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DISC1 2010—DISC1 and Endophenotypes

As part of our ongoing coverage of DISC1 2010, held 3-6 September 2010, in Edinburgh, the United Kingdom, we bring you a meeting missive from Rosie Walker, a graduate student at the University of Edinburgh.


17 September 2010. The third session of the conference—with David St Clair leading Part two of the Genotype and Phenotype theme—was opened on Saturday afternoon by Daniel Weinberger of the National Institute of Mental Health. Weinberger began by explaining the unique opportunity presented by DISC1 as a window into the brain mechanisms that underlie psychopathology: the DISC1 chromosomal abnormality observed in the Scottish t(1;11) is the only rare chromosomal abnormality that is not associated with any phenotype but psychopathology. Yet progress in understanding the function of DISC1 in the brain has been slow, and the study of DISC1 has been plagued by the same questions that are being asked in the psychiatric genetics field more generally: why have genes for psychiatric illness been so controversial? And why are clinical associations so weak? Weinberger answered both of these questions by stating that “genes do not encode for psychiatric illness,” and emphasized the benefits of studying intermediate phenotypes at the cellular and systems level where greater penetrance and, therefore, larger effect sizes are likely to be observed.

The validity of cognition as an intermediate phenotype for schizophrenia was highlighted using data from a recent collaboration between the NIMH group and Toulopoulou and colleagues at the Institute of Psychiatry, London (2010), which finds that a large proportion of the phenotypic correlation between schizophrenia and cognition is attributable to shared genetic effects. Weinberger proceeded to present results from a series of imaging experiments from his group that have investigated brain activity during working memory tasks. These experiments have revealed differences in hippocampal and prefrontal brain activation between carriers of different alleles of SNPs in DISC1 and other schizophrenia-associated genes (Callicott et al., 2005; Tan et al., 2008), as well as altered coupling between hippocampal and prefrontal regions in carriers of a schizophrenia risk variant in ZNF804A (Esslinger et al., 2009; see SRF related news story). Recent unpublished work from Weinberger's group has found an abnormal pattern of hippocampal and prefrontal coupling during a working memory task in patients with schizophrenia, and a phenotype intermediate between schizophrenic and control subjects in healthy siblings of schizophrenic patients. Decreased phase uncoupling observed in schizophrenic patients and, to a lesser extent, their healthy siblings, suggests that increased interference might diminish working memory function in these individuals.

Moving to intermediate phenotypes at the cellular level, Weinberger stated that genetic risk is ultimately due to cell morphology. In support of this claim, he cited another study from his group (Nakata et al., 2009), in which an altered distribution of short DISC1 transcripts were found in the brains of patients with schizophrenia.

Next to speak was William Hennah of the Academy of Finland. Continuing the theme of “Genotype and Phenotype,” Hennah spoke about genetic studies in the Finnish population. Recent studies in this population have supported the role of DISC1 interactors as risk factors for schizophrenia (Tomppo et al., 2009) and indicated the importance of studying interactions between SNPs for identifying conditional risk variants (Hennah et al., 2009). Further interaction analysis of DISC1 pathway genes is in progress, and unpublished work from Hennah’s group has identified genes, including the lactase (LCT) gene, that interact with specific DISC1 variants to increase levels of anhedonia, a personality trait associated with schizophrenia.

Next, Hennah turned to ongoing work with an obvious clinical application: the association of DISC1 pathway variants with psychoactive drug cessation, which is typically due to adverse side effects. Looking at 10-year prescription data, Hennah has found that cessation of several drugs is associated with variants across genes in the DISC1 pathway. It is hoped that by looking at overlaps between the associated genes, clues to drug functions will be revealed. Whilst these results are from a small sample and must be considered preliminary, they raise the interesting prospect of improved drug efficacy based on analysis of genotype at key markers in DISC1 pathway genes.

In the following talk, Svenja Trossbach of Heinrich-Heine University Düsseldorf, Germany, presented work investigating the link between DISC1 and nicotine. Increased smoking in schizophrenic patients compared to the general population is a well-documented phenomenon, and it has been shown that the schizophrenia-associated deficit in sensory gating, as measured by prepulse inhibition (PPI), can be ameliorated by nicotine (Kumari et al., 1997). It has been proposed that the effect of nicotine on PPI is mediated by α7 nicotinic acetylcholine receptors (α7 nAChR; Suemaru et al., 2004), the expression of which is regulated by neuregulin-1 (NRG1) type 3 (Hancock et al., 2008). Following on from recent work indicating that NRG1 regulates DISC1 (Seshadri et al., 2010; see SRF related news story), Trossbach asked whether there is a direct interaction between DISC1 and nicotine. Pull-down assays in rat hippocampal tissue and pharmacological studies in cultured cells yielded evidence consistent with an interaction. In humans, an antibody raised against the C-terminus of DISC1 indicated decreased DISC1 in peripheral blood mononuclear cells (PBMC) of schizophrenic patients compared with healthy controls. This decrease was further exacerbated by smoking, with schizophrenic smokers showing the lowest level of immunoreactivity. Trossbach concluded by stating that nicotine and the activity of nicotinic acetylcholine receptors has an effect on DISC1 expression, and raised the interesting prospect of using detection of DISC1 expression in PBMC as a biomarker for schizophrenia.

The session was brought to a close by Xianjin Zhou of the University of California, San Diego. In a recent publication, Zhou and colleagues identified a novel gene, named Boymaw, on chromosome 11 that is disrupted by the t(1;11) translocation found in the original Scottish family (Zhou et al., 2010). DISC1-Boymaw fusion transcripts generated to mimic the effect of the t(1;11) translocation were found to produce insoluble proteins in in vitro cell transfections, reminiscent of the insoluble DISC1 proteins found in the postmortem brains of some patients with schizophrenia (Leliveld et al., 2008). Zhou and colleagues are now in the process of generating chimeric mice carrying a conditional knock-in of the DISC1-Boymaw fusion gene, with the hope of producing a general model for assessing the consequences of insoluble DISC1.—Rosie Walker.

Comments on Related News


Related News: Schizophrenia-associated Variant in ZNF804A Gene Affects Brain Connectivity

Comment by:  James WaltersMichael Owen (SRF Advisor)
Submitted 3 June 2009
Posted 3 June 2009

Andreas Meyer-Lindenberg’s group examine the association between a single nucleotide polymorphism (SNP), rs1344706 in gene ZNF804A, recently identified as a risk factor for schizophrenia in a genome-wide association study (GWAS) (O'Donovan et al., 2008) and functional connectivity as measured by fMRI. The attraction of this polymorphism for a study of this kind is twofold. First, statistically speaking it is the most robust SNP association with schizophrenia reported to date. Second, because a single variant shows strong evidence for association, which is not the case for other reported associations, it is possible to specify a priori for the gene in question directional hypotheses in relation to potential neurocognitive correlates. This militates against the generation of false positives through the testing of multiple SNPs and haplotypes which has rendered problematic the interpretation of at least some previous genetic imaging studies (Walters and Owen, 2007). The function of ZNF804A is unknown but the fact that it contains a zinc finger domain suggests that it may be a transcription factor. It is hoped that the characterization of the actions of SNPs identified by GWAS will identify new pathogenic mechanisms of psychosis. One way in which this can be achieved is via approaches such as that taken in this article.

Esslinger et al. report variations in functional connectivity in 115 healthy individuals according to rs1344706 risk variant status. Given the association of ZNF804A with both schizophrenia and bipolar disorder they employed two fMRI tasks thought to be sensitive to altered function in these disorders: the N Back (2back) task is sensitive to deficits of dorsolateral prefrontal cortex (DLPFC) function in schizophrenia and an emotional face-matching task is linked with amygdala function and thought to be relevant to mood disorder. They compared the activity in these regions and functional connectivity (using time-series correlation) between the three rs1344706 genotype groups.

No differences between genotype groups were found for activation, but the authors did identify altered connectivity with the most activated DLPFC locale. Risk-allele carriers were shown to exhibit a lack of uncoupling of activity (increased functional connectivity) between the right DLPFC and left hippocampus during the 2-back task as well as decreased connectivity within right DLPFC and between right and left DLPFC. Risk variant carriers also showed wide ranging increased connectivity between right amygdala and other anatomical regions. The majority of these findings showed a risk allele dose effect.

The increased DLPFC/hippocampus functional connectivity in carriers of the risk allele is potentially the most interesting finding given that Meyer-Lindenberg’s group has previously shown that those with schizophrenia show increased functional connectivity between DLPFC and hippocampus during working memory (Meyer-Lindenberg et al., 2005). Notes of caution in this regard are that 1) the biological, anatomical or functional significance of fMRI determined functional connectivity is yet to be established and 2) other functional connectivity studies in schizophrenia have produced conflicting results Lawrie et al., 2002. Nonetheless, it is interesting that rs1344706 may affect co-ordination of activity between these two brain regions given their seeming importance in psychotic conditions. The significance of these findings to cognitive deficits and other symptom domains needs further investigation particularly as others have postulated dysconnectivity has more relevance to first rank psychotic symptoms (Stephan et al., 2009).

It is likely that genome-wide association approaches will continue to identify genes with unknown neural function and so approaches such as this are likely to be a valuable way of identifying the biological/neural pathways that involve these genes. It is also imperative that as in this study methodology is employed to allow for multiple testing and also that negative findings are reported. We would also suggest caution until these findings are replicated. As well as such approaches in humans, it is also important to investigate the effects of identified variants at other levels of analysis from gene expression to behavioural genetics work. Finally we find it reassuring that GWAS approaches seem to be successful in identifying risk variants whose functions can be investigated using methods such as that taken by Esslinger et al.

References:

O'Donovan MC, Craddock N, Norton N, et al. Identification of loci associated with schizophrenia by genome-wide association and follow-up. Nature Genetics. 2008;40(9):1053-1055. Abstract

Walters JT, Owen MJ. Endophenotypes in psychiatric genetics. Mol Psychiatry. 2007;12(10):886-890. Abstract

Meyer-Lindenberg AS, Olsen RK, Kohn PD, et al. Regionally Specific Disturbance of Dorsolateral Prefrontal-Hippocampal Functional Connectivity in Schizophrenia. Archives of General Psychiatry. 2005;62(4):379-386. Abstract

Lawrie SM, Buechel C, Whalley HC, Frith CD, Friston KJ, Johnstone EC. Reduced frontotemporal functional connectivity in schizophrenia associated with auditory hallucinations. Biological Psychiatry. 2002;51(12):1008-1011. Abstract

Stephan KE, Friston KJ, Frith CD. Dysconnection in Schizophrenia: From Abnormal Synaptic Plasticity to Failures of Self-monitoring. Schizophr Bull. 2009;35(3):509-527. Abstract

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Related News: Convergence Zone: NRG1 Signaling Linked to DISC1 Expression

Comment by:  Amanda Jayne Law, SRF Advisor
Submitted 19 April 2010
Posted 19 April 2010

The study of Seshadri, Sawa, and colleagues presents novel evidence of a potential biological link between two lead schizophrenia susceptibility genes, NRG1 and DISC1. The principal finding of the study is that NRG1 (EGFβ) regulates expression of a specific isoform of DISC1, mediated via ErbB2/3 but not ErbB4. The influence of NRG1 on expression of the DISC1 isoform was confirmed in a variety of in-vitro and in-vivo models. Specifically, the authors report (using Western blotting with the DISC1 antibodies: D27 and mExon3), that treatment with NRG1 (and NRG2), but not NRG3, increases levels of DISC1 immunoreactivity at 130 kDa in immature and mature rat primary neuron cultures. Interestingly, NRG1 (or NRG2) had no effect on expression of the previously reported full-length DISC1 immunoreactive bands of 100-105 kDa. Convincingly, reduction of the 130 kDa DISC1 band was observed in BACE1 -/- and NRG1 +/- mice, both of which have reduced NRG1 signaling. Taken together, these findings suggest that NRG1 signaling regulates expression of a unique 130 kDa DISC1 protein.

This is an important and thoughtful paper, but there are some details that raise questions about the interpretation of the results. Interestingly, two previous studies that characterized the D27 (and mExon3) antibody in mouse brain (Schurov et al., 2004; Ishizuka et al., 2007) failed to report the 130 kDa band described here. Ishizuka et al. reported that immunoprecipitation with the mExon3 antibody followed by detection with the D27 antibody recognized two primary signals (100 and 105 kDa), thought to correspond to full-length DISC1. In contrast, in the present study the authors report that immunoprecipitation of neuronal lysates using mExon3, followed by Western blotting with D27, consistently identifies an additional 130 kDa band (Fig. S2B), which is also present in the P0 mouse cortex (Fig 3C). Whilst it is not clear what accounts for these apparent differences in signal detection of the 130 kDa band using the same antibodies, factors such as species specificity (rat vs. mouse), tissue type, and developmental stage are likely relevant. Such factors are important considerations for future work. Similarly, it will be crucial to determine whether the 130 kDa band is present in human brain and how it relates to risk for schizophrenia. Of final note, the authors performed extensive experimentation in an attempt to confirm the identity of the 130 kDa band (including successful knockdown by a previously characterized RNAi to DISC1), but interestingly they fail to identify any DISC1 sequence in the 130 kDa signal using mass spectrometry (see discussion). In light of this, it is paramount that future studies determine exactly what the 130 kDa proposed DISC1 band represents (i.e., a novel splice isoform, post-transcriptionally modified protein, etc.), given that NRG1’s effects are specifically related to this variant.

In conclusion, this study provides intriguing evidence of a potential molecular link between NRG1 and DISC1, but at present, the interpretation of the results rests on an immunoblot band of unknown identify.

References:

Schurov IL, Handford EJ, Brandon NJ, Whiting PJ. Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment. Mol Psychiatry . 2004 Dec 1 ; 9(12):1100-10. Abstract

Ishizuka K, Chen J, Taya S, Li W, Millar JK, Xu Y, Clapcote SJ, Hookway C, Morita M, Kamiya A, Tomoda T, Lipska BK, Roder JC, Pletnikov M, Porteous D, Silva AJ, Cannon TD, Kaibuchi K, Brandon NJ, Weinberger DR, Sawa A. Evidence that many of the DISC1 isoforms in C57BL/6J mice are also expressed in 129S6/SvEv mice. Mol Psychiatry . 2007 Oct ; 12(10):897-9. Abstract

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Related News: Convergence Zone: NRG1 Signaling Linked to DISC1 Expression

Comment by:  Alexander Arguello
Submitted 3 May 2010
Posted 3 May 2010

This paper raises an interesting issue. It is unclear how an immuno band that has no DISC1 sequences can result from "alternative splicing or post-translational modification." Could someone provide a mechanistic account, at the molecular level, of how this may be possible? To support that this band is DISC1, at least some DISC1 sequence should have been detected. This issue could be related to the non-specific cross-reactivity of many DISC1 antibodies (see Kvajo et al., 2008 for a discussion) and now also raises the possibility of off-target effects of DISC1 RNAi.

Resolving these issues will be paramount for making meaningful insights into how variations in DISC1 contribute to psychotic disorders.

References:

Kvajo M, McKellar H, Arguello PA, Drew LJ, Moore H, MacDermott AB, Karayiorgou M, Gogos JA. A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition. Proc Natl Acad Sci U S A. 2008 May 13;105(19):7076-81. Abstract

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Related News: Convergence Zone: NRG1 Signaling Linked to DISC1 Expression

Comment by:  Saurav SeshadriAtsushi KamiyaEva AntonAkira Sawa (SRF Advisor)
Submitted 4 May 2010
Posted 4 May 2010

We are very glad to see Dr. Law’s thoughtful and very supportive comments on the work by Seshadri et al. We share the recognition, as we pointed out in the discussion of the paper, that identification of 130 kDa signal at the molecular level is an important future question. To confirm the authenticity of immunoreactivity, we tested if the 130 kDa signal is immunoprecipitated and immunoblotted by different DISC1 antibodies. Similar immunoreactive approaches have been used earlier to distinguish DISC1 isoforms, including a 71 kDa isoform in association with PDE4 (Millar et al., 2005; Chubb et al., 2008). Knockout mice deficient in DISC1 that we have recently generated (unpublished) were used for evaluating the specificity of several antibodies against DISC1 (Schurov et al., 2004; Ishizuka et al., 2007; Duan et al., 2007; Koike et al., 2006). Loss of this immunoreactivity by authentic shRNAs further supports this idea. The sequences of shRNAs are the same as those used in the study by Mao et al. (Mao et al., 2009) to demonstrate that DISC1 may be involved in progenitor cell proliferation.

Of note, mass spectrometry cannot be an ultimate confirmation, because with this technique it is hard to distinguish the signals from two adjacent or overlapped bands in Western blots of 1D gels, one of which is real and the other not. Therefore, regardless of our initial mass spectrometry analysis (even if one finds sequences of the target protein), validation with both immunoprecipitation and RNAi is required to draw a conclusion on the identity of 130 kDa signal. In the study by Seshadri et al., these two ways of validation were successfully made.

Furthermore, whether or not this 130 kDa isoform is also expressed in humans is a critical question. It is also very important to consider context-dependent expression of unique isoforms of genetic susceptibility factors. This unique form (130 kDa) is likely to be in that category; thus, as Dr. Law suggested, comparative analysis is very useful. Further analysis of the genesis, function, and processing of various DISC1 isoforms in the brain will be a worthy pursuit in the context of schizophrenia.

References:

Millar JK, Pickard BS, Mackie S, James R, Christie S, Buchanan SR, Malloy MP, Chubb JE, Huston E, Baillie GS, Thomson PA, Hill EV, Brandon NJ, Rain JC, Camargo LM, Whiting PJ, Houslay MD, Blackwood DH, Muir WJ, Porteous DJ. DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling. Science. 2005 Nov 18 ; 310(5751):1187-91. Abstract

Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. The DISC locus in psychiatric illness. Mol Psychiatry. 2008 Jan 1 ; 13(1):36-64. Abstract

Schurov IL, Handford EJ, Brandon NJ, Whiting PJ. Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment. Mol Psychiatry. 2004 Dec 1 ; 9(12):1100-10. Abstract

Ishizuka K, Chen J, Taya S, Li W, Millar JK, Xu Y, Clapcote SJ, Hookway C, Morita M, Kamiya A, Tomoda T, Lipska BK, Roder JC, Pletnikov M, Porteous D, Silva AJ, Cannon TD, Kaibuchi K, Brandon NJ, Weinberger DR, Sawa A. Evidence that many of the DISC1 isoforms in C57BL/6J mice are also expressed in 129S6/SvEv mice. Mol Psychiatry. 2007 Oct 1 ; 12(10):897-9. Abstract

Duan X, Chang JH, Ge S, Faulkner RL, Kim JY, Kitabatake Y, Liu XB, Yang CH, Jordan JD, Ma DK, Liu CY, Ganesan S, Cheng HJ, Ming GL, Lu B, Song H. Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell. 2007 Sep 21 ; 130(6):1146-58. Abstract

Koike H, Arguello PA, Kvajo M, Karayiorgou M, Gogos JA. Disc1 is mutated in the 129S6/SvEv strain and modulates working memory in mice. Proc Natl Acad Sci U S A. 2006 Mar 7 ; 103(10):3693-7. Abstract

Mao Y, Ge X, Frank CL, Madison JM, Koehler AN, Doud MK, Tassa C, Berry EM, Soda T, Singh KK, Biechele T, Petryshen TL, Moon RT, Haggarty SJ, Tsai LH. Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell. 2009 Mar 20 ; 136(6):1017-31. Abstract

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