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Mouse Models Explore Faulty Neuregulin Splitting and Signaling

22 September 2008. Four new mouse studies sketch how molecular alterations involving neuregulin1 (Nrg1) might lead to neurological and behavioral deficits in schizophrenia. In one, Bart De Strooper of the University of Leuven and Flanders Institute of Biotechnology, in Leuven, Belgium, and colleagues followed leads gained from their work on Alzheimer’s disease. Their July 15 PNAS report hints that turning off a gene that helps split proteins, including Nrg1, in the cell membrane produces cognitive deficits and neuropharmacological responses that parallel some of those seen in schizophrenia.

The other three studies zeroed in on a type of Nrg1 expressed mostly or totally in the nervous system. In the July 2 issue of the Journal of Neuroscience, a team led by David Talmage and Lorna Role, both of whom moved recently from Columbia University in New York City to Stony Brook University in Stony Brook, New York, conclude that type III Nrg1 plays crucial roles in brain structures involved in filtering sensory information and using short-term memory. Talmage's group also has a paper in the May 5 Journal of Cell Biology, suggesting that type III Nrg1 controls expression of α7 nicotinic acetylcholine receptors (nAChRs) along sensory neuron axons by acting as a receptor itself, instead of playing its better-known ligand role. Finally, in the September 10 issue of the Journal of Neuroscience, Role and colleagues link aspects of both these lines of research in describing a critical role for type III Nrg1, functioning as a receptor, in α7nAChR-mediated neurotransmission between hippocampus and striatum.

Complex molecular scissors
The gene that encodes Nrg1 has emerged as a leading schizophrenia candidate gene, notwithstanding the less-than-encouraging findings from the recent SchizophreniaGene meta-analysis (see SRF related news story). The protein facilitates processes that range from neural development to adult synaptic functioning, but only if split into the proper segments by a series of enzymes. One step involves snipping off a fragment that hangs outside the cell; this becomes the ligand for the ErbB4 receptor. Another occurs when the “stub” that remains lodged within the cell membrane subsequently undergoes splitting by γ-secretase, releasing it back into the cell.

The intramembrane protease γ-secretase may be best known for processing amyloid precursor protein into the amyloid-β that forms plaques in Alzheimer’s disease. In humans, the large γ-secretase complex includes one of the two forms of the protein Aph1, either the A or B variant. Mice carry an additional gene that encodes Aph1C, a twin of Aph1B; the two genes are typically referred to jointly as Aph1B/C.

De Strooper’s laboratory had been examining the role of the Aph1B/C-containing γ-secretase in the processing of amyloid precursor protein (Serneels et al., 2005). He and his colleagues wondered whether it also plays a part in the cleaving of Nrg1 and, hence, contributes to schizophrenia.

The causes of snipping snafus
In their new study, first author Tim Dejaegere and his coauthors examined the processing of Nrg1 in Aph1B/C knockout mice. They wanted to learn what Aph1B/C does; unlike Aph1A, it plays no crucial role in survival, so Aph1B/C KO mice survive to adulthood with no overt ill effects.

Dejaegere and colleagues found abnormal buildup of Nrg1 fragments in a number of brain areas in the knockout mice, suggesting that Aph1B/C is indeed critical for the proper cleaving of Nrg1. They identified the fragments not as the peptide that signals through ErbB4 receptors, but rather as the membrane-bound stub that should have been cleaved by Aph1B/C-γ-secretase.

In an interesting piece of supporting investigation, the researchers examined how deficits in Nrg1, as opposed to the γ-secretase complex, might lead to the same end. They focused on a particular single nucleotide polymorphism (SNP), in the coding region of the Nrg1 gene, reported to increase schizophrenia risk (Waiss-Bass et al., 2006). This SNP causes a valine-to-leucine substitution in the transmembrane region, where it might disrupt γ-secretase cleavage of the Nrg1 membrane-bound stub. Indeed, when Dejaegere and colleagues introduced this mutation to type III Nrg1 in otherwise normal cells in culture, they found above-normal accumulation of the stub.

Also of interest, in normal mice, the researchers found high levels of expression of Aph1B/C in the cerebral cortex, hippocampus, olfactory bulb, and cerebellum, but not the striatum. “Aph1B/C expression is enriched in brain areas with relevance for schizophrenia,” they write. They note that the areas with the most expression in the prefrontal cortex (PFC) and hippocampus, layer 5 and CA1, respectively, include those that send excitatory glutamatergic signals to the ventral striatum and other brain areas (for information on glutamate’s possible role in schizophrenia, see SRF current hypothesis by B. Moghaddam, as well as paper by D. Javitt cited below).

The consequences of snipping snafus
To illuminate the functions of Aph1B/C, the researchers compared knockout mice with their normal littermates using paradigms that often stand in for behavioral aspects of schizophrenia—specifically, tests of prepulse inhibition (PPI) and working memory. PPI, a common experimental paradigm for sensory gating, occurs when a weak stimulus lessens the startle response to a subsequent stronger stimulus. Studies have found PPI deficits in some patients with schizophrenia, suggesting that they cannot filter sensory information well. PPI tasks are often used to test putative animal models of schizophrenia pathology.

In the study by Dejaegere and colleagues, the Aph1B/C knockout mice showed PPI impairments, which disappeared after injection of haloperidol, a typical antipsychotic, or clozapine, an atypical antipsychotic. Since all current antipsychotic drugs block dopamine D2 receptors to alleviate psychosis, the team assessed dopaminergic functioning by administering amphetamine. This drug not only induces psychosis in otherwise healthy people, it worsens the symptoms of people with schizophrenia, apparently by promoting dopamine release in the ventral striatum.

The scientists found that the amphetamine revved up the Aph1B/C knockout mice more than their normal siblings. Furthermore, the knockouts showed evidence of excessive dopamine turnover in tissue from the ventral striatum. These findings dovetail with a prominent theory that puts dopamine hyperactivity in the mesolimbic pathway at the root of schizophrenia (see SRF current hypothesis). This pathway links the ventral tegmentum in the midbrain to the ventral striatum.

Dejaegere and colleagues also examined other possible neurotransmitter disturbances. “A hyperdopaminergic state of the ventral striatum can be a consequence of a primary PFC dysfunction of glutamatergic signaling,” they note. Since tests of working memory can gauge PFC disturbances, the researchers subjected the rodents to a working memory test in which recently presented information would help them navigate a maze. The knockout mice performed worse than their wild-type siblings.

To further explore glutamate’s role in the knockout mice’s behavior, the researchers injected both groups of mice with MK-801. This drug, used in a standard animal model of schizophrenia, blocks N-methyl-D-aspartate (NMDA) receptors. These glutamate receptors probably play a key role in memory, attention, and other cognitive functions (see SRF current hypothesis). Consistent with past research, MK-801 impaired PPI in normal mice, but it weakened it even more in the knockouts, to the point of nearly eliminating it. These findings relate the flawed filtering of sensory information to disturbed glutamatergic signaling involving NMDA receptors.

The investigators conclude, “Dysregulation of intramembrane proteolysis of Nrg1 could increase risk for schizophrenia and related disorders” by unleashing ripples through the glutamatergic and dopaminergic pathways. They view their data as supporting both the glutamate and dopamine hypotheses of schizophrenia.

In related work, some members of the research team have been exploring whether silencing Aph1B/C improves symptoms in a mouse model of Alzheimer’s disease. Their schizophrenia study, although it did not look at amyloid processing, warns that treating Alzheimer’s in such a way could raise schizophrenia risk. Hedging their bets, Dejaegere and colleagues write, “Because a wealth of data supports the contention that the development of schizophrenia depends on a neuronal insult early in life, it is possible that suppression of Aph1B/C activity in the brain in adulthood only will have no severe side effects at all.”

Portrait of a mouse with too little type III
Different gene promoters and ways of splicing result in over 15 isoforms of Nrg1. Enzyme-mediated cleavage snips most forms from the cell surface, enabling them to diffuse and affect distant targets. Only type III stays bound to the cell membrane, limiting its signaling to bordering cells ("juxtacrine" signaling”).

The uniqueness of type III, combined with its neuronal-specific expression, has intrigued the two closely collaborating groups of Talmage and Role for some time. In their recent study in the Journal of Neuroscience, led by first author Ying-Jiun Chen, currently of the University of California at San Francisco, they extended their research on its effects on neural circuits and behaviors that relate to schizophrenia. In addition, they sought to learn what regulates Nrg1 expression.

Chen and colleagues compared adult mutant mice that were heterozygous knockouts for type III Nrg1 with their wild-type siblings on structural, functional, and behavioral measures. They looked at heterozygotes because mice that produce no type III Nrg1 die at birth. The authors note that the heterozygotes had larger cerebral lateral ventricles than the wild-type mice. In addition, they had reduced density of dendritic spines, which receive excitatory input, on pyramidal neurons in the subiculum. These neurons convey processed information from the hippocampus to other brain areas such as the ventral striatum. The researchers liken these structural anomalies to those found in autopsy studies of humans with schizophrenia.

Further clues indicate that type III Nrg1 affects not only brain structure, but also function. In the heterozygotes, Chen and colleagues found evidence of low metabolism in hippocampal regions CA3 and CA1, the subiculum, and the medial prefrontal cortex. Such structural and functional changes might trigger abnormal behavior. On a maze test that required use of short-term memory and on an auditory PPI task, the heterozygotes performed worse than their wild-type peers. Interestingly, when the researchers had characterized the normal distribution of type III in the mouse brain, they found that it is "expressed in the medial prefrontal cortex, ventral hippocampus, and ventral subiculum, regions involved in the regulation of sensorimotor gating and short-term memory.”

In prior research, PPI deficits in mice and subjects with schizophrenia returned to normal after nicotine administration. Furthermore, many people with schizophrenia smoke, perhaps to self-medicate. After Chen and colleagues gave mice water laced with nicotine for six weeks, the heterozygotes performed as well as the wild-type mice.

Summing up their results, Chen and colleagues write, “Decreased expression of type III Nrg1 leads to structural, functional, and behavioral alterations that are related to schizophrenia.” This study, however, did not test whether the structural and functional changes directly affect behaviors related to schizophrenia in mice, let alone humans with the disease.

Backwards signaling targets nicotinic receptors
Complementing the work by Chen and associates, the remaining study from the Talmage and Role groups looked at the relationship between Nrg1 and nAChRs. Nicotine docks on these receptors, and past research hints that type III Nrg1 regulates expression of nAChR subunits, including α7. Research on animals, as well as people with schizophrenia and their relatives, links α7-containing nAChRs to sensory gating and to schizophrenia (for a literature review, see Martin and Freedman, 2007; see SRF related news story; SRF news story).

This other study, led by first author Melissa Hancock, investigated the molecular mechanisms by which type III Nrg1 affects presynaptic α7-containing nAChRs. When the researchers examined sensory neurons from embryos of type III Nrg1 knockout mice, they detected a significant reduction, relative to wild-type, of axonal α7nAChR surface expression. There was no overall reduction of this receptor subunit in the neurons, suggesting that trafficking to the cell membrane was specifically impaired.

The next challenge involved figuring out what role type III Nrg1 plays in α7 trafficking. In forward signaling, Nrg1 acts as a ligand for ErbB receptor tyrosine kinases (see SRF related news story). The Nrg1 fragment that is cleaved from the outside of the cell interacts with receptors on target cells some distance away. However, in this case, the researchers suspected that a different process, dubbed “back signaling” (Bao et al., 2003), might play a role. This presynaptic signaling occurs when the fragment that remains within the cell membrane acts as a receptor for ErbB, spurring the cell that expressed type III Nrg1 to fire.

To find out if type III Nrg1 controls nAChR expression by acting as a ligand or a receptor, Hancock and colleagues treated the neurons with extracellular domain from either ErbB4 (B4-ECD) or ErbB2 (B2-ECD). B4-ECD binds tightly with Nrg1; since B2-ECD does not, it served as a control substance. Only B4-ECD increased expression of α7 nAChRs along axons from the wild-type mice; neither substance did so on cells from the mutants.

When the researchers repeated the experiment with the addition of substances that hinder ErbB tyrosine kinase activity, α7 nAChRs still gathered along the axons. They inferred that type III controls the insertion of α7 nAChRs into the membrane by acting as a receptor for ErbB4 and not as a ligand that launches tyrosine kinase signaling.

The investigators found that the increased surface expression did not stem from ramped-up production of α7 nAChRs. Rather, back signaling appeared to shift existing nAChRs from caches inside the neuron to the axon membrane. It did so by activating a phosphatidylinositol 3-kinase signaling pathway that controls the movement of cell surface proteins. “These findings, in conjunction with prior results establishing that Type III Nrg1 back signaling controls gene transcription, demonstrate that Type III Nrg1 back signaling can regulate both short- and long-term changes in neuronal function,” Hancock and colleagues write.

Chimeras are different
The most recent installment in the Talmage-Role collaboration links aspects of both the Chen and Hancock papers. In a study published in the September 10 issue of the Journal of Neuroscience, Role, first author Chongbo Zhong, and colleagues examined the role of type III Nrg1 in synaptic transmission in a hippocampus-to-ventral striatum circuit, which they note, is "an important relay in sensory-motor gating."

The experimental method is a major technical challenge—creating a chimeric circuit by "hooking up" the output area of the hippocampus—ventral CA1 and subiculum—from a type III Nrg1 deficient mouse to the target region in the ventral striatum—the nucleus accumbens—from a normal mouse. This is all done in a cultured brain slice preparation, of course, where an excised piece of hippocampal tissue is allowed a day to accommodate itself to life in vitro before nucleus accumbens neurons from a different brain are added. Within a week in culture, the hippocampal neurons form synaptic connections with accumbal cells, and the researchers are able to record glutamatergic synaptic activity from the acumbal target cells in the chimeric circuits.

Whether the hippocampal cells came from mice with normal or reduced levels of type III Nrg1 made no difference in their effects on baseline postsynaptic activity of the nucleus accumbens cells. But when Zhong and colleagues added nicotine, they noticed a major difference. In wild-type-to-wild-type circuits, a single puff of nicotine boosted neurotransmission in the circuit for up to an hour. But when the hippocampal cells had reduced levels of type III Nrg1, nicotine produced only a brief enhancement of glutamatergic transmission in the circuit. "Alterations in this temporal profile might lead to deficits in sensory gating by altering glutamatergic transmission in corticostriatal circuits," the authors write.

Underlying this deficiency, the researchers report, is an 80 percent reduction in α7nACh receptors on the axons of the hippocampal cells. Zhong and colleagues were able to reverse this situation by incubating the hippocampal cells with the ErbB4 extracellular domain, supporting the evidence from the earlier paper by Hancock and colleagues that back signaling by type III Nrg1 controls the levels of α7nACh receptors on these axons.

These four new studies contribute to a framework that may eventually tie together the genes that encode Nrg1, ErbB4, and α7 as potential contributors to cognitive deficits in schizophrenia. They may also fuel suspicions that the pathways underlying schizophrenia and neurodegenerative diseases overlap.—Victoria L. Wilcox and Hakon Heimer.

Dejaegere T, Serneels L, Schäfer MK, Van Biervliet J, Horré K, Depboylu C, Alvarez-Fischer D, Herreman A, Willem M, Haass C, Höglinger GU, D’Hooge R, De Strooper B. Deficiency of Aph1B/C-gamma-secretase disturbs Nrg1 cleavage and sensorimotor gating that can be reversed with antipsychotic treatment. PNAS. 2008 July 15;105(28):9775-9780. Abstract

Hancock ML, Canetta SE, Role LW, Talmage DA. Presynaptic type III neuregulin 1-ErbB signaling targets alpha7 nicotinic acetylcholine receptors to axons. J Cell Biol. 2008 May 5;181(3):511-521. Abstract

Chen Y-J J, Johnson MA, Lieberman MD, Goodchild RE, Schobel S, Lewandowski N, Rosoklija G, Liu R-C, Gingrich JA, Small S, Moore H, Dwork AJ, Talmage DA, Role LW. Type III neuregulin-1 is required for normal sensorimotor gating, memory-related behaviors, and corticostriatal circuit components. J Neurosci. 2008 July 2;28(27):6872-6883. Abstract

Zhong C, Du C, Hancock M, Mertz M, Talmage DA, Role LW. Presynaptic type III neuregulin 1 is required for sustained enhancement of hippocampal transmission by nicotine and for axonal targeting of alpha7 nicotinic acetylcholine receptors. J Neurosci. 2008 Sep 10;28(37):9111-6. Abstract

Comments on Related News

Related News: Nicotinic Receptor Agonist Shows Promise in Pilot Study

Comment by:  Robert W. Buchanan
Submitted 5 July 2006
Posted 5 July 2006

In light of the limitations of first- and second-generation antipsychotics and other pharmacological agents for the treatment of cognitive impairments in schizophrenia, the demonstration of an acute benefit of DMXB-A for cognitive performance and sensory gating is of considerable potential interest. Patients with schizophrenia are characterized by a broad range of cognitive impairments (Nuechterlein et al., 2004). These impairments have been shown to be a major determinant of poor functional outcome (Green et al., 2004). The NIMH has made a substantial commitment to facilitate the development of new pharmacological treatments for cognitive impairments through their funding of the Measurement and Treatment Research to Improve Cognition in Schizophrenia (MATRICS) and Treatment Units for Research on Neurocognition and Schizophrenia (TURNS) initiatives. The MATRICS process selected the α7 nicotinic receptor as one of the top targets for the treatment of cognitive impairments in patients with schizophrenia (Geyer and Tamminga, 2004).

In the current study, 12 subjects with schizophrenia were administered DMXB-A, an α7 nicotinic receptor partial agonist. The study was designed to assess the proof of concept that increased stimulation of the α7 nicotinic receptor would enhance performance on a cognitive battery and improve sensory gating, as measured by the P50 dual click paradigm. There is extensive preclinical and clinical rationale for this approach, but few drugs are available to directly assess the efficacy of the approach. The results clearly support the benefit of this approach, but a number of questions will need to be addressed in future studies before the ultimate utility of this approach is known. The most important issue is whether the acute efficacy observed with essentially a single dose will translate to a persistence of an effect with chronic drug administration. Tachyphylaxis develops rapidly with repeated stimulation of the α7 nicotinic receptor. Preclinical data suggest that this may not be an issue with DMXB-A, but long-term exposure data are required to directly address this issue. Second, two DMXB-A doses were evaluated. The results were different between the two doses, but how different is unclear, because of the small sample size. Preclinical data suggest that α7 nicotinic receptor partial agonists may show an inverse u-shaped response curve. The loss of efficacy at higher doses underscores the importance and potential difficulty in delineating the most effective dose range. Third, subjects who used nicotine products in the last month were excluded from the study. Patients with schizophrenia who do not smoke cigarettes are in the minority, and perhaps represent less than a third of the total population. The question of whether the beneficial effect of the drug would generalize to patients who smoke cigarettes will eventually need to be evaluated. Finally, the most pronounced effect was observed for the RBANS attention index. This effect is consistent with previous studies of acute nicotine administration in patients with schizophrenia. Future studies will need to evaluate whether the effect of DMXB-A is largely limited to attention or whether it will have significant benefit for other cognitive domains.

In summary, the demonstration that the acute administration of DMXB-A produces improved performance on a neuropsychological battery is an important first step in developing a novel therapeutic approach for one of the most critical areas of schizophrenia therapeutics.


Nuechterlein KH, Barch DM, Gold JM, Goldberg TE, Green MF, Heaton RK. Identification of separable cognitive factors in schizophrenia. Schizophr Res. 2004; 72: 29-39. Abstract

Green MF, Kern RS, Heaton RK. Longitudinal studies of cognition and functional outcome in schizophrenia: implications for MATRICS. Schizophr Res. 2004;7 2: 41-51. Abstract

Geyer MA, Tamminga CA. Measurement and treatment research to improve cognition in schizophrenia: neuropharmacological aspects. Psychopharm. 2004; 174: 1-2.

View all comments by Robert W. Buchanan

Related News: Nicotinic Receptor Agonist Shows Promise in Pilot Study

Comment by:  Patricia Estani
Submitted 6 July 2006
Posted 6 July 2006
  I recommend the Primary Papers

Related News: Neuregulin, ErbB4 Drive Developmental Cell Fates

Comment by:  Cynthia Shannon Weickert, SRF AdvisorVictor Chong
Submitted 18 December 2006
Posted 18 December 2006

The study by Sardi et al. is truly remarkable. Their report of a novel ErbB4 cleavage-dependent mechanism regulating neuronal/astrocytic differentiation is groundbreaking, but their approach to unraveling and confirming this mechanism is more impressive. From their use of a yeast two-hybrid system in finding novel ErbB4 intracellular domain (E4ICD)-interacting factors to their meticulous experimental dissection of hypotheses and observations, the Corfas group has raised the bar in the investigation of mechanisms by which ErbB4 regulates neural precursor fates. In addition, the authors have shown that changes in E4ICD intracellular signaling pathways may produce cellular consequences distinct from those resulting from alterations in the activity of membrane-bound full-length ErbB4. More specifically, Sardi et al. illustrate that ErbB4 cleavage can regulate very early cell fates in the nervous system, while intact ErbB4 has mainly been examined in terms of its action at mature cortical synapses where its activation can dampen NMDA receptor function.

Recognition must also be given to Pat McCaffrey and Hakon Heimer, who provided an excellent summary of the article and highlighted the significance of the paper to schizophrenia. In the manuscript, disruptions in E4ICD signaling are discussed in terms of their relevance to Alzheimer disease. Since aberrant neuregulin-1-ErbB4 signaling has been implicated in schizophrenia, one could hypothesize that alterations in E4ICD-associated interactions and/or in the nuclear translocation of E4ICD complexes may contribute to schizophrenia pathology as well. Hence, it will be important to test whether either or both of these ErbB4-signaling streams are in fact altered not only in Alzheimer disease, but also in schizophrenia.

The difficulty in linking ErbB4 to neuropathological mechanisms underlying schizophrenia may be due in part to the limited information that exists on early developmental processes of this illness. In their paper, Sardi et al. suggest disruptions in ErbB4-dependent mechanisms may lead to premature astrogenesis that could contribute to Alzheimer disease-associated gliosis. However, in thinking about the relevance of altered E4ICD signaling in schizophrenia, elevated glial formation has not been observed in the schizophrenic brain. In fact, expression of the glial marker, GFAP, has been shown to be reduced in postmortem brains of subjects with this disorder (Johnston-Wilson et al., 2000; Webster et al., 2005). On the other hand, recent investigations suggest elevated prefrontal cortical feed-forward neuregulin-1-ErbB4 signaling in schizophrenia (Hahn et al., 2006), and this increase could lead to GFAP reductions possibly through the mechanism proposed by Sardi et al. if this elevated signaling translated to greater E4ICD cleavage. However, whether the findings of Sardi et al. in neural precursor cells extend to cells of the adult human brain remains to be explored. Nevertheless, the Corfas group has opened new avenues of research in the field of schizophrenia and has provided a framework for future studies on the role ErbB4 signaling in this disease.


Johnston-Wilson N.L., Sims C.D., Hofmann J.-P., Anderson L., Shore A.D., Torrey E.F. and Yolken R.H. (2000) Disease-specific alterations in frontal cortex brain proteins in schizophrenia, bipolar disorder, and major depressive disorder. Mol. Psychiatry. 5: 142-149. Abstract

Webster M.J., O'Grady J., Kleinman J.E. and Weickert C.S. (2005) Glial fibrillary acidic protein mRNA levels in the cingulate cortex of individuals with depression, bipolar disorder and schizophrenia. Neuroscience. 133: 453-461. Abstract

Hahn C.G., Wang H.Y., Cho D.S., Talbot K., Gur R.E., Berrettini W.H., Bakshi K., Kamins J., Borgmann-Winter K.E., Siegel S.J., Gallop R.J., Arnold S.E. (2006) Altered neuregulin 1-erbB4 signaling contributes to NMDA receptor hypofunction in schizophrenia. Nat. Med. 12: 824-828. Abstract

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Related News: Sweeping SchizophreniaGene Study Applies New Criteria to Finger Suspects

Comment by:  Stephen J. Glatt
Submitted 17 July 2008
Posted 21 July 2008
  I recommend the Primary Papers

The paper by Allen et al. is a tremendously useful addition to the fields of schizophrenia research, psychiatric genetics, and medical genetics. By efficiently summarizing a tremendous amount of work, Allen et al. have endeavored to provide a "state-of-the-art" summary that most of us, as individuals, struggle to accomplish; they have largely succeeded in their attempt. This manuscript, and the continual availability of the SZGene database, should long serve as invaluable resources for the increasingly complex task of building polygenic models of risk for schizophrenia. Furthermore, these methods, which were initially implemented in the AlzGene database, have clearly generalized quite successfully to SZGene and thus, should be easy enough to scale up to cover many other psychiatric disorders as well. In this way, the contribution to psychiatric genetics, and possibly other disorders outside of psychiatry, is crystalline.

Aside from the database, the contribution of the recent manuscript to the field of schizophrenia research is also tremendous. As pointed out by the authors, several of the significantly associated genes identified by their meta-analyses were never before studied in this manner, so a whole new set of top candidate genes was identified. This work also served to confirm the results of prior meta-analyses from my group and others, which is always reassuring. Application of the HuGENet criteria to grading the detected associations is useful as a heuristic, but it must be kept in mind that that while these criteria reflect a consensus, they also reflect a moving target. One difficulty in implementing grades (especially the "overall" grade) is analogous to difficulties often encountered in meta-analyses when rating the quality of studies, and that is the ambiguity of ratings. Thus, on a seven-point quality scale (or a three-letter-grade scale), a score can be arrived at by a variety of combinations of flaws or strengths, but similar scores may not (often do not) reflect identical strengths and weaknesses of the graded studies. For example, I, for one, am not certain that having a relatively low number of minor alleles reflected in a meta-analytic result (especially if it is a rare variant) is as big a decrement as the pooled OR dropping from significance when the initial study is omitted.

Nevertheless, I reiterate that the use of this heuristic grading system is helpful, but should be taken with a grain of salt. Overall, the paper and its conclusions are a great contribution to this field and warrant mass attention. The ultimate question, not yet addressed here but apparently on the horizon, is how well the emerging GWASs detect these "positive control" associations, or we might say how well these hypothesis-driven results stack up against new candidates to emerge from the high-throughput generation of novel hypotheses....

View all comments by Stephen J. Glatt

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: Unkind Cuts of NRG3 May Lead to Schizophrenia

Comment by:  Assen Jablensky
Submitted 15 September 2010
Posted 15 September 2010

Common or rare genetic variation in NRG3 influences risk for schizophrenia?
Emerging evidence implicating NRG3 as a likely susceptibility gene in population samples as diverse as the Ashkenazi Jews, Han Chinese, Australians of Anglo-Irish ancestry, and white Americans is certainly a “noteworthy” occurrence in schizophrenia genetics. The latest addition to the evidence (Kao et al., 2010) provides considerable support to earlier (Fallin et al., 2003; Wang et al., 2008) and recent findings of association of several polymorphisms (rs10883866, rs6584400, rs10748842) within a conserved linkage disequilibrium (LD) block in intron 1 of the NRG3 gene with a delusion-laden factor and a neurocognitive quantitative trait in the schizophrenia phenotype (Chen et al., 2009; Morar et al., 2010).

A fundamental contribution of the present study is the cloning and detailed characterization of full-length NRG3 transcripts from postmortem fetal, child, adolescent, and adult brain samples (whole brain, hippocampus, and dorsolateral prefrontal cortex). Sequencing of the cDNA clones and expression analysis revealed a complex picture of alternative splicing, abundance of developmentally regulated transcripts in schizophrenia brains, and, notably, increased expression of a fetal brain-derived clone (hFBNRG3), which introduces a premature stop codon resulting in a truncated protein and a possibly destabilized NRG3-ErbB4 signalling pathway. In their clinical collections (a family-based sample and a partially independent case-control sample), the authors report significant associations of rs10748842 (representing 12 SNPs located in the LD block within intron 1) with schizophrenia, with the PANSS (Positive and Negative Syndrome Scale) subscale score on delusion severity, as well as with the PANSS negative symptom load.

Overall, the findings from this investigation and the earlier studies appear to be in a broad agreement, converging on a plausible role of NRG3 in schizophrenia pathogenesis. However, there is a fly in the ointment: The associations found in the present study exhibit a risk allele reversal compared to previously reported results; namely, all significant associations are with the major, common alleles, rather than with the minor alleles, as in Chen et al. (2009) and Morar et al. (2010). While many reasons for genuine allele flipping can be invoked (multi-locus interactions, variation in local patterns of LD, environmental exposures, ethnic background differences—see Clarke and Cardon, 2010), the explanation for the flip in this particular context is not obvious, and NRG3 should remain on the examination bench. Even in the GWAS era, studies proceeding from biologically and clinically anchored hypotheses remain rewarding and potentially productive.


Chen PL, Avramopoulos D, Lasseter VK, McGrath JA, Fallin MD, Liang K-Y, Nestadt G, Feng N, Steel G, Cutting AS, Wolyniec P, Pulver AE, Valle D. Fine mapping on chromosome 10q22-q23 implicates Neuregulin 3 in schizophrenia. Am J Hum Genet. 2009;84:21-34. Abstract

Clarke GM, Cardon LR. Aspects of observing and claiming allele flips in association studies. Genet Epidemiol. 2010;34:266-74. Abstract

Fallin MD, Lasseter VK, Wolyniec PS, McGrath JA, Nestadt G, Valle D, Liang KY, Pulver AE. Genomewide linkage scan for schizophrenia susceptibility loci among Ashkenazi Jewish families shows evidence of linkage on chromosome 10q22. Am J Hum Genet. 2003;73:601-11. Abstract

Kao WT, Wang Y, Kleinman JE, Lipska BK, Hyde TM, Weinberger DR, Law AJ. Common genetic variation in Neuregulin 3 (NRG3) influences risk for schizophrenia and impacts NRG3 expression in human brain. Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15619-24. Abstract

Morar B, Dragovic M, Waters FAV, Chandler D, Kalaydjieva L, Jablensky A. Neuregulin 3 (NRG3) as a susceptibility gene in a schizophrenia subtype with florid delusions and relatively spared cognition. Mol Psychiatry. 2010 June 15. Abstract

Wang YC, Chen JY, Chen ML, Chen CH, Lai IC, Chen TT, Hong CJ, Tsai SJ, Liou YL. Neuregulin 3 genetic variations and susceptibility to schizophrenia in a Chinese population. Biol Psychiatry. 2008;64:1093-6. Abstract

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