Hyperphosphorylation of Tau induced by naturally secreted amyloid-β at nanomolar concentrations is modulated by insulin-dependent Akt-GSK3β signaling pathway

Abstract

Alzheimer disease (AD) is neuropathologically characterized by the formation of senile plaques from amyloid-β (Aβ) and neurofibrillary tangles composed of phosphorylated Tau. Although there is growing evidence for the pathogenic role of soluble Aβ species in AD, the major question of how Aβ induces hyperphosphorylation of Tau remains unanswered. To address this question, we here developed a novel cell coculture system to assess the effect of extracellular Aβ at physiologically relevant levels naturally secreted from donor cells on the phosphorylation of Tau in recipient cells. Using this assay, we demonstrated that physiologically relevant levels of secreted Aβ are sufficient to cause hyperphosphorylation of Tau in recipient N2a cells expressing human Tau and in primary culture neurons. This hyperphosphorylation of Tau is inhibited by blocking Aβ production in donor cells. The expression of familial AD-linked PSEN1 mutants and APP ΔE693 mutant that induce the production of oligomeric Aβ in donor cells results in a similar hyperphosphorylation of Tau in recipient cells. The mechanism underlying the Aβ-induced Tau hyperphosphorylation is mediated by the impaired insulin signal transduction because we demonstrated that the phosphorylation of Akt and GSK3β upon insulin stimulation is less activated under this condition. Treating cells with the insulin-sensitizing drug rosiglitazone, a peroxisome proliferator-activated receptor γ agonist, attenuates the Aβ-dependent hyperphosphorylation of Tau. These findings suggest that the disturbed insulin signaling cascade may be implicated in the pathways through which soluble Aβ induces Tau phosphorylation and further support the notion that correcting insulin signal dysregulation in AD may offer a potential therapeutic approach.

FIGURE 1.

Coculture system in this study. A, schematic illustration of coculture system. Donor HEK293 cells in the upper chamber were transiently transfected with various cDNA constructs to produce secreted Aβ. The recipient cells in the lower chamber are N2a cells stably expressing human Tau (4R1N) or primary neurons prepared from rat embryos. B, detergent lysates of N2a (lanes 1 and 2) and N2a cells stably expressing human Tau (4R1N) (lanes 3 and 4) were blotted with the anti-Tau antibodies Tau-1 and Tau-5. Note that exogenously expressed human Tau was markedly detectable in N2a cells expressing human Tau. C, the levels of Aβ in the medium of the donor cells transfected with APPswe or WT cDNA were compared with those in cerebrospinal fluid from AD patients by immunoblot analysis (mean age at lumbar puncture, 72 years; range, 60–86 years).

FIGURE 2.

Tau phosphorylation in N2a cells cocultured with donor cells secreting Aβ. A, donor HEK293 cells were mock-transfected or transiently transfected with APP WT or the APPswe mutant construct. The levels of Aβ secreted in the medium of the donor cells were analyzed using the anti-Aβ antibody 82E1. Detergent-extracted lysates of N2a cells were evaluated using the indicated antibodies. The level of phosphorylated Tau (p-tau) detected using the AT8 and AT180 antibodies significantly increased in the N2a cells cocultured with the donor cells expressing APPswe that generated a high level of Aβ. The levels of the dephosphorylated form of Tau detected using the Tau-1 antibody were comparable among the three types of donor cell. β-Actin was visualized for normalization of loading controls. B, the results of semiquantitative analysis of phosphorylated/total Tau by densitometry are shown as mean ± S.E. (error bars) (n = 4). *, p < 0.05; **, p < 0.01 by Tukey's test versus mock after ANOVA. C, donor HEK293 cells stably expressing WT PSEN1 or the D385A variant were transiently transfected with cDNA encoding APPswe. Note that hyperphosphorylation of Tau was markedly attenuated when the recipient cells were cocultured with the donor cells expressing PSEN1 D385A, which inhibits Aβ production. D, results of semiquantitative analysis of AT8/Tau-5 (total Tau) are shown (n = 3). **, p < 0.01 by Student's t test.

FIGURE 3.

Hyperphosphorylation of Tau by coculture of N2a cells with cells expressing PSEN1 mutants. A, donor HEK293 cells stably expressing WT PSEN1 or FAD-linked L166P and ΔT440 mutants were transiently transfected with cDNA encoding APP WT. The level of Aβ42 increased in the medium of the donor cells that stably expressed the L166P and ΔT440 mutants. Detergent-extracted lysates of N2a cells were evaluated using the indicated antibodies. Note that the hyperphosphorylation of Tau (p-tau) and accumulation of total Tau were observed in N2a cells cocultured with the donor cells stably expressing the PSEN1 mutants (L166P and ΔT440) that produced a high level of Aβ42. B, results of semiquantitative analysis of AT8/Tau-5 (total Tau) are shown (n = 4). *, p < 0.05 by Tukey's test versus WT PSEN1 after ANOVA. Error bars, S.E.

FIGURE 4.

Hyperphosphorylation of Tau induced by coculture of N2a cells with cells expressing APP oligomer mutation. A, donor HEK293 cells were transiently transfected with WT APP or the ΔE693 mutant, which induced the production of oligomer Aβ. Detergent-extracted lysates of N2a cells were analyzed using the indicated antibodies. The hyperphosphorylation of Tau (p-tau) and accumulation of total Tau were observed in the N2a cells cocultured with the donor cells transfected with the APP ΔE693 mutant. B, results of semiquantitative analysis of AT8/Tau-5 (total Tau) are shown (n = 4). *, p < 0.05 by Student's t test. Error bars, S.E.

FIGURE 5.

Insulin-dependent phosphorylation of Akt (Ser-473) and GSK3β (Ser-9). A, recipient N2a cells were cocultured with donor cells mock-transfected or transfected with APPswe for 24 h and treated with 1 μm insulin. Following the synchronized stimulation, pharmacological activations of pAkt (Ser-473) and pGSK3β (Ser-9) were determined using phosphospecific antibodies. The activations of pAkt and pGSK3β in the N2a cells cocultured with the donor cells expressing the APPswe mutant were insufficient compared with those in the mock-transfected donor cells. B, results of quantification of pAkt and pGSK3β normalized by their total amounts are shown as -fold changes over base lines. The solid line indicates values obtained from coculture with mock-transfected cells. The dotted line indicates values from coculture with APPswe-expressing cells. Results of three independent experiments are shown. *, p < 0.05; **, p < 0.01 by Student's t test. Error bars, S.E.

FIGURE 6.

Hyperphosphorylation of endogenous Tau in primary neurons. A, rat primary neurons were cocultured with donor cells that were mock-transfected or transiently transfected with cDNA encoding APPswe for 24 h. The primary neurons were also cultured in the presence of the γ-secretase inhibitor compound E (25 nm) during the course of coculture. Detergent-extracted lysates of the primary neurons were evaluated using the indicated antibodies. The hyperphosphorylation of endogenous Tau (p-tau) was observed in primary neurons cocultured with the donor cells transfected with the APPswe mutant. In the presence of compound E, the hyperphosphorylation of Tau in the primary neurons was attenuated. A mobility shift of Tau was observed by immunoblotting with the Tau-5 antibody in the primary neurons cocultured with the donor cells expressing APPswe. B, results of semiquantitative analysis of phosphorylated/total Tau are shown (n = 4). *, p < 0.05; **, p < 0.01 by Tukey's test versus mock and inhibitor treatment after ANOVA. Error bars, S.E.

FIGURE 7.

Hyperphosphorylation of endogenous Tau in primary neurons cocultured with cells expressing FAD-linked mutants. A, rat primary neurons were cocultured with donor cells stably expressing PSEN1 L166P and ΔT440, which were further transiently transfected with cDNA encoding WT APP. Detergent-extracted lysates of the primary neurons were evaluated using the indicated antibodies. The hyperphosphorylation of endogenous Tau (p-tau) was observed in the primary neurons cocultured with the donor cells stably expressing PSEN1 mutants. B, results of semiquantitative analysis of AT8/Tau-5 (total Tau) in the recipient primary neurons are shown (n = 4). *, p < 0.05 by Tukey's test versus WT after ANOVA. C, primary neurons were cocultured with donor cells transiently transfected with cDNA encoding WT APP or the ΔE693 mutant, which induced the production of the oligomeric form of Aβ species. Detergent-extracted lysates of the recipient primary neurons were analyzed using the indicated antibodies. The phosphorylation of endogenous Tau was significantly enhanced in the primary neurons cocultured with the donor cells expressing the APP ΔE693 mutant. D, results of semiquantitative analysis of AT8/Tau-5 (total Tau) in the recipient primary neurons are shown (n = 4). *, p < 0.05 by Student's t test. Error bars, S.E.

FIGURE 8.

Insulin-dependent phosphorylation of Akt (Ser-473) and GSK3β (Ser-9) in recipient primary neurons. A, rat primary neurons cocultured with donor cells mock-transfected or transiently transfected with cDNA encoding APPswe were stimulated with insulin (1 μm) to induce insulin signal cascade activation. Following the synchronized stimulation, the cell lysates of the primary neurons were collected at the indicated time points. The pharmacological activation of pAkt (Ser-473) and PGSK3β (Ser-9) was examined by immunoblot analysis using phosphospecific antibodies. The activation of pAkt and pGSK3β upon insulin stimulation was insufficient in the primary neurons cocultured with the donor cells expressing the APPswe mutant. B, results of quantification of pAkt and pGSK3β normalized by their total amount are shown as -fold changes over the base line. The solid line indicates values obtained from coculture with mock-transfected cells. The dotted line indicates values from coculture with APPswe-expressing cells. Results of three independent experiments are shown. *, p < 0.05 by Student's t test. Error bars, S.E.

FIGURE 9.

Protective effect due to enhanced insulin signal transduction against endogenous Tau phosphorylation. A, the recipient N2a cells with or without transient transfection of cDNA encoding the insulin receptor were cocultured with donor cells expressing APPswe. Detergent-extracted lysates of the recipient cells were analyzed using the indicated antibodies. Expression of insulin receptor in the presence of insulin reduced the level of phosphorylated Tau (p-tau) in the recipient cells. B, results of semiquantitative analysis of AT8/Tau-5 (total Tau) in the recipient Na2 cells are shown (n = 4). *, p < 0.05; **, p < 0.01 Tukey's test versus WT after ANOVA. C, rat primary neurons cocultured with donor cells transiently transfected with cDNA encoding APPswe were treated with vehicle only or with rosiglitazone (50 μm) in the presence of insulin (100 nm). Detergent-extracted lysates of the recipient cells were analyzed using the indicated antibodies. Treatment with rosiglitazone showed a protective effect against the hyperphosphorylation of Tau induced by Aβ secreted at physiologically relevant levels. D, results of semiquantitative analysis of AT8/Tau-5 (total Tau) in the recipient primary neurons are shown (n = 3). *, p < 0.05 by Tukey's test versus WT after ANOVA. Error bars, S.E.