NRG1 treatment to microglial cells induced phosphorylation of ERK1/2 and Akt without activating p38MAPK. a and b: Addition of NRG1 (10 nM) to resting microglial cells induced the phosphorylation of ERK1/2 as assessed by Western Blots. A representative membrane for one experiment is shown in a. In b we show the time-course of ERK1 (black bars) and ERK2 (grey bars) phosphorylation after NRG1 treatment (ratio of phospho-ERK over total ERK). There was a significant increase in ERK1 and 2 phosphorylation after 60 min of NRG1 treatment compared with resting state or control (ERK1: control versus 60 min NRG1 treatment P = 0.02 one-way ANOVA on ranks, ERK2: control versus 60 min NRG1 treatment P = 0.003 one-way ANOVA on ranks, n = 4). c and d: In the same way we assessed Akt phosphorylation after NRG1 treatment. In (c) a representative membrane of one experiment is shown. In (d) we show the time-course of Akt phosphorylation (ratio of phospho-Akt over total Akt) after NRG1 treatment of three independent experiments. There was a significant increase in Akt phosphorylation after 60 min of NRG1 treatment compared with resting state or control (P = 0.002, one-way ANOVA, Bonferroni post hoc test). e and f: NRG1 treatment in microglia did not elicit phosphorylation of p38MAPK (P = 0.7, t-test, n = 3). In e we show the time-course after NRG1 treatment where no phosphorylation of p38MAPK was seen. We tested if NRG1 could enhance p38MAPK phosphorylation of LPS treated microglia but could not observe any increase in phospho-p38 when treating LPS primed microglia with NRG1. In (f) a representative membrane is shown, in (g) we show quantification of phospho-p38 over p38 of 3 independent experiments (NRG1 vs. LPS or LPS+NRG1 P = 0.003, LPS vs. LPS+NRG1 P = 0.5, one-way ANOVA, Bonferroni post hoc test, n = 3). In h–j we show that only the MEK inhibitor U0126 could block ERK1 and 2 phosphorylation (h) and phosphorylation of ERK remained the same when cells were treated with the PI3K inhibitor Wortmannin (i). Quantification of three independent experiments is shown in j (for ERK 1 and 2 the phospho/total ratio was significantly different between NRG1 treated cells compared with NRG1 plus U0126 P < 0.05 but not between NRG1 and NRG1 plus Wortmannin, one-way ANOVA on rank, Dunn's Method). In k–m we show that only Wortmannin could block Akt phosphorylation (k) and U0126 did not affect Akt activation (l). Quantification of three independent experiments is shown in m (for Akt the phospho/total ratio was significantly different between NRG1 with NRG1 plus Wortmannin P = 0.04, but not between NRG1 and NRG1 plus U0126, ANOVA on Ranks, Dunn's method) *P < 0.05 Wort = Wortmannin. Error bars represent ± SEM.

NRG1 effect on microglial proliferation was dependant on MEK/ERK1/2 pathway. Proliferation was assessed by incubating microglia in medium supplemented with 5% FBS for 3 days and pulse-labeling for 16 hours with BrdU 10 μM. Microglia, were fixed and stained (Iba1 to label microglial cells in red, DAPI to label nuclei in blue, and BrdU to label proliferating nuclei in yellow). NRG1 10 nM treatment significantly increased the proportion of BrdU-positive microglial nuclei compared with control (a and b). This effect of NRG1 was significantly inhibited when cells were treated with the MEK inhibitor U0126 (d) but not when cells were treated with the PI3K inhibitor Wortmannin (c). In (e) we show quantification of four independent experiments. (NRG1 treatment compared with NRG1 plus U0126 P < 0.001, NRG1 compared with NRG1 plus Wortmannin P = 0.547, one-way ANOVA, Bonferroni post hoc test). Note that both inhibitors by their own did not change baseline levels of proliferation. Wort = Wortmannin. Scale bar: 50 μm. Error bars represent ± SEM.

NRG1 effect on microglial survival was dependant on PI3K/Akt and MEK/ERK pathways. Microglial survival was assessed by incubating the cells in “starving conditions” (i.e. medium without FBS) for 3 days. Microglia, were fixed and stained (Iba1 to label microglial cells in red and DAPI to label nuclei in blue). NRG1 10 nM treatment significantly increased the number of cells per well compared with control (a and b). This effect of NRG1 was significantly inhibited when cells were treated with the PI3K inhibitor Wortmannin (c) and with the MEK inhibitor U0126 (d). In (e) we show quantification of five independent experiments. (NRG1 vs. NRG1 + Wortmannin P < 0.001, NRG1 vs. NRG1 + U0126 P = 0.01, one-way ANOVA, Bonferroni post hoc test). Note that both inhibitors by their own did not change baseline levels of survival. Wort = Wortmannin. Scale bar: 100 μm. Error bars represent ± SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

NRG1 effect on microglial chemotaxis was dependant on PI3K/Akt pathway. The effects of NRG1 on microglial migration were studied using a Boyden chamber. The addition of NRG1 to the lower well of the chamber (b) increased microglial migration to the inner membrane surface compared with control (a) This effect of NRG1 was significantly inhibited when cells were pretreated for 1 hour with the PI3K inhibitor Wortmannin (c) but not when cells were pretreated with the MEK inhibitor U0126 (d). In e we show quantification of four independent experiments. (NRG1 treatment compared with NRG1 plus Wortmannin P < 0.001, NRG1 treatment compared with NRG1 plus U0126 P = 0.8, one-way ANOVA, Bonferroni post hoc test). Note that both inhibitors by their own did not change baseline levels of chemotaxis. Wort = Wortmannin. Scale bar: 100 μm. Error bars represent ± SEM. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Intrathecal administration of NRG1 for 4 consecutive days induced ERK1/2 phosphorylation in spinal microglia of naive animals. Representative sections are shown of control (saline in a–c) and NRG1 (in d–f) immunostained with an antibody that recognises the phosphorylated form of ERK1/2 (green) and Iba1 (red). In (g) we show quantification of the percentage of microglia (Iba1 positive cells) that were phospho-ERK positive. A significant increase in microglia presenting phospho-ERK was found when comparing control vs. NRG1 4 ng (P < 0.05, one-way ANOVA on ranks). We confirmed these results using WB. Phosphorylation of ERK 1 and 2 was significantly increased after NRG1 intrathecal injections (h). In (i) we showed the quantification of the ratio of phosphorylated to total ERK 1 and 2 (control vs. NRG1 ERK1 P = 0.02, ERK2 P < 0.001, t-test, n = 4 per group) **P < 0.001; *P < 0.05. Scale bar: 50 μm. Error bars represent ± SEM.

Inhibition of MEK1/2 could reverse NRG1-induced microgliosis and mechanical and cold pain-related hypersensitivity. NRG1 was administered intrathecally (4 ng given daily for 3 days) together with the MEK inhibitor U0126 (10 μg) or the inactive analogue U0124 (10μg). a and b: Dorsal horn of animals treated with NRG1 + U0124 (in a) or NRG1 + U0126 (in b) immunostained with Iba1. Note that U0126 but not U0124 could prevent NRG1 associated increase in numbers of microglia with an effector morphology. In c quantification of this response is shown. In d–k we assessed proliferation (pulse labeling with BrdU) after injections (Iba1 is shown in red, DAPI in blue, BrdU in yellow and in the last panel merged images are shown). d–g: Dorsal horn microglia from a NRG1 + U0124 treated animal. h–k: Dorsal horn microglia from a NRG1 + U0126 treated animal. In (l) quantification of all BrdU-positive microglia in the dorsal horn is shown. Again, U0126 but not U0124 prevented NRG1 induced increase in microglial proliferation. Mechanical (shown in m) and cold (shown in n) pain related hypersensitivity developed after NRG1 injections which were reversed by U0126 but not by U0124. Scale bars: a and b: 100 μm, d–k: 50 μm. *P <0.05, **P < 0.001. Error bars represent ± SEM.

Blocking NRG1-erbB signaling after L5 SNL reduced ERK1/2 phosphorylation in spinal cord microglia. Using immunohistochemistry (Iba1 in red, phospho-ERK in green) we observed that ERK was phosphorylated in spinal microglia following SNL. In a–c, a representative section of the ipsilateral dorsal horn of a vehicle treated animal is shown. In d–f, a representative section of an animal treated with PD168393 (erbB receptor inhibitor) is shown. Note the reduction of phospho-ERK positive microglia when this inhibitor is administered. We also used a NRG1 sequestering molecule (HBD-S-H4) that was injected intrathecally following L5 SNL and found similar results. In (g) we show the quantification of the percentages of phospho-ERK positive microglia in both experiments (vehicle vs. erbB inhibitor P < 0.001, t-test, n = 4; control vs. NRG1 antagonist P = 0.006, t-test, n = 4). To confirm these results we used WB analysis of the ipsilateral dorsal horn of animals treated with the erbB inhibitor. A representative blot is shown in (h) in where an increase in phosphorylated ERK1/2 is seen after nerve injury which was greatly decreased when using the erbB receptor inhibitor. In (i) we show quantification of the WB of four animals per group (vehicle vs. inhibitor: ERK1 and 2 P = 0.04, one-way ANOVA on ranks, Bonferroni post hoc) *P < 0.05, **P < 0.001. Scale bar: 50 μm. Error bars represent ± SEM. VEH: vehicle, INH: erbB2 inhibitor (PD168393), ANT: HBD-S-H4.

Microgliosis and mechanical and cold hypersensitivity following L5 SNL were significantly reduced by blocking MEK1/2 pathways. Animals underwent L5 SNL and were injected intrathecally daily with a 10 μg dose of the MEK inhibitor U0126 or its inactive analogue U0124. Three days after SNL animals treated with the inactive analogue U0124 demonstrated a dramatic increase in the number of dorsal horn microglia showing an effector morphology compared with naïve animals injected with saline. This microgliosis was significantly reduced when animals received the MEK inhibitor U0126. In (a) (SNL + U0124) and (b) (SNL + U0126) we show representative sections of the L5 spinal cord of these animals stained with Iba1 to label microglia. In (c) we show the quantification (n = 4 per group, P < 0.001 SNL + U0124 vs. SNL + U0126, one-way ANOVA, Bonferroni post hoc test). Similarly, increased microglial proliferation was found in SNL animals that were treated with the inactive MEK inhibitor and it was greatly reduced (by more than 70%) in animals treated with U0126. In d–k, we show representative section of L5 spinal cord immunostained for Iba1 (red), BrdU (yellow), and DAPI (blue). Panels d–g show an inactive analogue (U0124) treated animal and panels h–k show a MEK inhibitor treated animal, both 3 days after L5 SNL. Quantification of BrdU-positive nuclei in the dorsal horn is shown in (l) (n = 4 per group, P < 0.001 SNL + U0124 vs. SNL + U0126, one-way ANOVA, Bonferroni post hoc test). Mechanical (shown in m) and cold (shown in n) pain related hypersensitivity developed after L5 SNL which were partially prevented by U0126 but not by U0124 (**P < 0.001, *P < 0.05, RM two-way ANOVA, Bonferroni post hoc test, n = 7–8 for m and n). Scale bars, a and b 100 μm, c–j 25 μm.