Research ArticleAlzheimer’s Disease

β-amyloid redirects norepinephrine signaling to activate the pathogenic GSK3β/tau cascade

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Science Translational Medicine  15 Jan 2020:
Vol. 12, Issue 526, eaay6931
DOI: 10.1126/scitranslmed.aay6931
  • Fig. 1 α2AAR function is enhanced in patients with AD and animal models.

    (A) Membrane homogenates were prepared from postmortem prefrontal cortical tissues of patients with AD and control subjects. Bmax reflects α2AAR density. Emax reflects maximum α2AAR-mediated G protein activation in response to NE (applied with propranolol and prazosin to selectively activate α2AAR). For each set of experiments, an AD subject and a control subject were analyzed in parallel. **P < 0.01 by paired t test. (B) Changes in the adjusted z score for the mini-mental state (ZMMS) examination during the time period with or without clonidine usage were analyzed. ΔZMMS reflects average change in ZMMS in a year. **P < 0.01; ****P < 0.0001, by post hoc Sidak’s multiple comparisons test. (C) Average changes in the adjusted ZMMS score in patients with cognitive deficits during the time period with clonidine usage. Cognitive status code: 2, impaired not mild cognitive impairment (MCI); 3, MCI; and 4, dementia. ***P < 0.001 by one-way ANOVA. (D) Brain homogenates were prepared from nontransgenic (nTg) or APP/PS1 littermates at 7.5 months of age. G protein activation was measured in response to NE (with prazosin and propranolol to selectively activate α2AAR). **P < 0.01 by two-way ANOVA. (E) Sedation was measured by rotarod test in response to an α2AAR activator, UK14,304, in nTg, or APP/PS1 littermates at 7.5 to 8 months of age. **P < 0.01 by two-way ANOVA. (F) G protein activation in response to an A1R-selective activator, (R)-N6-(1-Methyl-2-phenylethyl)adenosine (R-PIA), in brain homogenates prepared from nTg or APP/PS1 littermates at 7.5 months of age. (G) α2AAR-mediated G protein activation in WT mouse brain homogenates in the presence or absence of AβO (100 nM, monomer equivalent). **P < 0.01 by two-way ANOVA. (H) α2AAR-mediated G protein activation in WT mouse brain homogenates in the presence of human TBS extracts with or without Aβ depletion. **P < 0.01, TBS extracts versus control by two-way ANOVA. All data are shown as means ± SEM. a.u., arbitrary units; GTP-γ-S, guanosine 5′-O-(3′-thiotriphosphate).

  • Fig. 2 O binds to an allosteric site of α2AAR with nanomolar affinity.

    (A and B) Human embryonic kidney (HEK) cells transfected with the empty vector or hemagglutinin (HA)–tagged α2AAR were incubated with AβO for 30 min. AβO bound to the surface of cells was detected by flow cytometry assays. ****P < 0.0001 by one-way ANOVA in (B). (C) Flow cytometry assays were performed with cells expressing HA-α2AAR after incubation with vehicle, monomer, or oligomer Aβ. (D) AβO and HA-α2AAR were detected by immunocytochemistry. Scale bars, 5 μm. (E) HA-α2AAR was immunopurified from HEK cells and incubated with increasing amounts of 5-Carboxyfluorescein (FAM)–labeled AβO or scrambled (scbd) Aβ42 peptide. (F) Saturation binding curves of FAM-AβO to different receptors expressed on the surface of intact HEK cells. (G) The docked Aβo-α2AAR complex model. Green, Aβ pentamer with hydrophobic C termini of monomers indicated in orange. Purple, the 3eL of α2AAR. Dashed black lines and orange lines indicate hydrogen bonds and hydrophobic contacts, respectively. (H) Binding of FAM-AβO to WT or α2AAR mutants, as indicated, expressed on the cell surface. (I) Binding of FAM-AβO (20 nM, monomer equivalent) to immunoisolated α2AAR in the presence of increasing concentrations of NE. All data are shown as means ± SEM. (J) Total brain lysates prepared from APP/PS1 or APP/PS1,α2AARHA/HA mice were subjected to coimmunoprecipitation assays using an HA antibody. The α2AARHA/HA allele harbors an HA tag at the N terminus of the endogenous α2AAR locus. APP/PS1 mouse brains were used as a negative control. Representative blots from multiple experiments are shown. IgG, immunoglobulin G; IP, immunoprecipitation.

  • Fig. 3 O redirects α2AAR signaling to activation of the GSK3β/tau cascade.

    (A) Representative blots and quantitation of protein kinase arrays. Array blots were incubated with lysates from Neuro2A cells expressing WT α2AAR with treatment as indicated. NE (400 nM) was applied with prazosin and propranolol to selectively activate α2AAR. Ctrl, positive controls for array blotting. *P < 0.05 by one-way ANOVA. (B) Primary cortical neurons (14 days in vitro) were stimulated as indicated for 30 min. V, vehicle; Clon, clonidine (1 μM). Representative Western blots of phospho-GSK3β (pGSK3β), total GSK3β, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). (C and D) Quantitation of changes in the ratio of pGSK3β to GSK3β. **P < 0.01 by one-way ANOVA Tukey’s multiple comparisons. *P < 0.05 by unpaired t test. (E) Neuro2A cells were cotransfected with WT α2AAR and a siRNA against GSK3β or scrambled (scbd) siRNA. Representative blots of tau phosphorylation are shown. (F to H) Mice that received bilateral intrahippocampal injection of AβO (100 pmol, monomer equivalent, each side) or vehicle were treated intraperitoneally with saline, idazoxan (3 mg/kg), lithium (300 mg/kg), or idazoxan and lithium. Twenty-four hours later, hippocampal lysates were analyzed by Western blot. Representative blots (F) and quantitation (G and H) of GSK3β and tau phosphorylation are shown. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA Tukey’s multiple comparisons. All data are shown as means ± SEM.

  • Fig. 4 Allosteric binding to the 3eL is required for AβO to induce activation of the GSK3β/tau cascade through α2AAR.

    Neuro2A cells expressing WT or 3eL-9A mutant α2AAR were treated with vehicle, NE (400 nM), AβO (100 nM, monomer equivalent), or NE and AβO. (A) Representative Western blots of pGSK3β at Ser9, total GSK3β, phospho-tau (ptau, detected by AT8 antibody), total tau, and GAPDH are shown. (B) Quantitation of changes in the ratio of pGSK3β to GSK3β. ****P < 0.0001 by two-way ANOVA post hoc Sidak’s multiple comparisons test. (C) Quantitation of changes in the ratio of ptau to tau. ****P < 0.0001 by two-way ANOVA Sidak’s multiple comparisons. (D) α2AAR-mediated G protein activation was measured by GTPγS binding assays using membrane homogenates prepared from cells expressing the WT or indicated mutant α2AAR.

  • Fig. 5 Blocking α2AAR in AD model mice with profound Aβ pathology reduces GSK3β activity, amyloid pathology, and tau hyperphosphorylation.

    Eight-month-old APP/PS1 and nontransgenic (nTg) littermate mice were treated with saline or idazoxan for 8 weeks, followed by a 1-week washout period. (A) Representative Western blots and (B) quantitation of phospho-GSK3β (pGSK3β) at Ser9 and total GSK3β in total cortical lysates. **P < 0.01 by unpaired Student’s t test. (C) Representative images and (D) quantitation of Aβ plaques (detected by 6E10 antibody) in the cerebral cortex and hippocampus of APP/PS1 mice subjected to treatments indicated. Scale bars, 500 μm. *P < 0.05 by unpaired t test. (E) Representative images and (F) quantitation of microglial cells (detected by Iba-1 antibody) in the cerebral cortex of APP/PS1 mice subjected to treatments were indicated. Scale bars, 100 μm. **P < 0.01 by unpaired t test. (G) Representative images of AT8 (for hyperphosphorylated tau) and an Aβ antibody staining in the cortex of nTg and APP/PS1 mice after the indicated treatment. Scale bars, 20 μm. (H) Quantitation of the intensity of AT8 signals plotted against the area of Aβ accumulations in the cortex. r2 = 0.84. Slope values for saline and idazoxan groups are 34.29 (±1.519) and 22.98 (±1.017), respectively. (I) Relative AT8 intensity normalized against the corresponding area of Aβ depositions. **P < 0.01 by unpaired t test.

  • Fig. 6 Blocking α2AAR in AD model mice with profound Aβ pathology ameliorates cognitive deficits.

    (A) Measurement of escape latency on each day in Morris water maze tests in APP/PS1 and nTg mice. ***P < 0.001, saline-treated APP/PS1 versus nTg mice; **P < 0.01, saline-treated versus idazoxan-treated APP/PS1 mice by two-way ANOVA. (B) Quantitation of the number of crosses of the target quadrant in probe trial. *P < 0.05 by one-way ANOVA post hoc Tukey’s multiple comparisons test. (C) Measurement of escape latency to the dark side in passive avoidance tests in 8-month-old WT and APP-KI mice. ***P < 0.001 by two-way ANOVA Tukey’s multiple comparisons test. (D) Measurement of escape latency to the dark side in passive avoidance tests in 8-month-old APP-KI mice treated with saline or idazoxan. **P < 0.01 and ****P < 0.0001 by two-way ANOVA Tukey’s multiple comparisons test. All data are shown as means ± SEM.

Supplementary Materials

  • stm.sciencemag.org/cgi/content/full/12/526/eaay6931/DC1

    Materials and Methods

    Fig. S1. α2AAR-mediated G protein activation in brain homogenates prepared from WT or APP-KI mice at 7.5 months of age.

    Fig. S2. α2AAR density measured by radioligand-binding assays in AD models.

    Fig. S3. α2AAR-mediated G protein activation and receptor density tested in brain homogenates prepared from nTg or APP/PS1 mice at 5 weeks of age.

    Fig. S4. Profiling of Aβ42 peptide oligomerization by fluorescent size-exclusion chromatography.

    Fig. S5. α2AAR-mediated G protein activation in WT mouse brain homogenates in the presence of human TBS extracts with or without Aβ depletion.

    Fig. S6. Cell-surface expression of HA-tagged receptors tested by fluorescence-activated cell sorting.

    Fig. S7. AβO was detected on the surface of cells expressing WT but not 3eL-9A mutant α2AAR.

    Fig. S8. Binding of an orthosteric ligand to WT or mutant α2AARs.

    Fig. S9. Full blots of the AKT pathway phosphorylation arrays.

    Fig. S10. Naturally secreted oligomeric Aβ induced GSK3β dephosphorylation/activation in neurons in the presence of clonidine.

    Fig. S11. Quantitation of tau phosphorylation and GSK3β expression.

    Fig. S12. Proposed model of Aβ hijacking NE signaling through α2AAR to induce activation of GSK3β/tau cascade.

    Fig. S13. Idazoxan treatment reduces Aβ pathology in APP-KI mouse brains.

    Fig. S14. Idazoxan treatment reduces GSK3β activity and tau hyperphosphorylation in APP-KI mouse brains.

    Fig. S15. Open-field and elevated zero maze tests in nTg and APP/PS1 mice.

    Fig. S16. Open-field and elevated zero maze tests in APP-KI mice.

    Table S1. Information of human samples used in Fig. 1A.

    Table S2. Extracted data used in Fig. 1 (B and C).

    Table S3. Information of antibodies used in this study.

    Data file S1. Raw data.

    References (2729, 31, 38, 4665)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. α2AAR-mediated G protein activation in brain homogenates prepared from WT or APP-KI mice at 7.5 months of age.
    • Fig. S2. α2AAR density measured by radioligand-binding assays in AD models.
    • Fig. S3. α2AAR-mediated G protein activation and receptor density tested in brain homogenates prepared from nTg or APP/PS1 mice at 5 weeks of age.
    • Fig. S4. Profiling of Aβ42 peptide oligomerization by fluorescent size-exclusion chromatography.
    • Fig. S5. α2AAR-mediated G protein activation in WT mouse brain homogenates in the presence of human TBS extracts with or without Aβ depletion.
    • Fig. S6. Cell-surface expression of HA-tagged receptors tested by fluorescence-activated cell sorting.
    • Fig. S7. AβO was detected on the surface of cells expressing WT but not 3eL-9A mutant α2AAR.
    • Fig. S8. Binding of an orthosteric ligand to WT or mutant α2AARs.
    • Fig. S9. Full blots of the AKT pathway phosphorylation arrays.
    • Fig. S10. Naturally secreted oligomeric Aβ induced GSK3β dephosphorylation/activation in neurons in the presence of clonidine.
    • Fig. S11. Quantitation of tau phosphorylation and GSK3β expression.
    • Fig. S12. Proposed model of Aβ hijacking NE signaling through α2AAR to induce activation of GSK3β/tau cascade.
    • Fig. S13. Idazoxan treatment reduces Aβ pathology in APP-KI mouse brains.
    • Fig. S14. Idazoxan treatment reduces GSK3β activity and tau hyperphosphorylation in APP-KI mouse brains.
    • Fig. S15. Open-field and elevated zero maze tests in nTg and APP/PS1 mice.
    • Fig. S16. Open-field and elevated zero maze tests in APP-KI mice.
    • Table S1. Information of human samples used in Fig. 1A.
    • Table S2. Extracted data used in Fig. 1 (B and C).
    • Table S3. Information of antibodies used in this study.
    • Legend for data file S1
    • References (2729, 31, 38, 4665)

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    Other Supplementary Material for this manuscript includes the following:

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