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

Chronic prostatic inflammation induces elevated autoantibody levels.

<p><b>A</b> Flow chart of the strategy used for the detection and cross-validation of autoantibody (AAB) signatures associated with chronic prostatic inflammation. Radical prostatectomy specimens were classified into two (high/low inflammation) groups based on the extent of immune cell infiltrations in the whole prostate. The corresponding pre-surgery blood serum samples were analyzed for autoantibodies (AAB) using a planar protein array (screening, n = 70). A cross-validation study testing the robustness of the identified AAB panel was based on the Luminex-bead protein array technology (cross-validation, n = 63). Statistical comparison of the serum autoantibody profiles in the low and high inflammation groups was used to identify and validate differentially abundant AABs. The prostate tissue expression patterns and the expression in different prostate cancer progression stages were established for three selected corresponding autoantigens (AAGs). <b>B</b> Bar chart for positively classified observations of the 15 most differentially detected autoantibodies in the high inflammation group compared to the low inflammation group. Data are expressed as percentage of total number of positive samples in each group. <b>C</b> Calculation of the fold change for each autoantibody revealed a significant increase of 165 antigens in high inflammation prostate cancer (upper right panel, p<0.05, fold change>2, Mann-Whitney Test) and a decrease of only one (upper left panel). <b>D</b> Graphical representation of the ten top ranked functional clusters assigned for inflammation associated autoantibodies using the DAVID functional annotation tool. The bar size corresponds to the percentage of identified corresponding genes related to a specific functional category (P<0.05).</p

Candidate autoantigens selected for analysis of their tissue expression pattern.

<p>Candidate autoantigens selected for analysis of their tissue expression pattern.</p

Clinical and pathological cohort characteristics.

<p>Clinical and pathological cohort characteristics.</p

Quantification of corresponding autoantigen levels in prostate tissue.

<p><b>A</b> Immunohistochemical stainings of representative tissue microarray spots from high and low inflammation patient cohorts. SPAST, STX18 and SPOP are expressed in the epithelium of benign (BE) and cancer (CA) areas of both cohorts. Quantitative analysis was performed using the HistoQuest immunohistochemistry analysis software (TissueGnostics). A score was calculated by multiplying staining intensity and percentage of positively stained cells. n = 25 per group. *P<0.05, Mann-Whitney Test. Bar, 100μm. <b>B</b> Quantification of the pan-lymphocyte marker CD45 (PTPRC) and autoantigen mRNA levels in high and low inflammation patient groups. n = 25 per group. ***P<0.001, Mann-Whitney Test. <b>C</b> Electropherograms of transcriptome (5’-3’) and exome (3’-5’) sequencing results depicting wild-type and D130H SPOP mutation sequences. High resolution melting curves for mutated and wild-type DNA: The purple melting curve of the sample consists of 50% mutant and 50% wild-type DNA.</p

Functional annotation of the top biomarker candidates.

<p>Functional annotation of the top biomarker candidates.</p