Aβ-induced deficits in axonal transport depend on GSK3β. (A–C) On DIV 10, neuronal cultures from wild-type mice were treated with nontargeting (NT) siRNA or siRNAs (siRNA-1 or siRNA-2) targeting GSK3β mRNA. GSK3β protein levels were determined by Western blotting on DIV 14. (A) Representative Western blot from a single gel that was scanned and digitally arranged. (B) Quantitation of Western blot signals. (C) mRNA levels were determined by RT-qPCR in replicate cultures on DIV 13. n = 3–12 samples per condition. ***, P < 0.001 versus no treatment (Dunnett’s test). (D) Primary hippocampal neurons from wild-type mice were treated with NT siRNA or anti-GSK3β siRNA on DIV 9–10. On DIV 13–14, the percentage of moving mitochondria in the axons was measured before (baseline) and during treatment with Aβ_1–42. n = 20–27 axons per group from three to four independent sessions. #, P < 0.05 (Kruskal-Wallis ANOVA, Dunn’s test); ***, P < 0.001 versus corresponding baseline (paired t test, Bonferroni). (E) Primary hippocampal neurons from wild-type mice were treated with the selective GSK-3 inhibitor SB 415286 (10 µM) or vehicle (Veh), followed by exposure to Aβ_1–42 oligomers. The percentage of moving mitochondria in the axons of Tau^+/+ neurons was measured before (baseline) and during these treatments. n = 23–27 axons per group from four independent sessions at DIV 13–14. #, P < 0.01 (Mann-Whitney rank-sum test); ***, P < 0.001 versus corresponding baseline (repeated measures ANOVA, Dunnett’s test). Data are means ± SEM.