PMC:3191169 / 5958-16239 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/3191169","sourcedb":"PMC","sourceid":"3191169","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3191169","text":"Materials and Methods\n\nAnimals\nSrf (flex1neo/flex1neo) and Camk2α-iCre mice were bred to obtain Srf mutants (Srf–/–; Camk2a-iCre) or control littermates (Srf –/–, Srf +/– or Srf +/–; Camk2α-iCre [35], [36]). Recombination induced by Cre recombinase expression via the Camk2α promoter starts just before birth and results in strong SRF down-regulation at time-points used to culture primary neurons [35], [36]. EphA7 mice were kindly provided by U. Drescher (King's College, London, UK; see [15]). Animal experiments and housing were approved by the local ethics committee (Einrichtung für Tierschutz, Tierärztlichen Dienst und Labortierkunde, Calwer Straße 7/4, 72076 Tübingen, Tübingen University; permit number: §4 Anzeige 15/10/2009).\n\nNeuronal cell culture\nP1 hippocampal or E17.5 cortical cultures were incubated in NMEM/B27 medium as described previously [36]. In brief, neurons (5×103–104) were cultured on poly-L-lysine (100 µg/ml; Sigma) and laminin (20 µg/ml; Gibco) coated coverslips (13 mm). Ephrin-A5-Fc (R\u0026D systems) or Fc alone (Sigma) was applied to the culture medium at 1 µg/ml, both pre-clustered with 10 µg/ml anti-human IgG Fc-specific (Sigma) for 30 mins at 37°C. BDNF was applied at 10 ng/ml (Fig. 1). Stimulation was performed for 45 min at 37°C (Fig. 1). For experiments with U-0126 (Cell Signaling), cells were pre-incubated with U-0126 at 10 µM for 1h before application of BDNF. For biochemistry (Fig. 2) and qRT-PCR, cortical neurons (approx. 106 cells/35 mm dish coated with poly-L-lysine at 10 µg/ml) were cultured for 3 div (days in vitro) prior to stimulation (see below). We used nucleofection (Amaxa, Cologne, Germany) to deliver a constitutively-active mutant of MEK1 [44] into cortical or hippocampal neurons.\n10.1371/journal.pone.0026089.g001 Figure 1 Ephrin-A5 treatment blocks BDNF-induced growth cone motility in an SRF-dependent manner.\nWild-type and SRF-deficient hippocampal neurons were stimulated with ephrin-A5 and/or BDNF as indicated for 45 min, followed by F-actin (red) and microtubule (green) staining. Individual growth cones are highlighted with arrows. (A-D) Wild-type neurons protruded multiple growth cones elaborating finger-like filopodia (A, higher magnification B). In Srf mutants (C, D), growth cones were rounded without any obvious filopodia. (E-H) BDNF enhanced growth cone area and filopodia number in wild-type neurons (E, F). BDNF required SRF gene activity to modulate filopodia number, as revealed by Srf mutant neurons remaining unaltered by BDNF treatment (G, H). (I–L) Ephrin-A5 induced growth cone collapse decreasing filopodia number and growth cone area (I, J). In SRF-deficient growth cones, ephrin-A5 application resulted in ring-like structures consisting of F-actin and microtubules (K, L). (M-P) Co-application of ephrin-A5 with BDNF in wild-type neurons counteracted BDNF-stimulated growth cone motility (M, N). Now, growth cones were collapsed (M, N) rather than increased in area as induced by BDNF alone (see E, F). In SRF-deficient neurons (O, P), growth cones protruded ring-like structures as seen with ephrin-A5 alone (K, L). (Q) Quantification of filopodia number/growth cone for the various treatments and genotypes. Statistical significance was calculated relative to wild-type/control condition. (R) The average growth cone area (wild-type and control condition set to 100%) was quantified. (S, T) Upon inhibition of MEK by U-0126, BDNF fails to increase filopodia number (S) and growth cone area (T). Scale-bar (A, C, E, G, I, K, M, O)  =  10 µm; (B, D, F, H, J, L, N, P)  =  2 µm.\n10.1371/journal.pone.0026089.g002 Figure 2 Ephrin-A5 suppresses ERK activation and inhibits BDNF-evoked ERK phosphorylation.\n(A) Cortical neurons were stimulated with ephrin-A5 for time-points indicated. EphA receptor activation (assessed by P-EphA3) suppressed ERK phosphorylation. At later time points (120–240 mins), EphA receptors were less active and ERK phosphorylation returned to control levels (“–”, untreated). Quantification of results depicts P-ERK levels relative to control treatment (set to1). (B) BDNF alone elevated P-ERK levels, whereas ephrin-A5 alone inhibited ERK phosphorylation. Ephrin-A5 inhibited BDNF-induced elevation of P-ERK. Neither EphA nor TrkB receptor activation was influenced by co-application of both substances. (C) Eph forward signaling requires phosphatases to suppress ERK phosphorylation. Neurons were pre-treated with phosphatase inhibitors (OA, ocadaic acid; SV, sodium vanadate; mix, commercially available phosphatase inhibitor mix). In the presence of either phosphatase inhibitor, ephrin-A5 failed to inhibit ERK phosphorylation. Quantification depicts P-ERK levels relative to control (without treatment and inhibitor set to 1) for OA and SV. (D) Ephrin-A5 does reduce BDNF-stimulated P-ERK levels in the presence of phosphatase inhibitors.\n\nBiochemistry\nCortical cultures were stimulated with 1 µg/ml clustered (see above) ephrin-A5-Fc and/or BDNF (10 ng/ml) in NMEM/B27 medium for the indicated time periods. Phosphatase inhibitors were applied for 15 mins prior to ephrin-A5-Fc and/or BDNF stimulation: sodium vanadate (200 µM; Sigma), okadaic acid (200 nM; Calbiochem), PhosStop (1x; Roche). Cells were lysed in 100 mM Tris pH 7.8, 150 mM NaCl, 1 mM EDTA, 1% Triton-X-100, 0.1% SDS and protease inhibitors (Roche). Samples were resolved on 10-12% SDS-PAGE, followed by transfer on PVDF membranes (Amersham). After blocking, first antibodies were applied overnight at 4°C: rabbit anti-ERK (1∶1000; Cell Signaling), rabbit anti-P-ERK (1∶1000; Cell Signaling), mouse anti-GAPDH (1∶50000), rabbit anti-TrkB (1∶1000; Santa Cruz), rabbit anti-P-TrkB (1∶1000; Cell Signaling), mouse anti-EphA4 (1∶750; BD Transduction Laboratories), P-EphA3 (1∶10000, gift of Dr. M. Greenberg, Harvard Medical School, Boston). Detection of first antibodies involved horseradish-peroxidase conjugated secondary antibodies (1∶5000) and the ECL Western Blotting Substrate (Pierce).\n\nQuantitative real-time PCR (qRT-PCR)\nCultures were stimulated with 2.5 ng/ml BDNF and/or 1 µg/ml pre-clustered ephrin-A5-Fc (see above). Total RNA was isolated with the RNeasy kit (Qiagen). Reverse transcription was performed with 1 µg RNA using reverse transcriptase (Promega) and random hexamers. qRT-PCR was performed on ABI PRISM 7700 Sequence Detector with the Power PCR SYBR green PCR master mix (Applied Biosystems). Expression was determined in relation to Gapdh RNA levels. Primer sequences can be obtained upon request.\n\nImmunocytochemistry\nCells were fixed for 15 minutes in 4% PFA/5% Sucrose/PBS, permeabilized for 5 minutes in 0.1% Triton-X-100/PBS and blocked for 30 minutes in 2% BSA/PBS. Primary antibodies were incubated overnight at 4°C as follows: mouse α-ß-tubulin (1∶5000; Sigma), mouse α-class III ß-tubulin (1∶1000; Covance), rabbit anti-ERK (1∶300; Cell Signaling), rabbit anti-P-ERK (1∶300; Cell Signaling). First antibodies were detected with Alexa 488 or 546-conjugated secondary antibodies (1∶1000; Molecular Probes). Cells were stained for F-actin with Texas Red-X Phalloidin (1∶100; Molecular Probes).\nFor visualization of P-ERK and Eph receptors, cultures were incubated with 1 µg/ml pre-clustered ephrin-A5 for 20 minutes, followed by fixation with 4%PFA/sucrose for 15 minutes. Ephrin-A5 bound to Eph receptors was visualized with anti-Fc Cy3 conjugated antibodies (1∶300 in block). Subsequently, P-ERK staining was performed as indicated above.\n\nImage acquisition and statistics\nPictures were acquired on a Zeiss Axiovert 200M or Zeiss LSM confocal microscope using an Axiocam camera and Axiovision software. We used 10x, 20x and 63x Zeiss objective lenses with numerical apertures of 0.3, 0.8 and 1.3 (oil), respectively. Pictures were further processed using Photoshop software (Adobe).\nFor data in Fig. 1 and Fig. S1, four independent experiments were performed. In each experiment 20 neurons/condition were analyzed. Filopodia number (\u003e1 µm) and growth cone surface was determined for all growth cones/neuron using Axiovision software. For quantification of neurite number and branches, only neurites of more than 30 µm and branches of more than 2 µm were included. In Fig. 2, three independent cultures/condition were analyzed using Image Quant software (Molecular Dynamics). To evaluate ERK nuclear translocation (Fig. 3), four independent experiments including 25 neurons/experiment/condition were analyzed. A ratio of cytoplasmic vs. nuclear P-ERK intensity was determined using Axiovision software. In qRT-PCR, RNA of at least three independent cultures/condition was harvested.\n10.1371/journal.pone.0026089.g003 Figure 3 Eph forward signaling prevents nuclear ERK translocation.\nWild-type hippocampal neurons were treated with guidance cues as indicated, followed by staining for P-ERK (green), DAPI (blue) and F-actin (red). Arrows point at the nucleus. (A-D) In untreated neurons, nuclear P-ERK levels were low. (E–H) BDNF enhanced P-ERK levels, which accumulated in and around the nucleus. (I–L) Ephrin-A5 did not increase nuclear P-ERK accumulation. However, P-ERK positive clusters were observed in the neurite (arrowhead in I). (M-P) Ephrin-A5, when applied along with BDNF, antagonized nuclear P-ERK entry evoked by BDNF. As with ephrin-A5 (I), P-ERK localized to neurites (arrowheads in M). (Q) Quantification of nuclear P-ERK. The ratio of P-ERK in the nucleus vs. cytosol was plotted against the three time-points of stimulation. Values of ratios for untreated neurons were set to 1. Statistical significance is provided relative to control (untreated) values. (R-U) Total ERK levels remained constant throughout the various treatments. (V) Hippocampal neurons stained for P-ERK localization. Ephrin-A5 stimulation induces clusters of P-ERK in neurites. (W) All Eph receptors bound by ephrin-A5 were labeled. Typically, Eph receptors were found in clusters localized to the neurites. (X) A merged image of (V) and (W), revealing co-localization of P-ERK and Eph receptors in hippocampal neurites. Scale-bar (A-P, R-X)  =  10 µm. Statistical significance was assessed by using a two-tailed t-test (Excel software) with *, **, *** representing P≤0.05, 0.01, 0.001. 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