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Genetics and Schizophrenia—Calcineurin Connection Grows

21 February 2007. A report published in the February 20 issue of PNAS suggests a genetic association between schizophrenia and a gene encoding the protein early growth response 3 (EGR3), which plays a key role in neuronal development. EGR3 is one of a family of transcription factors that regulate gene activity, and it is under the control of calcineurin, a protein that has previously been linked to schizophrenia. The finding, from the labs of Takeo Yoshikawa at the RIKEN Brain Science Institute, Saitama, Japan, and Susumu Tonegawa at MIT, strengthens the link between schizophrenia and calcineurin signaling.

Calcineurin, otherwise known as protein phosphatase 2B, is a key regulatory molecule, helping to modulate numerous signaling pathways by removing phosphate groups that have been added to proteins by various kinases. The addition of phosphate, or phosphorylation, is one of the primary mechanisms by which cellular processes are turned on and off, and phosphatases have evolved to reset those switches to ensure that cellular signaling does not get out of control. In neurons, calcineurin regulates phosphorylations elicited by both glutamatergic and dopaminergic signaling (see, for example, Nishi et al., 2002), making it particularly interesting to those studying schizophrenia since there is considerable evidence to suggest that both forms of neurotransmission may be dysfunctional in the disease.

The Tonegawa lab previously reported that genetic variations in the gene for the γ subunit of calcineurin associate with schizophrenia in U.S. families (see Gerber et al., 2003) and that knocking out the gene in the mouse forebrain causes abnormal behavior reminiscent of that seen in schizophrenia, including impaired prepulse inhibition and altered social activity (see Miyakawa et al., 2003). The latest evidence linking the phosphatase to schizophrenia comes from a study of calcineurin-related genes. First author Kazuo Yamada and colleagues analyzed single nucleotide polymorphisms (SNPs) in 124 Japanese schizophrenia families, focusing on 14 genes that have been implicated in calcineurin signaling, including calcineurin itself. The researchers confirmed the previously described association of the γ subunit of calcineurin, also called PPP3CC, with schizophrenia in American families. They also found an association with EGR3 and two of its family members, EGR2 and EGR4.

Because the EGR3 and PPP3CC genes lie very close together on chromosome eight, the researchers wondered if the two genes confer risk separately. To test this, they looked to see which variants of EGR3 are present in families that do not have the schizophrenia-linked variant of PPP3CC. That they found an association with two EGR3 SNPs in these families indicates that the two genes may independently contribute to the risk for schizophrenia.

To confirm the association with EGR3 the researchers turned to a wider case-control sample set. They found that two of four haplotypes, or groups of SNPs, are linked to the disease. In schizophrenia patients one of these haplotypes turned up more often than would be expected by chance, while the other was less frequent. Although statistically significant, the associations were not dramatic. The authors suggest that both calcineurin and EGR3 “may independently elicit a modestly increased risk for schizophrenia.”

Exactly how these genetic polymorphisms translate into disease risk is unclear. Yamada and colleagues analyzed postmortem brain samples to see if production of the calcineurin γ subunit or the EGR proteins is different from control samples. While they found no difference between levels of PPP3CC in schizophrenia and normal dorsolateral prefrontal cortex (see also Kozlovsky et al., 2006), they did find that all three EGRs were downregulated in the schizophrenia samples. In this regard it is noteworthy that the EGR3 SNP that seemed to confer the strongest risk is one that falls in a highly conserved intron. Because these non-coding sequences are not normally conserved, the authors wondered if this particular intron, and the SNP, may have some regulatory function. To test this, they measured the activity of the gene in cultured cells and found that when the SNP was present, production of the protein flagged. The findings are consistent with the idea that the EGR3 SNP somehow reduces production of the transcription factor in the brain.

While there is much more work to do to tease out any potential role of EGR3 in the pathology of schizophrenia, the authors make one other interesting observation: EGR3 can be regulated by the activity of neuregulin (see Jacobson et al., 2004), also implicated in schizophrenia (see SRF related news story), providing another potential genetic link between EGR3 and this complex disease.—Tom Fagan.

Yamada K, Gerber DJ, Iwayama Y, Ohnishi T, Ohba H, Toyota T, Aruga J, Minabe Y, Tonegawa S, Yoshikawa T. Genetic analysis of the calcineurin pathway identifies members of the EGR gene family, specifically EGR3, as potential susceptibility candidates in schizophrenia. PNAS 2007 Feb 20;104:2815-2820.

Comments on News and Primary Papers
Comment by:  Mary Reid
Submitted 26 February 2007
Posted 27 February 2007

Tom Fagan mentions that calcineurin regulates phosphorylations elicited by both glutamatergic and dopaminergic signaling. The activity of D-amino acid oxidase is increased in schizophrenia, and this also affects signaling through both these pathways. Is there any clinical benefit with the use of sodium benzoate which inhibits DAO activity?

He also mentions that EGR3 can be regulated by the activity of neuregulin. Interestingly, Roberts et al. suggest that BDNF, which is decreased in first-episode psychosis (Buckley et al., 2007), induces synthesis of EGR3 to regulate activity of GABRA4. Ma and colleagues (Ma et al., 2005) conclude that GABRA4 is involved in the etiology of autism and it has also has been implicated in nicotine dependence (Saccone et al., 2007).

Glorioso and colleagues (Glorioso et al., 2006) report changes in genes encoding early-immediate genes such as EGR1 and EGR2 and RGS4 which is involved in cellular signaling and has been implicated in schizophrenia following BDNF gene ablation. Several studies report that zinc increases BDNF expression. Does this give support to the zinc deficiency theory of schizophrenia?

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Comments on Related News

Related News: Functional Neuregulin Variant Linked to Psychosis, Abnormal Brain Activation and IQ

Comment by:  Amanda Jayne Law, SRF Advisor
Submitted 8 November 2006
Posted 8 November 2006

Convergent evidence supporting the role of a schizophrenia-associated polymorphic variant in the NRG1 gene (SNP8NRG1243177) with the regulation of cortical function and the development of psychosis
The study of Hall and colleagues describes association of a schizophrenia-related polymorphism in the NRG1 gene promoter (SNP8NRG1243177) with cortical and cognitive dysfunction and the emergence of psychotic symptoms in young individuals at high genetic risk for developing schizophrenia. We have previously demonstrated that the same polymorphism (SNP8NRG1243177) and a 22kb risk haplotype, including this SNP, predicts transcription levels of a novel isoform of the NRG1 gene (Type IV) in the brain of patients with schizophrenia (Law et al., 2006; see SRF related news story). The SNP resides in the NRG1 promoter region for the novel E187 exon (Type IV) and our investigations indicate that the SNP is central to a regulatory transcription factor binding domain. We previously suggested that a potential molecular mechanism behind the clinical association of NRG1 with schizophrenia (at least in the 5’ region of the gene) involves altered transcriptional regulation of the gene, which modifies to a small degree and in an isoform-specific fashion, the efficiency of NRG1 signaling effects on neural development and plasticity. We predicted that such effects may translate into altered adult brain function.

With this in mind, the study of Hall and colleagues provides a remarkable level of functional convergence suggesting a potential link between a molecular phenotype related to genetic risk at this loci (i.e., increased transcriptional regulation of the novel Type IV isoform, Law et al., 2006) and abnormal cortical development, function, and the subsequent manifestation of psychotic symptoms.

The major objective of the study was to determine the relationship between previously identified genetic variants in the 5’ region of NRG1 (Stefansson et al., 2002; see also Harrison and Law, 2006) with aspects of the schizophrenia phenotype (including decreased IQ, altered cortical function, and psychosis) in individuals who are at high risk of developing the disorder. Subjects were followed throughout the course of the study or until they developed schizophrenia. It is noteworthy that the incidence rate of developing schizophrenia was highest in subjects homozygous for the risk (T) allele at NRG1243177 (25 percent). Conversely, the occurrence of schizophrenia in non-risk C/C individuals was lower (15 percent), but still present, demonstrating the complex heterogeneous nature of the disease.

The study was performed on a modest sample of 79 high-risk individuals, 63 of whom fMRI data was available for. Firstly, brain activation patterns were determined by fMRI whilst individuals were performing the Hayling sentence completion task. Subjects who were homozygous for the risk T allele (T/T) at SNP8NRG1243177 exhibited decreased activation of Brodmann area 9 and the right temporo-occipital junction (Brodmann areas 39 and 19) when the activation during the task was compared to the resting state. However, unlike the medial prefrontal cortex, the difference in activation of the right temporal-occipital junction derived from the fact that T/T individuals had a “higher” resting activity compared to C/C individuals (as stated by the authors). Based on this observation, it is difficult to interpret which phenotype, in terms of cortical activation in this region, genetic risk at the allele is associated with—that is, is the risk variant associated with an overactive right temporo-occipital cortex at rest, or with decreased ability to further activate the region during demand?

In the supplementary notes, the authors address this issue, stating that a failure to deactivate the temporal cortex during rest may suggest that frontotemporal activity is disrupted in individuals homozygous for the T allele at SNP8NRG1243177. Furthermore, based on our studies, it would be important to see if genetic risk at SNP8NRG1243177 predicts hippocampal activation during a task that activates this area, allowing one to link the molecular changes in the hippocampus in schizophrenia, related to genetic risk at this SNP, to an outcome measure of brain function. Conversely, it would also be of use to determine whether NRG1 Type IV expression is altered in the brain areas implicated by Hall and colleagues.

Importantly, the study also shows that the genotype effects at SNP8NRG1243177 on cortical function are not related to medication status (all subjects were medication-free). Secondly, Hall and colleagues investigated the effects of the SNP8NRG1243177 risk allele on the development of psychotic symptoms in high-risk individuals. In a remarkable observation, 100 percent of individuals who had the risk T/T genotype developed psychotic symptoms, compared to less than 50 percent of C/C individuals, although the small sample size must be kept in mind. One interesting observation that is not readily apparent in the study is the fact that of the 12 T/T individuals who developed psychotic symptoms, only three of those (25 percent) developed schizophrenia before the end of the study. This may be due to the fact that others later went on to develop the disorder or that they developed other complex mental illnesses which include psychosis, such as bipolar disorder. (This is not clear from the study.) The association of genetic risk in the NRG1 gene and psychotic symptom development is consistent with the fact that genetic risk at NRG1 has been linked to psychosis in other brain diseases such as bipolar disorder and Alzheimer’s disease (see Harrison and Law, 2006). Finally, and perhaps most compelling, there is the observation that genetic risk at SNP8NRG1243177 is related to decreased IQ (measured by NART) in high-risk individuals.

Overall, the study of Hall and colleagues provides novel evidence that genetic variation in the NRG1 promoter, in particular a genetic variant that predicts altered expression of the NRG1 gene in the brain in schizophrenia (Law et al., 2006), is associated with abnormalities in cortical function and cognition and contributes to psychotic symptoms in individuals at high risk of developing the disease.

View all comments by Amanda Jayne Law

Related News: Functional Neuregulin Variant Linked to Psychosis, Abnormal Brain Activation and IQ

Comment by:  Nicholas Stefanis
Submitted 16 November 2006
Posted 16 November 2006

The readers might find our results (now in press) interesting in the context of the brilliant work by Law and colleaguesLaw et al (2006)and now Hall and colleagues. We examined the potential impact of 18 single nucleotide polymorphisms (SNPs) within the DTNBP1, NRG1, DAOA/G32 and DAAO genes, on cognition and self-rated schizotypy, in a representative population of 2,243 young male military conscripts. Single SNP and haplotype associations were evaluated. The risk allele of functional SNP8NRG243177 was associated with reduced spatial working memory capacity.

This is of particular interest since it has recently been reported that SNP8NRG243177 is a functional polymorphism, the risk allele (T) predicting higher levels of type IV NRG1 mRNA expression (Law et al., 2006), and associated with lower prefrontal (and temporal) activation and development of psychotic symptoms in high risk individuals for schizophrenia (Hall et al., 2006). If not a chance finding, our result constitutes the first independent confirmation that functional SNP8NRG243177 impacts aspects of human prefrontal brain function. Since spatial working deficits constitute an effective endophenotype for schizophrenia, this finding also suggests a mechanism by which this NRG1 variant may confer risk for the disorder at an information processing level. In contrast to Hall and colleagues, no association of SNP8NRG243177 with psychotic-like symptoms or IQ was detected in this study.

Nicholas C. Stefanis, Thomas A. Trikalinos, Dimitrios Avramopoulos, Nikos Smyrnis, Ioannis Evdokimidis, Evangelia E. Ntzani, John P. Ioannidis and Costas N. Stefanis. Impact of schizophrenia candidate genes on schizotypy and cognitive endophenotypes at the population level. Biological Psychiatry (in press).

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Related News: A Model Is a Model Is a Model of Mental Illness?

Comment by:  Kevin J. Mitchell
Submitted 7 November 2013
Posted 11 November 2013

Instead of asking whether a particular animal model is "valid" as a proxy for a particular psychiatric disorder, we should be asking, Is it useful? Can it tell us something we can't learn in humans? If we base that solely on supposed behavioral similarities, we haven't gotten very far—we might as well just be doing rodent psychoanalysis. What we are interested in is elucidating the underlying neurobiological abnormalities and the pathways from etiological factors to resultant pathophysiological states. Such states should be expected to affect behaviors in a species-specific manner—maybe there will be some surface similarity in the results between rodents and humans, but maybe not. Certainly, expecting any animal model to recapitulate the full profile of human symptoms associated with a particular psychiatric diagnostic category is asking too much—does any human patient model the entire spectrum of disease? If these diagnostic categories are really umbrella terms for hundreds of distinct genetic conditions, each with variable outcomes, then the focus in models should be more on the expression of particular symptom domains than on entire disease profiles.

Starting with strong etiological factors is a proven route to discovery of pathogenic mechanisms. As such, the SHANK3 duplication mice are more inherently relevant to disease than the calcineurin mice, which are an artificial transgenic line not directly representative of any human patient. Indeed, the genetic evidence implicating calcineurin in schizophrenia risk has effectively been superseded by negative results from very large GWASs (unless it has popped up again in the unpublished results of the PGC). It is, nevertheless, a very interesting genetic preparation that can be used to dissect circuit mechanisms of memory, which clearly are of relevance to several disease states. That really ought to be enough to garner a wide readership without resorting to claims of direct disease model validity.

View all comments by Kevin J. Mitchell

Related News: A Model Is a Model Is a Model of Mental Illness?

Comment by:  Barbara K. Lipska
Submitted 13 November 2013
Posted 15 November 2013

There is a classic catch-22 in an attempt to model schizophrenia (and other major mental disorders) as, on the one hand, the main purpose of creating a model is to discover the cause of illness (e.g., a genetic defect and the subsequent pathological processes underlying the disease), and on the other hand, it is unclear what to model because the etiology of schizophrenia is still not well understood. Many new models focus on genetic causes because of the strong evidence for heritability of mental illness and the recent discoveries of particular predisposing genes. It is also becoming clear that in most cases, no single gene is necessary or sufficient to cause the disease, but instead, common, low-penetrance genetic variants in more than one susceptibility gene, each contributing a small effect, act in combinations to increase the risk of illness. In some other cases it is possible that rare, but highly penetrant, mutations (i.e., point mutations, translocations, deletions) in single genes are responsible. It is also increasingly clear that there are interactions among susceptibility genes, and between genes and environmental factors that contribute to the risk for mental illness. Given all this, there is no doubt that the task of modeling schizophrenia in animals is formidably difficult.

It is further complicated by the fact that a gene-based animal model 1) may have to be related to a specifically human transcript and/or protein variant or variants artificially introduced into the animal; 2) will not exhibit abnormalities in all schizophrenia-related phenotypes (as animals will not have hallucinations or delusions); and 3) may require additional environmental manipulations to become fully penetrant at the behavioral level. We should thus perhaps accept the fact that a mouse model for an individual candidate gene will never be representative of the entire disorder, and at best it will reproduce either a subtype of the disorder or a particular aspect of a given phenotype. In that context, human cell-based models and studies of human brain tissues obtained postmortem from patients with mental illness (severely underutilized resources!) are perhaps better alternatives to gain insight into the origins and pathophysiology of these specifically human, challenging disorders.

View all comments by Barbara K. Lipska

Related News: A Model Is a Model Is a Model of Mental Illness?

Comment by:  Karoly Mirnics, SRF Advisor
Submitted 12 November 2013
Posted 15 November 2013
  I recommend the Primary Papers

I think that we are often making a mistake if we directly declare what disease are we modeling. There are no "valid" animal models of human schizophrenia or other major psychiatric disorders, and most likely, there will never be—the mouse is not a human, and has a quite different lifestyle! Furthermore, the mouse and the human genetic diversity are quite distinct. Thus, talking about modeling physiological and pathophysiological processes is much more correct. Understanding behavioral modulation by the various interneuronal subtypes, evaluating the role of gene X on cortical lamination, or assessing the effects of factor Y on neuronal outgrowth are all disease-relevant, essential studies.

View all comments by Karoly Mirnics