We found that both NRG1-ICD (53kDa) and full-length ErbB4 (180kDa) protein levels were elevated in PFC cytoplasmic fractions of schizophrenic patients relative to the other subject groups. These findings appear consistent with previous reports showing isoform-specific increases in PFC NRG1 and ErbB4 mRNA levels in schizophrenia (Hashimoto et al. 2004
;Law et al. 2007
;Silberberg et al. 2006
). Furthermore, none of the analyzed PFC NRG1 and ErbB4 immunoreactive fragments were altered in major depressed or bipolar subjects, suggesting the schizophrenia-associated PFC increases in NRG1-ICD and full-length ErbB4 protein levels were specific to schizophrenia. Hence, our results suggest the mechanism of NRG1 gene dysregulation in schizophrenia may be distinct from that in bipolar disorder since the NRG1 gene has been implicated in bipolar illness as well (Green et al. 2005
). We also found a significant positive correlation between NRG1-ICD and full-length ErbB4 levels exclusively in schizophrenic patients, but not in normal subjects. This preliminary observation may suggest that this NRG1 and ErbB4 relationship is unusually constrained in schizophrenic patients due to the disease process.
Our previous finding of schizophrenia-associated NRG1 mRNA increases (Hashimoto et al. 2004
;Law et al. 2006
) suggests elevated transcription may contribute to the increased PFC cytoplasmic NRG1-ICD levels in schizophrenia. However, the relation between NRG1 mRNA isoforms and the NRG1-ICD we detected is unclear. The elevations in PFC cytoplasmic NRG1-ICD could also indicate enhanced PFC NRG1 intracellular cleavage, which would have implications in NRG1 back signaling-mediated gene expression and cytoskeletal organization in schizophrenia. Specifically, the NRG1-ICD can repress apoptotic protein expression (Bao et al. 2003
) as well as bind and promote nuclear translocation of transcription factors like Eos, which enhances postsynaptic density-95 (PSD-95) transcription (Bao et al. 2004
). The NRG1-ICD also interacts with LIM kinase 1 in the cytoplasm where this association could regulate LIM kinase 1-mediated actin polymerization (Wang et al. 1998
). Hence, the schizophrenia-associated PFC cytoplasmic NRG1-ICD elevations may suggest augmentation of any of these NRG1-ICD-mediated events in the schizophrenic PFC. Alternatively, the elevated cytoplasmic NRG1-ICD levels could suggest defective NRG1-ICD-mediated nuclear translocation in the PFC of schizophrenic subjects, as we did not detect diagnostic changes in PFC nuclear NRG1-ICD levels.
Since we could not reliably measure full-length NRG1 in all subjects, it is difficult to predict whether increased synthesis or intracellular cleavage more likely explains the elevated PFC cytoplasmic NRG1-ICD. In the case of ErbB4, however, we could readily detect full-length ErbB4 and the smaller putative ErbB4 cleavage products. Hence, we were able to show that while PFC cytoplasmic full-length ErbB4 levels were elevated in schizophrenic subjects, PFC cytoplasmic ratios between 55 and 180kDa ErbB4 levels and between 21 and 180kDa ErbB4 quantities were reduced in schizophrenic patients. These findings, together with the previous observations of schizophrenia-associated ErbB4 mRNA increases (Law et al. 2007
;Silberberg et al. 2006
), may indicate PFC full-length ErbB4 synthesis is elevated with decreased intracellular cleavage of the receptor in schizophrenia.
Interestingly, several NRG1 and ErbB4 immunoreactive bands were found in both cytoplasmic and nuclear fractions of subjects in all diagnostic categories. This observation suggests that many NRG1- and ErbB4-derived proteins may translocate from the cytoplasm into the nucleus and that NRG1 and ErbB4 can regulate gene expression and/or nuclear protein shuttling, as hypothesized previously (Bao et al. 2004
;Sardi et al. 2006
). In addition, we detected the 180kDa ErbB4 protein in PFC nuclear fractions, supporting suggestions that full-length ErbB4 can enter the nucleus without being cleaved (Carpenter 2003
), though the function of full-length ErbB4 in neuronal nuclei is unknown. Since schizophrenic patients had PFC elevations in nuclear full-length ErbB4 compared to bipolar subjects and to depressed patients, the nuclear actions of the 180kDa protein in the PFC may be worthy of further investigation.
If our findings do reflect the schizophrenia-associated NRG1 and ErbB4 mRNA elevations reported previously, then the NRG1-ICD protein we detected would be expected to be derived from the type I isoform (Hashimoto et al. 2004
;Law et al. 2006
) while the full-length ErbB4 protein we observed would be expected to consist of JMa and/or Cyt1 domains (Law et al. 2007
;Silberberg et al. 2006
). Based on these assumptions, our observations would support previous evidence of enhanced PFC NRG1-ErbB4 signaling in schizophrenia (Hahn et al. 2006
) because the extracellular domains of both NRG1 type I and JMa-containing ErbB4 isoforms can be cleaved to activate membrane-bound ErbB4 and NRG1, respectively (Bao et al. 2003
). In addition, if the 180kDa ErbB4 protein is indeed derived from Cyt1-containing isoforms, which have phosphatidyl inositol 3 kinase (PI3K) sites, our findings may suggest that the increased full-length ErbB4 is associated with alterations in PI3K-regulated processes previously implicated in schizophrenia (Kalkman 2006
;Law et al. 2007
An alternative explanation for the observed concomitant PFC NRG1 and ErbB4 protein elevations in schizophrenia is that these increases are compensatory responses to defective NRG1-ErbB4 signaling. More specifically, if feed-forward NRG1-ErbB4 signaling (i.e. NRG1 to ErbB4) is intact, an up-regulation of both NRG1 type I and JMa-containing ErbB4 proteins would be expected to contribute to greater ErbB4-regulated downstream signaling events, including intracellular cleavage of the receptor. Yet, we found that full-length ErbB4 content increased with no alterations in the quantities of the smaller ErbB4 immunoreactive bands we detected in PFC cytoplasmic fractions of schizophrenic subjects. Moreover, the quantities of two of the detected potential ErbB4 intracellular cleavage fragments (i.e. at 21 and 55kDa) relative to full-length ErbB4 levels were significantly less in PFC cytoplasmic fractions of schizophrenic individuals. These reductions may indicate decreased PFC ErbB4 intracellular cleavage and hence abnormal feed-forward NRG1-ErbB4 signaling in these patients.
One limitation of our study is that we could not distinguish the particular variants of the detected NRG1 and ErbB4 immunoreactive bands due to the “pan” nature of the antibodies implemented. Furthermore, our observations conflict with those of Hahn et al., who showed no alterations in NRG1 or ErbB4 protein quantities in the PFC of schizophrenic patients (Hahn et al. 2006
). This discrepancy may be due to the age difference between our study group (mean age ~45y) and their relatively old cohort (mean age ~79y) that could possess aging-associated confounds (e.g. oxidative stress and DNA damage), which can influence protein levels (Lu et al. 2004
;Serrano and Klann 2004
). In addition, one study reported schizophrenia-associated reductions in PFC NRG1α protein (Bertram et al. 2007
). This observation, along with the fact our NRG1 antibody detects α and β isoforms, may suggest the increased NRG1-ICD is not derived from the α isoform. Thus, it appears precise schizophrenia-associated alterations in PFC NRG1 protein quantities may vary according to the NRG1 type measured.
In conclusion, we found that the PFC shows schizophrenia-specific alterations in the quantities of two interacting growth factor molecules. We also demonstrated NRG1 and ErbB4 alterations in distinct PFC intracellular compartments, involving both the NRG1-ICD and full-length ErbB4. These observations, along with the finding of novel NRG1 and ErbB4 immunoreactive bands, add to the complexity of our understanding of NRG1-ErbB4 signaling, particularly with respect to implications in schizophrenia. More studies are required to ascertain how the elevated NRG1 and ErbB4 immunoreactive proteins relate to the increased NRG1 and ErbB4 mRNA in the PFC of schizophrenic patients. Further research is also necessary to examine if the NRG1 and ErbB4 protein elevations in schizophrenia could be compensatory responses to aberrant NRG1-ErbB4 signaling. In addition, the PFC cell types associated with the observed schizophrenia-associated protein elevations need to be determined to understand the type of NRG1-ErbB4 signaling altered (paracrine, juxtacrine and/or autocrine) along with the neuronal circuits affected. These details may allow us to better understand the mechanism of concomitant NRG1 and ErbB4 protein elevations in the PFC as well as the consequences of our findings in relation to schizophrenia pathology and treatment.