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The disrupted-in-schizophrenia 1 (DISC1) gene was identified as a genetic risk factor for chronic mental illnesses (CMI) such as schizophrenia, bipolar disorder and severe recurrent depression. Insoluble aggregated DISC1 variants were found in the cingular cortex of sporadic, i.e. non-genetic, CMI patients. This suggests protein pathology as a novel, additional pathogenic mechanism, further corroborated in a recent transgenic rat model presenting DISC1 aggregates. Since the potential role of aggregation of DISC1 in sporadic CMI is unknown, we investigated whether DISC1 undergoes aggregation in cell culture and could spread between neuronal cells in a prion-like manner, as shown for amyloid proteins in neurodegenerative diseases. Co-culture experiments between donor cells forming DISC1 aggregates and acceptor cells showed that 4.5% of acceptor cells contained donor-derived DISC1 aggregates, thus indicatinganefficient transferinvitro. DISC1 aggregates were found inside tunnelling nanotubes (TNTs) and transfer was enhanced by increasing TNT formation and notably by dopamine treatment, which also induces DISC1 aggregation. These data indicate that DISC1 aggregates can propagate between cells similarly to prions, thus providing some molecular basis for the role of protein pathology in CMI.

In an effort to identify potential biological markers of major mental illness, we have previously proposed the identification of specific proteins which form insoluble aggregates in a condition-specific manner, as is the case for other neurological illnesses. To this effect, we have determined DISC1 (Disrupted in Schizophrenia 1) to specifically aggregate in a subset of patients with major mental illness as well as identifying a novel schizophrenia-related protein, CRMP1 (Collapsin Response Mediator Protein 1). This latter protein was identified through a process of raising antibodies against the pooled insoluble fractions of post mortem brain samples from schizophrenia patients and followed by epitope identification and confirmation using genetic techniques. In this study we pursue this epitope discovery paradigm further, revealing TRIO binding protein (TRIOBP) to be a major substrate of a monoclonal antibody with a high specificity to schizophrenia brain aggregomes. The TRIOBP gene encodes for several distinct protein species, however only those deriving from the 3’ end of the gene, as typified by the actin-bundling TRIOBP1, could be seen to form aggregates in neuroblastoma cell lines, either due to over-expression of the protein or spontaneously following differentiation of the cells. We have previously determined that the expression level of TRIOBP is dependent on a haplotype of the NDE1 (Nuclear Distribution Element 1) locus, a finding which we were able to replicate within schizophrenia cohorts. NDE1 is an important neurodevelopmental protein which has been linked to schizophrenia via genetic association studies, copy number variation and interaction with the DISC1 protein. Intriguingly, we find the TRIOBP and NDE1 proteins to also be interaction partners within the cell, suggesting that the NDE1 locus may alter the function of TRIOBP both at the transcriptional and protein level. Potential mechanisms for this will be discussed. Thus, in addition to identifying a potential new protein in schizophrenia pathology, we also reveal how it may fit into the wider picture of established mental illness related pathways.

The presence of aggregates of specific proteins in the brain can be used to characterize multiple neurodegenerative conditions, and arises as a result of imbalances in protein homeostasis during the development of the condition. We have previously proposed that similar protein deposits may arise in subsets of patients suffering from chronic mental illness, and were able to determine that proteins produced by two established schizophrenia risk factors, DISC1 and dysbindin, could be seen to aggregate specifically in the brains of a subset of patients with major mental illness. To detect other such proteins not previously implicated by genetics, we employed an antibody proteomics approach, identifying TRIOBP-1 as an additional protein which putatively shows schizophrenia-specific aggregation in the brain. The propensity for both over-expressed and endogenous TRIOBP-1 protein to aggregate was then confirmed in a variety of systems. The TRIOBP-1 protein is predicted to consist of an N-terminal Pleckstrin homology domain, as well as extensive predicted coiled-coil regions. In order to investigate the domain structure of TRIOBP-1 in more depth, and specifically in order to determine which elements of the protein were implicated in both its normal function and in its aggregation propensity, a large number of constructs were subcloned containing combinations of structural elements of TRIOBP-1. These were expressed in both neuroblastoma cell lines and in E. coli for recombinant protein production. In this manner, we have now identified a central segment of the protein, as being critical for the aggregation of the coiled-coil C-terminal segment of TRIOBP-1. The N-terminal section of TRIOBP-1 shows no such propensity, but was seen to be localized to the actin cytoskeleton by this same central region, implying a common underlying relationship between the aggregation and actin-binding features of TRIOBP-1. We have also found the first evidence that the N- and C-terminal portions of TRIOBP-1 may play distinct roles in the promotion of actin polymerization by TRIOBP-1. Experiments to confirm the relevance of TRIOBP-1 aggregation to schizophrenia in the wider population are currently underway. Through probing a protein misfolding event seemingly present in at least a subset of patients, we hope to determine both its potential utility as a biomarker, and more generally to understand a physiological element of chronic mental illness which has the potential to lie downstream of both genetic and environmental risk factors.

Disrupted in schizophrenia (DISC1) is a risk factor for chronic mental disease. In a previous proteomic study, we reported that knocking down DISC1 results in a sharp decrease in the levels of the neuropeptide precursor VGF (non-acronymic) and leads to reduced activation of cAMP response element-binding protein (CREB) and protein kinase B (AKT) in neurons. The main objective of this study is to complete the characterization of the route, or routes, involving AKT and CREB through which DISC1 modulates the expression of VGF. For that we explored known players upstream of AKT and the DISC1 binding partners glycogen synthase kinase-3 beta and Phosphodiesterase-4, which might in turn reach out to CREB in murine neuron primary culture. We found that DISC1 modulates the activation of Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). Furthermore, pharmacological inhibition of PI3K resulted in decreased expression of VGF. All this suggests that the PI3K/AKT pathway plays a role in mediating the effects of DISC1 silencing on VGF expression. Given the important roles of VGF in mental disease, and its drugability, the DISC1-VGF connection might prove to be important for efforts to develop new therapies for these diseases. Seventh Framework Programme EU-FP7 Instituto de Salud Carlos III (ISCIII) ERANET-NEURON DISCover

Neuregulin-1 (NRG1) and Disrupted-in-Schizophrenia-1 (DISC1) are promising susceptibility factors for schizophrenia. Both are multifunctional proteins with roles in a variety of neurodevelopmental processes, including progenitor cell proliferation, migration, and differentiation. Here, we provide evidence linking these factors together in a single pathway, which is mediated by ErbB receptors and PI3K/Akt. We show that signaling by NRG1 and NRG2, but not NRG3, increase expression of an isoform of DISC1 in vitro. Receptors ErbB2 and ErbB3, but not ErbB4, are responsible for transducing this effect, and PI3K/Akt signaling is also required. In NRG1 knockout mice, this DISC1 isoform is selectively reduced during neurodevelopment. Furthermore, a similar decrease in DISC1 expression is seen in β-site amyloid precursor protein cleaving enzyme–1 (BACE1) knockout mice, in which NRG1/Akt signaling is reportedly impaired. In contrast to neuronal DISC1 that was reported and characterized, expression of DISC1 in other types of cells in the brain has not been addressed. Here we demonstrate that DISC1, like NRG and ErbB proteins, is expressed in neurons, astrocytes, oligodendrocytes, microglia, and radial progenitors. These findings may connect NRG1, ErbBs, Akt, and DISC1 in a common pathway, which may regulate neurodevelopment and contribute to susceptibility to schizophrenia.