Research ArticleAlzheimer’s Disease

The amyloid-β oligomer Aβ*56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation

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Science Signaling  09 May 2017:
Vol. 10, Issue 478, eaal2021
DOI: 10.1126/scisignal.aal2021
  • Fig. 1 Coimmunoprecipitation and colocalization of Aβ*56 with the NMDAR subunit GluN1.

    (A) Coimmunoprecipitation of Aβ*56 with NMDAR subunits (GluN1, GluN2A, and GluN2B), AMPAR subunits (GluA1 and GluA2), α7-nicotinic acetylcholine receptor subunit (α7), mGluR5 (R5), or Ephrin B2 (B2) in membrane extracts from the forebrain of Tg2576 mice. Aβ was detected with 6E10. Blot is representative of three experiments (n = 6 mice per group). IP, immunoprecipitation; WB, Western blot; IgG, immunoglobulin G. (B and C) Western blots (B) and quantitation (C) of coimmunoprecipitation of Aβ*56 with GluN1 in membrane extracts from Tg2576 mice and a wild-type (WT) control. Antibody A11 was used to detect oligomeric Aβ. Data are means ± SD from n = 5 mice per group. P < 0.05 versus 5-month-old WT mice, P < 0.05 versus 7-month-old Tg2576 mice, by two-way analysis of variance (ANOVA) (F4,30 = 86.9203, P < 0.0001) followed by Student’s t test. Tg, transgenic; m, month. (D) Representative confocal images of Aβ*56 (green) binding to GluN1 (red) on WT or Prnp-null (Prnp−/−) primary cortical neurons. Neurons were also labeled for the dendritic neuronal marker microtubule-associated protein 2 (MAP2) (blue). n = 6 dishes per group. Veh., vehicle. (E) Software-assisted colocalization analysis of Aβ*56 and GluN1 on WT and Prnp-null neurons [six regions of interest (ROIs) per dish; n = 6 dishes per group]. (F) Western blots comparing GluN1 protein amounts in primary neurons and in HEK293 cells expressing GluN1. (G) Representative confocal images of Aβ*56 (A11; magenta) binding to HEK293 cells transfected with GluN1 (red) and/or GluN2B–enhanced green fluorescent protein (green). Arrowheads indicate colocalization between Aβ*56 and GluN1. n = 6 to 8 dishes per condition. Scale bars, 15 μm.

  • Fig. 2 Aβ*56 enhances synaptic NMDAR-dependent calcium transients in primary cultured neurons.

    (A) Representative confocal images for GCaMP6f-transfected neurons in the presence or absence of Aβ*56 at rest or after stimulation of synaptic NMDARs with Bic4AP. Scale bars, 20 μm. (B) Fluorescence responses of GCaMP6f-transfected neurons after synaptic NMDAR activation in the presence (red) or absence (black) of Aβ*56. Bold solid lines correspond to the average response; the flanking upper and lower gray shaded areas indicate SD. The black bar indicates the exposure of Bic4AP. A.U., arbitrary units. (C and D) Quantitation of the peak maximum GCaMP6f fluorescence (C) and area under the curve (D) in neurons exposed to Aβ*56 after simulation of synaptic NMDARs. F1,13 = 21.122 and F1,13 = 22.306, respectively. P < 0.05, Student’s t test; n = 6 to 9 cells per group. CTL, control. (E) Longitudinal fluorescence changes within GCaMP6f-transfected neurons in the absence of Aβ*56 (bars 1 to 3) or after a 15-min application of Aβ*56 (bars 5 to 9). Each bar corresponds to sequential bath stimulations. Histograms show means ± SD; one-way ANOVA (F8,51 = 9.4731, P < 0.0001) followed by Student’s t test, P < 0.05 versus Bic4AP (stimulation #2), P < 0.05 versus Bic4AP post-Aβ*56 (stimulation #5); n = 8 cells per group. (F) Mean Ca2+ responses in cortical neurons consecutively exposed to Bic4AP, Bic4AP post-Aβ*56 application, and Bic4AP + MK801. Histograms show means ± SD; one-way ANOVA (F2,40 = 14.7673, P < 0.0001) followed by Student’s t test, P < 0.05 versus Bic4AP, P < 0.05 versus Bic4AP post-Aβ*56; n = 16 responses per group.

  • Fig. 3 CaMKIIα is abnormally phosphorylated at Thr286 in the brain tissue of 7-month-old Tg2576 mice and in cortical neurons treated with Aβ*56.

    (A and B) Representative Western blots (A) and densitometry analysis (B) for pThr286-CaMKIIα and CaMKIIα in intracellular (IC) protein extracts of 4- and 7-month-old Tg2576 or age-matched WT and Tg5469 mice. Data are means ± SD; two-way ANOVA (F7,28 = 38.7825, P < 0.0001) followed by Student’s t test, P < 0.05 versus 4-month-old WT mice; n = 6 to 9 mice per group. (C) Confocal imaging analysis of pThr286-CaMKIIα abundance and subcellular localization in prefrontal cortex (PFC) and CA1 pyramidal neurons of 7-month-old WT and Tg2576 mice (n = 5 mice per group). Scale bars, 20 μm. (D and E) Representative Western blots (D) and quantitation (E) for pT286-CaMKIIα and CaMKIIα in membrane-bound (MB) protein extracts of 4-, 7-, 12-, and 16-month-old Tg2576 mice. Histograms show means ± SD; one-way ANOVA (F3,24 = 30.4023, P < 0.0001) followed by Student’s t test, P < 0.05 versus 4-month-old WT mice, P < 0.05 versus 7-month-old Tg2576 mice; n = 6 per group. (F and G) Western blots (F) and densitometry analysis (G) for pT286-CaMKIIα and total CaMKIIα in 12- to 14-DIV (day in vitro) primary mouse cortical neurons treated with vehicle or increasing concentrations of brain-derived Aβ*56 for 60 min. Histograms show means ± SD; ANOVA (F4,30 = 14.6822, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle, P < 0.05 versus 1 pM condition; n = 6 to 8 per group. (H and I) Western blot images (H) and quantitation (I) for pT286-CaMKIIα and total CaMKIIα in 12- to 14-DIV primary mouse cortical neurons treated with 2.5 pM brain-derived Aβ*56 for 1, 6, 8, 12, or 24 hours. Histograms show means ± SD; ANOVA (F4,34 = 17.4461, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle, P < 0.05 versus 1-hour condition; n = 6 per group. (J and K) Representative Imaris surface images (J) and quantitation (K) of the colocalization of pCaMKIIα with PSD-95 (yellow) with respect to MAP2 (blue) in neurons treated with vehicle or 2.5 pM Aβ*56 for 60 min. Histograms show means ± SD; Student’s t test, F1,14 = 37.339, P < 0.05 versus vehicle; n = 8 ROIs per group. Scale bars, 3 μm.

  • Fig. 4 Hyperphosphorylation and missorting profile of soluble tau species in young Tg2576 mice.

    (A and B) Representative Western blots (A) and quantitation (B) of soluble tau species detected in intracellular (IC)–enriched fractions from 4- and 7-month-old WT and Tg2576 mice. Histograms show means ± SD; two-way ANOVA (F2,21 = 67.6019, P < 0.0001) followed by Student’s t test, P < 0.05 versus age-matched WT mice, P < 0.05 versus 4-month-old Tg2576 mice; n = 6 to 9 mice per group. (C and D) Western blots (C) and densitometry analysis (D) of total soluble tau, PSD-95, and actin in membrane-bound (MB) protein extracts of Tg2576 mice at 4, 7, and 12 months of age. Histograms show means ± SD; one-way ANOVA (F2,18 = 19.7636, P < 0.0001) followed by Student’s t test, P < 0.05 versus 4-month-old Tg2576 mice, P < 0.05 versus 7-month-old Tg2576 mice; n = 6 to 9 mice per group. (E and F) Western blots (E) and quantitation (F) of total soluble tau in intracellular-enriched (I) or membrane extracts (M) of 4- and 7-month-old Tg2576 mice. Histograms show means ± SD; two-way ANOVA (F3,30 = 47.2095, P < 0.0001) followed by Student’s t test, P < 0.05 versus 4-month-old Tg2576 mice; n = 6 to 9 mice per group. (G) Representative confocal images of CA1 hippocampal neurons immunostained for MAP2 (blue), pSer202-tau (CP13; green), and pSer416-tau (red) revealed an aberrant accumulation and differential missorting of soluble tau species in 7-month-old Tg2576 mice. (H) Z-stack reconstruction from confocal images illustrating the colocalization for pSer202-tau and pSer416-tau (yellow), shown with the three-dimensional rendering of MAP2. n = 6 sections per animal; n = 3 to 6 animals per group. Scale bars, 20 μm (top and middle) or 10 μm (bottom) in (G) and 3 μm in (H).

  • Fig. 5 Selective tau hyperphosphorylation in primary neurons exposed to Aβ*56.

    (A and B) Western blots (A) and quantitation (B) of soluble tau species detected in mouse cortical neurons exposed to increasing concentrations of Aβ*56 for 60 min. Histograms show means ± SD; one-way ANOVA (F2,21 = 67.6019, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle-treated neurons, P < 0.05 versus 1 pM Aβ*56 condition; n = 6 to 8 dishes per treatment. (C and D) Western blots (C) and densitometry analysis (D) for total soluble tau detected with the antibody tau5, PSD-95, and actin in membrane-associated extracts from vehicle or Aβ*56-treated neurons. Histograms show means ± SD; one-way ANOVA (F2,21 = 67.6019, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle-treated neurons, P < 0.05 versus 1 pM Aβ*56 condition; n = 6 to 8 per group. (E and F) Western blots (E) and quantitation (F) for pS416-tau, total soluble tau detected with the antibody tau5, and actin in intracellular-enriched lysates of vehicle- or Aβ*56 (2.5 pM)–treated neurons. Histograms show means ± SD, P < 0.05 versus vehicle-treated neurons by t test; n = 6 dishes per group.

  • Fig. 6 Inhibiting CaMKII prevents Aβ*56-induced tau hyperphosphorylation at S416.

    (A and B) Western blots (A) and quantitation (B) for pCaMKIIα and total CaMKIIα in primary cortical neurons pretreated with the NMDAR uncoupling peptide tatNR2B9c for 15 min in the presence or absence of 2.5 pM Aβ*56. Histograms show means ± SD; one-way ANOVA (F3,14 = 252.0481, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle, P < 0.05 versus Aβ*56-treated neurons; n = 4 to 6 dishes per group. (C to E) Western blots (C) and densitometry analysis (D and E) for pSer202-tau, pSer416-tau, total tau, and actin in primary cortical neurons pretreated with the NMDAR uncoupling peptide tatNR2B9c for 15 min in the presence or absence of 2.5 pM Aβ*56. Total tau was detected with the antibody tau5. Histograms show means ± SD; one-way ANOVA (F3,14 = 22.6029, P < 0.0001, and F3,12 = 16.1364, P = 0.0009, respectively) followed by Student’s t test, P < 0.05 versus vehicle, P < 0.05 versus Aβ*56-treated neurons; n = 4 to 6 dishes per group. (F and G) Western blots (F) and quantitation (G) for pThr286-CaMKIIα and total CaMKII in primary cortical neurons pretreated with the CaMKII inhibitor tatCN21 in presence or absence of 2.5 pM Aβ*56. Histograms show means ± SD; two-way ANOVA (F3,35 = 25.0063, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle-treated neurons, P < 0.05 versus Aβ*56-treated neurons; n = 6 to 9 dishes per group. ANOVA results: Aβ*56 (F = 26.7966, P < 0.0001), tatCN21 (F = 16.2025, P = 0.0003), and Aβ*56 × tatCN21 interaction (F = 27.4058, P < 0.0001). (H to J) Western blots (H) and quantitation (I and J) for soluble pSer416-tau and total tau (as measured with the tau5 antibody) in mouse primary neurons pretreated with the CaMKII inhibitor tatCN21 in the presence or absence of 2.5 pM Aβ*56. Histograms show means ± SD; two-way ANOVA (F3,35 = 28.4569, P < 0.0001, and F3,35 = 24.8972, P < 0.0001, respectively) followed by Student’s t test, P < 0.05 versus vehicle-treated neurons, P < 0.05 versus Aβ*56-treated neurons; n = 6 to 9 dishes per group.

  • Fig. 7 CN21 pretreatment prevents the missorting of tau in cortical primary neurons exposed to Aβ*56.

    (A) Representative confocal images of primary mouse cortical neurons immunostained for MAP2 (blue), pThr286-CaMKIIα (green), and pSer416-tau (magenta) after treatment with 2.5 pM Aβ*56 for 60 min. n = 6 dishes per group. Scale bars, 30 μm. (B) Surface rendering of dendrites labeled with MAP2, pSer416-tau, and PSD-95, illustrating the cellular distribution of the pS416-tau/PSD-95 colocalization channel (yellow) with respect to MAP2 (blue) in neurons treated with vehicle, 2.5 pM Aβ*56, or tatCN21 pretreatment (15 min), followed by 2.5 pM Aβ*56 for 60 min. Scale bars, 3 μm. (C) Quantitation of the colocalization of pS416-tau with PSD-95 in mouse primary neurons exposed to vehicle, 2.5 pM Aβ*56, or tatCN21 pretreatment, followed by 2.5 pM Aβ*56. Histograms show means ± SD; one-way ANOVA (F2,20 = 67.7832, P < 0.0001) followed by Student’s t test, P < 0.05 versus vehicle, P < 0.05 versus Aβ*56-treated neurons; n = 8 ROIs per group. (D and E) Western blots (D) and quantitation (E) for soluble tau in membrane-associated lysates from neurons exposed to vehicle, Aβ*56, CN21, or Aβ*56 + CN21 using the pan tau-specific antibody tau5. Actin was used as internal standard. Histograms show means ± SD; one-way ANOVA (F3,12 = 197.3191, P < 0.0001) followed by Student’s t test; P < 0.05 versus vehicle, P < 0.05 versus Aβ*56-treated neurons; n = 4 dishes per group.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/478/eaal2021/DC1

    Fig. S1. Reverse coimmunoprecipitation of Aβ*56 with NMDAR subunits in Tg2576 and in human brain tissue.

    Fig. S2. Age-dependent accumulation of Aβ oligomers in Tg2576 mice.

    Fig. S3. Biochemical characterization of soluble Aβ species present in APP transgenic brain tissues.

    Fig. S4. Molecular and morphological characterization of primary cortical neurons.

    Fig. S5. The major pathways regulated by extrasynaptic NMDARs are not altered in 7-month-old Tg2576 mice.

    Fig. S6. Relationship between CaMKIIα activity and Aβ*56 expression in brain tissue from young Tg2576 mice.

    Fig. S7. The major pathways regulated by extrasynaptic NMDARs are not altered by endogenous Aβ oligomers in mouse cortical primary neurons after a 60-min exposure.

    Fig. S8. Aβ*56-induced translocation of pCaMKIIα to postsynaptic sites in primary cortical neurons.

    Fig. S9. CaMKIIα activation is not induced by low-n Aβ oligomers purified from APP transgenic mice.

    Fig. S10. Epitope and dephosphorylation tau assays confirm the presence of hyperphosphorylated tau conformers.

    Fig. S11. Aberrant phosphorylation of CaMKIIα and tau in young J20 mice expressing Aβ*56.

    Fig. S12. Temporal expression profiles of CDK5 adaptor proteins and GSK3 in young wild-type and Tg2576 mice.

    Fig. S13. Brain-derived Aβ dimers and trimers do not induce tau phosphorylation at Ser202.

    Fig. S14. Dose-dependent uncoupling of PSD95 and NMDAR GluN1 subunit induced by tatNR2B9c in primary neurons.

  • Supplementary Materials for:

    The amyloid-β oligomer Aβ*56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation

    Fatou Amar, Mathew A. Sherman, Travis Rush, Megan Larson, Gabriel Boyle, Liu Chang, Jürgen Götz, Alain Buisson, Sylvain E. Lesné*

    *Corresponding author. Email: lesne002{at}umn.edu

    This PDF file includes:

    • Fig. S1. Reverse coimmunoprecipitation of Aβ*56 with NMDAR subunits in Tg2576 and in human brain tissue.
    • Fig. S2. Age-dependent accumulation of Aβ oligomers in Tg2576 mice.
    • Fig. S3. Biochemical characterization of soluble Aβ species present in APP transgenic brain tissues.
    • Fig. S4. Molecular and morphological characterization of primary cortical neurons.
    • Fig. S5. The major pathways regulated by extrasynaptic NMDARs are not altered in 7-month-old Tg2576 mice.
    • Fig. S6. Relationship between CaMKIIα activity and Aβ*56 expression in brain tissue from young Tg2576 mice.
    • Fig. S7. The major pathways regulated by extrasynaptic NMDARs are not altered by endogenous Aβ oligomers in mouse cortical primary neurons after a 60-min exposure.
    • Fig. S8. Aβ*56-induced translocation of pCaMKIIα to postsynaptic sites in primary cortical neurons.
    • Fig. S9. CaMKIIα activation is not induced by low-n Aβ oligomers purified from APP transgenic mice.
    • Fig. S10. Epitope and dephosphorylation tau assays confirm the presence of hyperphosphorylated tau conformers.
    • Fig. S11. Aberrant phosphorylation of CaMKIIα and tau in young J20 mice expressing Aβ*56.
    • Fig. S12. Temporal expression profiles of CDK5 adaptor proteins and GSK3 in young wild-type and Tg2576 mice.
    • Fig. S13. Brain-derived Aβ dimers and trimers do not induce tau phosphorylation at Ser202.
    • Fig. S14. Dose-dependent uncoupling of PSD95 and NMDAR GluN1 subunit induced by tatNR2B9c in primary neurons.

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    Citation: F. Amar, M. A. Sherman, T. Rush, M. Larson, G. Boyle, L. Chang, J. Götz, A. Buisson, S. E. Lesné, The amyloid-β oligomer Aβ*56 induces specific alterations in neuronal signaling that lead to tau phosphorylation and aggregation. Sci. Signal. 10, eaal2021 (2017).

    © 2017 American Association for the Advancement of Science

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