25 June 2007. Several lines of study, in particular the assessment of gene expression in postmortem tissue, suggest that deficits in GABA neurotransmission may be intimately involved in the pathophysiology of schizophrenia. A trio of recent studies support this idea.
The most recent study, published in the June 12 issue of PNAS by Francine Benes at McLean Hospital, Belmont, Massachusetts, describes schizophrenia-specific, as well as bipolar disorder-specific, expression patterns of genes associated with the GABA synthesizing enzyme GAD67 in subregions and specific layers of hippocampus. The researchers suggest that such regional expression endophenotypes might help distinguish the roots of GABA-related dysfunction in schizophrenia from those of bipolar disorder.
In one of two earlier studies published online May 1 in Molecular Psychiatry, Richard Straub and colleagues at the NIMH also focused on GAD67. They report that inherited genetic polymorphisms in the gene for the enzyme are found more frequently in schizophrenia patients and their relatives. These results suggest that altered GABA levels may contribute to the disease, and they also hint that genetic variations affecting dopamine-based neurotransmission may exacerbate that pathology.
The second study in Molecular Psychiatry, led by David Lewis at the University of Pittsburgh, Pennsylvania, examines a wider range of genes that code for proteins related to GABA biology, specifically in dorsolateral prefrontal cortex (DLPFC). First author Takanori Hashimoto and colleagues report alterations in schizophrenia in genes involved in GABA synthesis, in genes for certain neuromodulators, and in genes that code for subunits of the GABAA receptor. This schizophrenia GABA "transcriptome" directs attention to specific subpopulations of DLPFC GABAergic neurons and raises the possibility of disease-related effects on both synaptic and extrasynaptic GABAergic signaling in DLPFC pyramidal neurons.
What's at the 5' End of Your GAD?
The evidence of GABAergic dysfunction in schizophrenia is multifaceted (as reviewed succinctly by Straub and colleagues in their introduction; see also Akbarian and Huang, 2006), and several studies have looked to genetic variations in the GAD67 gene, GAD1, to explain this, with mixed results to date (see entries in SchizophreniaGene. In their study, Straub and colleagues focused on genetic variations that are likely to affect GAD67 production, concentrating on single nucleotide polymorphisms (SNPs) near the promoter region, or on/off switch of the gene. They genotyped 19 polymorphisms in two independent data sets comprising parent-child trios. Data from the first, the NIMH’s Clinical Brain Disorders Branch’s sibling study database, which consists of samples of mixed ethnicity (though predominantly European American), revealed three SNPs that were significantly associated with schizophrenia, but only in female children. In contrast, data from the NIMH Genetics Initiative sample set (samples from only European American families) revealed six different SNPs that significantly associated with schizophrenia in females and yet another six SNPs that associate with the disease in males.
Because the genetic component of schizophrenia is predicted to be complex and dependent on variations in multiple genes, the researchers delved more deeply into the data, looking to see if any of the GAD1 variations are more common in individuals who have genetic variations in the catechol-O-methyltransferase (COMT) gene, which has also been linked to schizophrenia. One particular polymorphism in the COMT gene introduces either a methionine or valine at position 158 of the enzyme. The valine isoform is more active, and it has been suggested that this variant may increase susceptibility to schizophrenia because of more rapid degradation of dopamine in the brain. It is interesting, therefore, that when Straub and colleagues stratified the data by COMT genotype, they found additional GAD1 SNPs that associated with the disease—eight of the 19 GAD1 SNPs turned up a positive association with schizophrenia in families with the Val/Val genotype.
How might these GAD1 SNPs increase susceptibility for the disease? Given that they are located near the regulatory region of the gene, they might affect transcription and GAD67 levels. To test this, the researchers looked at GAD1 expression in postmortem brain samples. They found that one of the SNPs was associated with reduced levels of GAD1 mRNA in the DLPFC. How the other SNPs may affect the GAD1 gene or influence schizophrenia is unclear, but the authors found that genotype associated with cognitive performance. When they examined 15 different neurocognitive phenotypes, they found that 11 of them were associated with at least one of the 19 SNPs—there were both positive and negative associations. They also found that one of three SNPs tested associated with greater activation of the DLPFC, as judged by functional magnetic resonance imaging, during one of the cognitive tests.
The GABA-related Transcriptome
Hashimoto and colleagues looked more broadly at expression of GABA-related genes, using custom DNA arrays that detect 85 different GABA-related messenger RNAs, including those coding for GAD and other proteins involved in GABA synthesis, uptake, degradation, and binding. The researchers applied these microarrays to compare DLPFC gene expression profiles among 14 schizophrenia patient postmortem brain samples and age- and sex-matched control tissue.
The researchers report that expression of 10 of the 85 genes was significantly different in patient tissue. For all 10, expression was higher in control samples. These genes code for three categories of protein: neuropeptides released by GABA neurons; GABA receptor subunits; and presynaptic regulators of GABA. The last included GAD67—its mRNA levels were about 33 percent lower in schizophrenia samples. The researchers report the biggest difference was in expression of the neuropeptide somatostatin (SST), which was 1.6-fold lower in schizophrenia samples. Expression of two other neuropeptides, cholecystokinin (CCK) and neuropeptide Y (NPY), was also lower in patient samples. The researchers validated the microarray data with real-time quantitative PCR and in situ hybridization studies in an extended cohort of 23 pairs.
Gene expression changes in GAD67 were closely tracked by SST, NPY, and CCK expression. Since GABAergic neurons expressing CCK form a separate subpopulation from one coexpressing SST and NPY, Hashimoto and colleagues propose that schizophrenia-related deficits in GABAergic transmission could affect two distinct neuronal populations. These two populations are also distinct from the parvalbumin-containing GABAergic cells that the Lewis lab has reported on previously (which express less parvalbumin and no detectable GAD67 in schizophrenia; for review, see Lewis et al., 2005).
The researchers also report that expression of the δ subunit of the GABAA receptor, which is only found in extrasynaptic receptors, as well as of subunits found in synaptic receptors, are lower in the patient sample; they thus surmise that schizophrenia DLPFC pathophysiology features alterations not just in synaptic GABA neurotransmission, but also in signaling via GABA receptors located outside synapses. This further suggests, the authors write, the presence in schizophrenia of both "decreased synaptic (phasic) and extrasynaptic (tonic) inhibition … in pyramidal neuron dendrites."
Two Paths to GABA Dysfunction?
In their PNAS paper, Benes and colleagues describe the continuation of their work on GABAergic dysfunction in limbic cortical structures such as hippocampus and anterior cingulate gyrus, particularly in the context of comparing GABAergic pathophysiology in schizophrenia and bipolar disorder (for review, see Benes and Berretta, 2001). In their current study, they apply a targeted approach to postmortem tissue analysis, isolating microscopic samples of hippocampal tissue with laser-capture microdissection. The researchers then used gene expression profiling to find changes in a network of proteins linked to GAD67, which were found to be downregulated in hippocampus in both disorders in previous studies. For sampling, the researchers used postmortem hippocampal tissue from schizophrenia, bipolar disorder, and normal control brains—seven samples in each case.
Their findings highlight the value of laser-capture microdissection (LCM). While their analysis suggests no difference between GAD67 expression in schizophrenia versus control when total hippocampus was examined, and only a 1.8-fold reduction in bipolar disorder, robust differences emerged when microscopic samples were analyzed. In regions CA2 and CA3, and specifically in the stratum oriens, the second most superficial hippocampal layer, which is home to GABAergic neurons, they found GAD67 was almost 10-fold lower in bipolar disorder compared to control. The difference in schizophrenia samples—nearly threefold lower—was not so dramatic in stratum oriens, and was similar to decreases found in deeper hippocampal layers (stratum radiatum and stratum pyramidale) in both disorders. In region CA1, only one difference was detected in GAD67 expression between schizophrenia or bipolar groups and control—a threefold decrease in schizophrenia versus control in the stratum oriens.
Focusing on the stratum oriens, which featured expression abnormalities in both disorders, Benes and colleagues found that 18 of 25 GAD67 network genes have altered expression patterns in schizophrenia or bipolar disorder. Some are involved in neurotransmission, namely, glutamate receptor subunits, but transcription factors, cytokines, and chromatin modifying proteins were also in the mix. Interestingly, the expression profiles were not the same in schizophrenia and bipolar disorder, and the authors draw attention to several of these discrepancies—transcripts for proteins involved in epigenetic modification of genes were upregulated in schizophrenia but not bipolar disorder, whereas transcripts for cell differentiation factors were altered only in bipolar disorder. This leads the authors to suggest that, "a common cellular phenotype in SZ and BD, the decreased expression of GAD67 in GABAergic interneurons, may involve different underlying molecular mechanisms that are in part related to susceptibility genes for the respective two disorders, as well as activity-dependent changes arising from specific afferent inputs to these interneurons."—Tom Fagan.
Straub RE, Lipska BK, Egan MF, Goldberg TE, Callicott JH, Mayhew MB, Vakkalanka RK, Kolachana BS, Kleinman JE, Weinberger DR. Allelic variation in GAD1 (GAD67) is associated with schizophrenia and influences cortical function and gene expression. Molecular Psychiatry. 2007, May 1; online publication. Abstract
Hashimoto T, Arion D, Unger T, Maldonado-Aviles JG, Morris HM, Volk DW, Mirnics K, Lewis DA. Alterations in GABA-related transcriptome in the dorsolateral prefrontal cortex of subjects with schizophrenia. Molecular Psychiatry. 2007, May 1; online publication. Abstract
Benes FM, Lim B, Matzilevich D, Walsh JP, Subburaju S, Minns M. Regulation of the GABA cell phenotype in hippocampus of schizophrenics and bipolar. PNAS. 2007, June 4. Abstract