Tau deletion impairs intracellular β-amyloid-42 clearance and leads to more extracellular plaque deposition in gene transfer models

Abstract

Background: Tau is an axonal protein that binds to and regulates microtubule function. Hyper-phosphorylation of Tau reduces its binding to microtubules and it is associated with β-amyloid deposition in Alzheimer's disease. Paradoxically, Tau reduction may prevent β-amyloid pathology, raising the possibility that Tau mediates intracellular Aβ clearance. The current studies investigated the role of Tau in autophagic and proteasomal intracellular Aβ1-42 clearance and the subsequent effect on plaque deposition.

Results: Tau deletion impaired Aβ clearance via autophagy, but not the proteasome, while introduction of wild type human Tau into Tau-/- mice partially restored autophagic clearance of Aβ1-42, suggesting that exogenous Tau expression can support autophagic Aβ1-42 clearance. Tau deletion impaired autophagic flux and resulted in Aβ1-42 accumulation in pre-lysosomal autophagic vacuoles, affecting Aβ1-42 deposition into the lysosome. This autophagic defect was associated with decreased intracellular Aβ1-42 and increased plaque load in Tau-/- mice, which displayed less cell death. Nilotinib, an Abl tyrosine kinase inhibitor that promotes autophagic clearance mechanisms, reduced Aβ1-42 only when exogenous human Tau was expressed in Tau-/- mice.

Conclusions: These studies demonstrate that Tau deletion affects intracellular Aβ1-42 clearance, leading to extracellular plaque.

Figures

Figure 1

Inhibition of the proteasome or autophagy partially affects Aβ1-42 and p-Tau clearance. To dissect out the contribution of the proteasome from autophagy-lysosome in amyloid clearance, primary hippocampal neurons (DIV14) were infected with lentivirus plasmids, and then treated with 1 μl DMSO or autophagy modulators (Nilotinib or Bafilomycin-A1) and/or proteasome inhibitor (MG132). Histograms represent ELISA concentrations of A) Aβ1-42 and time course showing the distribution of intracellular and media B) Aβ1-42 and C) p-Tau Ser 396. D) p-Tau Ser 396 in the presence of modulators of autophagy and the proteasome. Insert). WB analysis on 4-12% NuPAGE SDS gel showing expression of the lentiviral tag V5, Aβ1-42, human Tau (HT7) and total Tau relative to actin. E) WB analysis on 10% NuPAGE SDS gel showing AT8 levels relative to actin. F) Histograms represent 20S proteasome activity assay in human M17 neuroblastoma cells. G) RT-PCR showing the effects of Nilotinib on lentiviral gene expression relative to GAPDH. # indicates significantly different to LacZ, Asterisk is significantly different to Aβ1-42 + DMSO or as indicated, bars are mean ± SEM, two-way ANOVA.

Figure 2

Tau deletion impairs autophagic clearance. Primary hippocampal neurons from WT and Tau−/− mice were infected with lentivirus plasmids and then treated with 1 μl DMSO or autophagy modulators (Nilotinib or Bafilomycin-A1) and/or proteasome inhibitor (MG132). Histograms represent ELISA concentrations of soluble and insoluble brain extracts of A) human Aβ1-42 and B) p-Tau Ser 396. WT (C57BL/6) and Tau−/− mice were injected with 1×106 multiplicity of infection (MOI) of lentiviral human Tau, Aβ1-42, Tau ± Aβ1-42 and adjusted with LacZ. All animals were treated 3 weeks post-injection with daily 10 mg/kg IP injection or 30 μL DMSO once a day for 3 (additional) consecutive weeks. Histograms represent ELISA concentrations of total brain extracts of C) human Aβ1-42 and D) p-Tau Ser 396. Asterisk is significantly different to Aβ1-42 + DMSO or as indicated, # indicates significantly different to Aβ1-42 + DMSO in WT mice. Bars are mean ± SEM, two-way ANOVA.

Figure 3

Boosting autophagy leads to Aβ1-42 clearance in WT but not Tau−/−mice. WT and Tau−/− mice were injected with lentiviral Tau ± Aβ1-42 for 3 weeks and treated I.P. with 10 mg/kg Nilotinib or DMSO once a day for 3 weeks. Brain tissues were fractionated to isolate AVs and human specific ELISA was performed. Histograms represent concentration of A) Aβ1-42, insert is WB on 4-12% SDS NuPAGE gel showing LC3 and LAMP-2a as AV markers, B) Aβ1-42 in AV20, and C) Aβ1-42 in lysosomal fractions in WT and Tau−/− mice. ELISA concentrations of Ser 396 Tau in D) AV10, E) AV20, and F) lysosomal fractions in WT and Tau−/− mice. WB analysis on 4-12% SDS NuPAGE gel of total brain extracts from WT mice showing G) V5 to verify equal expression of all lentiviruses, human Tau (HT7), total Tau, AT8 and AT180 relative to actin. H) shows p-Tau Ser 262, Ser 396 and autophagic markers Beclin-1 and LC3-I/II relative to actin. WB analysis on 4-12% SDS NuPAGE gel of total brain extracts from Tau−/− mice showing I) V5, human Tau (HT7), total Tau, AT8, and AT180 relative to tubulin. J) shows p-Tau Ser 262, Ser 396 and autophagic markers Beclin-1, LC3-I and LC3-II relative to actin. Asterisk indicates significantly different to Aβ1-42 + DMSO, bars are mean ± SEM, two-way ANOVA.

Figure 4

Aβ1-42 is more efficiently cleared in WT than Tau−/−mice. Staining of 20 μm thick coronal sections with p-Tau (AT8) and counterstained with DAB in A) LacZ + DMSO, B) Tau + DMSO, C) Tau + Nilotinib, D) Tau and Aβ1-42 + DMSO and E) Tau and Aβ1-42 + Nilotinib. Staining of hippocampus with AT8 and DAB in F) LacZ + DMSO, G) Tau + DMSO, H) Tau + Nilotinib, I) Tau and Aβ1-42 + DMSO and J) Tau and Aβ1-42 + Nilotinib in Tau−/− mice. Staining of 20 μm thick coronal sections with p-Tau (AT180) and DAB in K) LacZ + DMSO, L) Tau + DMSO, M) Tau + Nilotinib, N) Tau and Aβ1-42 + DMSO and O) Tau and Aβ1-42 + Nilotinib in WT mice. Staining of cortical sections in P) LacZ + DMSO, Q) Tau + DMSO, R) Tau + Nilotinib, S) Tau and Aβ1-42 + DMSO and T) Tau and Aβ1-42 + Nilotinib in Tau−/− mice. U) Histograms represent stereological quantification of p-Tau. Asterisk indicates significantly different to Aβ1-42 + DMSO, bars are mean ± SEM, two-way ANOVA.

Figure 5

Increased Aβ1-42 plaque deposition in Tau−/−mice. Staining of 20 μm thick coronal sections with 6E10 and DAB in wild type mice injected with lentiviral A) Aβ1-42 + DMSO, B) Aβ1-42 + Nilo, C) Aβ1-42 + Tau + DMSO, D) Aβ1-42 + Tau + Nilo and E) LacZ + DMSO in the hippocampus. Staining of 20 μm thick coronal sections with 6E10 and DAB in Tau−/− mice injected with lentiviral F) Aβ1-42 + DMSO, G) Aβ1-42 + Nilo, H) Aβ1-42 + Tau + DMSO, I) Aβ1-42 + Tau + Nilo and J) LacZ + DMSO in the cortex. Staining of 20 μm thick coronal sections with 6E10 and DAB in wild type mice injected with lentiviral K) Aβ1-42 + DMSO, L) Aβ1-42 + Nilo, M) Aβ1-42 + Tau + DMSO, N) Aβ1-42 + Tau + Nilo and O) LacZ + DMSO in the cortex. Staining of 20 μm thick coronal sections with 6E10 and DAB in Tau−/− mice injected with lentiviral P) Aβ1-42 + DMSO, Q) Aβ1-42 + Nilo, R) Aβ1-42 + Tau + DMSO, S) Aβ1-42 + Tau + Nilo and T) LacZ + DMSO in the cortex. Histograms represent U) stereological counting of Aβ1-42 positive cells and V) plaque number/mm2 in total brain. Asterisk is significantly different to control (Aβ1-42 + DMSO) or as indicated, bars are mean ± SEM, two-way ANOVA.

Figure 6

Tau deletion attenuates Aβ1-42-induced cell death. Cupric silver staining of 20 μm thick coronal sections in WT mice injected with lentiviral A) Aβ1-42 + DMSO, B) Aβ1-42 + Nilo, C) Aβ1-42 + Tau + DMSO, D) Aβ1-42 + Tau + Nilo and E) LacZ + DMSO. Silver staining in Tau−/− mice injected with lentiviral F) Aβ1-42 + DMSO, G) Aβ1-42 + Nilo, H) Aβ1-42 + Tau + DMSO, I) Aβ1-42 + Tau + Nilo and J) LacZ + DMSO. Histograms represent K) stereological quantification of silver-positive cells and L) caspase-3 activity. Asterisk is significantly different to Aβ1-42 + DMSO or as indicated, bars are mean ± SEM, two-way ANOVA.