The study of Seshadri, Sawa, and colleagues presents novel evidence of a potential biological link between two lead schizophrenia susceptibility genes, NRG1 and DISC1. The principal finding of the study is that NRG1 (EGFβ) regulates expression of a specific isoform of DISC1, mediated via ErbB2/3 but not ErbB4. The influence of NRG1 on expression of the DISC1 isoform was confirmed in a variety of in-vitro and in-vivo models. Specifically, the authors report (using Western blotting with the DISC1 antibodies: D27 and mExon3), that treatment with NRG1 (and NRG2), but not NRG3, increases levels of DISC1 immunoreactivity at 130 kDa in immature and mature rat primary neuron cultures. Interestingly, NRG1 (or NRG2) had no effect on expression of the previously reported full-length DISC1 immunoreactive bands of 100-105 kDa. Convincingly, reduction of the 130 kDa DISC1 band was observed in BACE1 -/- and NRG1 +/- mice, both of which have reduced NRG1 signaling. Taken together, these findings suggest that NRG1 signaling regulates expression of a unique 130 kDa DISC1 protein.

This is an important and thoughtful paper, but there are some details that raise questions about the interpretation of the results. Interestingly, two previous studies that characterized the D27 (and mExon3) antibody in mouse brain (Schurov et al., 2004; Ishizuka et al., 2007) failed to report the 130 kDa band described here. Ishizuka et al. reported that immunoprecipitation with the mExon3 antibody followed by detection with the D27 antibody recognized two primary signals (100 and 105 kDa), thought to correspond to full-length DISC1. In contrast, in the present study the authors report that immunoprecipitation of neuronal lysates using mExon3, followed by Western blotting with D27, consistently identifies an additional 130 kDa band (Fig. S2B), which is also present in the P0 mouse cortex (Fig 3C). Whilst it is not clear what accounts for these apparent differences in signal detection of the 130 kDa band using the same antibodies, factors such as species specificity (rat vs. mouse), tissue type, and developmental stage are likely relevant. Such factors are important considerations for future work. Similarly, it will be crucial to determine whether the 130 kDa band is present in human brain and how it relates to risk for schizophrenia. Of final note, the authors performed extensive experimentation in an attempt to confirm the identity of the 130 kDa band (including successful knockdown by a previously characterized RNAi to DISC1), but interestingly they fail to identify any DISC1 sequence in the 130 kDa signal using mass spectrometry (see discussion). In light of this, it is paramount that future studies determine exactly what the 130 kDa proposed DISC1 band represents (i.e., a novel splice isoform, post-transcriptionally modified protein, etc.), given that NRG1’s effects are specifically related to this variant.

In conclusion, this study provides intriguing evidence of a potential molecular link between NRG1 and DISC1, but at present, the interpretation of the results rests on an immunoblot band of unknown identify.

References:

Schurov IL, Handford EJ, Brandon NJ, Whiting PJ. Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment. Mol Psychiatry . 2004 Dec 1 ; 9(12):1100-10. Abstract

Ishizuka K, Chen J, Taya S, Li W, Millar JK, Xu Y, Clapcote SJ, Hookway C, Morita M, Kamiya A, Tomoda T, Lipska BK, Roder JC, Pletnikov M, Porteous D, Silva AJ, Cannon TD, Kaibuchi K, Brandon NJ, Weinberger DR, Sawa A. Evidence that many of the DISC1 isoforms in C57BL/6J mice are also expressed in 129S6/SvEv mice. Mol Psychiatry . 2007 Oct ; 12(10):897-9. Abstract

View all comments by Amanda Jayne Law

This paper raises an interesting issue. It is unclear how an immuno band that has no DISC1 sequences can result from "alternative splicing or post-translational modification." Could someone provide a mechanistic account, at the molecular level, of how this may be possible? To support that this band is DISC1, at least some DISC1 sequence should have been detected. This issue could be related to the non-specific cross-reactivity of many DISC1 antibodies (see Kvajo et al., 2008 for a discussion) and now also raises the possibility of off-target effects of DISC1 RNAi.

Resolving these issues will be paramount for making meaningful insights into how variations in DISC1 contribute to psychotic disorders.

References:

Kvajo M, McKellar H, Arguello PA, Drew LJ, Moore H, MacDermott AB, Karayiorgou M, Gogos JA. A mutation in mouse Disc1 that models a schizophrenia risk allele leads to specific alterations in neuronal architecture and cognition. Proc Natl Acad Sci U S A. 2008 May 13;105(19):7076-81. Abstract

View all comments by Alexander Arguello

We are very glad to see Dr. Law’s thoughtful and very supportive comments on the work by Seshadri et al. We share the recognition, as we pointed out in the discussion of the paper, that identification of 130 kDa signal at the molecular level is an important future question. To confirm the authenticity of immunoreactivity, we tested if the 130 kDa signal is immunoprecipitated and immunoblotted by different DISC1 antibodies. Similar immunoreactive approaches have been used earlier to distinguish DISC1 isoforms, including a 71 kDa isoform in association with PDE4 (Millar et al., 2005; Chubb et al., 2008). Knockout mice deficient in DISC1 that we have recently generated (unpublished) were used for evaluating the specificity of several antibodies against DISC1 (Schurov et al., 2004; Ishizuka et al., 2007; Duan et al., 2007; Koike et al., 2006). Loss of this immunoreactivity by authentic shRNAs further supports this idea. The sequences of shRNAs are the same as those used in the study by Mao et al. (Mao et al., 2009) to demonstrate that DISC1 may be involved in progenitor cell proliferation.

Of note, mass spectrometry cannot be an ultimate confirmation, because with this technique it is hard to distinguish the signals from two adjacent or overlapped bands in Western blots of 1D gels, one of which is real and the other not. Therefore, regardless of our initial mass spectrometry analysis (even if one finds sequences of the target protein), validation with both immunoprecipitation and RNAi is required to draw a conclusion on the identity of 130 kDa signal. In the study by Seshadri et al., these two ways of validation were successfully made.

Furthermore, whether or not this 130 kDa isoform is also expressed in humans is a critical question. It is also very important to consider context-dependent expression of unique isoforms of genetic susceptibility factors. This unique form (130 kDa) is likely to be in that category; thus, as Dr. Law suggested, comparative analysis is very useful. Further analysis of the genesis, function, and processing of various DISC1 isoforms in the brain will be a worthy pursuit in the context of schizophrenia.

References:

Millar JK, Pickard BS, Mackie S, James R, Christie S, Buchanan SR, Malloy MP, Chubb JE, Huston E, Baillie GS, Thomson PA, Hill EV, Brandon NJ, Rain JC, Camargo LM, Whiting PJ, Houslay MD, Blackwood DH, Muir WJ, Porteous DJ. DISC1 and PDE4B are interacting genetic factors in schizophrenia that regulate cAMP signaling. Science. 2005 Nov 18 ; 310(5751):1187-91. Abstract

Chubb JE, Bradshaw NJ, Soares DC, Porteous DJ, Millar JK. The DISC locus in psychiatric illness. Mol Psychiatry. 2008 Jan 1 ; 13(1):36-64. Abstract

Schurov IL, Handford EJ, Brandon NJ, Whiting PJ. Expression of disrupted in schizophrenia 1 (DISC1) protein in the adult and developing mouse brain indicates its role in neurodevelopment. Mol Psychiatry. 2004 Dec 1 ; 9(12):1100-10. Abstract

Ishizuka K, Chen J, Taya S, Li W, Millar JK, Xu Y, Clapcote SJ, Hookway C, Morita M, Kamiya A, Tomoda T, Lipska BK, Roder JC, Pletnikov M, Porteous D, Silva AJ, Cannon TD, Kaibuchi K, Brandon NJ, Weinberger DR, Sawa A. Evidence that many of the DISC1 isoforms in C57BL/6J mice are also expressed in 129S6/SvEv mice. Mol Psychiatry. 2007 Oct 1 ; 12(10):897-9. Abstract

Duan X, Chang JH, Ge S, Faulkner RL, Kim JY, Kitabatake Y, Liu XB, Yang CH, Jordan JD, Ma DK, Liu CY, Ganesan S, Cheng HJ, Ming GL, Lu B, Song H. Disrupted-In-Schizophrenia 1 regulates integration of newly generated neurons in the adult brain. Cell. 2007 Sep 21 ; 130(6):1146-58. Abstract

Koike H, Arguello PA, Kvajo M, Karayiorgou M, Gogos JA. Disc1 is mutated in the 129S6/SvEv strain and modulates working memory in mice. Proc Natl Acad Sci U S A. 2006 Mar 7 ; 103(10):3693-7. Abstract

Mao Y, Ge X, Frank CL, Madison JM, Koehler AN, Doud MK, Tassa C, Berry EM, Soda T, Singh KK, Biechele T, Petryshen TL, Moon RT, Haggarty SJ, Tsai LH. Disrupted in schizophrenia 1 regulates neuronal progenitor proliferation via modulation of GSK3beta/beta-catenin signaling. Cell. 2009 Mar 20 ; 136(6):1017-31. Abstract

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