In contrast to its once barren form, the table of potential causative genes for schizophrenia is now stocked to feast level (Straub and Weinberger, 2006). In keeping with the culinary theme, we suggest that this recent paper by Chang-Gyu Hahn and Hoau-Yan Wang is “a full course meal”!

Appetizer: An Important Biological Problem
If one assumes that alterations in NRG-1 account for at least some of the liability to developing schizophrenia, then it is only reasonable to look to the NRG-1 receptors for clues as to how and where NRG-1 may be acting. However, there are three known NRG-1 receptors that mediate a myriad of biological functions, almost all of which could be argued to be relevant to schizophrenia pathology. This paper draws our attention to one NRG-1 receptor, ErbB4, showing this receptor to be a probable NRG-1 partner in mediating this pathology. Recent studies provide further support that ErbB4 may be integral to the development of schizophrenia by demonstrating its gene to...  Read more

In contrast to its once barren form, the table of potential causative genes for schizophrenia is now stocked to feast level (Straub and Weinberger, 2006). In keeping with the culinary theme, we suggest that this recent paper by Chang-Gyu Hahn and Hoau-Yan Wang is “a full course meal”!

Appetizer: An Important Biological Problem
If one assumes that alterations in NRG-1 account for at least some of the liability to developing schizophrenia, then it is only reasonable to look to the NRG-1 receptors for clues as to how and where NRG-1 may be acting. However, there are three known NRG-1 receptors that mediate a myriad of biological functions, almost all of which could be argued to be relevant to schizophrenia pathology. This paper draws our attention to one NRG-1 receptor, ErbB4, showing this receptor to be a probable NRG-1 partner in mediating this pathology. Recent studies provide further support that ErbB4 may be integral to the development of schizophrenia by demonstrating its gene to be a potential susceptibility gene (Norton et al., 2006; Silberberg et al., 2006; Nicodemus et al., in press). So, genetic and neurobiological evidence suggest the authors selected their NRG-1 receptor wisely.

Main Course: A New Approach
The novel postmortem-stimulation approach used by Hahn and colleagues represents an important advance in the field of schizophrenia research. Through extensive validation of this protocol, this research group has paved the way for future experimentation into the molecular activation of proteins within the schizophrenic brain. More specifically, while previous studies have only been able to draw conclusions about the static state of the schizophrenic brain, this article has introduced a novel method for examining dynamic signaling systems in postmortem brains of patients with schizophrenia. For example, based on the finding that certain splice variant ErbB4 mRNAs are elevated in the prefrontal cortex of these individuals (Silberberg et al., 2006), one would assume that ErbB4 protein should also be elevated in these patients. But Hahn et al. demonstrate that schizophrenic individuals show only marginal increases in prefrontal cortical ErbB4 protein levels, which could suggest that ErbB4 protein plays little role in the pathology of schizophrenia. However, using the more dynamic postmortem-stimulation approach, the authors showed that ErbB4 signaling is, in fact, greatly enhanced in the prefrontal cortex of patients with this disease, leading to the alternative interpretation that ErbB4 protein may play significant roles in schizophrenia. In other words, this postmortem-stimulation protocol extends the examination of human postmortem brain protein from quantification to the functional level. We view this method as a powerful approach that will be important in translating genetic susceptibility into molecular mechanisms of the disease process. The postmortem-stimulation approach also gave rise to the observation that schizophrenic patients exhibit reduced prefrontal cortical NMDA receptor signaling capacity. This finding is highly significant because it is the first evidence directly linking reduced prefrontal cortical NMDA receptor function to schizophrenia. However, whether NRG-1-ErbB4 signaling is a major contributor to NMDA receptor hypofunction is debatable since the attenuation of NMDA receptor phosphorylation by NRG-1 appears proportionally similar between controls and schizophrenic patients.

Side Dish: Dealing with Antipsychotic Drugs
Since most patients with schizophrenia have received antipsychotic drugs and these agents can have profound impact on brain systems, it is essential to determine whether changes observed in the brains of patients with schizophrenia are secondary to antipsychotic drug exposure. To address this issue, the authors took two important steps. Firstly, Hahn et al. examined whether antipsychotic drug exposure affected prefrontal cortical ErbB4 expression or signaling in their human study group and found no correlation between antipsychotic drug treatment and either of these measured variables. Secondly, the authors examined antipsychotic drug effects on prefrontal cortical ErbB4 signaling in mice implanted with a haloperidol-containing bioabsorbable polymer, which has a number of advantages. For example, it allows for long-term treatment of the animals (12 weeks) while minimizing handling. This duration of exposure is arguably more appropriate than some schedules used to examine chronic effects of antipsychotic drugs in rodents. Remarkably, haloperidol treatment caused a reduction in ErbB4 signaling in the mice, suggesting that a decrease in ErbB4 signaling is associated with the therapeutic effects of antipsychotic agents. What may have been more informative is to show whether haloperidol had any effect on ErbB4 protein levels without NRG-1 treatment. In addition, the authors could have considered examining antipsychotic drug effect in mice whose ages were more reflective of those of the investigated human cohort, which consisted of elderly individuals (65-92 years). Furthermore, while their analysis of antipsychotic drugs on ErbB4 expression and signaling in postmortem brain was noteworthy, the authors only examined the effects of antipsychotic drugs taken in the final month before death in a very aged sample population. Thus, it is difficult to ascertain whether ErbB4 expression or signaling is not affected by lifetime antipsychotic drug treatment, which can result in cellular and molecular consequences that can remain long after termination of therapy.

Dessert: Challenging the Field
Of course, the first thing the field needs to do is attempt to replicate these findings in another cohort of patients with schizophrenia compared to controls. Careful attention to matching for age, PMI, and gender, etc., as was done in this study, is critical. We suggest that using a young cohort of patients would help rule out potential confounds such as associated dementia and interaction with the aging process. However, it is recognized that many other potential confounds will still remain in most studies comparing schizophrenics to unaffected controls. These confounds include suffering from years of an unremitting illness that compromises normal social and environmental stimulation, increased incidence of cigarette smoking among patients with schizophrenia, and years of antipsychotic drug exposure. When the finding of schizophrenia-associated increased ErbB4 signaling capacity is replicated, then the task at hand will be to determine how possible genetic changes in the DNA at the NRG-1 or ErbB4 locus (representing one etiological route) could lead to a “hyperactivatable” ErbB4.

Doggie Bag: Nagging Questions
One of the caveats we would like to raise in attempting to link molecular neurobiological changes found in schizophrenic brain tissue with possible changes in DNA is that causative variants in any one susceptibility gene are expected to occur only in a minority of schizophrenic patients. Most measures performed on postmortem schizophrenic brains are made on small sample sizes, which likely show much heterogeneity in terms of etiology. In other words, only a handful of patients in this study would be expected to have a faulty NRG-1 gene; yet this subpopulation shows alterations in ErbB4 signaling as a group. The logical extension of this observation may be that there are multiple routes by which ErbB4 could be “hyperactivatable” (i.e., not solely through NRG-1 genetic liability). To sort this out, we need to work from the gene forward, and thus there is a need to identify causative variants in susceptibility genes and to use these as starting points for basic mechanistic molecular and cellular studies.

Comment by Daniel Stewart and Kenneth Davis
The Corfas results are intriguing. Their findings confirm much of what we have either found or suspect in schizophrenia relating to white matter involvement. Demonstrations of OLIG2 interactions with ErbB4 in the cortex and with CNP in the striatum in schizophrenia from our team (Georgieva et al., 2006) fit well with this investigation in providing evidence for a link between a variety of potential etiologic oligodendrocyte-related mechanisms in schizophrenia. While in our study, we did not find interaction with NRG1 and OLIG2, it is important to note that differential expression of NRG1 might be found only at certain points in the timeline of disease development. Other recent support from our team for white matter involvement in schizophrenia comes from an investigation in which an SNP associated with CNP was found to be significantly correlated with schizophrenia (Peirce et al., 2006). Interestingly, Corfas’s group reports that...  Read more

Comment by Daniel Stewart and Kenneth Davis
The Corfas results are intriguing. Their findings confirm much of what we have either found or suspect in schizophrenia relating to white matter involvement. Demonstrations of OLIG2 interactions with ErbB4 in the cortex and with CNP in the striatum in schizophrenia from our team (Georgieva et al., 2006) fit well with this investigation in providing evidence for a link between a variety of potential etiologic oligodendrocyte-related mechanisms in schizophrenia. While in our study, we did not find interaction with NRG1 and OLIG2, it is important to note that differential expression of NRG1 might be found only at certain points in the timeline of disease development. Other recent support from our team for white matter involvement in schizophrenia comes from an investigation in which an SNP associated with CNP was found to be significantly correlated with schizophrenia (Peirce et al., 2006). Interestingly, Corfas’s group reports that when ErbB signaling is abolished in oligodendrocytes, myelin structure appears normal, but the myelin sheath is significantly thinner. This is in line with some of the ultrastructural findings of Uranova’s group and in rodent studies looking at MAG-deficient mice (both reviewed in Davis et al., 2003)—another downregulated myelin-related gene found in brains of schizophrenia patients.

Reductions in oligodendrocyte number on the order of 20 percent have been demonstrated in the brains of schizophrenia patients (Hof et al., 2002). Although this finding does not precisely parallel the findings in this investigation, the authors’ adroitly point out that this may be because the abnormalities they induced were during early oligodendrocyte and myelin expression, while it is possible that the abnormalities seen in the brains of schizophrenia patients occur relatively later in development, more likely during the second large wave of cortical myelination at the end of the second decade of life. The authors also point out that “defects in ErbB signaling in different cell types may contribute to different aspects of psychiatric symptoms.” This might also be the case in schizophrenia, giving rise to the myriad presentations of the disease, as might the fact that expression of both NRG1 and ErbB4 are susceptible to environmental insult.

Other important similarities between the authors’ findings and schizophrenia include that, even in light of these myelin abnormalities, gross brain volumes, as well as several other measures, remained normal. This buttresses the idea that in schizophrenia, myelin abnormalities might be at the root of the often unimpressive brain changes noted in schizophrenia on gross structural imaging. And finally, although speculative, the authors do note an intriguing set of behavioral abnormalities, some of which could mimic the social isolation and poor relatedness of schizophrenia, which is particularly remarkable given the increased susceptibility to amphetamines and the trends seen in DAT, D1, and D2 expression in this investigation.

Corfas’s findings are indeed exciting, and we commend his team on an eloquently designed and implemented investigation.

Neuregulin1 (NRG1) is the most promising risk factor for schizophrenia, and the study of the signaling of NRG1 and its receptor ErbB4 is very important in understanding the pathophysiology of the disease. Like other promising risk factors for schizophrenia, NRG1/ErbB4 is multifunctional with many molecular isoforms. NRG1/ErbB signaling plays a role both before and after birth. Furthermore, ErbB4 is expressed not only in neurons but also in other types of cells, such as oligodendrocytes.

To address context-dependent functions one by one, dominant-negative transgenic mice can be very useful. The advantage of dominant-negative transgenics is that we can knock down the endogenous function of our target molecules (in this work, ErbB4) in a temporally and spatially specific manner by utilizing a well-characterized promoter. In this outstanding study by Corfas and colleagues, they used the CNP promoter that confirms dominant-negative ErbB4 selectively in oligodendrocytes (but not in astrocytes and neurons) only after birth. This approach will be very useful in schizophrenia...  Read more

Neuregulin1 (NRG1) is the most promising risk factor for schizophrenia, and the study of the signaling of NRG1 and its receptor ErbB4 is very important in understanding the pathophysiology of the disease. Like other promising risk factors for schizophrenia, NRG1/ErbB4 is multifunctional with many molecular isoforms. NRG1/ErbB signaling plays a role both before and after birth. Furthermore, ErbB4 is expressed not only in neurons but also in other types of cells, such as oligodendrocytes.

To address context-dependent functions one by one, dominant-negative transgenic mice can be very useful. The advantage of dominant-negative transgenics is that we can knock down the endogenous function of our target molecules (in this work, ErbB4) in a temporally and spatially specific manner by utilizing a well-characterized promoter. In this outstanding study by Corfas and colleagues, they used the CNP promoter that confirms dominant-negative ErbB4 selectively in oligodendrocytes (but not in astrocytes and neurons) only after birth. This approach will be very useful in schizophrenia research.

The remarkable finding is that they observed alterations in dopamine-mediated neuronal networks and associated behaviors by disturbing NRG1/ErbB4 selectively in cells of oligodendrocyte lineage. Three important paradigms for schizophrenia (white matter pathology, dopamine, and a susceptibility gene) converge in this paper, and in this sense, I find it very exciting.

Does the effect of NRG1/ErbB4 signaling on myelination occur downstream of purinergic signaling? Fields suggests that adenosine is of primary importance in regulating early development of OPCs, where it stimulates differentiation and myelination (Fields, 2006). It's of interest that cAMP stimulates expression of neuregulin and cAMP levels in the lung are decreased in A2A adenosine receptor (22q11.2)-deficient mice (Tokita et al., 2001; Nadeem et al., 2007). Do you see reduced neuregulin levels in 22q11 deletion syndrome? Of particular interest is the study by Desai and colleagues reporting that signaling via the adenosine A2A receptor downregulates thrombospondin 1 (Desai et al., 2005). Perhaps overexpression of thrombospondin 1 may help explain the occular abnormalities in this syndrome (Wu et al., 2006; Forbes et al., 2007; Stalmans, 2005). Thrombospondins are also involved in synaptogenesis (Christopherson et al., 2005).

References:

Fields RD. Nerve impulses regulate myelination through purinergic signalling. Novartis Found Symp. 2006;276:148-58; discussion 158-61, 233-7, 275-81.

Tokita Y, Keino H, Matsui F, Aono S, Ishiguro H, Higashiyama S, Oohira A. Regulation of neuregulin expression in the injured rat brain and cultured astrocytes. J Neurosci. 2001 Feb 15;21(4):1257-64.

Nadeem A, Fan M, Ansari HR, Ledent C, Mustafa SJ. Enhanced airway reactivity and inflammation in A2A adenosine receptor deficient allergic mice. Am J Physiol Lung Cell Mol Physiol. 2007 Feb 9; [Epub ahead of print]

Desai A, Victor-Vega C, Gadangi S, Montesinos MC, Chu CC, Cronstein BN. Adenosine A2A receptor stimulation increases angiogenesis by down-regulating production of the antiangiogenic matrix protein thrombospondin 1. Mol Pharmacol. 2005 May;67(5):1406-13. Epub 2005 Jan 26. Comment in: Mol Pharmacol. 2005 May;67(5):1385-7.

Wu Z, Wang S, Sorenson CM, Sheibani N. Attenuation of retinal vascular development and neovascularization in transgenic mice over-expressing thrombospondin-1 in the lens. Dev Dyn. 2006 Jul;235(7):1908-20.

Forbes BJ, Binenbaum G, Edmond JC, Delarato N, McDonald-McGinn DM, Zackai EH. Ocular findings in the chromosome 22q11.2 deletion syndrome. J AAPOS. 2007 Apr;11(2):179-182. Epub 2006 Nov 30.

Stalmans I. Role of the vascular endothelial growth factor isoforms in retinal angiogenesis and DiGeorge syndrome. Verh K Acad Geneeskd Belg. 2005;67(4):229-76.

Christopherson KS, Ullian EM, Stokes CC, Mullowney CE, Hell JW, Agah A, Lawler J, Mosher DF, Bornstein P, Barres BA. Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis. Cell. 2005 Feb 11;120(3):421-33. Comment in: Cell. 2005 Feb 11;120(3):292-3.

View all comments by Mary Reid

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...  Read more

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