text: ERK signaling is involved in decreasing BDNF-induced gene activity via Eph receptors
  Data provided so far suggest that ephrin-A5 might counteract BDNF-mediated gene expression and growth cone responses via decreasing ERK activity and nuclear localization (Figs. 2 and 3). To test this hypothesis more directly, we analyzed whether Eph forward signaling can still suppress BDNF-stimulated IEG induction and growth cone collapse when ERK kinase activity was experimentally raised. For this, ERK activity was elevated by expressing constitutively-active MEK1 (CA-MEK1), an ERK upstream activator, in cortical neurons (Fig. S3). Subsequently, neurons were subjected to either an ephrin-A5 mediated growth cone collapse assay (Fig. 5) or stimulated with guidance cues for 20 minutes and IEG mRNA levels were quantified via qRT-PCR (Fig. 6).
  10.1371/journal.pone.0026089.g005 Figure 5
  Constitutively-active MEK1 blocks ephrin-A5 mediated growth cone collapse.
  Wild-type neurons were electroporated with vectors expressing GFP (control; A, C, E) or expressing a FLAG-tagged constitutively-active MEK1 (CA-MEK1; B, D, F). Neurons were treated with ephrin-A5-Fc for 30 min followed by visualization of GFP (A, C, E) or CA-MEK-1 (green in B, D, F), F-actin and betaIII tubulin. (A, B) Overexpression of CA-MEK1 (B) reduces mean neurite length compared to a control GFP-expressing neuron (A). (C-F) A control growth cone without ephrin-A5 application (C) typically protrudes multiple filopodia (arrow). CA-MEK-1 expressing growth cones (D) were indistinguishable from a GFP-expressing growth cone. Note that CA-MEK1 was expressed in a dot-like pattern in the growth cone (arrowheads in D and F). Upon ephrin-A5 application, a control growth cone (E) collapsed resulting in only few filopodia. In contrast, a CA-MEK1 expressing growth cone (F) was only partially collapsed and many filopodia structures were preserved after ephrin-A5 induction. (G) Quantification of average neurite length. (H, I) Quantification of growth cone area (H) and filopodia number/growth cone (I) in the four conditions. Ephrin-A5 induces a growth cone collapse, resulting in reduced growth cone area and filopodia number in GFP but not CA-MEK1 expressing neurons. Scale-bar (A, B) = 10 microm; (C-F) = 2 microm.
  10.1371/journal.pone.0026089.g006 Figure 6
  Ephrin-A5 suppresses BDNF-induced IEG responses via MAP kinase signaling.
  Wild-type cortical neurons, mock-electroporated (black bars) or with a vector expressing constitutively-active MEK1 (white bars), were treated with guidance cues for 20 minutes as depicted, followed by mRNA quantification. In mock-electroporated neurons, BDNF induced an IEG response of c-fos (A), Egr1 (B), Egr2 (C) and Arc (D). Co-application of ephrin-A5 and BDNF reduced mRNA levels of all four IEGs in mock-electroporated neurons (A-D). In contrast, expression of constitutively-active MEK1 prevented Eph forward signaling from suppressing BDNF-evoked IEG responses. This was most evident for the IEGs Egr1 (B) and Egr2 (C).
  First of all, we analyzed the impact of CA-MEK1 on neurite outgrowth and ephrin-A5 mediated growth cone responses (Fig. 5). The average neurite length of neurons overexpressing CA-MEK1 (Fig. 5B) was clearly reduced compared to control GFP expressing neurons (Fig. 5A; see quantification in (G)). Growth cones of GFP (C) and CA-MEK1 expressing neurons were similar and protruded many filopodia (arrows in Fig. 5C-F). CA-MEK1 expression in growth cones was confined to individual dot-like structures (arrowheads in Fig. 5D, F). Ephrin-A5 application on a GFP expressing control growth cone (Fig. 5E) resulted in a growth cone collapse as revealed by reduced overall growth cone area and filopodia number (quantified in Fig. 5H, I). In contrast, in neurons overexpressing CA-MEK1 (Fig. 5F), ephrin-A5 induced only a partial growth cone collapse. Thus, growth cone area and filopodia number were not reduced (see Fig. 5H, I). This result suggests that an ephrin-A5 induced signaling cascade targets MAP kinase signaling to exert a full growth cone collapse. This result is in contrast to a recent report [46] which excluded ERK signaling downstream of an ephrin-A5 mediated growth cone collapse.
  Next, we tested whether ephrin-A5 signaling suppresses ERK kinase signaling also to interfere with BDNF-mediated gene expression (Fig. 6). In agreement with previous results (Fig. 4), in mock-electroporated neurons, ephrin-A5 incubation reduced the BDNF-mediated up-regulation of c-fos (Fig. 6A), Egr1 (Fig. 6B), Egr2 (Fig. 6C) and Arc (Fig. 6D) mRNA. In contrast, in neurons over-expressing CA-MEK1, ephrin-A5 could not suppress BDNF-induced IEG responses to the same degree. Particularly Egr1 (Fig. 6B) and Egr2 (Fig. 6C) mRNA levels were almost identical when comparing neurons treated with BDNF alone and neurons with BDNF and ephrin-A5 together.
  Thus, ephrin-A5 represses gene activity elicited by BDNF at least in part via MAP kinases.
  Besides IEGs, we inspected the influence of both guidance cues on cytoskeletal genes, whose gene products might modulate cytoskeletal dynamics evoked by ephrin-As and/or neurotrophins (Fig. S4). Therefore, the filamentous actin (F-actin) stabilizing tropomyosins (Tpm1 and Tpm2), the F-actin cross-linker alpha-actinin 1 (Actn1) and the motor protein dynein light chain 1 (Dnal1) were analyzed. mRNA levels of other cytoskeletal genes were not altered by any of the guidance cues (i.e. cofilin, filamin A, mena, vinculin and smooth muscle actin; data not shown). Short-term stimulation with guidance cues did not result in major alterations of cytoskeletal mRNA levels except for upregulation of Tpm1 by ephrin-A5 (Fig. S4A). At 16h of incubation, both tropomyosin genes were induced by BDNF and ephrin-A5 alone and both together (Fig. S4B, D). Actn1 levels were induced by individual application of ephrin-A5 and BDNF and by co-application of both (Fig. S4F). A similar profile was observed for the dynein light chain (Fig. S4H).
  Taken together, cytoskeletal gene expression was modulated by ephrin-A5 and BDNF. In contrast to the antagonistic impact on the IEG gene response (Figs. 4 and 6), ephrin-A5 and BDNF resulted in rather synergistic action on cytoskeletal gene expression. This difference might be due to short-term (i.e. 20 minutes) vs. long-term (i.e. 16h) exposure of both guidance cues in the IEG and cytoskeletal gene response, respectively.
  
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