EGFR and ERK activation is required for sSORLA-dependent enhancement of neurite regeneration. EGFR inhibition abrogates EGF and sSORLA-dependent EGFR activation. A, Cultured cortical neurons were treated with EGF or sSORLA with or without the EGF inhibitor, AG-1478 as described in Materials and Methods. Lysates were then immunoblotted as indicated. B, pEGFR or pERK levels normalized to total EGFR or ERK levels in immunoblots from A were plotted compared with the untreated control (set to 1.0): left, control versus EGF (p = 0.0497), control versus sSORLA (p = 0.0315), sSORLA versus sSORLA/inhibitor (p = 0.0228), t test; right: F_(5,12) = 10.52, control versus sSORLA (p = 0.0047), EGF versus EGF/inhibitor (p = 0.0407), EGF versus inhibitor (p = 0.0335), sSORLA versus sSORLA/inhibitor (p = 0.0022), one-way ANOVA with Tukey's multiple comparisons. Characterizing the effects of EGFR inhibition on sSORLA-dependent enhancement of neurite regeneration. C, DIV6 cortical neurons were subjected to mechanical wound injury, and treated with EGF or sSORLA in the presence or absence of the EGF inhibitor AG-1478. After 16 h, neurons were fixed and stained for tubulin (green), F-actin (red), and with DAPI (blue). Scale bar, 60 μm. D, Images from C were quantified for neurite length from the wound border in all neurons (total neurite length, top) or averaged per image (average neurite length, bottom) (control, 201 neurites in 10 imaging fields; EGF, 150 neurites in 10 imaging fields; EGF with inhibitor, 166 neurites in 10 imaging fields; inhibitor, 156 neurites in 8 imaging fields; sSORLA, 177 neurites in 8 imaging fields; sSORLA with inhibitor, 169 neurites in 8 imaging fields; 3 coverslips from 2 independent dissections/3 cultures per treatment) (top: F_(5,1014) = 128.9; bottom: F_(5,48) = 69.98, control vs EGF, control vs sSORLA, EGF vs EGF/inhibitor, sSORLA vs sSORLA/inhibitor, p < 0.0001; bottom: one-way ANOVA with Tukey's multiple comparisons). E, WT DIV7 cortical neurons were treated with sSORLA (150 ng/ml), the MEK inhibitor PD98059 (100 μm), or sSORLA and PD98059 in combination for 16 h. Neuronal lysates were then subjected to immunoblot analysis as indicated, and pERK levels were quantified and normalized to total ERK (F_(3,16) = 11.62, control vs sSORLA, p = 0.0104; sSORLA vs PD98059, p = 0.0003; sSORLA vs sSORLA/PD98059, p = 0.0014, one-way ANOVA with Tukey's multiple comparisons). F, WT DIV7 cortical neurons were subjected to mechanical wounding, and exposed to sSORLA, PD98059, or sSORLA with PD98059 as in E. Neurons were fixed 16 h following wounding, stained for tubulin (green), F-actin (red), and DAPI (blue), and imaged by confocal microscopy. Scale bar, 20 μm. G, Images in F were quantified for total neurite length (from a minimum of 127 neurons/treatment; top) or averaged neurite length per imaged field (from a minimum of 9 fields/treatment; bottom): control, 180 neurites in 12 images, sSORLA, 140 neurites in 12 images; PD98059, 127 neurites in 11 images; sSORLA with PD98089, 134 neurites in 9 images; 3 coverslips from 2 independent dissections/3 cultures per treatment; top: F_(5,532) = 50.85, sSORLA versus control, sSORLA versus PD98059 and sSORLA versus sSORLA/PD98059 (p < 0.0001); bottom: F_(3,28) = 14, control versus sSORLA (p = 0.0008), sSORLA versus PD98059 and sSORLA versus sSORLA/PD98059 (p < 0.0001), one-way ANOVA with Tukey's multiple comparisons. B, E, Data are mean ± SE. D, G, Data are mean ± SD. Statistical significance: B, pERK/ERK, D, E, G, by One-Way ANOVA followed by Tukey's multiple comparisons; and B, pEGFR/EGFR, One-Way ANOVA (F_(5,12) = 4.625, p = 0.0139) was used in (B), where p-values were determined by Student's t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.