Differential ability of tau truncation mutants to reconstitute susceptibility to Aβ-induced axonal transport deficits in Tau^−/− neurons. (A) Schematic of full-length Tau-WT and truncation constructs generated. N-PRB, N terminus plus proline-rich and basic domain; RD-C, repeat domain and C terminus; RD, repeat domain alone; noRD, tau lacking the repeat domain. Numbers indicate amino acid positions in mouse 0N4R tau and numbers in parentheses are the corresponding amino acid positions in human 2N4R tau. (B) In the axons of Tau^−/− neurons transfected with plasmids encoding the indicated tau constructs, tau–tubulin binding was measured with the proximity ligation assay (PLA). Antibody combinations with source species in parentheses are indicated below each set of panels. Bar, 20 µm. (C) Quantification of the total proximity ligation assay signal (B, bottom), indicating the amount of tau that closely interacts with tubulin. A tau construct containing eight repeat domains (Tau-8RD), which has a higher tubulin binding affinity than wild-type tau (Preuss et al., 1997), was used as a positive control, and a no-primary tau antibody condition was used as a negative control. n = 37–256 axons per group. *, P < 0.05; **, P < 0.01 versus Tau-WT or for pairwise comparisons as indicated by brackets (Kruskal-Wallis ANOVA, Dunn’s test). (D) The percentage of moving mitochondria in the axons of Tau^−/− neurons transfected with empty plasmid or plasmids encoding the indicated tau constructs was measured before (baseline) and 10–60 min after adding Aβ_1–42 oligomers to the medium. Results are expressed relative to baseline (100%). n = 28–55 axons per construct recorded during three to five independent sessions at DIV 7–8 d. **, P < 0.01; ***, P < 0.001 versus corresponding baseline (paired t tests, Bonferroni); #, P < 0.05; ##, P < 0.01 (Kruskal-Wallis ANOVA, Dunn’s test). Data in C are medians and quartiles, and data in D are means ± SEM.