Verian Bader

Insolubility of Disrupted-in-Schizophrenia 1 Disrupts Oligomer-Dependent Interactions with Nuclear Distribution Element 1 and Is Associated with Sporadic Mental Disease

Disrupted-in-schizophrenia 1 (DISC1) and other genes have been identified recently as potential molecular players in chronic psychiatric diseases such as affective disorders and schizophrenia. A molecular mechanism of how these genes may be linked to the majority of sporadic cases of these diseases remains unclear. The chronic nature and irreversibility of clinical symptoms in a subgroup of these diseases prompted us to investigate whether proteins corresponding to candidate genes displayed subtle features of protein aggregation. Here, we show that in postmortem brain samples of a distinct group of patients with phenotypes of affective disorders or schizophrenia, but not healthy controls, significant fractions of DISC1 could be identified as cold Sarkosyl-insoluble protein aggregates. A loss-of-function phenotype could be demonstrated for insoluble DISC1 through abolished binding to a key DISC1 ligand, nuclear distribution element 1 (NDEL1): in human neuroblastoma cells, DISC1 formed expression-dependent, detergent-resistant aggregates that failed to interact with endogenous NDEL1. Recombinant (r) NDEL1 expressed in Escherichia coli selectively bound an octamer of an rDISC1 fragment but not dimers or high molecular weight multimers, suggesting an oligomerization optimum for molecular interactions of DISC1 with NDEL1. For DISC1-related sporadic psychiatric disease, we propose a mechanism whereby impaired cellular control over self-association of DISC1 leads to excessive multimerization and subsequent formation of detergent-resistant aggregates, culminating in loss of ligand binding, here exemplified by NDEL1. We conclude that the absence of oligomer-dependent ligand interactions of DISC1 can be associated with sporadic mental disease of mixed phenotypes.

Oligomer Assembly of the C-Terminal DISC1 Domain (640−854) Is Controlled by Self-Association Motifs and Disease-Associated Polymorphism S704C

Genetic studies have established a role of disrupted-in-schizophrenia-1 (DISC1) in chronic mental diseases (CMD). Limited experimental data are available on the domain structure of the DISC1 protein although multiple interaction partners are known including a self-association domain within the middle part of DISC1 (residues 403-504). The DISC1 C-terminal domain is deleted in the original Scottish pedigree where DISC1 harbors two coiled-coil domains and disease-associated polymorphisms at 607 and 704, as well as the important nuclear distribution element-like 1 (NDEL1) binding site at residues 802-839. Here, we performed mutagenesis studies of the C-terminal domain of the DISC1 protein (residues 640-854) and analyzed the expressed constructs by biochemical and biophysical methods. We identified novel DISC1 self-association motifs and the necessity of their concerted action for orderly assembly: the region 765-854 comprising a coiled-coil domain is a dimerization domain and the region 668-747 an oligomerization domain; dimerization was found to be a prerequisite for orderly assembly of oligomers. Consistent with this, disease-associated polymorphism C704 displayed a slightly higher oligomerization propensity. The heterogeneity of DISC1 multimers in vitro was confirmed with a monoclonal antibody binding exclusively to HMW multimers. We also identified C-terminal DISC1 fragments in human brains, suggesting that C-terminal fragments could carry out DISC1-dependent functions. When the DISC1 C-terminal domain was transiently expressed in cells, it assembled into a range of soluble and insoluble multimers with distinct fractions selectively binding NDEL1, indicating functionality. Our results suggest that assembly of the C-terminal domain is controlled by distinct domains including the disease-associated polymorphism 704 and is functional in vivo.

Convergence of Two Independent Mental Disease Genes on the Protein Level: Recruitment of Dysbindin to Cell-Invasive Disrupted-In-Schizophrenia 1 Aggresomes

BACKGROUND Both disrupted-in-schizophrenia 1 (DISC1) and dysbindin have been identified as schizophrenia candidate genes in independent genetic linkage studies. The proteins have been assigned distinct subcellular locations and functions. We investigated whether both proteins converge into a common pathway specific for schizophrenia or mental diseases. METHODS DISC1 and dysbindin were expressed as recombinant proteins with or without a fluorescent protein-tag in human or mouse neuroblastoma cells and as recombinant proteins in E. coli. Postmortem brains of patients with mental diseases from the Stanley Research Medical Institutes Consortium Collection were used to demonstrate molecular interactions in biochemically purified protein fractions. RESULTS First, upon overexpression in neuroblastoma cells, DISC1 formed aggresomes that recruited homologous soluble C-terminal DISC1 fragment or heterologous dysbindin. Domains involved in binding could be mapped to DISC1 (316-597) and dysbindin (82-173), indicating a specific interaction. In addition, recruitment was demonstrated when externally added, purified DISC1 aggresomes penetrated recipient cells after coincubation. Second, a direct interaction between soluble DISC1 protein and dysbindin was demonstrated in a cell free system using E. coli-expressed proteins. Third, co-aggregation of DISC1 and dysbindin was demonstrated in postmortem brains for a subgroup of cases with chronic mental disease but not healthy control subjects. CONCLUSIONS A direct interaction of soluble and insoluble DISC1 protein with dysbindin protein demonstrates convergence of so far considered independent mental disease genes by direct molecular interaction. Our findings highlight protein aggregation and recruitment as a biological mechanism in mental disease.

Misassembly of full-length Disrupted-in-Schizophrenia 1 protein is linked to altered dopamine homeostasis and behavioral deficits

Disrupted-in-schizophrenia 1 (DISC1) is a mental illness gene first identified in a Scottish pedigree. So far, DISC1-dependent phenotypes in animal models have been confined to expressing mutant DISC1. Here we investigated how pathology of full-length DISC1 protein could be a major mechanism in sporadic mental illness. We demonstrate that a novel transgenic rat model, modestly overexpressing the full-length DISC1 transgene, showed phenotypes consistent with a significant role of DISC1 misassembly in mental illness. The tgDISC1 rat displayed mainly perinuclear DISC1 aggregates in neurons. Furthermore, the tgDISC1 rat showed a robust signature of behavioral phenotypes that includes amphetamine supersensitivity, hyperexploratory behavior and rotarod deficits, all pointing to changes in dopamine (DA) neurotransmission. To understand the etiology of the behavioral deficits, we undertook a series of molecular studies in the dorsal striatum of tgDISC1 rats. We observed an 80% increase in high-affinity DA D2 receptors, an increased translocation of the dopamine transporter to the plasma membrane and a corresponding increase in DA inflow as observed by cyclic voltammetry. A reciprocal relationship between DISC1 protein assembly and DA homeostasis was corroborated by in vitro studies. Elevated cytosolic dopamine caused an increase in DISC1 multimerization, insolubility and complexing with the dopamine transporter, suggesting a physiological mechanism linking DISC1 assembly and dopamine homeostasis. DISC1 protein pathology and its interaction with dopamine homeostasis is a novel cellular mechanism that is relevant for behavioral control and may have a role in mental illness.

Aggregation of the protein TRIOBP-1 and its potential relevance to schizophrenia.

We have previously proposed that specific proteins may form insoluble aggregates as a response to an illness-specific proteostatic dysbalance in a subset of brains from individuals with mental illness, as is the case for other chronic brain conditions. So far, established risk factors DISC1 and dysbindin were seen to specifically aggregate in a subset of such patients, as was a novel schizophrenia-related protein, CRMP1, identified through a condition-specific epitope discovery approach. In this process, antibodies are raised against the pooled insoluble protein fractions (aggregomes) of post mortem brain samples from schizophrenia patients, followed by epitope identification and confirmation using additional techniques. Pursuing this epitope discovery paradigm further, we reveal TRIO binding protein (TRIOBP) to be a major substrate of a monoclonal antibody with a high specificity to brain aggregomes from patients with chronic mental illness. TRIOBP is a gene previously associated with deafness which encodes for several distinct protein species, each involved in actin cytoskeletal dynamics. The 3′ splice variant TRIOBP-1 is found to be the antibody substrate and has a high aggregation propensity when over-expressed in neuroblastoma cells, while the major 5′ splice variant, TRIOBP-4, does not. Endogenous TRIOBP-1 can also spontaneously aggregate, doing so to a greater extent in cell cultures which are post-mitotic, consistent with aggregated TRIOBP-1 being able to accumulate in the differentiated neurons of the brain. Finally, upon expression in Neuroscreen-1 cells, aggregated TRIOBP-1 affects cell morphology, indicating that TRIOBP-1 aggregates may directly affect cell development, as opposed to simply being a by-product of other processes involved in major mental illness. While further experiments in clinical samples are required to clarify their relevance to chronic mental illness in the general population, TRIOBP-1 aggregates are thus implicated for the first time as a biological element of the neuropathology of a subset of chronic mental illness.

Simultaneous effects on parvalbumin-positive interneuron and dopaminergic system development in a transgenic rat model for sporadic schizophrenia.

To date, unequivocal neuroanatomical features have been demonstrated neither for sporadic nor for familial schizophrenia. Here, we investigated the neuroanatomical changes in a transgenic rat model for a subset of sporadic chronic mental illness (CMI), which modestly overexpresses human full-length, non-mutant Disrupted-in-Schizophrenia 1 (DISC1), and for which aberrant dopamine homeostasis consistent with some schizophrenia phenotypes has previously been reported. Neuroanatomical analysis revealed a reduced density of dopaminergic neurons in the substantia nigra and reduced dopaminergic fibres in the striatum. Parvalbumin-positive interneuron occurrence in the somatosensory cortex was shifted from layers II/III to V/VI, and the number of calbindin-positive interneurons was slightly decreased. Reduced corpus callosum thickness confirmed trend-level observations from in vivo MRI and voxel-wise tensor based morphometry. These neuroanatomical changes help explain functional phenotypes of this animal model, some of which resemble changes observed in human schizophrenia post mortem brain tissues. Our findings also demonstrate how a single molecular factor, DISC1 overexpression or misassembly, can account for a variety of seemingly unrelated morphological phenotypes and thus provides a possible unifying explanation for similar findings observed in sporadic schizophrenia patients. Our anatomical investigation of a defined model for sporadic mental illness enables a clearer definition of neuroanatomical changes associated with subsets of human sporadic schizophrenia.

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimers disease (Aβ, tau), Parkinsons Disease (α-synuclein), Huntingtons disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms. Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for Aβ6, tau7-9, α-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders. Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic α-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

Functional Proteomics Backed by Genetics and Reverse Genetic Engineering: A Novel Successful Approach to Identify Schizophrenia Subsets

Abstract Background Disruption of proteostasis is a common cellular phenotype after a gentic or exogenous lesion of postmitotic neurons. In the most extreme examples, the neurodegenerative diseases, proteostasis disturbance leads to microscopically visible protein deposits. However, it is reasonable to assume that also in other chronic brain conditions, for example mental illnesses like residual schizophrenia or chronic depression, proteostasis occurs, even though clearly not accompanied by massive neuronal loss. The hypothesis of my laboratory is therefore to investigate the dsturbance of proteostasis in the context of chronic mental illnesses like schizphrenia, exemlified by the occurrence of protein pathology, ie. proteins insoluble in ionic detergents. Methods Post mortem brains from patients with schizophrenia, bipolar disorder, depression or healthy controls were obtained from the Stanley Research Foundation (Consortium collection; n = 60), and the insoluble proteome purified by biochemical fractionation which involves several ultracentrifugation steps, the last in cold ionic detergent. The insoluble proteome of each patient was then either immunoblotted for candidate genes or pooled by diagnosis (n = 15) and injected into mice for the generation of monoclonal antibodies that would selectively recognize only schizophrenia brains but not healthy brain (epitope discovery); epitopes of such antibodies were determined on protein arrays. Finally, we also performed proteomics of the insoluble proteome by LC-MS/MS. For positive hits, genetic studies were performed to gather independent evidence. Results For the rare candidate protein Disrupted-in-Schizophrenia 1 (DISC1), we could show insolubility in a subset of patients with mental illness crossing clinical diagnoses. When we modeled in a novel transgenic rat modestly overexpressing human full length DISC1, we observed disruption of dopamine homeostasis with amphetamine hypersensitivity, aberrant dopamine reuptake (Trossbach et al., 2016, Molecular Psychiatry), and aberrant dopamine as well as interneurron neuroanatomy, validating the functional notion of DISC1 protein pathology for chronic mental illness. Using epitope discovery we identified two candidate proteins, CRMP1 and TRIOBP1 as aggregated in subsets of cases with chronic mental illness. Systematic proteomics of the insoluble proteome of schizophrenia yielded more novel candidates like NKCC1 that are currently validated. Discussion Protein pathology is a novel way of classifying chronic mental illnesses such as schizophrenia, complementing genetic analyses. Specific signatures of insoluble proteins correspond to a disease-specific failure in proteostasis. More specifically, insoluble DISC1 assemblies seem to regulate dopamine homeostasis, a brain central metabolic disturbance during psychosis