Tau reduction prevents Aβ-induced axonal transport deficits by blocking activation of GSK3β - PubMed (original) (raw)
Tau reduction prevents Aβ-induced axonal transport deficits by blocking activation of GSK3β
Keith A Vossel et al. J Cell Biol. 2015.
Abstract
Axonal transport deficits in Alzheimer's disease (AD) are attributed to amyloid β (Aβ) peptides and pathological forms of the microtubule-associated protein tau. Genetic ablation of tau prevents neuronal overexcitation and axonal transport deficits caused by recombinant Aβ oligomers. Relevance of these findings to naturally secreted Aβ and mechanisms underlying tau's enabling effect are unknown. Here we demonstrate deficits in anterograde axonal transport of mitochondria in primary neurons from transgenic mice expressing familial AD-linked forms of human amyloid precursor protein. We show that these deficits depend on Aβ1-42 production and are prevented by tau reduction. The copathogenic effect of tau did not depend on its microtubule binding, interactions with Fyn, or potential role in neuronal development. Inhibition of neuronal activity, N-methyl-d-aspartate receptor function, or glycogen synthase kinase 3β (GSK3β) activity or expression also abolished Aβ-induced transport deficits. Tau ablation prevented Aβ-induced GSK3β activation. Thus, tau allows Aβ oligomers to inhibit axonal transport through activation of GSK3β, possibly by facilitating aberrant neuronal activity.
© 2015 Vossel et al.
Figures
Figure 1.
Tau ablation, γ-secretase modulation, and NMDAR blockade each ameliorates deficits in anterograde axonal transport of mitochondria in Aβ-producing primary hippocampal neurons from hAPP-J20 mice. (A and B) Anterograde (A) and retrograde (B) axonal transport in neurons from mice of the indicated genotypes. n = 25–51 axons from three to five mice and three to six independent sessions for each genotype at DIV 10–14. ***, P < 0.001 versus Tau+/+ or as indicated by bracket (Dunnett’s test). (C) Levels of Aβ1-x and Aβ1–42 in the medium, measured by ELISA, were roughly equivalent to 4 and 0.55 nM of Aβ monomer, respectively. n = 4–9 wells from three to five mice per genotype at DIV 14. (D) Aβ levels in DIV 14 medium from hAPP/Tau+/+ neurons treated with a GSM (BMS-893204; 100 nM final concentration) from DIV 1–14, relative to Aβ levels in replicate cultures treated with vehicle (DMSO; 0.001% final concentration). n = 5–6 wells from four mice per treatment. ***, P < 0.001 versus vehicle (arbitrarily defined as 1.0) by one-sample t test. (E) Axonal transport in neurons of the indicated genotypes treated with GSM (100 nM) or vehicle (Veh; DMSO) over 12–14 d. n = 23–29 axons from three mice per genotype and treatment from three independent sessions at DIV 12–14. ***, P < 0.001 (Dunnett’s test). (F) Axonal transport in neurons of the indicated genotypes before (baseline) and after treatment with the selective NMDAR antagonist D-AP5 (100 µM final concentration; for 1 h) at DIV 12–14. n = 22–24 axons from three mice for each genotype at DIV 12–14. **, P < 0.01 versus Tau+/+ baseline (Dunnett’s test); ###, P < 0.001 (paired t test, Bonferroni). Data are means ± SEM.
Figure 2.
Knocking down tau with lentivirus-shRNA prevents deficits in axonal transport of mitochondria in wild-type (Tau+/+) primary hippocampal neurons caused by exposure to Aβ oligomers. (A) Neurons were transduced with lentivirus expressing anti-tau shRNA and EGFP (Lenti-shTau-GFP; left, monochrome; right, green) on DIV 0 and transfected with a mitochondrial marker (mito-RFP; middle, monochrome; right, red) at DIV 6. Arrowheads indicate mitochondria in axon. Bar, 20 µm. (B) Total tau levels in neurons transduced with Lenti-shTau-GFP (shTau) or a similar construct expressing a scrambled shRNA (shScr) were determined by Western blot analysis with the Tau-5 antibody. Top, representative blot; bottom, quantitation of blot signals. Mean Tau/actin ratios in scrambled shRNA–expressing neurons were arbitrarily defined as 1.0. n = 6–7 wells from four to five experiments per condition at DIV 14. *, P < 0.05 (unpaired t test). (C) The percentage of mitochondria moving anterogradely or retrogradely relative to all mitochondria in the axons of neurons transduced as in A and B before (baseline) and 10–60 min after adding vehicle (Veh) or Aβ1–42 oligomers (final concentration equivalent to 2-µM monomer) to the medium. n = 25–40 axons recorded during three to four independent sessions at 12–14 DIV. ***, P < 0.001 (paired t test, Bonferroni). Data are means ± SEM.
Figure 3.
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 Aβ1–42 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.
Figure 4.
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 Aβ1–42 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.
Figure 5.
Aβ-induced deficits in axonal transport depend on neuronal activity, but are not associated with obvious rises in [Ca2+]i. (A) Primary hippocampal neurons from wild-type mice were treated with TTX (1 µM) to silence neuronal activity or with vehicle (Veh), followed by exposure to Aβ1–42 oligomers. The percentage of moving mitochondria in the axons of Tau+/+ neurons was measured before (baseline) and during these treatments. n = 22–26 axons per treatment combination from three independent sessions at DIV 12–14. ##, P < 0.01 as indicated by bracket (unpaired t test); ***, P < 0.001 versus corresponding baseline (repeated measures ANOVA, Dunnett’s test). (B–E) [Ca2+]i levels in primary hippocampal neurons from Tau+/+ and Tau−/− mice were measured with Fura-2 (340:380 ratio) by live fluorescence microscopy at baseline (2 min) and after adding Aβ1–42 oligomers (for 40 min) or KCl (50 mM final concentration for 5 min) to the medium. (B) Representative images of the cultures. Bars, 100 µm. (C–E) Quantitation of [Ca2+]i relative to baseline levels (arbitrarily defined as 1.0). Aβ increased the variance in [Ca2+]i in both genotypes compared with vehicle-treated Tau+/+ neurons. **, P < 0.01 (Levene’s test). Kruskal-Wallis ANOVA, followed by Dunn’s post hoc test confirmed that the sevenfold rise in [Ca2+]i induced by KCl in both genotypes was significant compared with vehicle-treated Tau+/+ neurons (**, P < 0.01) and revealed no differences between genotypes for Aβ and KCl. (C and D) n = 356–1,002 neurons per genotype from 5–11 independent sessions at DIV 12–14. (E) n = 63–356 neurons per genotype from two to five independent sessions at DIV 12–14. Data in A are means ± SEM. Data in C are means ± SD. Plots in D and E show medians, quartiles, and ranges.
Figure 6.
Aβ-induced deficits in axonal transport do not depend on interactions between tau and Fyn. (A) Diagram of 0N4R mouse tau indicating the Y18F point mutation that should prevent phosphorylation of tau by Fyn (Lee et al., 2004) and the PxxP to AxxA substitutions in the seventh proline-directed region (AxxA7) that should interfere with binding of tau to Fyn (Lee et al., 1998). Numbers in parentheses indicate corresponding amino acid positions in human 2N4R tau. (B) 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 Aβ1–42 oligomers to the medium. Results are expressed relative to baseline (100%). n = 19–37 axons per construct recorded during three to five independent sessions at DIV 7–8. ##, P < 0.01 versus empty (Kruskal-Wallis ANOVA, Dunn’s test); ***, P < 0.001 versus corresponding baseline (paired t tests, Bonferroni). Data are means ± SEM.
Figure 7.
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 Aβ1–42. 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 Aβ1–42 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.
Figure 8.
Tau reduction prevents Aβ-induced activation of GSK3β. (A–C) Phosphorylation of GSK3β at serine 9 (p-GSK3β), a modification that inhibits GSK3β activity (Sutherland et al., 1993), and total GSK3β (t-GSK3β) and GAPDH or actin levels in Tau+/+ and Tau−/− neurons were determined by Western blot analysis after treatment of neuronal cultures with vehicle (Veh), Aβ1–42 oligomers (30 min), or the phosphoinositide 3-kinase inhibitor wortmannin (WM; 0.1 µM, 30 min). (A) Representative Western blot from a single gel that was scanned and digitally arranged. (B) Quantitation of the p-GSK3β/t-GSK3β ratio for each treatment. Aβ decreased the ratio (i.e., increased GSK3β activity) in Tau+/+, but not Tau−/−, neurons, whereas WM decreased the ratio in both types of neurons. (C) Quantitation of t-GSK3β levels revealed no significant difference between vehicle-treated Tau+/+ and Tau−/− neurons (t test). n = 7–18 wells per condition from three to six independent experiments at DIV 14. ***, P < 0.001 versus vehicle in the same genotype (Dunnett’s test). Data are means ± SEM.
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