RSK1 promotes mammalian axon regeneration by inducing the synthesis of regeneration-related proteins
Fig 7
RSK1 promotes axon regeneration through BDNF and IGF1.
(A, B) RT-qPCR analysis of the expression of BDNF (A) or IGF1 (B) in neurons infected with AAV expressing control shRNA (Con-sh), RSK1-shRNA2 (RSK1-sh2), RSK1 (RSK1-OE), or empty AAV (Con-OE) for 7 days (N.S., not significant, mean ± SEM, 1-way ANOVA, Bonferroni post hoc test, n = 3 biologically independent experiments). (C, D) Quantification of secreted BDNF (C) or IGF1 (D) levels in the supernatant of neurons without AAV infection (M) or neurons infected with AAV expressing Con-sh, RSK1-sh2, RSK1-OE, or Con-OE for 7 days followed by replating for 16 hours as determined by ELISA (mean ± SEM, 1-way ANOVA, Bonferroni post hoc test, n = 5 biologically independent samples). (E) Timeline for neutralization antibody incubation of DRG neurons infected with RSK1-OE or Con-OE followed by replating. After replating, the neurons infected with AAV-Con-OE were not incubated with any antibody (Con), and the neurons infected with AAV-RSK1-OE were incubated with no antibody (R), antibody IgG (R+IgG), BDNF neutralizing antibody (R+B), IGF1 neutralizing antibody (R+I), or both BDNF and IGF1 neutralizing antibodies (R+B+I). Antibody details can be found in S8 Table. (F) Representative images of DRG neurons treated as in (E). Scale bar, 100 μm. (G) Quantification of the total and the longest neurite outgrowth per neuron relating to (F) (mean ± SEM, 1-way ANOVA, Tukey post hoc test, n = 4 biologically independent experiments, approximately 50 cells/experiment on average). The data underlying all the graphs shown in the figure are included in S1 Data. DRG, dorsal root ganglion; ELISA, enzyme-linked immunosorbent assay; OE, overexpression; RSK1, ribosomal S6 kinase 1; RT-qPCR, reverse transcription quantitative real-time PCR; SEM, standard error of the mean.