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DISC1 and Neuronal Migration: The Amyloid Precursor Protein Connection

20 September 2010. In the world of disrupted-in-schizophrenia 1 (DISC1) biology, things are getting curiouser and curiouser. According to a study published in the Journal of Neuroscience on August 4, DISC1 protein, a key suspect in schizophrenia, interacts with amyloid precursor protein (APP), the darling of Alzheimer disease research. The study from Dennis Selkoe's lab at Harvard University shows that the two proteins team up to direct migration of newborn neurons in the developing brain.

The new work places DISC1 downstream of APP, and shows that APP is required for DISC1 to localize to the centrosome, a microtubule organizing center which DISC1 must reach for the cell to migrate correctly. "The idea is that APP at the cell surface may act as kind of a scaffolding factor where it binds DISC1, and another protein called Disabled-1," says first author Tracy Young-Pearse, who has just started her own research lab at Harvard.

There have been whiffs of a connection between the molecular players of Alzheimer disease and schizophrenia before, mainly involving β-site amyloid precursor protein cleaving enzyme 1 (BACE1). In addition to helping turn APP into amyloid-β protein—the main component of the amyloid plaques indicative of Alzheimer's—BACE1 also cleaves neuregulin-1 (NRG1; see SZGene entry), a top suspect in schizophrenia. One study described schizophrenia-like behaviors in an Alzheimer's mouse model missing BACE1 (see SRF related news story), and another found that BACE1-induced changes in NRG1 levels could regulate an isoform of DISC1 (see SRF related news story). Other intriguing tidbits include a link between γ-secretase, another APP processing enzyme, and NRG1-Erb4 signaling (see SRF related news story); binding between DISC1 and APLP, a protein similar to APP (Millar et al., 2003); and an association between a single nucleotide polymorphism in DISC1 and late-onset Alzheimer disease (Beecham et al., 2009).

By exploring how APP normally works, rather than on what goes wrong with it in Alzheimer disease, Young-Pearse first discovered a role for APP in cell migration while a postdoc in Selkoe's lab (Young-Pearse et al., 2007). Despite massive amounts of research into how APP is cleaved and processed to form the amyloid-β protein, not many people had bothered to study what APP normally does, she says.

She found that knocking down APP levels in cortical precursor cells resulted in neurons that did not migrate correctly: instead of progressing up into the cortical plate where they belong, they got stuck in the lower intermediate zone.

While finishing up this initial study, a talk by Akira Sawa alerted Young-Pearse to a potential connection between DISC1 and APP: his slides showing migration defects when DISC1 is knocked down (see SRF related news story) looked remarkably like her own APP knockdown data, she says.

Despite the connection she turns up between APP and DISC1 in the new study, Young-Pearse is careful to draw a distinction between APP's role in migration, and its involvement in the manufacture of amyloid-β protein. "We don’t think this role of APP in migration is necessarily linked to Alzheimer disease," she says. "I'm more interested in what APP and migration could be doing in schizophrenia."

DISC1 to the rescue
Young-Pearse and colleagues began by replicating Sawa's DISC1 results. In utero electroporation of shRNA that inhibits DISC1 into cortical precursor cells of rat embryos led to a clear migration defect, with new neurons unable to make it into the cortical plate. This defect could be rescued by overexpressing full-length DISC1, and further experiments established the critical DISC1 regions: the C-terminal half of the protein, containing both the self-association binding site and the NDEL1 binding site for normal migration. Notably, a truncated version of DISC1 corresponding to the human translocation breakpoint led to the abnormal migration pattern.

Because of the similar migration defects in DISC1 and APP knockdown experiments, the researchers next asked if one protein could compensate for the other. Co-electroporating the DISC1 shRNA along with APP did not rescue the migration defect. But strikingly, adding DISC1 could rescue APP knockdown. While APP knockdown limits migration to the cortical plate to only about 10 percent of electroporated cells, with extra DISC1 on board, nearly 40 percent of electroporated cells made it. The C-terminal portion of DISC1 containing the self-association site and the NDEL1 site could also rescue APP knockdown, getting over 60 percent of electroporated cells into the cortical plate; the other, N-terminal half could not. Because DISC1 can spur normal migration without APP around, Young-Pearse and colleagues conclude that it must be acting downstream of APP.

This functional interaction stems from a physical one, as shown by a series of co-immunoprecipitation experiments. DISC1 directly binds to APP, and this interaction takes place between the cytoplasmic side of APP and the N-terminal half of DISC1. APP spans the membrane, with a large portion sticking out of the cell, and its smaller DISC-binding portion inside of the cell. Though DISC1 localizes to different regions within a cell, it did co-immunoprecipitate with APP in isolated membrane fractions of the cell—as expected if DISC1 transiently interacts with APP.

Centered on the centrosome
To begin to understand how the APP-DISC1 interaction mucks up migration, the researchers tracked what happened to DISC1 inside the cell when APP was knocked down. DISC1 levels were not changed, but where it ended up within the cell did. Normally DISC1 staining forms a tight knot within a neuron, and it colocalizes with other centrosome markers. When the researchers knocked APP down, however, this seemed to set DISC1 adrift, leaving DISC1 staining diffusely spread throughout the cell.

The researchers offer a working model for their results that casts APP as a kind of meeting place for DISC1 and its binding partners that are critical for migration—lissencephaly protein 1 (LIS1) and nuclear distribution factor E homolog like-1 (NDEL1) (see SRF related news story; SRF news story). The DISC1-LIS1-NDEL1 complex then leaves APP and travels to the centrosome, enabling migration. With APP missing, DISC1 drifts aimlessly throughout the cell, unable to connect with its partners. Adding extra DISC1 rescues this situation because flooding the cell with DISC1 increases the chances that it will bump into its binding partners. This idea explains the odd combination of results showing that the N-terminal portion of DISC1 binds to APP, but the C-terminal portion of DISC1 can rescue APP knockdown.

So is the schizophrenia research community ready to welcome APP into its fold of molecules? Though it's unclear whether there are APP gene variants associated with schizophrenia (Forsell et al., 1995), this APP-DISC1 connection helps to elucidate DISC1's role in migration. Understanding this is arguably one of the best avenues available for getting at the perturbations during brain development that predispose to schizophrenia.—Michele Solis.

Reference:
Young-Pearse TL, Suth S, Luth ES, Sawa A, Selkoe DJ. Biochemical and functional interaction of disrupted-in-schizophrenia 1 and amyloid precursor protein regulates neuronal migration during mammalian cortical development. J Neurosci. 2010 Aug 4; 30:10431-10440. Abstract

Comments on Related News


Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Anil Malhotra, SRF Advisor
Submitted 21 November 2005
Posted 21 November 2005

The relationship between DISC1 and neuropsychiatric disorders, including schizophrenia, schizoaffective disorder, and bipolar disorder, has now been observed in several studies. Moreover, a number of studies have demonstrated that DISC1 appears to impact neurocognitive function. Nevertheless, the molecular mechanisms by which DISC1 could contribute to impaired CNS function are unclear, and these two papers shed light on this critical issue.

Millar et al. (2005) have followed the same strategy that they so successfully utilized in their initial DISC1 studies, identifying a translocation that associated with a psychotic illness. In contrast to DISC1, in which a pedigree was identified with a number of translocation carriers, this manuscript is based upon the identification of a single translocation carrier, who appears to manifest classic signs of schizophrenia, without evidence of mood dysregulation. Two genes are disrupted by this translocation: cadherin 8 and phosphodiesterase 4B (PDE4B). The researchers' elegant set of experiments provides compelling biological evidence that PDE4B interacts with DISC1 and suggests a mechanism mediated by cAMP for DISC1/PDE4B effects on basic molecular processes underlying learning, memory, and perhaps psychosis. It remains possible that PDE4B (and DISC1) are proteins fundamentally involved in cognitive processes, and that the observed relationship to psychotic illnesses represents a final common pathway of neurocognitive impairment. This would be consistent with data from our group (Lencz et al., in press) demonstrating that verbal memory impairment specifically predicts onset of psychosis in at-risk subjects. Similarly, Burdick et al. (2005) found that our DISC1 risk genotypes (Hodgkinson et al., 2004) were associated with impaired verbal working memory. Finally, Callicott et al. (2005) found that a DISC1 risk SNP, Ser704Cys, predicted hippocampal dysfunction, an SNP which we (DeRosse et al., unpublished data) have also found to link with the primary psychotic symptoms (persecutory delusions) manifested by the patient in the Millar et al. study. This body of evidence supports the notion that these proteins play fundamental roles in the key clinical manifestations of schizophrenia.

Kamiya et al. (2005) provide another potential mechanism for these effects, suggesting that a DISC1 mutation may disrupt cerebral cortical development, hinting that studies examining the role of DISC1 genotypes on brain structure and function in the at-risk schizophrenia pediatric patients may be fruitful.

Taken together, these papers add considerable new data suggesting that DISC1 plays a key role in the etiology of schizophrenia, and places DISC1 at the forefront of the rapidly growing body of schizophrenia candidate genes.

References:
Burdick KE, Hodgkinson CA, Szeszko PR, Lencz T, Ekholm JM, Kane JM, Goldman D, Malhotra AK. DISC1 and neurocognitive function in schizophrenia. Neuroreport 2005; 16(12):1399-1402. Abstract

Callicott JH, Straub RE, Pezawas L, Egan MF, Mattay VS, Hariri AR, Verchinski BA, Meyer-Lindenberg A, Balkissoon R, Kolachana B, Goldberg TE, Weinberger DR. Variation in DISC1 affects hippocampal structure and function and increases risk for schizophrenia. Proc Natl Acad Sci USA 2005; 102(24): 8627-8632. Abstract

Hodgkinson CA, Goldman D, Jaeger J, Persaud S, Kane JM, Lipsky RH, Malhotra AK. Disrupted in Schizophrenia (DISC1): Association with schizophrenia, schizoaffective disorder, and bipolar disorder. Am J Hum Genet 2004; 75:862-872. Abstract

Lencz T, Smith CW, McLaughlin D, Auther A, Nakayama E, Hovey L, Cornblatt BA. Generalized and specific neurocognitive deficits in prodromal schizophrenia. Biological Psychiatry (in press).

View all comments by Anil Malhotra

Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Angus Nairn
Submitted 29 December 2005
Posted 31 December 2005
  I recommend the Primary Papers

This study describes an interesting genetic link between PDE4B (phosphodiesterase 4B) and schizophrenia that may be related to a physical interaction with DISC1 (disrupted in schizophrenia 1), another gene associated with the psychiatric disorder. The study is highly suggestive of a role for the PDE4B/DISC1 complex in schizophrenia. However, the mechanistic model suggested by the authors whereby DISC1 sequesters PDE4B in an inactive state seems overly speculative, given the results presented in this paper and in prior studies that have examined the regulation of PDE4B by phosphorylation in the absence of DISC1.

View all comments by Angus Nairn

Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Patricia Estani
Submitted 2 January 2006
Posted 2 January 2006
  I recommend the Primary Papers

Related News: Lis1 Acts as Middleman for Actin and Microtubules

Comment by:  Akira Sawa, SRF Advisor
Submitted 12 January 2006
Posted 12 January 2006

I found the paper by Kholmanskikh and colleagues, which proposes a novel role for LIS1 in neuronal motility by bridging calcium signaling to Cdc42, of great interest for schizophrenia research. LIS1 was originally identified as the causative gene for lissencephaly, but cascades that include LIS1 may have implications for schizophrenia. Several groups, including ours, have reported that a candidate gene product for schizophrenia, DISC1, forms a protein complex with LIS1 (Brandon et al., 2004; Kamiya et al., 2005).

My collaborators, Brian Kirkpatrick and Rosy Roberts, have observed and presented data that DISC1 immunoreactivity is enriched in some (but not all) of the postsynaptic densities, where Rho-family GTPases, such as Cdc42, also occur and regulate synaptic functions (Society for Neuroscience Meeting, 2004). Many of us agree that schizophrenia is, at least in part, a disorder of synapses. Taken all together, it may be useful to have a working hypothesis that a candidate susceptibility gene product for schizophrenia, DISC1, may have an additional role in regulating synaptic functions via Rho-family GTPases, probably in some association with LIS1.

This paper may attract researchers on schizophrenia in another context. The authors used D-serine as a trigger of calcium signaling via activation of the NMDA receptor. Although several genes coding for proteins that are involved in synthesis and degradation of D-serine have been associated with schizophrenia, pathophysiological roles for D-serine remain to be elucidated. In this sense, the impact of D-serine in activation of Cdc42 and synaptic morphology may have implications for schizophrenia research. Personally speaking, it is the most interesting point in this paper that the authors use D-serine in this type of experiment.

View all comments by Akira Sawa

Related News: Messing with DISC1 Protein Disturbs Development, and More

Comment by:  Ali Mohammad Foroughmand
Submitted 16 December 2006
Posted 16 December 2006
  I recommend the Primary Papers

Related News: DISC1 Delivers—Genetic, Molecular Studies Link Protein to Axonal Transport

Comment by:  Akira Sawa, SRF Advisor
Submitted 12 January 2007
Posted 12 January 2007

Although DISC1 is multifunctional, its role for neurite outgrowth has been substantially characterized for the past couple of years (Ozeki et al., 2003; Miyoshi et al., 2003; Kamiya et al., 2006). These studies indicated that DISC1 is involved in neurite outgrowth by more than one mechanism, such as interactions with NUDEL/NDEL1 and FEZ1.

These two papers from Kaibuchi’s lab provide further understanding of how DISC1 is involved in neuronal outgrowth. Kaibuchi’s group identified kinesin heavy chain of kinesin-1 as a novel interactor of DISC1. In their papers, a novel role for DISC1, to link kinesin-1 (microtubule-dependent and plus-end directed motor) to several cellular molecules, including NUDEL, LIS1, 14-3-3, and Grb2, is reported. DISC1 and kinesin-1 are, therefore, responsible to sort Grb2 to the distal part of axons where Grb2 functions as an adaptor and plays a role in NT-3-induced phosphorylation of ERK1/2. This mechanism well explains our previous work, led by Ryota Hashimoto, reporting that knockdown of DISC1 expression results in decreased levels of phosphorylation of ERK1/2 and Akt in primary cortical neurons (Hashimoto et al., 2006).

The interaction of DISC1 and kinesin-1 may also be interesting from the perspective of psychiatric genetics. First, the mechanism proposed in one of the papers (Taya et al., 2007) supports the notion that the C-terminal truncated DISC1 fragment—that occurs due to the balanced translocation in an extended Scottish family—functions as a dominant-negative. Second, the domain of DISC1 responsible for kinesin-1 is overlapped with the haplotype block region(s) that are positive in more than one association study of DISC1 and major mental illnesses.

View all comments by Akira Sawa

Related News: DISC1 Delivers—Genetic, Molecular Studies Link Protein to Axonal Transport

Comment by:  Luiz Miguel Camargo (Disclosure)
Submitted 13 January 2007
Posted 13 January 2007

Two recent back-to-back papers, published this month in Journal of Neuroscience, highlight the value of protein-protein interactions in determining the biological role of a key schizophrenia risk factor, DISC1, in processes that are important for the proper development of neurons.

Key questions need to be addressed once having established a set of interactors for a given protein. First, where do these proteins interact on the target molecule? Second, do these interactions take place at the same time (i.e., do they form a complex)? Third, in what context do these interactions occur (temporal, tissue/cell compartment, signaling), and, fourth, are the biological processes of the interacting molecules affected/regulated by the protein of interest? The Kaibuchi lab, as exemplified in the works by Taya et al. and Shinoda et al., elegantly address some of these questions in the context of DISC1 interactions with Grb2, Nudel (NDEL1), 14-3-3ε, and kinesin-1. The key findings of these papers are as follows:

1. Identification of the interaction sites, or more importantly, which part of DISC1 is involved in particular processes, for example, that axon elongation is dependent on the N-terminal, but not the C-terminal portion of DISC1. This suggests that the DISC1 role in axon elongation is mediated by interactions with the N-terminal portion of DISC1 that could be competed for by the truncated protein in a dominant negative fashion (Camargo et al., 2007).

2. Although a protein may have many interacting partners, such as DISC1, these interactions may not occur at the same time. For example, DISC1 is able to form a ternary complex with kinesin-1 and NDEL1 or with kinesin-1 and Grb2. However, a ternary complex of DISC1-Grb2-NDEL1 is not possible as Grb2 and NDEL1 may be competing for the same interaction site on DISC1.

3. Protein interactions may occur in certain cellular compartments, in the case of DISC1, the cell body and the distal part of axons.

4. Neurotrophin-induced axon elongation requires DISC1.

These papers confirm some of the hypotheses raised by the interactions that we have recently derived for DISC1 and some of its interacting partners (see Camargo et al., 2007). From the DISC1 interactome, we concluded that DISC1 may affect key intracellular transport mechanisms, such as those regulated by kinesins, and that DISC1 may be downstream of neurotrophin receptors, via its interaction with SH3BP5, an adaptor protein, which we found to interact with SOS1, a guanine exchange factor that binds Grb2 and responds to signaling of neurotrophin receptors. These observations have been validated by Taya et al. and Shinoda et al. and demonstrate the value of the DISC1 interactome in understanding the role of DISC1, and as a valuable resource to the wider community.

The molecular function of DISC1, as defined by its structure, still remains elusive, requiring a more dedicated effort on this front. The good news is that, via its protein-protein interactions, significant progress on the role of DISC1 in key biological processes has been achieved, as illustrated by the work of different labs (Brandon et. al., 2004; Millar et al., 2005; Kamiya et al., 2005; and now by Shinoda et al. and Taya et al.).

View all comments by Luiz Miguel Camargo

Related News: Down to BACE-ics—Old Mouse a New Model for Schizophrenia?

Comment by:  Victor ChongCynthia Shannon Weickert (SRF Advisor)
Submitted 23 May 2008
Posted 23 May 2008

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

View all comments by Victor Chong
View all comments by Cynthia Shannon Weickert

Related News: Convergence Zone: NRG1 Signaling Linked to DISC1 Expression

Comment by:  Amanda Jayne Law, SRF Advisor
Submitted 19 April 2010
Posted 19 April 2010

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

Related News: Convergence Zone: NRG1 Signaling Linked to DISC1 Expression

Comment by:  Alexander Arguello
Submitted 3 May 2010
Posted 3 May 2010

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

Related News: Convergence Zone: NRG1 Signaling Linked to DISC1 Expression

Comment by:  Saurav SeshadriAtsushi KamiyaEva AntonAkira Sawa (SRF Advisor)
Submitted 4 May 2010
Posted 4 May 2010

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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