The Disrupted in Schizophrenia 1 (DISC1) gene was found mutated when studying a chromosomal translocation t(1;11)(q42.1;q14.3) in a Scottish family; this translocation correlated with cases of schizophrenia, bipolar disorder and major depression [1
]. Further studies also found that the truncation of this gene in an American family segregated with cases of schizophrenia [3
Since the discovery of this translocation, many groups have invested their efforts in understanding the role of DISC1 protein, with the hope of revealing new mechanisms that could explain the neurobiology behind mental disease. Therefore, DISC1 was proposed to be involved in diverse processes such as neurogenesis [4
], synapse regulation [6
], neurite outgrowth [6
], and neural migration and proliferation [13
]. Also, yeast two hybrid experiments [16
] and other molecular studies have revealed several important interacting partners of DISC1 including GSK3β [5
], PDE4B [17
], Rac1 [8
], Girdin [18
] or TNIK [9
] among others. Thus, DISC1 might act as a molecular scaffold, providing cohesion and coordination among different biological events in the brain [19
To acquire a deeper understanding of the mechanisms of action of DISC1, several proteomic analyses have been conducted to specifically address the role of the truncated isoform of DISC1 on the cellular proteome of neural cells [20
]. In this study, we decided to specifically address the role of DISC1 loss of function, for that we carried out an unbiased proteomic analysis in DISC1-silenced neurons.
We report that DISC1 alters the expression of many relevant proteins related to neurodevelopment and synaptic function, reinforcing the idea that DISC1 is a key molecular link bridging neurodevelopmental functions with the regulation of synaptic formation and neurosignaling processes.
We have taken advantage of a well-established murine primary neuron DISC1 knock-down experimental system [8
] to carry out an unbiased proteomic analysis and thus, identify proteins which have their expression affected by DISC1.
The results of our analysis highlight the importance of DISC1 both in neurodevelopment and synaptic regulation. Both functions have been already ascribed to DISC1; however, this study describes new important routes to explore, as the effect DISC1 silencing on the expression of CRMP family of proteins. This could be a powerful mechanism to further investigate considering the relevance this family of proteins has in the neurobiology of mental disease [27
Furthermore, DISC1 knockdown resulted in a neurite outgrowth deficit in RA-treated SH-SY5Y cells. Previous studies have reported an impaired neurite outgrowth in cell models that overexpress mutant isoforms of DISC1 [11
] and an increase of neurite outgrowth was seen in PC12 cells that overexpress DISC1 [42
]. Therefore, our study reinforces the idea that the loss of function of DISC1 is critical for proper regulation of neurite outgrowth. In this direction, other studies have previously shown DISC1 silencing affected neurite outgrowth using PC12 cells [14
]. We have to consider neurite outgrowth in PC12 cells is a result of two processes, neural differentiation and subsequent neurite extension, so the effects of silencing may be interpreted as measuring an effect on either/both processes. In contrast, SH-SY5Y cells are already neuronal and forming neurites, so we could compare neurite length and the effect is specific to neurite outgrowth.
At the same time, we have found that several proteins that participate in synaptic membrane trafficking and synapse formation are altered in DISC1 silenced neurons, such as syntaxin 7, MUNC-18, cadherin-13, and Rab proteins (Table 1
), but we cannot conclude whether trafficking is up- or downregulated in our system. Previous studies have shown that DISC1 enhances the transport of synaptic vesicles, therefore we could expect that knocking down DISC1 expression produced an attenuated vesicle transport in primary cortical neurons [43
Summarizing, our study shows that DISC1 works as an important modulator of proteins that are directly involved both in neurodevelopment and in adult synaptic regulation, representing a unifier factor of two seemingly different categories.
4. Materials and Methods
Commercial antibodies specific for the following proteins were used: CRMP-2, p(Thr514)CRMP-2, Stathmin, p(Ser38)Stathmin (1:1000; Cell Signaling Technology, Danvers, MA, USA); tubulin, GAPDH (1:5000; Sigma-Aldrich, St. Louis, MO, USA); the human DISC1-specific antibody 14F2 has been previously described [44
]; the mouse DISC1-specific antibody D27 was a kind gift from Merck (Kenilworth, NJ, USA). Goat anti-rabbit (1:2000; Dako Cytomation, Glosstrup, Denmark), sheep anti-mouse (1:5000; GE Healthcare Amersham Bioscience, Uppsala, Sweden) and donkey anti-goat (1:2000; Santa Cruz Biotechnologies, Santa Cruz, CA, USA) were used as secondary antibodies.
4.2. Cell Culture
SH-SY5Y neuroblastoma cells (European Collection of Cell Cultures, Salisbury, UK) were maintained in 1:1 Earle’s Balanced Salt Solution (EBSS)- F12HAM (Sigma Aldrich) with 15% fetal bovine serum (FBS) (Gibco, Life Technologies, Gaithesburg, MD, USA), 1% Glutamine (Gln) (Sigma Aldrich), 1% non-essential amino acids (NEAA) (Sigma Aldrich), and 1% Penicillin-Streptomycin (P/S) (Invitrogen). 293FT cells (Invitrogen) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma Aldrich) with 10% FBS, 1% sodium pyruvate (Sigma Aldrich), 1% NEAA, 1% Gln, and 1% P/S.
Murine cortex and hippocampal primary neurons were prepared from 14–15 days embryos (see below ethical statement). Pregnant dams were killed by cervical dislocation in accordance with institutional guidelines for care and use of animals. The embryos were maintained and dissected in PBS Ca/Mg (Invitrogen) supplemented with 33 mM glucose. Pooled tissue was mechanically dissociated, treated with trypsin (Invitrogen) and DNaseI (Roche Applied Science, Mannheim, Germany) and resuspended in Neurobasal medium (Invitrogen) supplemented with 50X B27 (Invitrogen), 0.55g/100mL glucose (Sigma Aldrich), 42 mg/100 mL sodium bicarbonate (Sigma Aldrich), 1% P/S and 1% glutamine. The cells were plated on poly-D-lysine (Sigma Aldrich) coated Petri dishes. Cultures were maintained in serum free medium at 37 °C in 95% air/5% CO2.
4.3. Ethics Statement
Animal experiments were carried out in accordance with the European Union Council Directive 86/609/EEC, and were approved by the University of Santiago de Compostela Ethics Committee (protocol 15005AE/12/FUN 01/PAT 05/JRR2, 5 January 2012).
4.4. DISC1 Silencing
For DISC1 knock-down in murine primary neurons, we chose a validated shRNA construct developed by Akira Sawa’s group (DISC1 RNAi #1) that has been shown to specifically decrease the amount of DISC1 in cortical neural cell cultures [8
]. The commercial pLK0.1-puro non-mammalian shRNA control construct from Sigma Aldrich (reference: SHC002) was used as a scramble control. Lentiviruses were produced by calcium phosphate triple co-transfection of shRNA (see Table S3 and Figure S1 in Supporting Information
), VSVG and ΔR8.9 constructs into 293FT packaging cells. Virus-containing medium was collected 48 h after transfection, and added (10 mL of lentiviral solution/3 × 106
neurons) to the medium of primary neurons at 7 DIV. The medium was changed 24 h after infection, and incubation continued for 72 h.
In SH-SY5Y cells, DISC1 was silenced using commercial Mission®
shRNA lentiviral transduction particles (Sigma Aldrich, reference NM_018662) containing two alternative PLKO.1-Puro-CMV shRNA plasmids (Table S2 in Supporting Information
pLKO.1-puro non-mammalian shRNA particles (reference: SHC002V) were used as control. Stable cell lines were generated for any of these constructs after selection with puromycin as previously described [37
4.5. Sample Preparation for Proteomic Studies
Cells (confluent 100 mm plates) were washed twice with cold PBS and solubilized in lysis buffer (20 mM HEPES, 2 mM EGTA, 1 mM DTT, 1 mM sodium orthovanadate, 1% Triton X-100, 10% Glycerol, 2 µM leupeptin, 400 µM PMSF, 50 µM β-glycerophosphate, 100 µg/mL Trasylol). The cells were scraped on ice for 10 min, incubated on ice for 30 min with periodic vortexing every 5 min and centrifuged for 20 min at 14,000 g, 4 °C. The supernatant was saved and the pellet discarded. The protein content was determined using the BCA protein assay kit (Pierce Chemical). Proteins were precipitated with 60% trichloroacetic acid (TCA) in acetone. After 2–3 acetone washes, proteins were dissolved in 500 μL of 2D sample buffer (5 M urea, 2 M thiourea, 2 mM tributyl-phosphine, 65 mM DTT, 65 mM CHAPS, 0.15 M NDSB-256, 1 mM sodium vanadate, 0.1 mM sodium fluoride, and 1 mM benzamidine). Ampholytes (Servalyte 4–7) were added to the sample to a final concentration of 1.6% (v/v).
4.6. Proteomic Studies
The primary neuron cell lysates were subjected to two-dimensional gel electrophoresis (2-DE). Protein quantitation was performed with the Coomassie plus protein reagent (Thermo Scientific, Asheville, NC). Five hundred micrograms of protein were loaded onto each gel to allow detection of low abundance proteins. Four gels per study group (DISC1 knock-down and control) were compared. Immobilized pH gradient (IPG) strips (4–7, 24 cm, GE Healthcare, Uppsala, Sweden) were rehydrated in the sample, and isoelectric focusing (IEF) was performed in a Multiphor (GE Healthcare) for 85 kVh at 17 °C. Following focusing, the IPG strips were immediately equilibrated for 15 min in 4 M urea, 2 M thiourea, 130 mM DTT, 50 mM Tris pH 6.8, 2% w/v SDS, 30% v/v glycerol. Later, the strips were placed for 15 min in the same buffer, in which DTT was replaced by 4.5% iodoacetamide (Sigma Aldrich). The IPG strips were placed on top of the second dimension gels and embedded with 0.5% melted agarose. Proteins were separated in the second dimension by SDS-polyacrylamide gel electrophoresis (PAGE) on 10% gels at run conditions of 10 °C, 20 mA per gel for 1 h, followed by 40 mA per gel for 4 h by using an Ettan Dalt 6 system (GE Healthcare). Following electrophoresis, gels were fixed in 10% methanol/7% acetic acid for 1 h, and stained overnight with Sypro Ruby fluorescent dye (Lonza, Switzerland). After staining, gels were washed for 1 h in 10% methanol/7% acetic acid, and scanned in a Typhoon 9410 (GE Healthcare).
4.7. Differential Image Analysis
Image analysis was performed with the Ludesi REDFIN 3 Solo software (Ludesi, Malmö, Sweden). The integrated intensity of each of the spots was measured, and the background corrected and normalized. Differential expression of proteins was defined on the basis of ≥2-fold change between group averages and p < 0.05.
4.8. Mass Spectrometric Analysis
Spots of interest were carefully excised and subjected to in-gel digestion with trypsin [45
]. Tryptic digests were analyzed using a 4800 MALDI-TOF/TOF analyzer (Applied Biosystems). Dried peptides were dissolved in 4 µL of 0.5% formic acid. Equal volumes (0.5 µL) of peptide and matrix solution, consisting of 3 mg alpha-cyano-4-hydroxycinnamic acid (α-CHCA) dissolved in 1 mL of 50% acetonitrile in 0.1% trifluoroacetic acid, were deposited using the thin layer method, onto a 384 Opti-TOF MALDI plate (Applied Biosystems). MS spectra were acquired in reflectron positive-ion mode with a Nd:YAG, 355 nm wavelength laser, averaging 1000 laser shots and using at least three trypsin autolysis peaks as internal calibration. All MS/MS spectra were performed by selecting the precursors with a relative resolution of 300 (FWHM) and metastable suppression. Automated analysis of mass data was achieved by using the 4000 Series Explorer Software V3.5. MS and MS/MS spectra data were combined through the GPS Explorer Software v3.6. Database search was performed with the Mascot v2.1 search tool (Matrix Science, London, UK) screening SwissProt (release 56.0). Searches were restricted to mouse taxonomy allowing carbamidomethyl cysteine as a fixed modification and oxidized methionine as potential variable modification. Both the precursor mass tolerance and the MS/MS tolerance were set at 30 ppm and 0.35 Da, respectively, allowing 1 missed tryptic cleavage site. All spectra and database results were manually inspected in detail using the above software. Protein scores greater than 56 were accepted as statistically significant (p
< 0.05), considering positive the identification when protein score CI (confidence interval) was above 98%. In case of MS/MS spectra, total ion score CI was above 95%.
4.9. SDS-PAGE and Western Blotting
A total of 50 µg of protein was mixed with Laemmli sample buffer (BioRad), heated at 100 °C for 10 min, spun, and the supernatant loaded on a 7.5% SDS-PAGE gel. Samples were subjected to electrophoresis and transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The conditions of the electrophoresis were 200 V, 1 h. Electrophoresis was performed using a Mini-PROTEAN 3 cell electrophoresis system (BioRad). The transfer was performed in a Trans-blot SD semi-dry transfer cell (BioRad) using the following conditions: 0.8 mA/cm2, 90 min. The PVDF membranes were blocked in 5% non-fat milk in PBS-0.1% Tween solution overnight at 4 °C, then 4 washes of 5 min with PBS-0.1% Tween20 were performed, and the membrane was incubated with the primary antiserum (in 5% BSA in PBS-0.1%Tween20) for 1 h at room temperature, washed again and incubated with the peroxidase-conjugated secondary antibody (in PBS-0.1% Tween20), and subjected to 4 washes of 5 min each with PBS-0.1% Tween20. Finally the membrane was incubated with the chemiluminescence solution Luminata Forte Western HRP substrate (Merck Millipore). To develop the membranes Hypercassette (GE Healthcare) and Amersham Hyperfilm ECL (GE Healthcare) were used.
4.10. Ingenuity Pathway
Ingenuity Pathway Analysis software (Ingenuity Systems, CA, USA) was used to investigate interactions between all the 48 identified proteins. Interactive pathways were generated to observe potential direct and indirect relations among the differentially expressed proteins. To test the enriched pathways we consider as settings direct and indirect relationships that were experimentally observed.
4.11. Neurite Outgrowth Assays
Stable SH-SY5Y cell lines generated using TRCN0000118997 (Silenced 1), TRCN0000119000 (Silenced 4) and non-target shRNAs were cultured for 7 and 14 days in medium containing 10 µM retinoic acid (RA) (Sigma Aldrich). To analyze neurite outgrowth, images of live cells were taken under a microscope and processed using Image J software (http://rsb.info.nih.gov/ij
). Cells with and without neurites longer than two cell bodies were counted in photomicrographs of the differentiated control and DISC1-silenced cells.
4.12. Immunocytochemistry of SH-SY5Y Cells
Retinoic acid-treated cells were fixed in paraformaldehyde and immunostained for β3-tubulin and nuclei were visualized using DAPI, as previously described by the authors of [46
4.13. Statistical Analysis
One-way ANOVA was employed in the proteomic analysis to determine statistically significant differences between groups of samples. For each spot ID, ANOVA p-value was calculated using the quantified and normalized spots volumes for the matched spot in each of the images. Differential expression of proteins was defined on the basis of ≥2-fold change between group averages and p < 0.05.
In the neurite outgrowth assay, three fields of up to 100 cells were analyzed for each condition and the experiment was performed twice. Statistical analysis was performed using a non-parametric unpaired Mann-Whitney U-test (two-tailed); results were considered significant with p < 0.05.