Transfection with wild-type mouse tau makes primary hippocampal neurons from Tau^−/− mice susceptible to Aβ-induced deficits in axonal transport. (A) Neuronal cultures from Tau^−/− mice were transfected with a cDNA encoding Tau-WT at DIV 6, fixed the next day, and immunostained for total tau (EP2456Y, white) or colabeled for the dendritic marker MAP2 (red) and for phosphorylated (PHF-1) or dephosphorylated (Tau-1) tau (green [grn]) in axons (left). To compare tau expression in transfected Tau^−/− neurons with the expression of endogenous tau in Tau^+/+ neurons, a small number of Tau^+/+ neurons was co-cultured with untransfected Tau^−/− neurons, followed by fixation on DIV 14 and immunostaining as in the left panels (middle). Cultures of untransfected Tau^−/− neurons served as a negative control (right). (B–E) The intensity of immunostaining for total tau in the axon (B), total tau in the soma (C), and phosphorylated (D) and dephosphorylated (E) tau in the axon was compared by fluorescence microscopy in Tau^+/+ neurons co-cultured with Tau^−/− neurons (endogenous [Endog] tau) and in Tau^−/− neurons transfected (Transf) with mouse tau. n = 109–144 (B and C) and n = 36–71 (D and E) neurons per group. *, P < 0.05; ***, P < 0.001 (Mann-Whitney rank-sum test). (F) To measure axonal transport in live cells, Tau^−/− neurons were transfected with EGFP (green), a mitochondrial marker (mito-RFP; red), and mouse tau. Tau was detected by immunostaining with the EP2456Y antibody (blue) after fixation. All images in F were taken after fixation. (G) The percentage of moving mitochondria in the axons of Tau^−/− neurons transfected with empty plasmid or a plasmid encoding Tau-WT was measured before (baseline) and 10–60 min after adding Aβ_1–42 oligomers to the medium. n = 37 axons per group recorded during three to five independent sessions at DIV 7–8. ***, P < 0.001 versus corresponding baseline (paired t test, Bonferroni). Bars, 20 µm. Plots in B–E show medians, quartiles, and ranges. Data in G are means ± SEM.