Alzheimer's amyloid-β oligomers rescue cellular prion protein induced tau reduction via the Fyn pathway

Abstract

Amyloid-β (Aβ) and tau are the pathogenic hallmarks in Alzheimer's disease (AD). Aβ oligomers are considered the actual toxic entities, and the toxicity relies on the presence of tau. Recently, Aβ oligomers have been shown to specifically interact with cellular prion protein (PrP(C)) where the role of PrP(C) in AD is still not fully understood. To investigate the downstream mechanism of PrP(C) and Aβ oligomer interaction and their possible relationships to tau, we examined tau expression in human neuroblastoma BE(2)-C cells transfected with murine PrP(C) and studied the effect under Aβ oligomer treatment. By Western blotting, we found that PrP(C) overexpression down-regulated tau protein and Aβ oligomer binding alleviated the tau reduction induced by wild type but not M128V PrP(C), the high AD risk polymorphic allele in human prion gene. PrP(C) lacking the Aβ oligomer binding site was incapable of rescuing the level of tau reduction. Quantitative RT-PCR showed the PrP(C) effect was attributed to tau reduction at the transcription level. Treatment with Fyn pathway inhibitors, Fyn kinase inhibitor PP2 and MEK inhibitor U0126, reversed the PrP(C)-induced tau reduction and Aβ oligomer treatment modulated Fyn kinase activity. The results suggested Fyn pathway regulated Aβ-PrP(C)-tau signaling. Overall, our results demonstrated that PrP(C) down-regulated tau via the Fyn pathway and the effect can be regulated by Aβ oligomers. Our study facilitated the understanding of molecular mechanisms among PrP(C), tau, and Aβ oligomers.

Figure 1

The morphology, assembly, and cellular binding of Aβ oligomers. (A) The morphology of freshly prepared and oligomeric Aβ42 was observed by TEM. Insets are the zoom-in images. Scale bars are 100 nm. (B) The assembly of freshly prepared and oligomeric Aβ42 were examined by Tris-tricine SDS-PAGE and blotted with anti-Aβ antibody 6E10. Molecular mass markers and estimated assembly of Aβ are shown. (C, D) The cellular binding of Aβ42 oligomers to PrP^C expressing cells. Freshly prepared and oligomeric biotinylated Aβ42 (0–2500 nM) were added to PrP^C WT (C) and M128V (D) expressing neuroblastoma, and the bound Aβ were stained by BCIP/NBT chromogenic stain through biotin and streptavidin–alkaline phosphatase interaction. (E) Flow cytometry analysis for the binding of the Aβ oligomers. Oligomeric biotinylated Aβ42 (0–2500 nM) were added to PrP^C WT and M128V expressing neuroblastoma, and the bound Aβ were stained by FITC-conjugated streptavidin. (F) The binding affinity of the Aβ oligomers (0–2500 nM) to PrP^C WT and M128 V expressing neuroblastoma cells. The mean fluorescence intensity from FITC was plotted against Aβ oligomer concentrations.

Figure 2

Tau expressions were reduced in PrP^C WT and PrP^C M128V expressing neuroblastoma cells, and the reduction can be rescued by Aβ42 oligomer treatment in PrP^C WT expressing cells. (A) BE(2)-C cells were transfected with empty vectors or vectors containing murine PrP^C WT or M128V cDNAs. The total lysates were immunoblotted with prion antibody 6D11, total tau antibody tau-5, phosphorylated tau antibody AT-8, and β-actin antibody. (B) The quantified and normalized tau-5/β-actin values. Eight replicas were performed. The mock group was used for normalization. All values are presented as mean ± SEM. (C) The quantified and normalized phosphorylated tau (AT8)/β-actin values. Four replicas were performed. The mock group was used for normalization. All values are presented as mean ± SEM. (D) The quantified and normalized phosphorylated tau (AT8)/tau-5 values. Four replicas were performed. The mock group was used for normalization. All values are presented as mean ± SEM. (E) BE(2)-C were transfected with empty vectors or vectors containing murine PrP^C WT or M128V cDNAs for 1 day and treated with different concentrations of Aβ42 oligomers for an additional day. The total lysates were immunoblotted with tau-5, AT-8, and β-actin antibodies. (F) The quantified and normalized values of tau-5/β-actin. Six replicas were performed. The mock group without Aβ oligomer treatment was used for normalization. All values are presented as mean ± SEM. (G) The quantified and normalized values of phosphorylated tau (AT8)/β-actin. Four replicas were performed. The mock group without Aβ oligomer treatment was used for normalization. All values are presented as mean ± SEM. (h) The quantified and normalized phosphorylated tau (AT8)/tau-5 values. Four replicas were performed. The mock group was used for normalization. All values are presented as mean ± SEM. All statistical analyses were performed by Student’s t test, (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001, and one-way ANOVA, (##) P < 0.01.

Figure 3

The rescuing effect of Aβ oligomers on tau reduction was diminished in PrP^C Δ95–110 expressing neuroblastoma after Aβ42 oligomer treatment. (A) BE(2)-C cells were transfected with empty vectors or vectors containing murine PrP^C WT or Δ95–110 and treated with different concentrations of Aβ42 oligomers for 1 day. The total lysates were immunoblotted with tau-5 and β-actin antibodies. (B) The quantified and normalized values of tau-5/β-actin from the Western blots. Four replicas were performed. The mock group without Aβ oligomer treatment was used for normalization. All values are presented as mean ± SEM. (C) BE(2)-C cells were transfected with empty vectors or vectors containing murine PrP^C M128V or its Δ95–110 mutant and treated with different concentrations of Aβ42 oligomers for 1 day. The total lysates were immunoblotted with tau-5 and β-actin antibodies. (D) The quantified and normalized values of tau-5/β-actin from the Western blots. Three replicas were performed. The mock group without Aβ oligomer treatment was used for normalization. All values are presented as mean ± SEM. All statistical analyses were performed by Student’s t test, (∗) P < 0.05, (∗∗∗) P < 0.001, and one-way ANOVA, (##) P < 0.01.

Figure 4

The mRNA level of tau was reduced in PrP^C expressing neuroblastoma cells and can be rescued after Aβ oligomer treatment in PrP^C WT expressing cells. qPCR of MAPT in mock group and PrP^C WT or M128V expressing cells with and without Aβ42 oligomer treatment was performed. (A) BE(2)-C cells were transfected with empty vectors or vectors containing murine PrP^C WT or M128V cDNAs. The mRNA levels of tau gene MAPT and GAPDH were examined. The MAPT/GAPDH ratios from nine replicas were plotted after normalization with the mock group. All values were presented as mean ± SEM. (B) BE(2)-C cells were transfected with empty vectors or vectors containing murine PrP^C WT or M128 V cDNAs and treated with different concentrations of Aβ42 oligomers for 1 day. The MAPT/GAPDH ratios after Aβ42 oligomer treatment were plotted. The data from three replicas were normalized to the mock group without Aβ42 oligomer treatment. All values are presented as mean ± SEM. All statistical analyses were performed by Student’s t test, (∗) P < 0.05, (∗∗) P < 0.01, and one-way ANOVA, (##) P < 0.01.

Figure 5

The PrP^C induced tau reduction can be rescued by Fyn kinase inhibitor. (A) BE(2)-C cells were transfected with the empty vectors or vectors containing PrP^C with and without 1 μM Src kinase inhibitor PP2 or non-Fyn kinase inhibitor PP3 as a negative control. The total lysates were immunoblotted with the total tau antibody tau-5 and β-actin antibody. (B) The quantified and normalized values of tau-5/β-actin with PP2 or PP3 treatments. More than four replicas were performed. The data were normalized to the mock group without treatment. All values are presented as mean ± SEM. The statistical analyses were performed by Student’s t test, (∗) P < 0.05, (∗∗) P < 0.01. (C) The tau, phosphorylated Fyn, and total Fyn levels under dose-dependent PP2 treatment in mock group and PrP^C WT expressing cells. Different PP2 concentrations ranging from 0 to 10 μM were employed. (D,E) The quantified and normalized values of tau-5/β-actin (D) and phosphorylated Fyn/Fyn (E) with dose-dependent PP2 treatment. Four replicas were performed. The data were normalized to the mock group without treatment. All values are presented as mean ± SEM. The statistical analyses were performed by Student’s t test, (∗∗) P < 0.01, and one-way ANOVA, (##) P < 0.01; (###) P < 0.001.

Figure 6

The PrP^C induced tau reduction can be rescued by MEK inhibitor. (A) BE(2)-C cells were transfected with the empty vectors or vectors containing PrP^C with and without different concentrations of MEK inhibitor U0126. The final U0126 concentrations were 0, 0.1, 1, and 10 μM. The total lysates were immunoblotted with the total tau antibody tau-5 and β-actin antibody. (B) The quantified and normalized values of tau-5/β-actin with dose-dependent U0126 treatment. Three replicas were performed. The data were normalized to the mock group without treatment. All values are presented as mean ± SEM. The statistical analyses were performed by Student’s t test, (∗∗) P < 0.01, and one-way ANOVA, (#) P < 0.05.

Figure 7

Aβ oligomer treatment modulated Fyn kinase activity. (A) BE(2)-C cells were transfected with the empty vectors or vectors containing PrP^C WT with and without Aβ42 oligomer treatment (0, 0.1, 1 μM) for 2 h or 1 day. The total lysates were immunoblotted with tau-5, phospho-SFK, Fyn, and β-actin antibodies. (B) The quantified and normalized values of phosphorylated Fyn/Fyn with dose-dependent Aβ oligomer treatment. More than three replicas were performed. The data were normalized to the mock group without treatment. All values are presented as mean ± SEM. The statistical analyses were performed by Student’s t test, (∗) P < 0.05; (∗∗) P < 0.01, and one-way ANOVA, (#) P < 0.05; (##) P < 0.01.

Figure 8

Illustration of Aβ–PrP^C–tau signaling via Fyn pathway. Molecules involved in the Aβ–PrP^C–tau signaling and the relative subcellular localizations are indicated.