The use of high throughput tools such as microarrays has broadened the understanding of gene expression alterations in several brain regions in major mental illnesses such as schizophrenia. These regions include the PFC [1
], cerebellum [9
], amygdala [14
], hippocampus [10
], as well as the cingulate [13
], temporal [9
], parietal [13
], enterohinal [7
] and occipital [13
] cortices. Interestingly, the STG, a region reported to be dysfunctional in schizophrenia has the most pronounced changes in gene expression when compared to most of the other regions implicated in schizophrenia [13
The number of genes expressed in the cerebral cortex has been largely unreported in previous gene expression studies of the PFC, temporal, parietal and occipital cortices. Although significantly altered gene expression has been reported for these areas, identifying the approximate number of genes expressed, and identifying similar expression patterns across regions, could lead to furthering our understanding of the functional and biological similarities and differences between cortical areas. In this study, 8737 of the 19000 genes tested were detectable in the STG in 4 or more of the 7 matched pairs studied, this is in contrast to our study of the amygdala, where only 5394 genes were detected using the same 19000 gene microarray [14
]. 4667 genes were expressed in both the amygdala and STG. Although the STG connects with the amygdala, the functions of the two regions are quite distinct which may be reflected in the difference in numbers of genes expressed. Mirnics and colleagues (2000) reported an average of 3735 genes expressed in the PFC of 9 individuals with schizophrenia, which is less than half that for the STG. However the microarray used by Mirnics and colleagues (2000) contained 7000 gene transcripts and the criteria to define genes that are expressed different from this study. Therefore, if tested using a larger gene set (e.g. 19000) the number of genes expressed in the PFC may be much higher.
One of the most consistent results across post-mortem brain studies is a trend towards overall down-regulation of genes in schizophrenia when compared to healthy controls [4
]. The average expression of the gene transcripts measured in this study showed an overall trend towards down-regulation in the 7 matched pairs of STG tissue which was similar to that previously reported for other brain regions in schizophrenia. This global down-regulation across many brain regions, particularly in the cerebral cortex including the STG, may contribute to the generalized deficiencies seen in individuals with schizophrenia, such as poor performance on cognitive tasks, reduced MMN, pre-pulse inhibition (PPI) and eye-tracking dysfunction. Although the biological cause underpinning these deficiencies is yet to be identified, overall down-regulation of gene expression in many brain regions may be the first steps towards explaining these phenomena.
The number of genes with altered expression in the STG identified by SAM and a 1.5 fold change cut-off in this study was remarkably similar to that reported by Katsel and colleagues (2005) using a high stringency criteria. This study identified 216 genes down-regulated and 85 genes up-regulated whereas Katsel and colleagues (2005) reported 185 genes down-regulated and 88 genes up-regulated in the same region in 22 schizophrenia and control samples. Although the genes identified in each study could not be directly compared.
The dysregulation of gene expression observed in the STG also highlighted a number of key cellular pathways including neurotransmission, particularly glutamate signaling, neurodevelopment and neuronal differentiation and presynaptic function. Indeed, there is building evidence for dysfunction of glutamate synapses in the STG and temporal lobe in general. For example, increased density of the N-methyl-D-aspartate (NMDA) glutamate receptor was reported by Nudmamud and colleagues [34
] using ligand binding studies and Le Corre and colleagues [35
] used in situ
hybridization to show increased density of the NMDA NR1 subunit splice variant in the STG from subjects with schizophrenia. In addition, Eastwood and Harrison (2001) [36
] reported that complexin II mRNA (expressed in excitatory neurons) was reduced in the dorsolateral PFC and the superior temporal cortex, and complexin I mRNA (expressed in inhibitory neurons) was decreased in the superior temporal cortex in schizophrenia. Furthermore, several of the schizophrenia candidate genes (e.g. glutamate receptor, metabotropic 3 (GRM3), G72, D-amino acid oxidase (DAAO), proline dehydrogenase oxidase 1 (PRODH), neuregulin 1 (NRG1) and protein phosphatase 3 catalytic subunit gamma isoform (PPP3CC)) are thought to be directly or indirectly linked with glutamatergic transmission via NMDA receptors [37
In this current study of the STG, the IPLA2γ gene was significantly up-regulated. IPLA2γ is an isoform of IPLA2 expressed in many tissue types, including the brain [39
]. IPLA2, a key enzyme for phospholipid degradation, is vital for the maintenance and formation of cellular membranes [40
]. IPLA2 is involved in neurotransmission via modulation of phosphorylation of the AMPA glutamate receptors. Endogenous IPLA2 activity limits phosphorylation on serine sites of the AMPA receptor GluR1 subunit, resulting in IPLA2 control over AMPA mediated synaptic transmission in the hippocampus [41
]. There are, however, limited reports of IPLA2 function in other brain regions and the control over AMPA-mediated transmission is yet to be studied in areas such as the STG. IPLA2 is thought to be involved in learning and memory via alterations in hippocampal plasticity [43
], deficiencies of which are key indicators of schizophrenia. Contrary to most reports being related to hippocampal tissue, IPLA2 protein expression has previously been reported to be increased by up to 45% in post-mortem temporal cortex [44
] and in blood serum of individuals with schizophrenia [45
], thus suggesting IPLA2 is worthy of further study.
Furthermore, the microarray analysis also showed that other genes involved in glutamate signaling were altered in the STG in patients with schizophrenia. These include, glutamate receptor, ionotropic, N-methyl D-aspartate 2B (GRIN2B) subunit, also known as NR2B, that had significantly up-regulated expression, and two glutamate receptor interacting proteins glutamate receptor N-methyl D-asparate-associated protein 1 (GRINA) and glutamate receptor interacting protein 2 (GRIP2) showing significant down-regulated expression in schizophrenia subjects. Furthermore, Lin-7b, which was downregulated in the STG of schizophrenia patients by microarray analysis, is highly enriched in post-synaptic densities (PSD) in association with PSD95/NMDA receptor complexes [47
]. More specifically, Lin-7b is a PSD-95/Dlg/ZO-1 (PDZ) domain containing protein, which increases the channel activity of the NR1-NR2B glutamate receptor [48
]. Thus, the evidence from this study supports dysfunction of glutamate transmission in the STG in the pathophysiology of schizophrenia.
Alteration to neurodevelopment and neuronal differentiation in the STG is supported by the significant up-regulation of ATBF1 by microarray and real-time PCR analysis in the STG tissue from individuals with schizophrenia compared to the non-psychiatric controls. This gene is highly expressed in the central nervous system, in particular dopaminergic neurons during neuronal differentiation [49
]. The expression of ATBF1 results in suppression of the nestin gene and activation of the Neurogenic differentiation 1 (NEUROD1) gene, which are responsible for specific neuronal differentiation [50
]. NEUROD1 was up-regulated in a gene expression study of the middle temporal gyrus (MTG) in schizophrenia [11
], which may be a direct result of ATBF1 up-regulation similar to that observed in this study. Overexpression of ATBF1 in neuroblastoma cell lines produces cell cycle arrest, which is thought to occur in vivo as the result of nuclear localization of ATBF1 in differentiating neurons [50
]. Therefore, overexpression of ATBF1 in the STG of patients with schizophrenia might reflect deficits in neuronal differentiation and neurodevelopment
There have been a number of reports indicating dysregulation of presynaptic function genes [51
]. Indeed, our previous study of the amygdala [14
] highlighted dysfunction of the genes coding for proteins in the cytomatrix active zone, a specialized region of the synapse involved in the regulation of vesicle release. RIMS2 codes for a protein that forms part of the cytomatrix of the active zone of synapses and was observed to be upregulated in the amygdala in schizophrenia [14
]. The STG directly connects to the limbic system, of which the amygdala is a constituent. In this study RIMS2 was significantly up-regulated by 1.72 fold, similar to the 2.37 fold change reported in the amygdala. In contrast, Mirnics et al (2001a) reported a downregulation of presynaptic function genes, although those genes were not investigated in this study. RIMS2 modulates Ca2+
-triggered exocytosis and may be overexpressed in the STG as a compensatory mechanism to overcome other presynaptic function deficiencies.
Regulator of G-protein signaling 4 (RGS4) is a schizophrenia candidate gene that was identified by Mirnics and colleagues [30
] and has subsequently been reported in linkage [52
] and convergent functional genomics studies [53
]. The relative expression level of RGS4 mRNA in the STG from the cohort used in this study has been previously reported as significantly down-regulated by an average fold change of -1.93 by relative real-time PCR [31
], which was confirmed in the current study as a significant down-regulation of -1.79 fold by microarray analysis. This down-regulation of RGS4 expression in the STG is in accordance with the decreased expression previously reported in the PFC, motor and visual cortices in schizophrenia [30
] further establishing RGS4 as a schizophrenia candidate gene.
Changes in gene expression in PBLs are emerging as biological reflections of altered expression in the brain as described in [32
]. Consistent with previous studies [54
] 3 genes, MTMR9, EWSR1 and NFκβα were altered in the same direction in the post-mortem STG tissue and the peripheral blood lymphocytes (PBLs) from a separate cohort of individuals with schizophrenia identified in a previous study [32
]. Whether this may lead to the use of PBLs in the development of a biological basis for identifying individuals with schizophrenia awaits further investigation.
The findings of this study and previous gene expression studies involving post-mortem brain tissue are limited by the availability of tissue and the potential of mRNA expression alterations being due to mRNA degradation from variables such as brain pH and PMI [55
]. To control for this potential bias in this study, post-mortem brain tissue from individuals with schizophrenia and non-psychiatric controls were carefully screened and matched for brain pH and PMI in addition to brain hemisphere, age and gender. The tissue pH for pairs 3, 8 and 10 were not as closely matched as the remaining pairs, however there was no correlation between lower pH in the patients, numbers of genes expressed, levels of gene expression on the microarrays or in the relative levels of gene expression measured by real-time PCR. Even though all the variables thought to influence RNA integrity and gene expression were controlled for as much as possible in this study, one cannot rule out the possibility that differences in pH could cause changes in gene expression in post-mortem brain tissue.
In addition to this, another variable which may influence post-mortem tissue gene expression is anti-psychotic medication. The large range of chlorpromazine equivalents (CPE) dosages across the cohort as well as the medication history of one individual being unknown resulted in the inability to perform correlation analyses between anti-psychotic medications and altered gene expression, therefore the influence of anti-psychotic medications on the genes with altered expression in the STG requires further investigation.