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Polymorphisms and Schizophrenia—The Ups and Downs of Neuregulin Expression

21 April 2006. Since single nucleotide polymorphisms (SNPs) in the gene for neuregulin 1 (NRG1) were linked to increased risk for schizophrenia, scientists have struggled to explain exactly how those SNPs might relate to the disease at a biological level. The problem is that the SNPs do not lie in the coding region of the gene, but upstream. This has led to suggestions that the nucleotide changes may alter expression of neuregulin. Now, in an April 25 PNAS paper, currently available online (and first reported in our Neurosciences 2005 coverage), researchers report that they have addressed that theory in a large sample of postmortem brains. Amanda Law and colleagues from Oxford University, England, and the National Institutes of Health, Bethesda, Maryland, have analyzed SNPs and measured NRG1 isoform expression in postmortem hippocampal samples from more than 80 controls and 40 schizophrenia patients. So does aberrant expression of NRG1 explain why these SNPs increase risk for the disease? The answer appears to be yes…and no.

Law and colleagues measured hippocampal expression of four major isoforms of neuregulin (type I – IV). In a subset of samples (53 controls and 38 patients), they found that mRNA for the type I isoform is slightly, though significantly higher in patient tissue. On average, that slight increase is 34 percent, though deviations from the mean are quite large. The authors report finding a similar relationship when they examined samples from the full cohort (n = 84 for controls, n = 44 for patients). None of the other three isoforms showed any expression differences between patient and controls. Researchers from the same group have previously reported that type I NRG1 is also increased slightly in the prefrontal cortex in the disease (see Hashimoto et al., 2004). Both the prefrontal cortex and the hippocampus have been linked to the psychopathology of the disease.

When Law and colleagues examined the effects of the risk alleles on type I NRG1 expression, they found that the relationship was quite complex and not easily reconciled with disease susceptibility. The authors found that one of the original five SNPs identified as increasing risk for schizophrenia (see Stefansson et al., 2002) did seem to alter gene expression but not in the same way in patients and controls. Patients homozygous for the risk allele of this SNP (SNP8NRG221132) had slightly higher type I expression than patients who were heterozygous for the allele—this difference was not significant. But in control cases the trend was in the opposite direction—those homozygous for the high-risk allele actually had significantly lower expression of type I NRG1. “At present, the relevance of and an explanation for these findings is obscure,” write the authors in their supplementary discussion, which can be found at the PNAS website. But one possibility that they put forth is that other factors driving increased expression of NRG1 in schizophrenia patients may be masking the effect of the high-risk allele seen in normal controls. If this turns out to be true, then the risk allele (a guanine nucleotide instead of adenine) would be predicted to decrease NRG1 expression.

In the case of type IV NRG1, the opposite seems to be the case. The authors found that increased expression of the type IV isoform was associated with the risk allele for a second SNP, SNP8NRG243177. The presence of the high-risk thymine instead of cytosine at this position was correlated with higher levels of type IV NRG1 in both patients and controls, and there is evidence of a dose effect: T/T homozygotes have highest expression followed by C/T heterozygotes and C/C homozygotes. Though this trend appeared more pronounced in patients, the authors did not find any strong relationship between SNP genotype and diagnosis of schizophrenia. Nevertheless, in the total cohort (patients and controls), the difference between type IV NRG1 expression in the two homozygote groups reached statistical significance, indicating that the risk allele does increase type IV NRG1 expression. Given that the risk allele has similar effects in both patients and normal controls, additional factors must contribute to pathology in patients.

How might these SNPs affect expression of the different isoforms of neuregulin? The authors note that both lie in nucleotide motifs that are recognized by a variety of transcription factors. SNP8NRG221132, for example, lies in a region recognized by serum response factor (SRF), and the high-risk allele is predicted to remove binding of this transcription factor and therefore reduce expression of neuregulin. The presence of SNP8NRG243177, on the other hand, is predicted to increase binding of high-mobility group box protein-1, which would fit with the increased expression of type IV NRG1 that appears to be elicited by this SNP. The differential effects of the SNPs on different isoforms may be related to the complex alternative splicing of this gene, which is governed by alternative promoter usage.

But the situation is slightly more complex, because SNP8NRG243177 also lies in a second SRF-binding motif and is predicted to abolish SRF binding also. All told, the interplay among high-risk SNPs, differential promoter usage, NRG1 expression, and schizophrenia is turning out to be extremely complex. Nonetheless, the authors conclude “…that the mechanism behind the clinical association of NRG1 with schizophrenia is altered transcriptional regulation, which modifies, probably to a small degree and in an isoform-limited fashion, the efficiency of NRG1 signaling effects on neural development and plasticity.”—Tom Fagan.

Reference:
Law AJ, Lipska BK, Weickert CS, Hyde TM, Straub RE, Hashimoto R, Harrison PJ, Kleinman JE, Weinberger DR. Neuregulin 1 transcripts are differentially expressed in schizophrenia and regulated by 5’ SNPs associated with the disease. PNAS April 25, 2006;103:6747-6752. Abstract

Comments on News and Primary Papers
Comment by:  William Carpenter, SRF Advisor (Disclosure)
Submitted 22 April 2006
Posted 22 April 2006
  I recommend the Primary PapersComment by:  Stephan Heckers, SRF Advisor
Submitted 29 April 2006
Posted 29 April 2006
  I recommend the Primary Papers

The gene Neuregulin 1 (NRG1) on chromosome 8p has been identified as one of the risk genes for schizophrenia. It is unclear how the DNA sequence variation linked to schizophrenia leads to abnormalities of mRNA expression. This would be important to know, in order to understand the downstream effects of the neuregulin gene on neuronal functioning in schizophrenia.

Law and colleagues explored this question in post-mortem specimens of the hippocampus of control subjects and patients with schizophrenia. This elegant study of the expression of four types of NRG1 mRNA (types I-IV) is exactly what we need to translate findings from the field of human genetics into the field of schizophrenia neuropathology. The findings are complex and cannot be translated easily into a model of neuregulin dysfunction in schizophrenia. I would like to highlight two findings.

First, the level of NRG1 type I mRNA expression was increased in the hippocampus of schizophrenia patients. This confirms an earlier study of NRG1 mRNA expression in schizophrenia. It remains to be seen how this change in NRG1 type I mRNA expression relates to the finer details of neuregulin dysfunction in schizophrenia.

Second, one single nucleotide polymorphism (SNP8NRG243177) of the risk haplotype linked to schizophrenia in earlier studies predicts NRG1 type IV mRNA expression. The SNP determines a binding site for transcription factors, providing clues for how DNA sequence variation may lead, via modulation of mRNA expression, to neuronal dysfunction in schizophrenia. It is exciting to see that we can now test specific hypotheses of molecular mechanisms in the brains of patients who have suffered from schizophrenia. The study by Law et al. is an encouraging step in the right direction.

View all comments by Stephan HeckersComment by:  Bryan Roth, SRF Advisor
Submitted 5 May 2006
Posted 5 May 2006
  I recommend the Primary Papers

I think this is a very interesting and potentially significant paper. It is important to point out, however, that it deals with changes in mRNA abundance rather than alterations in neuregulin protein expression. No measures of isoform protein expression were performed, and it is conceivable that neuregulin isoform protein expression could be increased, decreased, or not changed. A second point is that although statistically significant changes in mRNA were measured, they are modest.

Finally, although multiple comparisons were performed, the authors chose not to perform Bonferroni corrections, noting in the primary paper that, "Correction for random effects, such as Bonferroni correction, would be an excessively conservative approach, particularly given that we have restricted our primary analyses to planned comparisons (based on strong prior clinical association and physical location of the SNPs) of four SNPs and a single haplotype comprised of these SNPs. Because the SNPs are in moderate LD, the degree of independence between markers is low and, therefore, correcting for multiple testing would result in a high type II error rate. The prior probability and the predictable association between the deCODE haplotype and expression of NRG1 isoforms (especially type IV, which is its immediate physical neighbor) combined with the LD between SNPs in this haplotype makes statistical correction for these comparisons inappropriate. Nevertheless, our finding regarding type IV expression and the deCODE haplotype and SNP8NRG243177 requires independent replication."

It will thus be important to determine if these changes in neuregulin mRNA isoform abundance are mirrored by significant changes in neuregulin isoform protein expression and if the findings can be independently replicated with other cohorts.

View all comments by Bryan RothComment by:  Patricia Estani
Submitted 9 June 2007
Posted 10 June 2007
  I recommend the Primary Papers

Comments on Related News


Related News: SfN 2005: Cortical Deficits in Schizophrenia: Have Genes, Will Hypothesize

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

Related News: Neuregulin Partner ErbB4 Spices Up Genetic Associations

Comment by:  Amanda Jayne Law, SRF Advisor
Submitted 22 February 2006
Posted 22 February 2006
  I recommend the Primary Papers

The study of Ghashghaei and colleagues provides a remarkable insight into the function of neuregulin 1 (NRG1), and NRG2 in adult neurogenesis. The study demonstrates that NRG1(2)/ErbB4 signaling influences the proliferation, differentiation, organization, and migration of adult neural progenitor cells in the subventricular zone (SVZ) and rostral migratory stream (RMS), in a ligand- and cell-dependent fashion. Using immunohistochemistry, Ghashghaei and colleagues first demonstrate that NRG1, NRG2, and ErbB4 are expressed by distinct cell types in the SVZ and RMS, notably ErbB4 and NRG1 by polysialylated neural cell adhesion molecule positive (PSA-NCAM+) neuroblasts, and ErbB2/3/4 by a subset of GFAP+ cells. These observations extend the group's previous studies of NRG1 and ErbB4 in the RMS (Anton et al., 2004). In their current study, Ghashghaei went on to examine the effects of exogenous infusion of NRG1 and NRG2 on neurogenesis in the RMS of adult mice. Interestingly, NRG1 was shown to decrease the initiation of neuroblast migration from the SVZ to the RMS by inducing the rapid aggregation of cells in the SVZ. The consequence of this rise in NRG1 was a decrease in the number of PSA-NCAM+ cells in the RMS and GABA+ cells in the olfactory bulb, demonstrating that ectopic or elevated expression of NRG1 prevents differentiation and migration of neurons from the adult SVZ to the RMS.

The study is particularly interesting in terms of the role of NRG1/ErbB4 signaling in directional cell migration. Flames et al. (2004) recently reported that NRG1 (specifically the Ig containing family of isoforms, e.g., Types I, II and IV; for review, see Harrison and Law, 2006) functions as a long distance chemoattractant for ErbB4 positive GABAergic interneurons migrating from the medial ganglionic eminence to the developing cortex. The observation that NRG1 is a chemoattractant in other brain regions may appear somewhat contradictory to the findings of Ghashghaei, which suggest that in-vivo NRG1 actually inhibits migration of neurons from the SVZ (at least when introduced ectopically). However, it would seem that these two findings are actually consistent. Ghashghaei and colleagues ectopically infused NRG1 into the lateral ventricles of adult mice. The subsequent aggregation of cells in the SVZ demonstrates that NRG1 indeed acts as a chemoattractant, not in an obvious manner by inducing the cells to migrate away, but simply by "attracting" them to aggregate or "clump" where they are (subsequently preventing migration to the RMS). So in fact, both the studies of Flames and Ghashghaei show that NRG1 is chemotactic to specific populations of neurons and cells, whether it is expressed at a distance and cells preferentially migrate toward it, or in the immediate environment and cells are attracted to migrate to, or stay in its vicinity.

In the past few years, NRG1 and ErbB4 have both been identified as potential susceptibility genes for schizophrenia. The aim now is to determine the molecular and biological mechanisms by which the genes confer risk for the disease. In terms of schizophrenia, we have previously demonstrated that the Type I isoform of NRG1 is elevated in the hippocampus (and prefrontal cortex; see Hashimoto et al., 2004) in the disease and that expression of the novel Type IV isoform is related to disease-associated sequence variants within the NRG1 gene (Law et al., 2006). Furthermore, we have recently demonstrated that these changes are accompanied by altered expression of specific isoforms of the ErbB4 receptor, consistent with that of Silberberg et al., 2006 (Law et al., 2005). Ghashghaei and colleagues provide the first direct evidence that ectopic or elevated expression of NRG1 in the brain can perturb cell migration. In light of this and other evidence, our findings in schizophrenia may translate into altered neuronal migration, cortical development and possibly neurogenesis in the disease.

At present, the exact links between altered NRG1/ErbB4 signaling and the pathophysiology of schizophrenia are unknown and potentially numerous (i.e., synaptogenesis, neurotransmitter function, neuronal migration, differentiation, glia formation and function, myelination). Studies such as that of Ghashghaei et al. provide insight into the normal role of NRG1/ErbB4 signaling in neurodevelopment and the adult brain which is essential if we are to understand the pathogenic role of the NRG1 gene and its receptors in disease.

References:

Anton ES, Ghashghaei HT, Weber JL, McCann C, Fischer TM, Cheung ID, Gassmann M, Messing A, Klein R, Schwab MH, Lloyd KC, Lai C. Receptor tyrosine kinase ErbB4 modulates neuroblast migration and placement in the adult forebrain. Nat Neurosci. 2004 Dec;7(12):1319-28. Epub 2004 Nov 7. Abstract

Flames N, Long JE, Garratt AN, Fischer TM, Gassmann M, Birchmeier C, Lai C, Rubenstein JL, Marin O. Short- and long-range attraction of cortical GABAergic interneurons by neuregulin-1. Neuron. 2004 Oct 14;44(2):251-61. Abstract

Hashimoto et al., 2004, Mol. Psychiatry 9, 299-307.

Law et al (a) 2006. Neuregulin 1 (NRG1) transcripts are differentially expressed in schizophrenia and regulated by 5’ SNPs associated with the disease. PNAS

Also See SfN 2005 SRF research news: Cortical Deficits in Schizophrenia: Have Genes, Will Hypothesize

Law 2005, SNPing away at NRG1 and ErbB4 gene expression in schizophrenia Neuropsychopharmacology, vol. 30, Supplement 1.

View all comments by Amanda Jayne Law

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.

References:
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).

View all comments by Nicholas Stefanis

Related News: Neuregulin and Schizophrenia—Functional Failure Fingers Risk Allele

Comment by:  Ali Mohamad Shariaty
Submitted 14 July 2007
Posted 14 July 2007

It is really a fascinating article which is a step towards understanding the molecular mechanisms underlying phenotypes of schizophrenia. Relating genotypes to phenotypes is really necessary for untangling the puzzle of a complex disorder. However, when a regulatory SNP interferes with normal binding of a transcription factor, is it understood that the transcription factor should play a role in brain and therefore in the molecular pathology of schizophrenia? Is there any direct role for involvement of serum response factor (SRF) in brain development or any neurological process?

View all comments by Ali Mohamad Shariaty

Related News: Neuregulin and Schizophrenia—Functional Failure Fingers Risk Allele

Comment by:  Amanda Jayne Law, SRF Advisor
Submitted 14 July 2007
Posted 15 July 2007

In response to Ali Mohamad Shariaty’s comment: Serum response factor (SRF) plays a key role in regulating the transcription of a number of genes involved in brain development. Genetic manipulation of SRF has revealed a direct role for it as a regulator of cortical and hippocampal function (e.g., Etkin et al., 2006) influencing both learning and memory. At the cellular level SRF has been shown to regulate dendritic morphology and neuronal migration. Therefore, SRF is indeed an important neurodevelopmental molecule, mediated via its regulation of genes, such as NRG1. Genetic variations that are predicted to interfere with SRF binding (such as the SNP characterized in our study) may affect critical aspects of brain development and function that contribute to schizophrenia. Since SRF regulates the expression of a number of genes, beyond that of NRG1, its involvement in schizophrenia is likely mediated “indirectly” via its effects on the regulation of genes associated with the disorder.

References:

Etkin A, Alarcón JM, Weisberg SP, Touzani K, Huang YY, Nordheim A, Kandel ER. A role in learning for SRF: deletion in the adult forebrain disrupts LTD and the formation of an immediate memory of a novel context. Neuron. 2006 Apr 6;50(1):127-43. Abstract

View all comments by Amanda Jayne Law

Related News: Neuregulin and Schizophrenia—Functional Failure Fingers Risk Allele

Comment by:  Robert Hunter
Submitted 17 July 2007
Posted 17 July 2007
  I recommend the Primary Papers

Related News: Down to BACE-ics—Old Mouse a New Model for Schizophrenia?

Comment by:  Victor ChongCynthia Shannon Weickert (SRF Advisor)
Submitted 23 May 2008
Posted 23 May 2008

The findings of Savonenko et al. (2008) are an impressive addition to the growing evidence supporting a role for neuregulin-1 (NRG1) in schizophrenia pathology. The authors not only revealed a novel relationship between schizophrenia-like behavior and the loss of BACE1 proteolytic function, but also showed that this association results from disruption of BACE1-mediated NRG1 cleavage. These observations support the notion that aberrant processing of NRG1 may contribute to the development of schizophrenia-like phenotypes, providing a basis for examining other NRG1-cleaving pathways in the context of schizophrenia. Savonenko et al. were thorough in their behavioral assessment of the BACE1 mutant mice, convincingly showing that these animals exhibit schizophrenia-related behaviors that could be exacerbated by psychostimulants and improved by antipsychotic drug treatment.

What remains unclear, however, is the relationship between the NRG1/ErbB4 protein findings in the BACE1 mutant mouse brain and those previously reported in the schizophrenic human brain. For example, the authors reported reductions in ErbB4-PSD95 coupling in the BACE1 mutant mouse, whereas Hahn et al. (2006) demonstrated increased ErbB4-PSD95 interaction in the prefrontal cortices of schizophrenic patients. In addition, our recent investigation found elevated prefrontal cortical levels of both NRG1 C-terminal fragment (ICD) and full-length ErbB4 protein in schizophrenic subjects (Chong et al., 2008), while Savonenko et al. showed decreased NRG1 C-terminal fragment levels with no alterations in ErbB4 protein in the BACE1 mutant mouse cortex. On the other hand, the lack of variations in overall cortical ErbB4 in these mice may correspond to the findings of Hahn et al. (2006) who reported no alterations in prefrontal cortical ErbB4 protein levels in schizophrenic subjects.

These seemingly conflicting results could suggest that any imbalance in cortical NRG1 signaling, whether increased or diminished, may lead to schizophrenia. Indeed, studies have suggested that improper tuning of other cortical signaling systems, particularly those of dopamine, can contribute to cognitive deficits associated with this disease (Vijayraghavan et. al, 2007). Optimal synaptic function may display “inverted-U” shaped response to NRG1-ErbB4 activity as proposed by Role and Talmage (2007). Alternatively, the authors speculated that some of the discrepancies between the findings in the BACE1 mutant mice and those observed in the schizophrenic humans may be due to differences in the duration of NRG1 signaling modification between the animals and the patients, who had a lifetime of mental illness. One way to examine the validity of this suggestion is to look at cortical ErbB4-PSD95 coupling and NRG1/ErbB4 protein levels in the BACE1 mutant mice at different developmental and adult time points. This approach could test whether these animals at later stages in life display alterations in cortical ErbB4-PSD95 interactions and/or in NRG1/ErbB4 protein levels comparable to those seen in schizophrenic subjects of the human studies, which primarily consisted of adults beyond middle age. Also of interest would be to create NRG1 and ErbB4 gain-of-function mutants where the timing of over-expression could be controlled.

Given the significance of NRG1 signaling/cleavage in the BACE1 mutant mouse schizophrenia-like phenotypes, it may also be important to consider pathways leading to changes in ErbB4 C-terminal fragment levels in schizophrenia etiology. A recent paper by Walsh et al. (2008) demonstrated that at least one schizophrenic patient in their study has a gene deletion encompassing the C-terminal intracellular kinase domain of ErbB4, and we have found decreases in ErbB4 C-terminal fragments relative to full-length ErbB4 in the frontal cortex of schizophrenic subjects (Chong et al., 2008). These observations together with those of Savonenko et al. raise interesting questions regarding how molecular alterations in NRG1 signaling and cleavage may impact ErbB4 signaling and cleavage and whether changes in NRG1 and/or ErbB4 could be primary or secondary to the schizophrenia disease process.

In summary, Savonenko et al. have provided a novel avenue to probe NRG1 function and processing in relation to schizophrenia pathology. They have also introduced BACE1 as a potentially important schizophrenia susceptibility molecule that to our knowledge has not been directly investigated in subjects with schizophrenia and may be worth studying in the brain tissues of these patients. In addition, it would be interesting to examine how the schizophrenia-related traits of the BACE1 mutant mice compare with those of other NRG1 mutant mice such as the heterozygous NRG1 transmembrane knock-out mice (Stefansson et al., 2002). Such an investigation could provide insight into whether similar NRG1 signaling deficiencies underlie the schizophrenia-like phenotypes of these animal models.

References:

Hahn CG, Wang HY, Cho DS, Talbot K, Gur RE, Berrettini WH, Bakshi K, Kamins J, Borgmann-Winter KE, Siegel SJ, Gallop RJ, Arnold SE. (2006) Altered neuregulin 1-erbB4 signaling contributes to NMDA receptor hypofunction in schizophrenia. Nat Med. 12:824-8. Abstract

Chong VZ, Thompson M, Beltaifa S, Webster MJ, Law AJ, Weickert CS. (2008) Elevated neuregulin-1 and ErbB4 protein in the prefrontal cortex of schizophrenic patients. Schizophr Res. 100:270-80. Abstract

Vijayraghavan S, Wang M, Birnbaum SG, Williams GV, Arnsten AF. (2007) Inverted-U dopamine D1 receptor actions on prefrontal neurons engaged in working memory. Nat Neurosci. 10:376-84. Abstract

Role LW, Talmage DA (2007) Neurobiology: new order for thought disorders. Nature. 448:263-5. Abstract

Walsh T, McClellan JM, McCarthy SE, Addington AM, Pierce SB, Cooper GM, Nord AS, Kusenda M, Malhotra D, Bhandari A, Stray SM, Rippey CF, Roccanova P, Makarov V, Lakshmi B, Findling RL, Sikich L, Stromberg T, Merriman B, Gogtay N, Butler P, Eckstrand K, Noory L, Gochman P, Long R, Chen Z, Davis S, Baker C, Eichler EE, Meltzer PS, Nelson SF, Singleton AB, Lee MK, Rapoport JL, King MC, Sebat J. (2008) Rare structural variants disrupt multiple genes in neurodevelopmental pathways in schizophrenia. Science. 320:539-43. Abstract

Stefansson H, Sigurdsson E, Steinthorsdottir V, Bjornsdottir S, Sigmundsson T, Ghosh S, Brynjolfsson J, Gunnarsdottir S, Ivarsson O, Chou TT, Hjaltason O, Birgisdottir B, Jonsson H, Gudnadottir VG, Gudmundsdottir E, Bjornsson A, Ingvarsson B, Ingason A, Sigfusson S, Hardardottir H, Harvey RP, Lai D, Zhou M, Brunner D, Mutel V, Gonzalo A, Lemke G, Sainz J, Johannesson G, Andresson T, Gudbjartsson D, Manolescu A, Frigge ML, Gurney ME, Kong A, Gulcher JR, Petursson H, Stefansson K. (2002) Neuregulin 1 and susceptibility to schizophrenia. Am J Hum Genet. 71:877-92. Abstract

View all comments by Victor Chong
View all comments by Cynthia Shannon Weickert

Related News: Gene Expression Study May Open Window on Brain Development

Comment by:  Barbara Lipska
Submitted 15 June 2009
Posted 15 June 2009

In this very important and innovative study, Sestan and colleagues report a transcriptome-wide survey across multiple brain regions of the fetal mid-gestation brain. They show dramatic differences in expressed transcripts, including alternative splice variants, between brain regions, and most surprisingly, between several cortical regions. The authors have undertaken an ambitious task of further characterizing differentially expressed genes by functional clustering and co-expression clustering and comparing the results with genes identified through neurobiological experiments. They have also performed extensive validation using several additional fetal brains. Most interestingly, the authors showed that differentially expressed genes are more frequently associated with human-specific evolution of putative cis-regulatory elements. For this, they have identified genes that are near highly conserved non-coding sequences (CNSs) and found that the genes that are differentially expressed between the regions are more frequently near human-specific accelerated evolution CNSs.

The weakness of the study is a very small number of fetal brains (four) and the fact that they range in age from 18 to 23 weeks of gestation. During these several weeks of fetal life, the brain undergoes dramatic developmental changes and expression of many genes, either increases or decreases steeply. Thus, it would be critical to fully characterize these changes across fetal age. It is also crucial to explore genetic influences on fetal gene expression as it appears that in adult brain both gene expression and splicing are strongly genetically regulated. The authors have made an important contribution to our understanding of development of human brain, and further research of this type will generate the data that would help in better understanding of human brain disorders. In particular, genetic-expression effects in human brain across the entire lifespan, including fetal period, may help identify molecular mechanisms whereby candidate genes increase risk for developing the disorder. Using expression levels of transcripts and their splicing characteristics as intermediate phenotypes may yield statistically positive associations and improved understanding of the mechanisms that lead to neurodevelopmental disorders such as autism and schizophrenia, as they are the most proximal phenotypes to the risk alleles.

View all comments by Barbara Lipska

Related News: Gene Expression Study May Open Window on Brain Development

Comment by:  Karoly Mirnics, SRF Advisor
Submitted 15 June 2009
Posted 15 June 2009

This outstanding study reinforces how much we still do not understand about human brain development and function! It is just mind-boggling that the mid-fetal human brain expresses more than three quarters of the human genome, and that region-specific splicing appears to be an absolutely critical feature of the developing brain. Interestingly, the structural and functional interhemispheric differences do not appear to be related to gene expression differences in mid-fetal life, but rather, either they develop independently of gene expression patterns, or they are developing at later stages of cortical maturation, perhaps in a postnatal activity-driven pattern.

So, how is this developmental expression machinery related to various neurodevelopmental disorders, such as schizophrenia? Is usage of an "inappropriate" splice variant sufficient to alter the neuronal phenotypic development to a degree that would predispose the brain to developing a disease? Are environmental insults capable of disrupting this finely tuned, region-specific splicing machinery? As this is a likely possibility, we must rethink the existing disease-related gene expression findings in the context of the present study, and accept that our previous gene expression measurements may have been too crude to uncover some of the most meaningful changes that are potentially hallmarks of various brain disorders. Furthermore, as the genes that show the most widespread regional use of splice variants can be essential for proper neuronal migration or connectivity, one can argue that these genes should be the primary targets for evaluation in the various regions of postmortem tissue of diseased individuals.

Finally, there is also a minor, cautionary note arising from this study. The fact that the Affymetrix U133 and the Exon array results showed a correlation of R2 >0.5 is encouraging, but underscores that platform-dependence of the findings remains a significant interpretational challenge. Some platforms will be better suited to identify certain gene expression changes, while others will have a greater power to reveal a different (but also potentially valid!) set of mRNA alterations.

View all comments by Karoly Mirnics

Related News: Researchers Model Susceptibility to Schizophrenia in a Petri Dish

Comment by:  Alan Mackay-Sim
Submitted 13 April 2011
Posted 13 April 2011

With a heritability of 50 percent, schizophrenia is very clearly a disease of disturbed biology, but to dissect the biological contribution to its etiology, researchers need relevant, patient-derived cell models. Ideally, we need cell models that can tell us how schizophrenia cell biology leads to an altered brain. Induced pluripotent stem (iPS) cells are genetically engineered cells, from a patient's cells (e.g., fibroblasts), that resemble embryonic stem cells, that can be used to generate neurons. There is much excitement that they will be useful as models for many brain disorders and diseases. Two new papers in Molecular Psychiatry and Nature report on applying iPS cell technology to schizophrenia by generating iPS cells from patients with a DISC1 mutation (Chiang et al., 2011) and from patients selected with a high likelihood of a genetic component to disease (Brennand et al., 2011).

When specific genes are implicated, then animal models can provide breakthroughs by determining the cellular functions of the implicated genes and their mutations. Although schizophrenia lacks single commonly mutated genes of large effect, some candidate genes, such as DISC1, are being identified in some families. This is now a very hot area for research that is identifying the role of this gene at the cellular level and in animal models. As such candidate genes are identified and their functions are ascertained, it will be essential to demonstrate their direct relevance in schizophrenia through patient-derived cellular models. In this regard, a new tool has emerged in the recent letter to Molecular Psychiatry reporting the generation of induced pluripotent cells from two patients with DISC1 mutation (Chiang et al., 2011). This preliminary study did not report a disease-associated phenotype in these iPS cells.

A disease-associated phenotype is best identified by comparing iPS cells from patients and controls, as now demonstrated by Brennand et al. (2011). This work is a significant new contribution to the field because it has demonstrated differences in the biology of neurons derived from patients and controls. As proof of principle, they have identified differences in the way patient neurons branch (they have fewer branches) and connect with each other (they connect to fewer other neurons). Most importantly, the patient neurons had normal physiological properties. That is to say, their physiology was not different from controls. These are interesting and important distinctions that are a reassuring proof of principle for this model, suggesting that the etiology of schizophrenia derives from altered connectivity of neuronal circuits and not from basic neuronal functions. This fits with the postulated “neurodevelopmental hypothesis” of schizophrenia. Patient neurons also had decreased levels of synaptic proteins (PSD95, glutamate receptor), which is consistent with “synaptic hypotheses” of schizophrenia. These are early days yet, but this cell model already demonstrates how a relevant cell model can provide a path for unifying etiological hypotheses.

Another aim for developing cell models of schizophrenia is to use them for drug discovery. Patient-control differences in cell functions can be the basis for screening chemical compounds that ameliorate this difference. Here, too, Brennand et al. (2011) demonstrate proof of principle by showing that loxapine treatment of the patient neurons increased their connectivity towards control levels. Only loxapine, of five antipsychotic drugs tested, had this effect, but the results are a clear sign of the utility of such cells for drug screening to find new potential drug candidates.

These two papers are a great start to using iPS cells as models of schizophrenia.

References:

Chiang CH, Su1Y, Wen Z, Yoritomo N, Ross CA, Margolis RL, Song H, Ming G-I. (2011) Integration-free induced pluripotent stem cells derived from schizophrenia patients with a DISC1 mutation. Molecular Psychiatry advance online publication, 22 February 2011. Abstract

Brennand KJ, Simone A, Jou1 J, Gelboin-Burkhart C, Tran N, Sangar S, Li Y, Mu Y, Chen G, Yu D, McCarthy S, Sebat J, Gage FH (2011). Modeling schizophrenia using human induced pluripotent stem cells. Nature.

View all comments by Alan Mackay-Sim

Related News: Researchers Model Susceptibility to Schizophrenia in a Petri Dish

Comment by:  Akira Sawa, SRF Advisor
Submitted 13 April 2011
Posted 13 April 2011

I fully appreciate the efforts of Brennand and colleagues as pioneers. Indeed, this is great work. Like any pioneering work, this paper will be both applauded and criticized. The strength of the paper is in providing ways for us to analyze iPS cells and derived neurons. The multifaceted approach taken in this study will be a great platform for many investigators.

Schizophrenia is, clinically, a very heterogeneous condition, but for the past several years, basic scientists have tended to oversimplify the disorder. It is also true that this trend makes the neurobiology of schizophrenia move productively forward in some ways. I believe that the new tools for studying the biology of schizophrenia, such as iPSC-derived neurons, will teach us how difficult it is to draw simplified pathways for the disorder. Nonetheless, some common pathway(s) may be identified in the future, I optimistically hope.

Based on the great experimental procedures that this paper provides, many other groups may need to address whether or not these data are reproducible or not in “general” cases of schizophrenia. In such studies, the most important issue is to examine detailed clinical information of the subjects in comparison with this study.

View all comments by Akira Sawa

Related News: Neuregulin and ErbB4: Synaptic Jacks of All Trades

Comment by:  Michael CahillPeter Penzes
Submitted 29 April 2011
Posted 29 April 2011
  I recommend the Primary Papers

The recent study by Pitcher et al. provides a novel mechanism linking NRG1/ErbB4 activity to the suppression of NMDAR activity in a manner requiring Src kinase inhibition. The study uses biochemical manipulation of Src activation, as well as studies on cells lacking Src, to examine the role for Src kinase on the effects of NRG1 on NMDAR responses in pyramidal neurons. Overall, the study provides convincing evidence indicating that Src inhibition by NRG1 is an important contributor to the effects of NRG1 on NMDAR pyramidal neuronal hypofunction. The effect of NRG1 and ErbB4 on Src family kinase activation remains complex. Previous studies have found that NRG1 can activate Src, and that inhibition of Src family kinases can block some of the effects of NRG1 on cells, including cellular migration and proliferation (Eckert et al., 2009; Grossmann et al., 2009). Moreover, ErbB4 activity is able to activate fyn when overexpressed in heterologous cells, and NRG1 treatment activates fyn in cells expressing endogenous ErbB4 (Bjarnadottir et al., 2007). Recent findings have similarly found that ErbB4 can activate Src kinases in heterologous cells and indicate that Src family kinase activation, particularly that of fyn, has a role in regulating the effects of NRG1 on interneuron morphology through RhoGEF activity (Cahill et al., 2011).

The findings of Picher et al. indicating that NRG1 can suppress Src kinase are not incompatible with these previously discussed studies, however. Indeed, studies have found that Src kinases can be both activated and inhibited by NRG1 treatment in a cyclic manner (Eckert et al., 2009), suggesting that the duration of NRG1 activity is an important consideration. The effects of NRG1 on pyramidal neuronal structure and/or function also seem to differ depending on the length of NRG1 treatment, as studies have found that chronic NRG1 or ErbB4 activity can promote synaptic structure and/or function (e.g., Barros et al., 2010; Li et al., 2007), whereas short-term NRG1 treatment is detrimental to pyramidal neuronal function (e.g., Wen et al., 2010), indicative of the importance of treatment duration to the functional consequences on neurons. The location of the examined effects is also an important consideration, as biochemical and morphological effects in pyramidal neurons and interneurons might differ following NRG1 treatment, potentially due to differences in ErbB4 expression profiles in these cells (Vullhorst et al., 2009). Given the links of NRG1/ErbB4 to schizophrenia, understanding how short-term and long-term activity of these molecules regulates both interneuron and pyramidal neuron function is of special importance, and merits further studies.

References:

Eckert JM, Byer SJ, Clodfelder-Miller BJ, Carroll SL. Neuregulin-1 beta and neuregulin-1 alpha differentially affect the migration and invasion of malignant peripheral nerve sheath tumor cells. Glia . 2009 Nov 1 ; 57(14):1501-20. Abstract

Grossmann KS, Wende H, Paul FE, Cheret C, Garratt AN, Zurborg S, Feinberg K, Besser D, Schulz H, Peles E, Selbach M, Birchmeier W, Birchmeier C. The tyrosine phosphatase Shp2 (PTPN11) directs Neuregulin-1/ErbB signaling throughout Schwann cell development. Proc Natl Acad Sci U S A . 2009 Sep 29 ; 106(39):16704-9. Abstract

Bjarnadottir M, Misner DL, Haverfield-Gross S, Bruun S, Helgason VG, Stefansson H, Sigmundsson A, Firth DR, Nielsen B, Stefansdottir R, Novak TJ, Stefansson K, Gurney ME, Andresson T. Neuregulin1 (NRG1) signaling through Fyn modulates NMDA receptor phosphorylation: differential synaptic function in NRG1+/- knock-outs compared with wild-type mice. J Neurosci . 2007 Apr 25 ; 27(17):4519-29. Abstract

Cahill ME, Jones KA, Rafalovich I, Xie Z, Barros CS, Müller U, Penzes P. Control of interneuron dendritic growth through NRG1/erbB4-mediated kalirin-7 disinhibition. Mol Psychiatry . 2011 Apr 12. Abstract

Eckert JM, Byer SJ, Clodfelder-Miller BJ, Carroll SL. Neuregulin-1 beta and neuregulin-1 alpha differentially affect the migration and invasion of malignant peripheral nerve sheath tumor cells. Glia . 2009 Nov 1 ; 57(14):1501-20. Abstract

Barros CS, Calabrese B, Chamero P, Roberts AJ, Korzus E, Lloyd K, Stowers L, Mayford M, Halpain S, Müller U. Impaired maturation of dendritic spines without disorganization of cortical cell layers in mice lacking NRG1/ErbB signaling in the central nervous system. Proc Natl Acad Sci U S A . 2009 Mar 17 ; 106(11):4507-12. Abstract

Li B, Woo RS, Mei L, Malinow R. The neuregulin-1 receptor erbB4 controls glutamatergic synapse maturation and plasticity. Neuron . 2007 May 24 ; 54(4):583-97. Abstract

Wen L, Lu YS, Zhu XH, Li XM, Woo RS, Chen YJ, Yin DM, Lai C, Terry AV, Vazdarjanova A, Xiong WC, Mei L. Neuregulin 1 regulates pyramidal neuron activity via ErbB4 in parvalbumin-positive interneurons. Proc Natl Acad Sci U S A . 2010 Jan 19 ; 107(3):1211-6. Abstract

Vullhorst D, Neddens J, Karavanova I, Tricoire L, Petralia RS, McBain CJ, Buonanno A. Selective expression of ErbB4 in interneurons, but not pyramidal cells, of the rodent hippocampus. J Neurosci . 2009 Sep 30 ; 29(39):12255-64. Abstract

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