17 February 2005. The last few years have seen a flurry of activity around neuregulin 1 (NRG1), with several studies finding or confirming that polymorphisms in the gene are linked to schizophrenia in Icelandic (see Stefannson et al., 2002; Williams et al., 2003), Irish (see Corvin et al., 2004), Chinese (see Yang et al., 2003), and Dutch (see Bakker et al., 2004) populations. But exactly how neuregulin 1 contributes to pathology is unclear. The protein is a large complex molecule involved in many biological activities, such as neuronal migration, synaptogenesis, myelination, and cell-to-cell communication. In the human brain, neuregulin 1 comes in as many as 15 different isoforms that may contain transmembrane, immunoglobulin, or epidermal growth factor (EGF) domains, to name just a few. While any or all of these motifs may be involved in schizophrenia pathology, the EGF domain may be particularly relevant (for review, see Harrison and Law, 2006 and SRF related meeting story). Two recent papers in the American Journal of Medical Genetics report that polymorphisms in the ErbB4 tyrosine kinase, a receptor for neuregulin’s EGF domain, are also linked to schizophrenia. In addition, a paper in PNAS explores the role of neuregulins and ErbB4 in neuronal differentiation and migration during adult neurogenesis.
Of ErbB4 SNPs and haps
In the January issue of the journal, Michael O’Donovan, Wales School of Medicine, Cardiff, and colleagues reported that a combination of specific polymorphisms in the neuregulin and ErbB4 genes increases susceptibility to schizophrenia. First author Nadine Norton and colleagues first sequenced the gene in DNA samples obtained from 14 unrelated patients to determine if there were any single nucleotide polymorphisms (SNPs) that could be linked to the disease. The researchers identified 15 SNPs. Two of these were in the coding regions of the gene, and the remaining 13 were found in flanking introns.
Norton and colleagues next tested over 600 patients and controls (unrelated Caucasians from the UK or Ireland) but found that there was no strong association between any of the 15 SNPs and schizophrenia. However, they did find that individuals who were heterozygous for one SNP (IVS12-15C>T) had a significantly higher risk for schizophrenia if they also tested positive for the so-called Icelandic neuregulin 1 haplotype—a combination of one SNP and two specific microsatellite sequences in the NRG1 gene. “Our data require independent replication, but tentatively suggest that NRG1 may mediate its effects on schizophrenia susceptibility through functional interaction with ErbB4,” write the authors.
The second paper published in the March edition of the journal, and currently available online, supports the neuregulin-ErbB4 connection. Ruth Navon from Tel Aviv University, Israel, and colleagues also set out to test if ErbB4 polymorphisms have any impact on the development of schizophrenia. First author Gilad Silberberg and colleagues selected nineteen ErbB4 SNPs from the human haplotype map database (see SRF related news story) and compared these sequences with those obtained from 59 Ashkenazi schizophrenia patients and 130 matched controls. They found that three of the SNPs, which are found in a block that encompasses exon 3, were differentially represented in the case and control samples. The results were highly significant and led the authors to conclude that neuregulin 1 and its receptor, ErbB4, are involved in the pathophysiology of schizophrenia.
Exciting as these findings may be, there is still the question of how the two genes conspire to increase the likelihood of developing the disease. Both research groups focused on expression of ErbB4 as a likely mechanism. Norton and colleagues used allelic expression assays, in which one allele of a heterozygote serves as an internal control for measuring the expression of the other, to determine expression levels of ErbB4 in 26 different individuals. They found that there was an almost twofold variation, suggesting that cis-acting polymorphisms may indeed regulate the expression of the protein in the human brain.
Silberberg and colleagues tried a slightly different approach, using real-time DNA amplification techniques to measure expression levels of specific isoforms. They found that ErbB4 containing either the cytoplasmic tail-1 (CYT-1) or the juxtamembrane-a (JM-a) domain was significantly elevated (1.8- and 1.3-fold, respectively) in dorsolateral prefrontal cortex samples taken from patients at postmortem. On the other hand, transcripts containing CYT-2 or JM-b domains were expressed at similar levels in patients and controls.
A developing story for schizophrenia?
If changes in expression of ErbB4 and or neuregulin can explain, even partly, the susceptibility to schizophrenia, the obvious question then becomes, what pathways and molecular interactions are perturbed by that overexpression (Harrison and Law, 2006)? One developmental interaction that may be particularly interesting was just reported by Eva Anton and colleagues in the February 7 PNAS. First author Troy Ghashghaei and colleagues report that neuregulins 1, 2, and ErbB4, play a role in the proliferation and migration of neural precursors in the subventricular zone (SVZ) of the brain. Examining the adult neurogenesis seen in the subventricular zone of mice, the researchers found that ErbB4 and the two neuregulins were robustly expressed in a variety of cells in the SVZ. While the receptor is mainly found on migratory neuroblasts, it also appears in astrocytes and ependymal cells. The authors found NRG2 in neuroblasts and a subset of astrocytes, though NRG1 was found almost exclusively in neuroblasts.
Figure1. In the photo on the left, normally migrating neuronal precursors form a bright green river flowing from the subventricular zone along the rostral migratory stream toward the olfactory bulb. In the next photo, application of neuregulin 1 has disrupted this stream. (Photo courtesy of Troy Ghashghaei, Eva Anton, and colleagues.)
To examine what role these proteins play in neurogenesis, the researchers added exogenous NRG1 or NRG2 to the SVZ of either wild-type animals or those that had the ErbB4 receptor knocked out. This allowed them to distinguish specific ErbB4-mediated action of the two neuregulins. They found that NRG1 stimulates aggregation of precursor cells and decreases neuroblast migration from the SVZ. NRG2, on the other hand, seems to stimulate proliferation and migration of neuroblasts and increases γ-aminobutyric acid neurons in the olfactory bulb. Examination of the cellular organization of the SVZ in the ErbB4-null mice showed that disrupting neuregulin-ErbB4 signaling disrupts different cell types in the SVZ, which may ultimately retard the generation and migration of neurons to their final destinations.
Figure 2. In the photo on the left, normal astrocyte processes stretch away from the ventricular surface, but in the next photo, from ErbB4-deficient mice, the fibers appear to have lost their way. (Photo courtesy of Troy Ghashghaei, Eva Anton, and colleagues.)
The fact that these three proteins may be involved in neuronal migration is of interest, given that schizophrenia is now widely believed to have a significant developmental component. In fact, Ghashghaei and colleagues posit that “abnormal NRG1 signaling in the SVZ and the resultant changes in neuroblast generation and placement may alter the neural circuitry of the brain and contribute to neurodevelopmental disorders such as schizophrenia.”—Tom Fagan.
Norton N, Moskvina V, Morris DW, Bray NJ, Zammit S, Williams NM, Williams HJ, Preece AC, Swyer S, Wilkinson JC, Spurlock G, Kirov G, Buckland P, Waddington JL, Gill M, Corvin AP, Owen MJ, O’Donovan MC. Evidence that interaction between neuregulin 1 and its receptor ErbB4 increases susceptibility to schizophrenia. Amer. J. Med. Genet. Part B. February, 2006;141B:96-101. Abstract
Silberberg G, Darvasi A, Pinkas-Kramarski R, Navon R. The involvement of ErbB4 with schizophrenia: Association and expression studies. Amer. J. Med. Genet. Part B. February, 2006;141B:142-148. Abstract
Ghashghaei HT, Weber J, Pevny L, Schmid R, Schwab MH, Kent Lloyd KC, Eisenstat DD, Lai C, Anton ES. The role of neuregulin-ErbB4 interactions on the proliferation and organization of cells in the subventricular zone. PNAS. February 7, 2006;103:1930-1935. Abstract