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Inducing Schizophrenic Behavior? Researchers Roll Out New DISC1 Mouse

17 September 2007. Schizophrenia researchers may be turned on by a new transgenic mouse. In the September Molecular Psychiatry, Mikhail Pletnikov and colleagues at Johns Hopkins University report that they have engineered mice with a mutant human DISC1 (hDISC1) transgene that can be turned on simply by feeding the mice an antibiotic. Many researchers believe that DISC1, or disrupted in schizophrenia, is the most promising of a growing list of schizophrenia susceptibility genes, and the new mouse should help them study how the timing of mutant DISC1 expression might influence the pathophysiology underlying schizophrenia and related diseases.

DISC1 was discovered in an extended Scottish family with a history of mental illness (see SRF related news story). In affected family members a chromosomal rearrangement results in the truncation of the DISC1 gene. Pletnikov and colleagues mimicked this natural genetic rearrangement, inserting a truncated human DISC1 gene, under the control of the tetracycline responsive element (TRE), into normal mice. The well-known TRE element is turned on by the tetracycline transactivator, tTA. The researchers crossed the DISC1 transgenic animals with a second transgenic line harboring a tTA gene driven by the neuron-specific calcium-calmodulin kinase II (CaMKII) promoter. In double transgenic offspring, neuron-produced tTA activates the mutant DISC1 gene. The whole system can be turned off by simply adding doxycycline to mouse chow, since doxycycline prevents activation of the TRE by tTA.

In keeping with the neuron-specific expression of CaMKII, Pletnikov and colleagues found that human DISC1 expression was restricted to mouse forebrain where it turned up as early as embryonic day 15 (ED 15). Human DISC1 was absent from the brainstem and the cerebellum, and was not detected in astrocytes or microglia, the other two major cell types in the brain. The mutant DISC1 appeared to have no effect on early neurodevelopment, since the architecture and morphology of the brain was normal, as was body weight and breeding and nesting behavior. However, at 9 months the volume of the brain lateral ventricles was significantly increased in double transgenic mice compared to those expressing tTA alone. The growth of lateral ventricles is also a feature of a transgenic mouse recently engineered by Akira Sawa’s group at Johns Hopkins (see SRF related news story), suggesting that this is a feature common to DISC1 mouse models. The authors believe this is due to a decrease in dendritic arborization, rather than neurodegeneration. In support of this, Pletnikov and colleagues found that there was significant loss of neurite complexity in primary cortical neurons isolated from the double transgenic mice and an associated decrease in levels of SNAP-25, a pre-synaptic protein marker. The authors also report that a putative dominant-negative mechanism of the neuronal effects of mutant human DISC1 could be related to its interaction with endogenous mouse DISC1, leading to decreased levels of mouse DISC1 and LIS1, a DISC1 protein partner. Both proteins are involved in neurodevelopment. Barbara Lipska and colleagues at the National Institutes of Health, Bethesda, Maryland, have also found reductions in LIS1 in brain tissue taken from people with schizophrenia (see SRF related news story).

The researchers subjected the mice to a range of tests to judge if they might have behavioral changes that relate to schizophrenia. Unlike the mice developed in the Sawa lab, these mice were no different from controls when tested for prepulse inhibition of the acoustic startle response. They also appeared to have normal olfaction and showed no differences in anxiety level. Male double transgenic mice did show greater spontaneous locomotor activity than controls, however, and they engaged more frequently in aggressive social interactions than controls. In contrast, female mice had some difficulty in a water maze test of spatial memory. It is not clear why these behaviors were sex-dependent, though there are significant sex differences in schizophrenia. One potential weakness of the model, the authors note, is that the mice were bred in a hybrid genetic background, which is likely to increase phenotypic variability. Nevertheless, “the opportunity to regulate expression of mutant hDISC1 is an advantage of the model, facilitating study of the timing of the effects of mutant DISC1 on brain and behavior development,” write the authors.—Tom Fagan.

Pletnikov MV, Ayhan Y, Nikolskaia O, Xu Y, Ovanesov M, Huang H, Mori S, Moran TH, Ross CA. Inducible expression of mutant human DISC1 in mice is associated with brain and behavioral abnormalities reminiscent of schizophrenia. Molecular Psychiatry. 2007. Abstract

Comments on News and Primary Papers
Comment by:  John RoderSteven Clapcote
Submitted 17 September 2007
Posted 17 September 2007

This is a useful model from Pletnikov, Ross, and colleagues, but like all models, it has some limitations. Since DISC1 is known to have a strong role in development and physiology, the development of inducible mutants is necessary to separate the two.

In the TeT-off system used in the paper, mice must be treated with doxycycline for their entire lives to keep the expression of this gene off. Doxycycline must be used at high levels and may have side effects when used this long. The TeT-on system is better because doxycycline is only used transiently for 1 week for maximum induction then washed away. The TeT-on system is also available for the same promoter used in the paper, that of the CaMKII gene.

The phenotype of reduced neurite length was obtained from in vitro neuron cultures, which are prone to artifacts. There are ways of labeling these neurons in vivo for measuring neurite length and spines. The brain phenotype was obtained by MRI. There are ways, such as adding manganese, of enhancing active pathways. This has been done in the bird brain to map song pathways.

The behavioral phenotype was similar to the recent paper from the Sawa group (Hikida et al., 2007) in that it also analyzed a transgenic mouse expressing the same C-terminal truncation of the human DISC1 gene, using the same CaMKII promoter. An important difference in the findings was a reduction of murine DISC1 (50 percent at protein level) in the Pletnikov et al. mice but none in the Sawa group mice. This issue is important because of a recent paper in Cell by the Song group (Duan et al., 2007). In that paper, RNAi was used to reduce wild-type native murine DISC1. Individual neurons with targeted DISC1 knockdown showed accelerated neurite development, greater synapse formation and enhanced excitability. Hippocampal granule cells showed accelerated morphological integration resulting in mispositioning. Unfortunately, in the Song paper they analyzed only cells with complete or no knockdown of DISC1. Partial knockdown vectors were made that achieved 75 percent reduction at the protein level but were not analyzed. Only then would it be possible to compare these morphological data with those from Pletnikov et al., which was a 50 percent reduction. Another difference was that the Song group found that DISC1 needed to interact with Nudel. Pletnikov et al. found normal levels of Nudel in the mice but lower LIS1, which could explain the brain development phenotype.

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Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Anil Malhotra, SRF Advisor
Submitted 21 November 2005
Posted 21 November 2005

The relationship between DISC1 and neuropsychiatric disorders, including schizophrenia, schizoaffective disorder, and bipolar disorder, has now been observed in several studies. Moreover, a number of studies have demonstrated that DISC1 appears to impact neurocognitive function. Nevertheless, the molecular mechanisms by which DISC1 could contribute to impaired CNS function are unclear, and these two papers shed light on this critical issue.

Millar et al. (2005) have followed the same strategy that they so successfully utilized in their initial DISC1 studies, identifying a translocation that associated with a psychotic illness. In contrast to DISC1, in which a pedigree was identified with a number of translocation carriers, this manuscript is based upon the identification of a single translocation carrier, who appears to manifest classic signs of schizophrenia, without evidence of mood dysregulation. Two genes are disrupted by this translocation: cadherin 8 and phosphodiesterase 4B (PDE4B). The researchers' elegant set of experiments provides compelling biological evidence that PDE4B interacts with DISC1 and suggests a mechanism mediated by cAMP for DISC1/PDE4B effects on basic molecular processes underlying learning, memory, and perhaps psychosis. It remains possible that PDE4B (and DISC1) are proteins fundamentally involved in cognitive processes, and that the observed relationship to psychotic illnesses represents a final common pathway of neurocognitive impairment. This would be consistent with data from our group (Lencz et al., in press) demonstrating that verbal memory impairment specifically predicts onset of psychosis in at-risk subjects. Similarly, Burdick et al. (2005) found that our DISC1 risk genotypes (Hodgkinson et al., 2004) were associated with impaired verbal working memory. Finally, Callicott et al. (2005) found that a DISC1 risk SNP, Ser704Cys, predicted hippocampal dysfunction, an SNP which we (DeRosse et al., unpublished data) have also found to link with the primary psychotic symptoms (persecutory delusions) manifested by the patient in the Millar et al. study. This body of evidence supports the notion that these proteins play fundamental roles in the key clinical manifestations of schizophrenia.

Kamiya et al. (2005) provide another potential mechanism for these effects, suggesting that a DISC1 mutation may disrupt cerebral cortical development, hinting that studies examining the role of DISC1 genotypes on brain structure and function in the at-risk schizophrenia pediatric patients may be fruitful.

Taken together, these papers add considerable new data suggesting that DISC1 plays a key role in the etiology of schizophrenia, and places DISC1 at the forefront of the rapidly growing body of schizophrenia candidate genes.

Burdick KE, Hodgkinson CA, Szeszko PR, Lencz T, Ekholm JM, Kane JM, Goldman D, Malhotra AK. DISC1 and neurocognitive function in schizophrenia. Neuroreport 2005; 16(12):1399-1402. Abstract

Callicott JH, Straub RE, Pezawas L, Egan MF, Mattay VS, Hariri AR, Verchinski BA, Meyer-Lindenberg A, Balkissoon R, Kolachana B, Goldberg TE, Weinberger DR. Variation in DISC1 affects hippocampal structure and function and increases risk for schizophrenia. Proc Natl Acad Sci USA 2005; 102(24): 8627-8632. Abstract

Hodgkinson CA, Goldman D, Jaeger J, Persaud S, Kane JM, Lipsky RH, Malhotra AK. Disrupted in Schizophrenia (DISC1): Association with schizophrenia, schizoaffective disorder, and bipolar disorder. Am J Hum Genet 2004; 75:862-872. Abstract

Lencz T, Smith CW, McLaughlin D, Auther A, Nakayama E, Hovey L, Cornblatt BA. Generalized and specific neurocognitive deficits in prodromal schizophrenia. Biological Psychiatry (in press).

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Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Angus Nairn
Submitted 29 December 2005
Posted 31 December 2005
  I recommend the Primary Papers

This study describes an interesting genetic link between PDE4B (phosphodiesterase 4B) and schizophrenia that may be related to a physical interaction with DISC1 (disrupted in schizophrenia 1), another gene associated with the psychiatric disorder. The study is highly suggestive of a role for the PDE4B/DISC1 complex in schizophrenia. However, the mechanistic model suggested by the authors whereby DISC1 sequesters PDE4B in an inactive state seems overly speculative, given the results presented in this paper and in prior studies that have examined the regulation of PDE4B by phosphorylation in the absence of DISC1.

View all comments by Angus Nairn

Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Patricia Estani
Submitted 2 January 2006
Posted 2 January 2006
  I recommend the Primary Papers

Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Ali Mohammad Foroughmand
Submitted 16 December 2006
Posted 16 December 2006
  I recommend the Primary Papers

Related News: Modeling Schizophrenia Phenotypes—DISC1 Transgenic Mouse Debuts

Comment by:  David J. Porteous, SRF AdvisorKirsty Millar
Submitted 2 August 2007
Posted 2 August 2007

Several genetic studies point to involvement of DISC1 in major psychiatric illness, including schizophrenia and bipolar disorder, but to date the only causal variant that has been definitively identified is the translocation between human chromosomes 1 and 11 that co-segregates with major mental illness in a large Scottish family and which directly disrupts the DISC1 gene (Millar at al., 2000). It has been speculated that a truncated form of DISC1 may be expressed from the translocated allele and, if so, that this could exert a dominant-negative effect, but there is no such evidence from studies of the translocation cases. Rather, the evidence from studies of lymphoblastoid cell lines carrying the translocation suggests that haploinsufficiency is the most likely disease mechanism in this family (Millar et al., 2005). The unresolvable caveat to this, of course, is that it has not been possible to determine whether this is true also for the brain. Moreover, it is far from certain that any productive product from the translocation chromosome would be a perfectly truncated protein encoded by all the remaining exons, as opposed to an exon-skip isoform, with or without a hybrid protein component borrowing sequence information from chromosome 11. What does seem likely from other human studies is that additional genetic mechanisms, including missense mutations, altered expression, and possibly also copy number variation, play a role in the generality of DISC1 as a risk factor.

The evidence in support of DISC1 as an important biological determinant across a spectrum of major mental illness has now received a further boost from the study in PNAS by Hikida et al. The Sawa lab describes a transgenic approach where a truncated human DISC1 protein is expressed from a CAMKII promoter. The truncation was designed to mimic the hypothetical truncation arising from the Scottish translocation. This forebrain-specific promoter confers preferential expression of the transgene at neonatal stages, as distinct from the expression of the endogenous protein, which is abundant from embryonic development into adulthood. This model therefore permits investigation of the effect of the truncated protein in the forebrain within a specific developmental window, against a background of endogenous mouse DISC1 expression. Since there is no evidence for production of a truncated protein from the translocated allele, the relevance of this model to psychiatric illness remains unclear. However, on the positive side and from a functional perspective, dominant-negative effects as a consequence of expressing the truncated protein have already been demonstrated in cultured cells, altering the subcellular distribution of DISC1 and interaction with DISC1 partner proteins. Moreover, expression of the truncated form of DISC1 mimics downregulation of DISC1 in vivo, inhibiting migration of neurons in the developing mouse cortex (Kamiya et al., 2005). Thus, this model has the genuine potential to enhance our understanding of the biology of DISC1.

This is, in fact, the third study describing mice expressing modified DISC1 alleles. In the first study, Gogos and colleagues (Kioke et al., 2006) studied the effects of a modified DISC1 allele carrying a spontaneous 25 bp deletion in exon 6 that is present in all 129 mouse strains (Koike et al., 2007; see SRF related news story). This allele additionally has an artificial stop codon in exon 8 and a downstream polyadenylation signal. After back-crossing this mutagenised version of the 129 allele onto a C57Bl6 background, they reported a deficit in an assay of working memory in both heterozygous and homozygous mutants, but other standard behavioral tests were unaltered or unreported, and there were no anatomical, electrophysiological, or pharmacological studies included. In the second study, one led by the Roder laboratory, Toronto, we described two mouse strains with missense mutations in exon 2, Q31L and L100P (Clapcote et al., 2007). Reductions in brain volume, deficits in a range of standard behavioral tests, and responses to pharmacological treatments were reported, which can be summarized as consistent with the 100P mutants displaying schizophrenia-like phenotypes and the 31L mutants, mood disorder-like phenotypes. There is a frustrating dearth of consistent biomarkers for schizophrenia, but one of the best replicated findings is by brain imaging of enlarged ventricles in schizophrenia (also, but less markedly, in bipolar disorder). Adding to the observations of Clapcote et al., arguably the most striking claim by Hikida et al. is for altered ventricular brain volume and reduced brain laterality following neonatal transgenic overexpression of truncated DISC1. Additionally, behavioral tests were reported that overlap in part with those reported earlier by Clapcote et al. That three studies all describe behavioral abnormalities consistent with modeling components of schizophrenia is reassuring. That there are clear differences, too, between the phenotypes should come as no surprise either, given the important differences in terms of genetic lesion and developmental expression. Other mouse models are in the pipeline and they, too, will be welcomed. Indeed, this is very much what is needed for the field to move forward. But we should do so with some caution, paying careful attention to the specific nature of the models, what they can and cannot tell us about DISC1 biology, and what they may or may not tell us about the human condition. Although none of these models relates directly to a known causal variant, it appears that the mouse models concur with the human genetic studies in suggesting that there are likely to be several routes by which DISC1 can perturb brain function leading to characteristics of human mental illness. What we need now is for the human genetic studies to catch up with the mouse so that defined pathognomic variants can be modeled.

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Related News: Modeling Schizophrenia Phenotypes—DISC1 Transgenic Mouse Debuts

Comment by:  John Roder
Submitted 2 August 2007
Posted 2 August 2007

A new mouse model from the Sawa lab strengthens the evidence for the candidate gene DISC1 playing a role in psychosis and mood disorders. This important paper is the first to address one potential disease mechanism, that of a dominant-negative effect. Expression of the C-terminal deletion of human DISC1—which represented the original rearrangement found by the Porteous group in the Scottish families with schizophrenia and depression—in transgenic mice driven by the α CaMKII promoter, first described by Mark Mayford when a postdoctoral fellow in the Kandel lab, leads to mice showing behaviors consistent with schizophrenia and depression, with enlarged lateral ventricles. Since the Sawa group expressed the human C-terminal truncation in mouse with no change in mouse DISC1 levels, they feel this supports a dominant-negative mechanism. More direct experiments are required. For example, create a null mutant mouse for DISC1 and express the full-length and truncated human DISC1 under the influence of their own promoter in transgenic mice using human BACs. Full-length human DISC1 would, hopefully, rescue the null. If so, then a mixture of full-length and truncated DISC1 proteins could be tried. No rescue by the mixture of full-length and truncated proteins would suggest a dominant-negative mechanism.

The Porteous group has shown no detectable DISC1 protein in lymphoblasts from the patients, and put forward the following implicit model. The C-terminal truncation of DISC1 makes the protein unstable and sensitive to degradation, a plausible alternative notion. In my opinion both are likely in different schizophrenia patients with perturbations in DISC1, most of which are alterations other than the C-terminal truncation. Some have SNPs that lead to as yet uncharacterized disease. It has been shown by the Sawa lab that mice with a partial RNAi knockdown of DISC1 show perturbations in brain development, and if aged to 8-12 weeks these mice might have shown behavioral and neuropathological hallmarks of schizophrenia and depression. There is currently no null mutation in the mouse that would address this issue, although DISC1 is one of the genes being targeted in the NIH knockout mouse project. Fortunately, there are now several mouse models—the more the better. The Gogos lab has a 25bp deletion in exon 6 that removes some, but not all isoforms. The Roder lab used a reverse genetic screen of an ENU archive to generate two missense mutants in non-conserved amino acids. The phenotypes of all these lines are nicely summarized in the Sawa paper. This work represents a step forward in our understanding of the DISC1 gene.

View all comments by John Roder