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 phenotypeswere generated. Fromthese,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

Endothelial LRP1 transports amyloid-β1-42 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-beta (A beta) brain accumulation and drives Alzheimer's disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in A beta 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 A beta 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 (Slo1c1-CreER(Tz) Lrp1(fl/fl) mice) and used these mice to accurately evaluate LRP1-mediated A beta BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [I-125] A beta(1-42). Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma A beta levels and elevated soluble brain A beta, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic AD elimination via the BBB. Together, our results suggest that receptor-mediated A beta BBB clearance may be a potential target for treatment and prevention of A beta brain accumulation in AD

Aggregation of the Protein TRIOBP-1 and Its Potential Relevance to Schizophrenia

<div><p>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. <i>TRIOBP</i> 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.</p></div

TRIOBP splice variants and their potential to form aggregates.

<p>(<b>A</b>) Relative positions of the major splice variants of TRIOBP, using the mouse nomenclature. Approximate chromosomal positions of the transcripts on human chromosome 22 and mouse chromosome 15 are indicated. (<b>B</b>) Schematic of the predicted structure of the TRIOBP-1 protein, with putative Pleckstrin homology (PH) domain and predicted coiled-coils indicated. Below are shown predicted “hot spots”, with high potential for forming protein aggregates. These were identified through analysis with six aggregation prediction paradigms from four independent servers. Hot spots were defined as stretches of 5 or more consecutive amino acids each of which was predicted to be aggregated by 3 (shown in yellow), 4 (orange) or 5 (red) of these 6 methods. (<b>C</b>) Equivalent schematic of TRIOBP-4, with two previously described repeat motifs indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111196#pone.0111196-Bao1" target="_blank">[11]</a>. The protein is predicted to have an entirely disordered structure.</p

The TRIOBP-1 splice variant forms aggregates, while TRIOBP-4 does not.

<p>(<b>A</b>) GFP-fused TRIOBP-1 and TRIOBP-5 form aggregates when over-expressed in SH-SY5Y, while GFP-TRIOBP-4 does not. GFP shown in green, actin cytoskeleton revealed by fluorescent phalloidin is shown in red, DAPI-stained nuclei shown in blue. Scale bars: 20 µm. (<b>B</b>) Similarly, GFP-TRIOBP1 forms aggregates when over-expressed in rat cortical neurons (harvested at embryonic day 18, transfected at 13 days <i>in vitro</i>, fixed after 14 days <i>in vitro</i>), while TRIOBP-4 does not. GFP shown in green, neuron specific β3-tubulin antibody TUJ1 shown in red. Scale bars: 20 µm. (<b>C</b>) Upon transfection into SH-SY5Y (left panel) or rat primary cortical neurons (transfected after 13 days <i>in vitro</i> and lysed 24 hours later, right panel), over-expressed GFP-TRIOBP-1, labelled with black arrows, is seen by Western blot to be in the purified aggregated fraction. Endogenous TRIOBP can also be seen, particularly in the cortical neuron blot in which the transfection was less effective (red arrow). (<b>D</b>) Three sets of rat cortical neurons were lysed at 21 days <i>in vitro</i> and their aggregomes purified revealing the presence of TRIOBP-1 (black arrow), long variants such as TRIOBP-5 (red arrows) and shorter splice variants of the <i>TRIOBP</i> 3′ region (blue arrows) to be consistently present in this insoluble fraction. Based on the antibody used, such shorter variants would be predicted to be those which share amino acid sequence with the C-terminal half of TRIOBP-1. In all Western blots, aggregomes are enriched 10-fold relative to lysates.</p

Antibody 6H11 detects TRIOBP-1 as a major epitope.

<p>(<b>A</b>) While the 6H11 antibody is able to detect both the long and short variants of CRMP1 when over-expressed in NLF neuroblastoma cells (black arrows), it also shows strong affinity to an additional 70 kDa species. (<b>B</b>) Binding strength of the schizophrenia aggregome-specific antibody 6H11 at differing dilutions to recombinant TRIOBP-1 on a protein array. Two separate preparations of the antibody from a hybridoma cell line are shown. (<b>C</b>) 6H11 recognises recombinant TRIOBP-1 protein fused to MBP (black arrow) but not recombinant MBP alone (red arrow). Some breakdown products are also visible. (<b>D</b>) Using Western blot secondary antibodies which emit at two distinct wavelengths, it can be seen that the major 70 kDa species detected by antibody 6H11 (green) coincides exactly with the major band detected by a polyclonal antibody against the C-terminus of TRIOBP-1/5 (red, 70 kDa band labelled with a black arrow). 6H11 does not recognise a 40 kDa TRIOBP species (red arrow). 6H11 thus recognises TRIOBP-1, most likely at an epitope within the N-terminal half of the protein.</p

The effect of TRIOBP expression on Neuroscreen-1 cells.

<p>(<b>A</b>) Examples of NS-1 cells transfected with GFP alone (n = 181), GFP-TRIOBP-1 (n = 86) or GFP-TRIOBP-4 (n = 86). Transfected cells are indicated by white asterisks. Total cell body is visualised in red using the TUJ1 antibody, scale bars: 20 µm. (<b>B</b>) NS-1 cells transfected with GFP-TRIOBP1 show significantly longer cell bodies than those expressing GFP alone. Expression of GFP-TRIOBP-4 causes a more modest increase in length compared to GFP alone. (<b>C</b>) NS-1 cells transfected with GFP-TRIOBP1 show significantly wider cell bodies than those expressing GFP alone. (<b>D</b>) There is no significant difference in the degree of cell body elongation of NS-1 cells transfected with either GFP alone, GFP-TRIOBP1 or GFP-TRIOBP4. (<b>E</b>) Sholl analysis of NS-1 neurite growth following transfection with GFP, GFP-TRIOBP-1 or GFP-TRIOBP-4. The mean number of neurites per cell reaching a range of distances from the cell body is displayed for each transfection type. Only the first 160nm are shown as less than 5% of cells displayed neurites longer than this. Longest neurite recorded was 280 nm. Black asterisks show lengths at which the expressed protein type has a significant effect on neurite number by the Kruskal-Wallis one-way analysis of variance, while red asterisks indicate that in addition GFP-TRIOBP-1 has a significant effect over GFP by the Mann-Whitney U test, after correction for multiple testing. In all graphs, *: p<0.05, **: p<0.01, ***: p<0.001, ****: p<0.0001.</p

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

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-β1-42 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-beta (Abeta) brain accumulation and drives Alzheimer&#039;s disease (AD) pathology. However, due to conflicting reports on the involvement of LRP1 in Abeta 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 Abeta 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-CreERT2 Lrp1fl/fl mice) and used these mice to accurately evaluate LRP1-mediated Abeta BBB clearance in vivo. Selective deletion of Lrp1 in the brain endothelium of C57BL/6 mice strongly reduced brain efflux of injected [125I] Abeta1-42. Additionally, in the 5xFAD mouse model of AD, brain endothelial-specific Lrp1 deletion reduced plasma Abeta levels and elevated soluble brain Abeta, leading to aggravated spatial learning and memory deficits, thus emphasizing the importance of systemic Abeta elimination via the BBB. Together, our results suggest that receptor-mediated Abeta BBB clearance may be a potential target for treatment and prevention of Abeta brain accumulation in AD