28 November 2007. Based on linkage and genetic association evidence, DTNBP1, the human gene for the dysbindin protein, is generally cited as one of the most promising candidates for a schizophrenia susceptibility gene (see O’Tuathaigh et al., 2007; Riley and Kendler, 2006; Norton et al., 2006). Two recent reports add functional clues about the potential role of the gene in the disease.
In one study, led by Gary Donohoe of Trinity College, Dublin, in collaboration with the laboratory of John Foxe at the Nathan Kline Institute in Orangeburg, New York, schizophrenia patients who were carriers of a dysbindin risk haplotype showed significant deficits in early visual processing as measured by event-related potentials (ERPs). In a second study, Richard Straub and colleagues from the Genes Cognition and Psychosis program led by Daniel Weinberger at the National Institute of Mental Health in Bethesda, Maryland, knocked down dysbindin in neurons in culture by RNA interference. This increased the number of dopamine D2 receptors (DRD2) on the cell surface, which lead to excessive DRD2 signaling. DRD2 is a primary target of antipsychotic drugs, and the authors characterize their results as “the first demonstration of a schizophrenia susceptibility gene exerting a functional effect DRD2 signaling, a pathway that has long been implicated in the illness.”
The DTNBP1 (dystrobrevin binding protein 1) gene on chromosome 6 was first associated with schizophrenia in Irish pedigrees in 2002 (Straub et al., 2002), and it has since been explored in many case-control and family-based studies in diverse populations (see overview and meta-analyses for DTNBP1 in SchizophreniaGene). Postmortem studies of patients with schizophrenia have revealed reduced levels of DTNBP1 mRNA in the prefrontal cortex and midbrain (Weickert et al., 2004), as well as the hippocampus (Weickert et al., 2007). The precise functions of DTNBP1’s protein product, dysbindin, are unknown, but it is widely expressed in the brain, reduced in brains from schizophrenics, and thought to be involved in signaling at both pre- and post-synaptic sites, with particularly prominent expression at glutamatergic synapses (Talbot et al., 2004; 2006).
One known function of dysbindin is its crucial role in the protein complex known as BLOC-1 (biogenesis of lysosome-related organelles complex 1), which is thought to be involved in the membrane trafficking and degradation of synaptic vesicles via an endosomal-lysosomal pathway. In addition to DTNBP1, the BLOC-1 complex gene BLOC1S3 has been reported to be associated with schizophrenia (Morris et al., 2007). Another recent clue comes from a study at Osaka University of the dysbindin knockout mouse sandy (sdy), which showed significantly higher ratios of homovanillic acid (a dopamine metabolite) to dopamine in the cortex and hippocampus, an indication of high dopamine turnover that may be caused by increased dopamine transmission in these regions (Murotani et al., 2007).
Effects on a sensory endophenotype
The CTCTAC and C-A-T haplotypes and several SNPs in DTNBP1 have recently been associated with measures of IQ, cognitive decline, and spatial working memory in schizophrenia (Burdick et al., 2007; Zinkstok et al., 2007; Donohoe et al., 2007), but the recent study by Donohoe and colleagues, published online October 16 in Biological Psychiatry, is one of the first to explore whether dysbindin risk variants have effects on sensory processing in schizophrenic patients.
In this study, the researchers conducted reaction-time experiments while obtaining continuous EEG measures from 26 individuals meeting DSM-IV criteria for schizophrenia, 14 of whom were carriers of the C-A-T dysbindin risk haplotype. In each experimental block, subjects were presented with 100 “isolated-check” stimuli (an 8 8 array of gray squares on a white background) interspersed with 40 line drawings of two different, but similar-looking, animals. Subjects were asked to press a button upon seeing a target animal, a task that ensured that they attended carefully to all stimuli, including the isolated-check stimuli.
The researchers’ real aim was to measure the amplitude of the so-called P1 ERP, an “automatic” response seen in occipital and parietal sensory regions between 75 and 110 milliseconds after the presentation of visual stimuli. The P1 has been put forth as a promising endophenotype for schizophrenia, as several studies (e.g., Yeap et al., 2006) have shown that both schizophrenic patients and their unaffected relatives show deficits in the P1 response. Parieto-occipital cortical circuits involved in early visual processing depend on glutamate, and the Donohoe group postulated that the P1 response would reflect DTNBP1-related deficits in glutamatergic signaling.
The reaction-time task was irrelevant to the P1 response, so the group only analyzed P1 responses to the isolated-check stimuli. They found significant overall deficits in the P1 response in subjects carrying the C-A-T risk haplotype, and particularly pronounced P1 deficits in posterior brain regions in the risk group.
In conclusion, the authors write, “the reduced P1 [response] associated with the dysbindin risk haplotype . . . presents functional confirmation of its deleterious effect on brain activity, making it likely to be part of the neurobiology of schizophrenia.”
A link to dopamine signaling?
In the in vitro study led by Straub and first author Yukihiko Iizuka, SH-SY5Y human neuroblastoma cells and primary cortical neurons from embryonic day 18 rats were transfected with DTNBP1 siRNA. This was quite effective at tamping down dysbindin expression: in both cell types, immunocytochemical measures showed dysbindin reductions of 60 percent and 70 percent, respectively, compared to control cells transfected with random siRNA.
When the researchers measured basal levels of surface expression of DRD2 with flow cytometry, they found approximately 30 percent increases in the receptor in transfected cells compared to controls. To determine whether this basal level overexpression was accompanied by a block of dopamine-induced receptor internalization, which is a characteristic regulatory mechanism of DRD2 signaling, Iizuka and colleagues treated control and transfected cells with dopamine. In control cells, dopamine exposure reduced surface expression of DRD2 by 18 percent, but there was no change in cells transfected with DTNBP1 siRNA. Confocal microscopy of dopamine-treated cells corroborated these results. In contrast, no difference in surface expression or blockade of internalization of D1 (DRD1) receptors was observed. Notably, similar effects on DA receptors were found when expression of the protein muted, dysbindin's binding partner in the BLOC-1 complex, was knocked down with siRNA.
To explore the functional consequences of the observed surface overexpression of DRD2, the NIMH team treated DTNBP1 siRNA-transfected rat primary neurons with the DRD2 agonist quinpirole. Normally, DRD2 activates the Gi protein, which inhibits adenylate cyclase, thereby reducing production of cAMP and phosphorylation of the cAMP response element binding protein (CREB). When stimulated with quinpirole, cells transfected with DTNBP1 siRNA showed significantly reduced CREB phosphorylation, indicating that overexpression of surface DRD2 had indeed resulted in excessive intracellular signaling downstream of the Gi inhibition of adenylate cyclase. This increase was blocked by administration of the DRD2 antagonist haloperidol, suggesting that the downstream effects of DTNBP1 siRNA were via DRD2.
Because robust compensatory mechanisms that regulate DRD2 expression are likely to be in place by adulthood, Iizuka and colleagues stress that the DRD2 overexpression they observed may exert important effects during development that could contribute to the adult schizophrenic phenotype. In particular, they cite a study of transgenic mice in which behavioral impairments induced by DRD2 overexpression during development persisted even when this overexpression was reversed in the adult (see SRF related news story).
“The novel result reported here,” they write, is that the dysbindin deficiency and the resultant block of DRD2 internalization they observed “can increase the level of cell surface DRD2 and enhance the strength of DRD2 signaling while leaving DRD1 levels unchanged. This may be one of the mechanisms underlying some of the dopaminergic disturbances implicated in schizophrenia that are benefited by drugs that antagonize DRD2."—Peter Farley and Hakon Heimer.
Donohoe G, Morris DW, De Sanctis P, Magno E, Montesi JL, Garavan HP, Robertson IH, Javitt DC, Gill M, Corvin AP, Foxe JJ. Early visual processing deficits in dysbindin-associated schizophrenia. Biol Psychiatry. 2007 Oct 16. Abstract
Iizuka Y, Sei Y, Weinberger DR, Straub RE. Evidence that the BLOC-1 protein dysbindin modulates dopamine D2 receptor internalization and signaling but not D1 internalization. J Neurosci. 2007 Nov 7;27(45):12390-5. Abstract