Figure 6: Activation of the JNK or Wnt pathways rescues detrimental Nrf2-dependent alterations in neuronal maturation.

(a–f) Expression of Nrf2 occludes the deleterious effects of DN-βCat or Rac-N17 on neuronal development. Neurons were transfected on DIV4 with the indicated plasmids, plus GFP to visualize the cells, and studied at DIV7. Where used, quantities of DN-βCat, Rac-N17 and Nrf2 were always kept constant, and control plasmid used to ensure that total quantity of transfected DNA was also constant. (a,d) Sholl analysis of neurons transfected as indicated. *P<0.05, two-way analysis of variance (ANOVA), plus Tukey’s post-hoc test: *indicates difference between equivalent ‘Con’ and ‘Nrf2’ conditions. 15–24 cells analysed within n=3 independent experiments. Note that the same ‘Con’ and ‘Nrf2’ conditions are shown for a and d because the DN-βCat and Rac-N17 experiments were performed at the same time, with the data separated for easier visualization. (b,c,e,f) Dendrite length (b,e) and branch tip number (c,f) were calculated in neurons transfected with the indicated plasmids. *P<0.05, two-way ANOVA with Tukey’s post-hoc test; 15–24 cells analysed per condition within n=3 experiments. (g–r) Activation of Wnt, JNK or Rac signalling rescues the detrimental impact of Nrf2 expression on neuronal morphology. DIV4 neurons were transfected with the indicated plasmids and Sholl, dendritic length and branch tip analysis performed as previously. (g,k,o) Sholl analysis: *P<0.05, two-way ANOVA with Bonferronni’s post-hoc test. *A significant rescue, comparing the Nrf2 only condition to Nrf2+βCatS33Y or RacQ61L, or JNK1a (n=4, 32–36 cells analysed in total per condition). (h,i,l,m,p,q) *P<0.05, one-way ANOVA with Dunnett’s post-hoc test (n=4, 32–36 cells analysed in total per condition). (j,n,r) Example images of neurons analysed. Scale bar, 50 μm.