TRIOBP-1 Protein Aggregation Exists in Both Major Depressive Disorder and Schizophrenia, and Can Occur through Two Distinct Regions of the Protein.

The presence of proteinopathy, the accumulation of specific proteins as aggregates in neurons, is an emerging aspect of the pathology of schizophrenia and other major mental illnesses. Among the initial proteins implicated in forming such aggregates in these conditions is Trio and F-actin Binding Protein isoform 1 (TRIOBP-1), a ubiquitously expressed protein involved in the stabilization of the actin cytoskeleton. Here we investigate the insolubility of TRIOBP-1, as an indicator of aggregation, in brain samples from 25 schizophrenia patients, 25 major depressive disorder patients and 50 control individuals (anterior cingulate cortex, BA23). Strikingly, insoluble TRIOBP-1 is considerably more prevalent in both of these conditions than in controls, further implicating TRIOBP-1 aggregation in schizophrenia and indicating a role in major depressive disorder. These results were only seen using a high stringency insolubility assay (previously used to study DISC1 and other proteins), but not a lower stringency assay that would be expected to also detect functional, actin-bound TRIOBP-1. Previously, we have also determined that a region of 25 amino acids in the center of this protein is critical for its ability to form aggregates. Here we attempt to refine this further, through the expression of various truncated mutant TRIOBP-1 vectors in neuroblastoma cells and examining their aggregation. In this way, it was possible to narrow down the aggregation-critical region of TRIOBP-1 to just 8 amino acids (333-340 of the 652 amino acid-long TRIOBP-1). Surprisingly our results suggested that a second section of TRIOBP-1 is also capable of independently inducing aggregation: the optionally expressed 59 amino acids at the extreme N-terminus of the protein. As a result, the 597 amino acid long version of TRIOBP-1 (also referred to as "Tara" or "TAP68") has reduced potential to form aggregates. The presence of insoluble TRIOBP-1 in brain samples from patients, combined with insight into the mechanism of aggregation of TRIOBP-1 and generation of an aggregation-resistant mutant TRIOBP-1 that lacks both these regions, will be of significant use in further investigating the mechanism and consequences of TRIOBP-1 aggregation in major mental illness.

Disruption of cellular proteostasis by H1N1 influenza A virus causes α-synuclein aggregation.

Neurodegenerative diseases feature specific misfolded or misassembled proteins associated with neurotoxicity. The precise mechanisms by which protein aggregates first arise in the majority of sporadic cases have remained unclear. Likely, a first critical mass of misfolded proteins starts a vicious cycle of a prion-like expansion. We hypothesize that viruses, having evolved to hijack the host cellular machinery for catalyzing their replication, lead to profound disturbances of cellular proteostasis, resulting in such a critical mass of protein aggregates. Here, we investigated the effect of influenza virus (H1N1) strains on proteostasis of proteins associated with neurodegenerative diseases in Lund human mesencephalic dopaminergic cells in vitro and infection of Rag knockout mice in vivo. We demonstrate that acute H1N1 infection leads to the formation of α-synuclein and Disrupted-in-Schizophrenia 1 (DISC1) aggregates, but not of tau or TDP-43 aggregates, indicating a selective effect on proteostasis. Oseltamivir phosphate, an antiinfluenza drug, prevented H1N1-induced α-synuclein aggregation. As a cell pathobiological mechanism, we identified H1N1-induced blocking of autophagosome formation and inhibition of autophagic flux. In addition, α-synuclein aggregates appeared in infected cell populations connected to the olfactory bulbs following intranasal instillation of H1N1 in Rag knockout mice. We propose that H1N1 virus replication in neuronal cells can induce seeds of aggregated α-synuclein or DISC1 that may be able to initiate further detrimental downstream events and should thus be considered a risk factor in the pathogenesis of synucleinopathies or a subset of mental disorders. More generally, aberrant proteostasis induced by viruses may be an underappreciated factor in initiating protein misfolding.

Disrupted in Schizophrenia 1 regulates the processing of reelin in the perinatal cortex.

Disrupted in Schizophrenia 1 (DISC1) is a prominent gene in mental illness research, encoding a scaffold protein known to be of importance in the developing cerebral cortex. Reelin is a critical extracellular protein for development and lamination of the prenatal cortex and which has also been independently implicated in mental illness. Regulation of reelin activity occurs through processing by the metalloproteinases ADAMTS-4 and ADAMTS-5. Through cross-breeding of heterozygous transgenic DISC1 mice with heterozygous reeler mice, which have reduced reelin, pups heterozygous for both phenotypes were generated. From these, we determine that transgenic DISC1 leads to a reduction in the processing of reelin, with implications for its downstream signalling element Dab1. An effect of DISC1 on reelin processing was confirmed in vitro, and revealed that intracellular DISC1 affects ADAMTS-4 protein, which in turn is exported and affects processing of extracellular reelin. In transgenic rat cortical cultures, an effect of DISC1 on reelin processing could also be seen specifically in early, immature neurons, but was lost in calretinin and reelin-positive mature neurons, suggesting cell-type specificity. DISC1 therefore acts upstream of reelin in the perinatal cerebral cortex in a cell type/time specific manner, leading to regulation of its activity through altered proteolytic cleavage. Thus a functional link is demonstrated between two proteins, each of independent importance for both cortical development and associated cognitive functions leading to behavioural maladaptation and mental illness.

Biophysical insights from a single chain camelid antibody directed against the Disrupted-in-Schizophrenia 1 protein.

Accumulating evidence suggests an important role for the Disrupted-in-Schizophrenia 1 (DISC1) protein in neurodevelopment and chronic mental illness. In particular, the C-terminal 300 amino acids of DISC1 have been found to mediate important protein-protein interactions and to harbor functionally important phosphorylation sites and disease-associated polymorphisms. However, long disordered regions and oligomer-forming subdomains have so far impeded structural analysis. VHH domains derived from camelid heavy chain only antibodies are minimal antigen binding modules with appreciable solubility and stability, which makes them well suited for the stabilizing proteins prior to structural investigation. Here, we report on the generation of a VHH domain derived from an immunized Lama glama, displaying high affinity for the human DISC1 C region (aa 691-836), and its characterization by surface plasmon resonance, size exclusion chromatography and immunological techniques. The VHH-DISC1 (C region) complex was also used for structural investigation by small angle X-ray scattering analysis. In combination with molecular modeling, these data support predictions regarding the three-dimensional fold of this DISC1 segment as well as its steric arrangement in complex with our VHH antibody.

Endothelial LRP1 transports amyloid-ß(1-42) across the blood-brain barrier.

According to the neurovascular hypothesis, impairment of low-density lipoprotein receptor-related protein-1 (LRP1) in brain capillaries of the blood-brain barrier (BBB) contributes to neurotoxic amyloid-ß (Aß) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Aß transport and the expression of LRP1 in brain endothelium, the role of LRP1 at the BBB is uncertain. As global Lrp1 deletion in mice is lethal, appropriate models to study the function of LRP1 are lacking. Moreover, the relevance of systemic Aß clearance to AD pathology remains unclear, as no BBB-specific knockout models have been available. Here, we developed transgenic mouse strains that allow for tamoxifen-inducible deletion of Lrp1 specifically within brain endothelial cells (Slco1c1-CreER(T2) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated Aß BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Aß(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Aß levels and elevated soluble brain Aß, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Aß elimination via the BBB. Together, our results suggest that receptor-mediated Aß BBB clearance may be a potential target for treatment and prevention of Aß brain accumulation in AD.

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.

Immunohistochemical characterization of novel monoclonal antibodies against the N-terminus of amyloid ß-peptide.

Abstract Amyloid ß-peptide (Aß) is a key molecule in Alzheimer's disease (AD). Reliable immunohistochemical (IHC) methods to detect Aß and Aß-associated factors (AAF) in brain specimens are needed to determine their role in AD pathophysiology. Formic acid (FA) pre-treatment, which is generally used to enable efficient detection of Aß with IHC, induces structural modifications within the Aß, as well as in AAF. Consequently, interpretation of double IHC stainings becomes difficult. Therefore, serial stainings of two newly produced monoclonal antibodies (mAbs) VU-17 and IC16 and two other mAbs (6E10 and 3D6) were performed with four different pre-treatments (no pre-treatment, Tris/EDTA, citrate and FA) and additionally six IHC characteristics were scored: diffuse/compact/classic plaques, arteries with cerebral Aß angiopathy, dyshoric angiopathy, capillaries with dyshoric angiopathy. Subsequently, these stainings were compared with IHC procedures, which are frequently used in a diagnostic setting, employing mAbs 4G8 and 6F/3D with FA pre-treatment. IHC Aß patterns obtained with VU-17 and, IC16 and 3D6 without the use of FA pre-treatment were comparable to those obtained with 4G8 and 6F/3D upon FA pre-treatment. Omission of FA pre-treatment gives the advantage to allow double IHC stainings, detecting both Aß and AAF that otherwise would have been structural modificated upon FA pre-treatment.

Proteomic, genomic and translational approaches identify CRMP1 for a role in schizophrenia and its underlying traits.

Schizophrenia is a chronic illness of heterogenous biological origin. We hypothesized that, similar to chronic progressive brain conditions, persistent functional disturbances of neurons would result in disturbed proteostasis in the brains of schizophrenia patients, leading to increased abundance of specific misfolded, insoluble proteins. Identification of such proteins would facilitate the elucidation of molecular processes underlying these devastating conditions. We therefore generated antibodies against pooled insoluble proteome of post-mortem brains from schizophrenia patients in order to identify unique, disease-specific epitopes. We successfully identified such an epitope to be present on collapsin-response mediator protein 1 (CRMP1) in biochemically purified, insoluble brain fractions. A genetic association analysis for the CRMP1 gene in a large Finnish population cohort (n = 4651) corroborated the association of physical and social anhedonia with the CRMP1 locus in a DISC1 (Disrupted-in-schizophrenia 1)-dependent manner. Physical and social anhedonia are heritable traits, present as chronic, negative symptoms of schizophrenia and severe major depression, thus constituting serious vulnerability factors for mental disease. Strikingly, lymphoblastoid cell lines derived from schizophrenia patients mirrored aberrant CRMP1 immunoreactivity by showing an increase of CRMP1 expression, suggesting its potential role as a blood-based diagnostic marker. CRMP1 is a novel candidate protein for schizophrenia traits at the intersection of the reelin and DISC1 pathways that directly and functionally interacts with DISC1. We demonstrate the impact of an interdisciplinary approach where the identification of a disease-associated epitope in post-mortem brains, powered by a genetic association study, is rapidly translated into a potential blood-based diagnostic marker.

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions.

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.

Disrupted-in-Schizophrenia-1 expression is regulated by beta-site amyloid precursor protein cleaving enzyme-1-neuregulin cascade.

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 beta-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.

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.

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.