Fig. 7
mTOR-independent activator prevents neurite retraction and clears protein accumulation in OPTN(E50K) hPSC-RGCs. A Representative images of soma area in wild-type, OPTN(E50K), and trehalose-treated OPTN(E50K) hPSC-RGCs. Scale bar 25 μm. B–D Representative neurite tracing images of wild-type, OPTN(E50K), and trehalose-treated OPTN(E50K) hPSC-RGCs. Scale bar: 200 μm. E–H Quantitative analysis of neurite parameters demonstrated protection of neurites in OPTN(E50K) hPSC-RGCs after trehalose treatment for 2 weeks, as measured by the soma area (n = 3 biological replicates using WT n = 30, E50K n = 30, and E50K-trehalose n = 30 technical replicates, One-way ANOVA, Tukey post hoc test. ****p < 0.0001, ns = not significant, p > 0.05) E, number of primary neurites (n = 3 biological replicates using WT n = 15, E50K n = 15, and E50K-trehalose n = 15 technical replicates; One-way ANOVA, Tukey post hoc test. ****p < 0.0001, ***p < 0.001, ns = not significant, p > 0.05) F, total neurite length (n = 3 biological replicates using WT n = 15, E50K n = 15, and E50K-trehalose n = 15 technical replicates; One-way ANOVA, Tukey post hoc test. **p < 0.01, ns = not significant, p > 0.05) G, and Sholl analysis (n = 3 biological replicates using WT n = 15, E50K n = 15, and E50K-trehalose n = 15 technical replicates; One-way ANOVA, Tukey post hoc test. WT vs E50K: **p = 0.0095; WT vs E50K(trehalose): ns = not significant, p = 0.81; E50K vs E50K(trehalose): **p = 0.0012) H. I–N Immunostaining displayed the decrease of OPTN puncta in OPTN(E50K) hPSC-RGCs after trehalose treatment. Scale bar: 10 μm. O, P Quantification of OPTN puncta in hPSC-RGCs (n = 3 biological replicates using WT n = 51, E50K n = 60, and E50K-trehalose n = 61 technical replicates; One-way ANOVA, Tukey post hoc test. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = not significant, p > 0.05). Q–T Western blot and the relative protein expression demonstrated the recovery of changes to LC3-II and pS6Ser240/244 expression in OPTN(E50K) hPSC-RGCs after trehalose treatment. (n = 5 for each WT, E50K, and E50K with trehalose treatment; One-way ANOVA, Tukey post hoc test. LC3-I: WT vs E50K: p = 0.41, WT vs E50K(trehalose): p = 0.91, E50K vs E50K(trehalose): p = 0.23. LC3-II: WT vs E50K: p = 0.03, WT vs E50K(trehalose): p = 0.99, E50K vs E50K(trehalose): p = 0.03. pS6: WT vs E50K: p = 0.048, WT vs E50K(trehalose): p = 0.95, E50K vs E50K(trehalose): p = 0.029.). Data are all represented as mean values ± SEM