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. 2014 Oct 21;9(10):e111196.
doi: 10.1371/journal.pone.0111196. eCollection 2014.

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

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Free PMC article

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

Nicholas J Bradshaw et al. PLoS One. .
Free PMC article

Abstract

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.

Conflict of interest statement

Competing Interests: AL and SM are employed by Protagen AG, Dortmund, Germany, however there are no patents, patent applications or products, either current or in development, to declare in relation to the results of this work. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Antibody 6H11 detects TRIOBP-1 as a major epitope.
(A) 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) 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. (C) 6H11 recognises recombinant TRIOBP-1 protein fused to MBP (black arrow) but not recombinant MBP alone (red arrow). Some breakdown products are also visible. (D) 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.
Figure 2
Figure 2. TRIOBP splice variants and their potential to form aggregates.
(A) 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) 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. (C) Equivalent schematic of TRIOBP-4, with two previously described repeat motifs indicated . The protein is predicted to have an entirely disordered structure.
Figure 3
Figure 3. The TRIOBP-1 splice variant forms aggregates, while TRIOBP-4 does not.
(A) 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) Similarly, GFP-TRIOBP1 forms aggregates when over-expressed in rat cortical neurons (harvested at embryonic day 18, transfected at 13 days in vitro, fixed after 14 days in vitro), while TRIOBP-4 does not. GFP shown in green, neuron specific β3-tubulin antibody TUJ1 shown in red. Scale bars: 20 µm. (C) Upon transfection into SH-SY5Y (left panel) or rat primary cortical neurons (transfected after 13 days in vitro 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). (D) Three sets of rat cortical neurons were lysed at 21 days in vitro 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 TRIOBP 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.
Figure 4
Figure 4. Increased aggregation of TRIOBP-1 in post-mitotic cell culture.
(A) Western blots of total lysates and purified aggregome fractions from SH-SY5Y and NMB cells which have been harvested during proliferation (“Prolif.”, samples 1–3) or following six days differentiation (“Diff.”, samples 4–6), along with their corresponding insoluble aggregome fractions. Antibody staining reveals endogenous TRIOBP-1 to be present to a greater extent in the aggregome fraction in differentiated, non-mitotic cells. In order to allow clear viewing of both the TRIOBP-1 in the lysates and aggregomes, the same image is shown at two different lengths of exposure. GADPH is shown as a loading control. (B) An antibody which detects both TRIOBP-1 and TRIOBP-5 (green) demonstrates the presence of bright punctate endogenous TRIOBP structures in SH-SY5Y cells which have been differentiated, but not in cells which are still proliferating, consistent with an increase in TRIOBP-1 aggregation in these cells. Images were taken using the same microscope settings for direct comparison. Actin is visualised by phalloidin in red and DAPI in blue, scale bars: 20 µm. (C) Western blot of CL4 epithelial cell lysates and aggregomes taken from cell culture layers of varying confluency (approximate percentage of surface area covered with cells indicated). TRIOBP staining shows that endogenous TRIOBP-1 as well as shorter splice variants have a much higher aggregation propensity as the cells become confluent and mitosis becomes rarer.
Figure 5
Figure 5. Aggregation of TRIOBP-1 does not occur via its Pleckstrin homology domain.
(A) GST-TRIOBP-1 lacking its Pleckstrin homology domain (ΔPH) forms aggregates when expressed in SH-SY5Y neuroblastoma cells in the same manner as the full length construct. GFP shown in green, actin cytoskeleton revealed by fluorescent phalloidin (red), DAPI-stained nuclei shown in blue. (B) Western blot of three SH-SY5Y lysates transfected with full length (FL, samples 1–3, shown with a black arrow) or ΔPH GFP-TRIOBP-1 (samples 4–6, shown with a red arrow) along with their purified aggregome fraction, reveals no apparent difference in aggregation of TRIOBP-1 following removal of its Pleckstrin homology domain. Endogenous TRIOBP protein is also visible. (C) In CL4 epithelial cells, both full length and ΔPH GFP-TRIOBP-1 form insoluble aggregates while GFP-TRIOBP4 does not. GFP shown in green, actin cytoskeleton revealed by fluorescent phalloidin (red), DAPI-stained nuclei shown in blue. In all Western blots, aggregomes are enriched 10-fold relative to the lysates. In all microscopy images, scale bars: 20 µm.
Figure 6
Figure 6. The effect of TRIOBP expression on Neuroscreen-1 cells.
(A) 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) 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. (C) NS-1 cells transfected with GFP-TRIOBP1 show significantly wider cell bodies than those expressing GFP alone. (D) 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. (E) 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.

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References

    1. Kendler KS (2013) What psychiatric genetics has taught us about the nature of psychiatric illness and what is left to learn. Mol Psychiatry 18: 1058–1066. - PubMed
    1. Cavallucci V, D'Amelio M, Cecconi F (2012) Aβ Toxicity in Alzheimer's Disease. Molecular Neurobiology 45: 366–378. - PubMed
    1. Bertram L, Lill CM, Tanzi RE (2010) The Genetics of Alzheimer Disease: Back to the Future. Neuron 68: 270–281. - PubMed
    1. Korth C (2012) Aggregated proteins in schizophrenia and other chronic mental diseases: DISC1opathies. Prion 6: 134–141. - PMC - PubMed
    1. Leliveld SR, Bader V, Hendriks P, Prikulis I, Sajnani G, et al. (2008) Insolubility of Disrupted-in-Schizophrenia 1 disrupts oligomer-dependent interactions with Nuclear Distribution Element 1 and is associated with sporadic mental disease. J Neurosci 28: 3839–3845. - PMC - PubMed

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This work was supported by the Alexander von Humboldt Foundation (www.humboldt-foundation.de), the Fritz Thyssen Foundation (www.fritz-thyssen-stiftung.de) and the Forschungskommission of the Medical Faculty of the Heinrich Heine University (www.medizin.hhu.de/dekanat/gremien-und-kommissionen/kommissionen/forschungskommission.html) to NJB. EU-FP6 (“cNEUPRO”, ec.europa.eu/research/fp6) to CK and SM, and the Stanley Medical Research Institute (02R-186, Baltimore, Maryland, United States of America, www.stanleyresearch.org), the Brain Behavior and Research Foundation (NARSAD Independent Investigator Award #20350, bbrfoundation.org/II), NEURON-ERANET (“DISCover”, BMBF 01EW1003, www.neuron-eranet.eu/en/196.php) and EU-FP7 (MC-ITN “IN-SENS” #607616, ec.europa.eu/research/mariecurieactions/about-mca/actions/itn/index_en.htm) to CK. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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