiling technologies in suicide research will be discussed.

12.2. TECHNOLOGICAL AND METHODOLOGICAL APPROACHES AND CONSIDERATIONS

Microarray technology was first developed almost two decades ago by researchers at Stanford University, who were able to simultaneously quantify the expression of 45 Arabidopsis genes using cDNA printed onto glass slides (Schena et al. 1995). In the years since, microarray technology has made huge advancements and can now be used to measure the expression of thousands of gene transcripts in organisms of all classes of life. This technology quickly became an exciting tool, not only for expanding our understanding of processes underlying cellular function, but as a means to identify pathological gene expression changes occurring in disease and in relation to phenotypes such as suicidal behaviors. At the same time, the ability to analyze and interpret microarray findings has faced many challenges and required the development of new statistical methods, including those to address the fact that gene expression can vary over several orders of magnitude, as well as analytical methods to deal with issues related to multiple testing. Furthermore, strategies for proper experimental design have represented important considerations, particularly given the cost of microarray technology. In spite of these challenges, high-throughput gene expression arrays have become an important resource in suicide research, and 15 studies examining the overall transcriptome in suicide completers have been performed to date. In order to better appreciate the findings arising from high-throughput gene expression studies, an understanding of the laboratory methods and technologies, statistical analysis strategies, and experimental design is required. This section will first give a brief overview of the microarray technologies and statistical approaches that have been employed to assess gene expression patterns related to suicide, followed by a discussion of methods that have been used to validate these findings. Following this, aspects related to sample and tissue selection, as well as their inherent limitations, will be explored.

12.2.1. Microarray Technology and Analysis

To date, all microarray studies examining suicide completers have used one-color microarrays, most commonly platforms produced by Affymetrix, which consist of hundreds of thousands of short (typically 25 base pairs) oligonucleotides. Biotinylated complementary RNA produced from mRNA extracted from biological samples is hybridized with the array, and gene expression is represented by the intensity of fluorescence at each probe following immunochemical treatment. The levels of mRNA carrying each sequence of interest are interrogated by a set of probes (probe set), and data from each probe are combined to generate an overall expression value for that sequence. Illumina BeadChip microarrays, which have also been used in suicide research, use similar methods, with the main difference being the use of larger probes. In both cases, probes have typically been designed to target the 3′ end of mRNA molecules in order to reduce the impact of quality and processing biases. Following hybridization and measurement of fluorescence, several post-processing steps are performed, including quality assessment, normalization of intensity values across chips, and combination of probe intensity values (Miron and Nadon 2006). Although substantial efforts have been put into the development of suitable computational methods for these steps, there remains considerable debate regarding the most appropriate algorithms (Steinhoff and Vingron 2006). To date, the most commonly used analysis methods have been the Microarray Analysis Suite produced by Affymetrix and Robust Multiarray Average (Irizarry et al. 2003). While a variety of statistical approaches have been used to identify gene expression patterns specific to suicide, these have all been required to address the multiple testing issue arising from the vast amount of data generated in these experiments. Although it is generally accepted that standard methods to correct for multiple testing, such as the Bonferroni correction, are excessively stringent for high-throughput studies, it is understood that failing to sufficiently correct will inevitably produce a large number of false positive results. The most typical methods that have been used in suicide research have involved either prespecified fold change and P-value cutoffs or false discovery rate corrections.

Technical validation (i.e., confirming that the technology has properly measured mRNA levels) has also generally been performed following the identification of genes displaying differential expression. The current “gold-standard” method of validation is quantitative real-time polymerase chain reaction (qRT-PCR), although in the past less precise methods such as semiquantitative RT-PCR and immunohistochemistry have also been employed. While initially it was believed that a significant correlation of mRNA expression with protein measurements was also an essential aspect of validation, it has now become better recognized that mRNA and protein levels represent distinct aspects of cellular functioning, and that a lack of correlation between these measurements is more often due to the presence of multiple levels of gene regulation. In addition, molecular processes such as alternative splicing or cell type–specific expression can also result in a failure to validate differences in RNA levels if experiments assess different transcripts from those evaluated by a particular probe set, or use RNA extracted from nonidentical tissue samples.

Beyond technical validation, scientific validation is an important consideration in gene expression studies in suicide and other complex phenotypes, where differences in expression may result from confounding factors that may not be directly related to the phenotype being investigated. Greater confidence in the scientific relevance of gene expression findings can be obtained by replication in independent samples. Resources, such as the Stanley Neuropathology Consortium Integrative Database (SNCID), which holds a large body of gene expression and other information obtained from a well-characterized psychiatric sample, have allowed researchers to readily determine how well their results could be extended to other populations (Kim and Webster 2010b). Moreover, the wealth of information available for these subjects has allowed the combination of expression data with other neurobiological information (Kim and Webster 2010a,b), thus allowing even greater knowledge regarding the nature of dysregulated expression to be obtained. The preselection of genes or probe sets prior to statistical analysis based on knowledge regarding a particular biological pathway (Lalovic et al. 2010; Morita et al. 2005) or chromosomal region (Fiori 2009) has also been used as a means to increase the likelihood that positive results represent biologically relevant findings. In addition to protein measurements, the biological importance of specific genes in suicide has been further investigated through genetic association studies (Sequeira et al. 2006; Yanagi et al. 2005).

12.2.2. Experimental Design

Sample selection represents the most important consideration in microarray studies, as this will determine the ability to detect small magnitude differences in gene expression, as well as allow these results to be properly interpreted in reference to specific research questions. Both the size of the overall sample and the number of samples within each experimental group are essential aspects of experimental design when attempting to identify differential gene expression in general. In order to determine how meaningful and relevant the results are to suicide, both the method by which experimental groups are defined and the ability to identify and address confounding variables play essential roles. Finally, the tissue that is examined will also partially determine which biological pathways are found to be differentially expressed in relation to suicide.

As a result of both the cost of microarray technologies and issues inherent in sample recruitment, sample sizes have generally been small, ranging from 6 (Yanagi et al. 2005) to 90 (Kim et al. 2007) subjects overall, with generally between 10 and 15 subjects within each experimental group. This has posed a significant problem when attempting to identify gene expression changes specific to suicide, as suicide completers represent a heterogeneous group, particularly in terms of psychiatric diagnosis, drug and alcohol use, and gender. Differentiating the effects of suicide from those related to comorbid psychiatric disorders has required careful selection of experimental groups and has typically been approached in one of three ways:

1. Grouping together all suicides irrespective of diagnosis and comparing them to a non-suicide control group
2. Grouping suicide completers within Axis I disorders (particularly major depression, bipolar disorder, or schizophrenia) and then separately comparing each group to the control group
3. Comparing, within a group of subjects with specific Axis I disorders, individuals who died by suicide with those who died of other causes

Confounding factors, including the use of alcohol, medication, or other drugs, are an inherent concern in studies of psychiatric disorders, and have the potential to influence the interpretability of findings in terms of suicide. Unfortunately, these issues have only been addressed in a few studies: approaches have included drug or alcohol use as a covariate in statistical analyses (Kim et al. 2007; Klempan et al. 2009c; Sequeira et al. 2007, 2009), performing a gene expression study in a separate sample of alcohol abusers (Sequeira et al. 2009), and examining the effects of drugs in animals in order to assess their potential influence on gene expression patterns in humans (Ernst et al. 2009; Sequeira et al. 2009). Gender also represents a confounding variable that has not been well addressed. Although gender plays a significant role in the susceptibility and presentation of suicidal behaviors, very few studies have been able to assess the effects of gender, largely as a consequence of difficulties in recruiting female samples due to the much lower rate of suicide completion in females. Accordingly, studies have either typically used exclusively male samples in order to avoid gender-specific effects or examined gender samples in which the numbers of female subjects have been insufficient to fully examine the influence of gender.

Finally, the selection of the biological sample to examine is also an important factor to consider when designing studies to address specific research questions. To date, all studies examining suicide have used postmortem brain tissues. Given their implication in psychiatric disorders, studies have largely focused on tissues obtained from the prefrontal cortex (Brodmann areas [BA] 8, 9, 10, 11, 44, 45, 46, and 47) or limbic areas (amygdala, hippocampus, BA 24, and BA 29). More recently additional regions, including the motor cortex, temporal cortex, thalamus, hypothalamus, and nucleus accumbens, have also been examined (Ernst et al. 2009; Sequeira et al. 2009). The quality of the brain tissue is also a source of confounding variables. Tissue pH, which can be influenced by both antemortem and postmortem factors, can affect RNA quality, as well as have specific effects on gene expression, including those implicated in psychiatric disorders (Vawter et al. 2006). In addition to being influenced by pH, RNA quality can be influenced by other postmortem variables and laboratory-specific factors, such as sample storage and RNA extraction methods, and must be carefully monitored as the RNA degradation state can directly impact gene expression measurements.

12.3. FINDINGS ARISING FROM MICROARRAY STUDIES

When microarray studies were first undertaken as a means to investigate psychiatric phenotypes, it was believed that they would both confirm and expand on information regarding genes and pathways previously implicated in psychiatry, as well as identify new systems that are involved in their pathology. Interestingly, the majority of microarray studies examining suicide have largely failed to identify altered expression of genes related to the expected pathways. Rather, the microarray studies to date have highlighted many pathways that were previously not suspected to be involved in the neurobiology of suicide, including additional systems related to neurotransmission, stress response, and cellular functioning. Although difficulties in obtaining sufficient numbers of non-suicide psychiatric controls have made it difficult to extricate the gene expression changes associated with suicidal behaviors from those pertaining to underlying psychiatric disorders, the consistency of many findings across samples from different research groups has provided good support for their involvement in suicide.

12.3.1. Neurotransmission

Synaptic transmission in the central nervous system (CNS) underlies much of what makes us who we are and is the main site of the action of the psychopharmaceutical agents currently in use. Consequently, monoaminergic neurotransmission has been the most extensively studied system in suicide research. Interestingly, however, although numerous studies over the last few decades have consistently identified altered functioning of monoaminergic systems in suicide completers, microarray studies have provided only minimal evidence for the dysregulation of genes involved in serotonergic or noradrenergic neurotransmission in suicide completers. Instead, gene expression studies have highlighted the involvement of the glutamatergic and γ-aminobutyric acid (GABA)-ergic neurotransmitter systems, as well as genes that regulate neurotransmission in general. Interestingly, altered expression of genes related to GABA and glutamate signaling are among the strongest findings arising from microarray studies examining suicide, strongly emphasizing the importance of these two pathways in the pathology of suicide.

12.3.1.1. Glutamate

Glutamate is the primary excitatory neurotransmitter in the brain and acts at four classes of receptors: the ionotropic α-amino-3-hydroxy-5-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), kainate, and N-methyl-d-aspartate (NMDA) receptors, as well as the metabotropic glutamate receptors (Conn and Pin 1997; Dingledine et al. 1999). Glutamate is synthesized from either glucose obtained from the tricarboxylic acid (TCA) cycle or from glutamine, which is synthesized by glial cells and taken up by neurons (Daikhin and Yudkoff 2000). Glutamate transmission is terminated by reuptake into neurons or astrocytes, which then convert it to glutamine to be resynthesized into glutamate by the enzyme glutaminase (GLS) (Daikhin and Yudkoff 2000).

A number of receptor binding studies have been used to examine glutamate transmission in the brains of suicide completers, but have generated largely negative findings. However, alterations in genes related to glutamatergic signaling have consistently emerged from microarray studies, providing strong evidence for a role of this system in suicide. Microarray studies have identified alterations in the levels of several glutamate receptors, including NMDA-like receptor 1A (GRINL1A), NMDA receptor 2A (GRIN2A), AMPA receptors 1–4 (GRIA1, GRIA2, GRIA3, and GRIA4), metabotropic glutamate receptor 3 (GRM3), and kainate receptor 1 (GRIK1) (Klempan et al. 2009c; Sequeira et al. 2009; Thalmeier et al. 2008). Additionally, downregulated expression has been observed for glutamate–ammonia ligase (glutamine synthetase) (GLUL), the enzyme responsible for removing glutamate from synapses, GLS, and glial high-affinity glutamate transporters SLC1A2 and SLC1A3 (Kim et al. 2007; Klempan et al. 2009c; Sequeira et al. 2009). GRIA3 is particularly interesting as it has been associated with a number of psychiatric conditions including bipolar disorder, schizophrenia, and citalopram treatment–emergent suicidal ideation (Gécz et al. 1999; Laje et al. 2007; Magri et al. 2008; O’Connor et al. 2007). Interestingly, several of these genes are localized to glia, which lends support for the involvement of dysregulated astroglial functioning in suicide, which will be discussed in a later section.

12.3.1.2. γ-Aminobutyric Acid

GABA is the primary inhibitory neurotransmitter in the CNS and plays many important roles, including cortical development, synaptic plasticity, neurogenesis, and stress responses (Ge et al. 2007; Li and Xu 2008; Nugent and Kauer 2008; Radley et al. 2009). GABA acts upon two classes of receptors: ionotropic GABA_A receptors and metabotropic GABA_B receptors. The metabolism of GABA is intricately tied to that of glutamate, which is the precursor for GABA synthesis by glutamic acid decarboxylase (GAD). Additionally, following its release into synapses, GABA is transported into astrocytes and converted to glutamine (Bak et al. 2006).

Although many studies have investigated GABA levels as well as the number and function of GABA receptors, the overall results have been inconsistent. Nonetheless, dysregulated expression of GABAergic genes has been among the most consistent findings arising from microarray studies of suicide completers. Altered expression of numerous GABA receptor subunits have been observed across prefrontal and limbic brain regions, including GABA_A, α1 (GABRA1), GABA_A, α4 (GABRA4), GABA_A, α5 (GABRA5), GABA_A, β1 (GABRB1), GABA_A, β3 (GABRB3), GABA_A, δ (GABRD), GABA_A, γ-1 and γ-2 (GABRG1 and GABRG2), GABA_B, β2 (GABBR2), and GABA_C, ρ1(GABRR1), as well as GABA_A receptor-associated protein-like 1 (GABARAPL1), and a GABA transporter (SLC6A1) (Choudary et al. 2005; Kim et al. 2007; Klempan et al. 2009c; Sequeira et al. 2007, 2009). Additionally, one study from our group found that 16% and 36% of the probe sets annotated as involved in GABAergic signaling were significantly differentially expressed in suicide completers in BA 44 and 46, respectively (Klempan et al. 2009c). Microarray findings have been reinforced by candidate gene expression studies, which have identified significant decreases in the expression of several GABA_A receptor subunits in the frontopolar region of suicide completers, and interestingly, there appear to be differences in the interrelations between different GABA_A subunits across the brain of depressed suicide completers relative to controls (Merali et al. 2004; Poulter et al. 2010).

12.3.1.3. Synaptic Structure and Function

In addition to factors related to specific neurotransmitter systems, synaptic transmission is highly regulated by presynaptic factors controlling the vesicle-mediated release of neurotransmitters. Synapse-related proteins have many different roles, including the docking and fusion of synaptic vesicles with the cellular membrane, as well as vesicle packaging and recycling. Considerable evidence is now emerging implicating this system in suicide. Altered expression of numerous synapse-related genes have been identified in suicide completers, including vesicle-associated membrane protein 3 (VAMP3), synaptotagmin I (SYT1), synaptotagmin IV (SYT4), synaptotagmin V (SYT5), synaptotagmin XIII (SYT13), synaptophilin (SNPH), synaptophysin-like protein (SYPL), synapsin II (SYN2), synaptosomal-associated protein (23kDa) (SNAP23), synaptosomal-associated protein (25 kDa) (SNAP25), synaptosomal-associated protein (29kDa) (SNAP29), synaptic vesicle glycoprotein 2B (SV2B), and synaptopodin 2 (SYNPO2) (Klempan et al. 2009c; Sequeira et al. 2006, 2007, 2009). These findings may partially explain how monoaminergic neurotransmission may be altered in suicide completers when the processes involved in monoamine metabolism and reception remain intact.

12.3.2. Stress Systems

One of the most widely used models to conceptualize risk for mental disorders, which has also been adopted in suicide research, is based on the notion of a stress– diathesis interaction. In the case of suicidal behaviors, this model assumes that suicide results from the combination of stressors and predisposing factors. Molecular systems involved in stress response have thus been investigated for their roles in suicide, as they are intricately tied to both stress and predisposition. The two major stress systems in humans include the autonomic nervous system, which is largely influenced by factors influencing the catecholamines, and the hypothalamic– pituitary–adrenal axis system. Although metabolic analyses and candidate gene studies have provided evidence to support a role for these systems in suicide, relatively few related genes have been identified in microarray studies. In contrast, microarray studies highlighted the importance of a third stress pathway, the polyamine system, whose role in suicide had not been previously suspected.

12.3.2.1. Polyamines

The polyamines are ubiquitous aliphatic molecules comprising agmatine, putrescine, spermidine, and spermine. The polyamine system has been identified in all organisms and plays an important role in numerous essential cellular functions, including growth, division, and signaling cascades, as well as stress responses at both the cellular and behavioral levels (Gilad and Gilad 2003; Minguet et al. 2008; Rhee et al. 2007; Seiler and Raul 2005; Tabor and Tabor 1984). Its involvement in schizophrenia and behavioral stress responses has been investigated for several decades (Fiori and Turecki 2008); however, it was not suspected to be involved in suicide until micro-array studies identified spermidine/spermine N1-acetyltransferase (SAT1) as one of the genes displaying the strongest and most consistently altered expression in suicide completers (Sequeira et al. 2006). Evidence for its downregulation in suicide completers has now been extended to additional brain regions and populations, and dysregulated expression of other polyamine-related genes, including spermine synthase (SMS), spermine oxidase (SMOX), ornithine decarboxylase antizymes 1 and 2 (OAZ1, OAZ2), S-adenosylmethionine decarboxylase (AMD1), arginase II (ARG2), and ornithine aminotransferase-like 1 (OATL1), has also been identified (Fiori et al. 2011; Guipponi et al. 2009; Klempan et al. 2009b,c; Sequeira et al. 2007).

Although the mechanism by which altered polyamine levels can influence risk for suicide or other psychiatric disorders is not yet clear, several mechanisms have been proposed. First, the polyamines can influence neurotransmission through several systems, including the catecholamines (Bastida et al. 2007; Bo et al. 1990; Hirsch et al. 1987; Ritz et al. 1994), glutamate (Williams 1997), GABA (Brackley et al. 1990; Gilad et al. 1992; Morgan and Stone 1983), and nitric oxide (Galea et al. 1996), each of which may be involved in psychiatric disorders. Agmatine itself is believed to act as a neurotransmitter through imidazoline receptors, α2-adrenoceptors, nicotinic acetylcholine receptors, and serotonin 3 receptors: this theory is supported by its storage in synaptic vesicles and capacity to be released upon depolarization (Reis and Regunathan 2000). Interestingly, alterations in imidazoline receptor binding sites have also been implicated in depression and anxiety, and their importance in modulating behavioral stress responses has become accepted (Halaris and Piletz 2003, 2007; Piletz et al. 2000). Second, the importance of neurogenesis in the development and treatment of psychiatric disorders has been recognized (Dranovsky and Hen 2006; Jacobs et al. 2000), and it is therefore of great interest that manipulation of the polyamine system produces significant effects on both CNS development and adult brain neurogenesis (Malaterre et al. 2004; Seiler 1981), and that variations in polyamine levels are associated with both pro- and anti-apoptotic effects in neuronal cells (Harada and Sugimoto 1997; Sparapani et al. 1997). Several studies have found relationships connecting the neurogenic and neuroprotective effects of the polyamines with stress and depression (Li et al. 2006; Zhu et al. 2007, 2008), providing evidence that dysregulation of the polyamine system may act in part through these mechanisms. Finally, both agmatine and putrescine, as well as the polyamine precursor S-adenosylmethionine, demonstrate anxiolytic and antidepressant effects in animal and human studies (Bressa 1994; Gong et al. 2006; Lavinsky et al. 2003; Zeidan et al. 2007; Zomkowski et al. 2002, 2004, 2005, 2006), which could be particularly relevant for suicide completers with comorbid mood or anxiety disorders.

12.3.3. Cellular Function

Both synaptic neurotransmission and stress response pathways rely heavily on proper cellular functioning, and both the systems described earlier, as well as many other neurobiological pathways implicated in suicide, can be influenced by pathological alterations to cellular processes. Indeed, microarray studies have demonstrated alterations in several essential components of cellular functioning, including growth factor signaling and energy metabolism. Moreover, these studies have provided strong evidence for cell type–specific alterations in gene expression, pointing toward altered glial functioning as an important pathological component underlying suicidal behavior.

12.3.3.1. Growth Factors

Growth factors are cellular signaling molecules that play essential roles in proliferation, differentiation, and survival (Pawson 1994). Growth factor receptors are found at the cell surface, and following binding of their ligands, they dimerize and activate the tyrosine kinase activity of their intracellular domain, resulting in autophosphorylation that allows the intracellular domains to interact with proteins involved in various second messenger systems (Pawson 1994). Microarray studies have provided evidence for altered expression of genes associated with two classes of growth factors: the neurotrophins and fibroblast growth factor (FGF).

The neurotrophin brain-derived neurotrophic factor (BDNF) plays an important role in neuronal growth and survival through its binding to the receptor neurotrophic tyrosine kinase, type 2 (NTRK2). Numerous studies have implicated this pathway in depression (Brunoni et al. 2008; Chen et al. 2001; Sen et al. 2008), and evidence from microarray studies has suggested that it also plays a role in suicide. Although gene expression of BDNF itself has not been found to be altered in suicide completers, decreased expression of NTRK2 has been observed in several brain regions (Ernst et al. 2009; Kim et al. 2007; Sequeira et al. 2007). Interestingly, this decrease appears to be due to the downregulation of one specific NTRK2 isoform, TrkB.T1, which is expressed exclusively in astrocytes (Ernst et al. 2009).

Alterations in the expression of components of FGF signaling pathways have also been consistently observed in suicide. There are over 20 FGF ligands in humans, which act upon four different tyrosine kinase receptors (FGFR1–4), where they play important roles in cellular proliferation and differentiation during development, as well as in neuronal signal transduction into adulthood (Ornitz and Itoh 2001). Several microarray studies have identified altered expression of genes involved in FGF signaling, including FGFR2 and FGFR3, in suicide completers (Ernst et al. 2008; Kim et al. 2007). Similar to BDNF, many components of the FGF system, including FGFR2 and FGFR3, show glial-specific expression.

12.3.3.2. Energy Metabolism

Processes regulating the production and usage of adenosine triphosphate (ATP) are essential for maintaining adequate energy stores for cellular functions. It is thus of interest that altered expression of several ATP-related genes has been observed in microarray studies of suicide completers. One study found that TCA cycle and ATP-related genes were significantly associated with gene expression differences between depressed and non-depressed suicide completers, and suggested that some defects may be markers for suicidal behavior in the context of depression (Klempan et al. 2009c). Another study identified downregulated expression of the Na⁺/K⁺-ATPase α3 subunit (ATP1A3) in the prefrontal cortex of suicide completers with diagnoses of depression, bipolar disorder, or schizophrenia, indicating that altered energy metabolism in suicide completers is not exclusive to depression (Tochigi et al. 2008). It is also of interest that recent microarray studies by our group examining mood-disordered suicide completers found altered expression of four genes related to creatine metabolism, another important source of high-energy phosphate groups (Fiori et al. 2011).

12.3.3.3. Glial Cells

Glial cells play many distinct roles in the CNS, including the development and maintenance of the nervous system, processing of synaptic transmission, regulation of cerebral blood flow, and immune responses in the brain (Araque 2008; Gehrmann et al. 1995; Koehler et al. 2009; Pfrieger 2009). Although traditional research in suicide and other psychiatric disorders largely ignored glial cells, histopathological studies have demonstrated alterations in glial cell densities and numbers in psychiatric disorders, and microarray studies demonstrating altered expression of glial-specific genes have provided further evidence to support a role for altered glial functioning in suicide.

Astrocytes form the largest group of cells in the CNS and have multiple functions, including roles in the synthesis, release and uptake of neurotransmitters, development of synapses, and formation of the blood–brain barrier (Fiacco et al. 2009; Montana et al. 2006; O’Kusky and Colonnier 1982; Stevens 2008). Many microarray studies have supported the involvement of dysregulated astrocyte functioning in suicide and have identified altered expression of several astrocyte-specific genes including FGFR2, FGFR3, GLUL, S100 calcium-binding protein beta (S100B), and TrkB.T1 (Ernst et al. 2009; Kim et al. 2007; Klempan et al. 2009c; Sequeira et al. 2009). Given the important role of astrocytes in glutamate neurotransmission, it has been proposed that they may also play a role in the alterations of this system in suicide (Sequeira et al. 2009).

While the best-known function of oligodendrocytes is their role in axon myelination, they also participate in neurotransmission, synaptic function, neuronal development, and neuronal survival (Deng and Poretz 2003). Oligodendrocyte-specific genes have demonstrated altered expression in brains of suicide completers, including membrane glycoprotein M6B (GPM6B), S100B, and quaking homolog, KH domain RNA binding (mouse) (QKI) (Fiori et al., 2011; Klempan et al. 2009a,c). Interestingly, GPM6B was recently shown to interact with and alter the surface expression of the serotonin transporter, indicating the importance of oligodendrocytes in monoaminergic neurotransmission (Fjorback et al. 2009). Furthermore, given the importance of myelination in neural transmission, cognition, and brain development, it is not surprising that alterations in oligodendrocyte function may be associated with suicide (Fields 2008).

12.4. FUTURE AVENUES FOR GENE EXPRESSION STUDIES

Although our understanding of the neurobiology of suicide has greatly increased within the last few decades, we are still far from developing an integrated picture of how specific biological and neurochemical alterations interact to confer risk for suicidal behaviors and how this knowledge can be used to chemically treat these behaviors. The majority of studies performed to date have focused on genes and proteins of interest, and while these studies continue to be important, they are not sufficient to address these issues. Microarrays have played an essential role in highlighting new molecular pathways involved in suicidal behavior, yet cannot explain either the origin of these alterations or the nature of their short- and long-term effects on brain function. However, continued advances in the fields of both molecular biology and high-throughput technologies can provide the means to answer these questions. By combining gene expression data with information obtained through other measures, such as clinical, epigenetic, genomic, proteomic, or metabolic studies, a more comprehensive view of these processes will be possible. Also, by using more powerful technologies, such as RNA-Seq—next-generation sequencing of the transcriptome—we will be able to gain more precise insight into gene expression profiles associated with the suicide process. In addition, continued sample collection and collaborations between research groups will allow for a greater capacity to investigate the effects of variables such as medication, gender, and the environment, as well as to properly differentiate the effects of suicide from those of Axis I disorders.

12.4.1. Gene Function

Although it is clear that specific alterations in gene expression underlie the suicide process, the functional impact of these alterations remains largely unknown and represents an essential step in understanding the pathological effects of gene expression differences. Several methods are now available to better characterize these effects. As mentioned earlier, gene expression microarray studies of suicide completers have used arrays that are primarily designed to measure the expression of the 3′ end of mRNAs. However, many newer arrays have been developed that allow the expression of each exon within a gene to be quantified. As the functional importance and diversity of alternative splicing has become well recognized over the last decade, the use of exon arrays is an important next step in suicide research. These arrays will allow the identification of genes that show specific splicing differences, thereby improving our understanding of how the function of these genes may be altered in suicide. Similarly, as mentioned earlier, the advance in sequencing methods now allow for the implementation of techniques to directly sequence and quantify the transcriptome, and techniques such as RNA-Seq are powerful alternatives to micro-array studies. In addition, although high-throughput methods to examine the proteome have not advanced as quickly as other fields, the technology continues to be improved. In the future, integrating mRNA expression with protein expression data will allow for a better understanding of the functional effects of alterations in gene expression, and how they may ultimately lead to suicide. Finally, while neuroimaging techniques and knowledge regarding the cell type–specific expression of genes have been invaluable in studying the roles of neurons and glia in suicide, they represent somewhat crude approaches for analyzing gene expression differences between different cell types. Protocols for laser capture microdissection have now been optimized for postmortem brain tissues (Pietersen et al. 2009), which will be invaluable in better characterizing the specific roles played by neurons and glia in suicide.

12.4.2. Gene Regulation

Identifying mechanisms involved in gene regulation is also an important step in understanding how pathological changes in gene expression arise; moreover, they can represent potential molecular targets for the development of new treatments. By integrating gene expression data with that obtained from platforms assessing single nucleotide polymorphisms (SNPs) across the genome, essential information regarding the regulation of gene expression can be obtained. Genetic regions that are associated with gene expression differences may represent specific functional genetic variants, or may be due to larger chromosomal alterations, known as copy number variations (CNV), which have been associated with psychiatric disorders including bipolar disorder, schizophrenia, and autism (Cook and Scherer 2008). Epigenetic modifications are also important regulators of gene expression. These modifications can be environmentally influenced, particularly through physiological and behavioral stressors, and it is believed that they may mediate the interaction between the genome and the environment in conferring risk for suicide (Tsankova et al. 2007). Two important epigenetic modifications that have been examined in the context of suicide are DNA methylation and posttranslational histone modifications. Although the epigenetic studies of suicide to date have only focused on genes of interest, examination at a larger scale is now possible using comparative hybridization arrays, large-scale, genome-based deep sequencing, and chromatin immunoprecipitation-based methods. MicroRNAs represent a third mechanism for the epigenetic modification of gene expression. These short, single-stranded RNA molecules bind to specific mRNA molecules and target them for degradation, and have been implicated in other psychiatric conditions (Abu-Elneel et al. 2008; Chen et al. 2009; Hansen et al. 2007). Examination of each of these three epigenetic mechanisms using high-throughput methods, in conjunction with gene expression microarrays, will allow the systematic investigation of epigenetic effects that may be involved in suicide risk. Furthermore, as epigenetic markings are potentially reversible, this is an exciting area of investigation that creates the opportunity for therapeutic intervention.

12.4.3. Other Considerations

Given the large gender differences in the rates and presentations of suicidal behaviors, gender is an essential consideration in studies of suicide. To address this issue, sample recruitment practices and statistical analyses must be adjusted in order to identify gender-specific factors. Additionally, there is a need for increased sample sizes as well as the availability of well characterized and appropriately selected control groups in order to enable the identification of phenotype-specific gene expression changes—a particularly important challenge in neurobiological studies of suicide. Finally, developing a more comprehensive view of the means by which psychopharmacological agents affect gene expression is an important step in understanding the mechanisms by which they exert their therapeutic effects, which will greatly assist in the development of better treatments directed toward suicidal behaviors.

12.5. CONCLUSIONS

Genomic gene expression profiling has provided an invaluable tool for obtaining a global view of gene expression changes underlying the suicide process, and allowed us to widen our focus beyond the pathways classically studied in suicidal behaviors. While the general lack of evidence supporting alterations in these classical pathways is perplexing, this is a strong indication that gene expression differences represent only one facet of the neurobiological alterations occurring in this complex phenotype. In the future, by integrating gene expression data with that interrogating other molecular and clinical variables, we will be able to obtain a more thorough understanding of the underlying mechanisms involved in the etiology and pathology of suicide, which may ultimately lead toward improved methods to treat and prevent suicidal behaviors.

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