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

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

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 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

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

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

<div><p>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. V_HH 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 V_HH domain derived from an immunized <i>Lama glama</i>, 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 V_HH-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 V_HH antibody.</p></div

Mapping the binding site of the anti-DISC1 V_HH B5 antibody on the DISC1 protein.

<p>(A) The binding epitope of the anti-DISC1 V_HH B5 antibody was mapped by western blot using truncated DISC1 protein constructs. (+) and (-) indicate positive and negative control, respectively. All constructs were detected using an anti-His tag antibody, whereas only the constructs covering the 691–715 segment were detected using the anti-DISC1 V_HH B5 antibody. The multiple bands with higher apparent mass observed in each lane correspond to SDS-resistant oligomers, which are a typical feature of DISC1 protein fragments [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191162#pone.0191162.ref019" target="_blank">19</a>]. (B) Detection of human and mouse DISC1 protein in the lysates of transfected NLF cells by western blot, using either anti-rodent Disc1 C-term antibody, control MEM media, anti-DISC1 V_HH B5 antibody or anti-human DISC1 14F2 antibody. The four samples tested are untransfected NLF cell lysate, mouse Disc1 transfected NLF cell lysate, human DISC1 transfected NLF cell lysate, recombinant human DISC1 (50 ng). (C) Alignment of the human DISC1 epitope (residues 691–715) with the corresponding region of mouse Disc1.</p

Parameters derived from the SAXS data (MM, molecular mass).

<p>Parameters derived from the SAXS data (MM, molecular mass).</p

<i>Ab initio</i> SAXS reconstructions and molecular models for the DISC1^691-836 protein, the V_HH domain, and their complex.

<p>The hypothetical DISC1 model (upper left) features a single domain containing three longer and three shorter α-helices. Similar to the terminal segments, the two extended loops (tilde symbols) are suggested to be poorly ordered. The positions of functionally important residues S704 and S713 are indicated in a close-up view. Also note the proline-rich motif (ball-and-stick representation, marked by a hash), which is predicted to be readily accessible for protein-protein interactions. The 691–715 region, suggested to participate in the epitope for the nanobody, is highlighted in gold while cloning artifacts at the termini are colored dark gray. The model of the V_HH antibody (lower left) displays a canonical immunoglobulin fold, including a conserved disulfide bridge (ball-and-stick model, marked by an asterisk). The CDR segments, as defined by the Chothia criteria [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191162#pone.0191162.ref060" target="_blank">60</a>], are highlighted in cyan. Finally, the DISC1^691-836 fragment and the nanobody are predicted to interact in a complex (right) featuring a 1:1 stoichiometry.</p

Investigation of DISC1-V_HH complex formation by SAXS.

<p>(A) Primary scattering data (Intensity <i>I</i> as a function of momentum transfer <i>q</i>, the latter being defined as 4π sinθ / λ) for the V_HH domain (blue), the DISC1^691-836 protein (red), and their complex (yellow). The inset shows the linear regions of the respective Guinier plots at low <i>q</i> values, as suggested by <i>AUTORG</i>; the slope of the linear fit is related to the radius of gyration of the respective sample. (B) Normalized Kratky plot of the data shown in (A). A peak at low <i>qR</i>_<i>g</i> values is considered typical of folded globular proteins. (C) Distance distribution functions derived from the data shown in (A), using the indirect Fourier transformation implemented in <i>DATGNOM</i>. For each species, the intersection with the positive abscissa corresponds to the maximum particle diameter. A.u., arbitrary units.</p