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

Abstract

The brain noradrenergic system is critical for normal cognition and is affected at early stages in Alzheimer's disease (AD). Here, we reveal a previously unappreciated direct role of norepinephrine signaling in connecting β-amyloid (Aβ) and tau, two key pathological components of AD pathogenesis. Our results show that Aβ oligomers bind to an allosteric site on α_2A adrenergic receptor (α_2AAR) to redirect norepinephrine-elicited signaling to glycogen synthase kinase 3β (GSK3β) activation and tau hyperphosphorylation. This norepinephrine-dependent mechanism sensitizes pathological GSK3β/tau activation in response to nanomolar accumulations of extracellular Aβ, which is 50- to 100-fold lower than the amount required to activate GSK3β by Aβ alone. The significance of our findings is supported by in vivo evidence in two mouse models, human tissue sample analysis, and longitudinal clinical data. Our study provides translational insights into mechanisms underlying Aβ proteotoxicity, which might have strong implications for the interpretation of Aβ clearance trial results and future drug design and for understanding the selective vulnerability of noradrenergic neurons in AD.

Fig. 1.. α_2AAR function is enhanced in AD patients and animal models.

(A) Membrane homogenates were prepared from postmortem prefrontal cortical tissues of AD patients and control subjects. Bmax reflects α_2AAR density. Emax reflects maximum α_2AAR-mediated G protein activation in response to NE (applied with propranolol and prazosin to selectively activate α_2AAR). 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 examination (ZMMS) 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 z-score for the mini–mental state examination (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; 4=Dementia. ***, p<0.001 by one-way AVOVA. (D) Brain homogenates were prepared from non-transgenic (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 α_2AAR). **, p<0.01 by two-way ANOVA. (E) Sedation was measured by rotarod test in response to an α_2AAR activator, UK14,304, in nTg or APP/PS1 littermates at 7.5–8 months of age. **, p<0.01 by two-way ANOVA. (F) G protein activation in response to an A1R-selective activator, R-PIA, in brain homogenates prepared from nTg or APP/PS1 littermates at 7.5 months of age. (G) α_2AAR-mediated G protein activation in WT mouse brain homogenates in the presence or absence of Aβ_O (100nM, monomer equivalent). **, p<0.01 by two-way ANOVA. (H) α_2AAR-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 vs. control by two-way ANOVA. All data are shown as mean±SEM.

Fig. 2.. Aβ_O binds to an allosteric site of α_2AAR with nanomolar affinity.

(A and B) HEK cells transfected with the empty vector or HA-tagged α_2AAR were incubated with Aβ_O for 30 min. Aβ_O 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-α_2AAR after incubation with vehicle, monomer or oligomer Aβ. (D) Aβ_O and HA-α_2AAR were detected by immunocytochemistry. Scale bar, 5μm. (E) HA-α_2AAR was immunopurified from HEK cells and incubated with increasing amounts of FAM-labeled Aβ_O or scrambled (scbd) Aβ42 peptide. (F) Saturation binding curves of FAM-Aβ_O to different receptors expressed on the surface of intact HEK cells. (G) The docked Aβ_O-α_2AAR complex model. Green, Aβ pentamer with hydrophobic C termini of monomers indicated in orange. Purple, the 3eL of α_2AAR. Dashed black lines and orange lines indicate hydrogen bonds and hydrophobic contacts, respectively. (H) Binding of FAM-Aβ_O to WT or α_2AAR mutants, as indicated, expressed on the cell surface. (I) Binding of FAM-Aβ_O (20nM, monomer equivalent) to immunoisolated α_2AAR in the presence of increasing concentrations of NE. All data are shown as mean±SEM. (J) Total brain lysates prepared from APP/PS1 or APP/PS1,α_2AAR^HA/HA mice were subjected to co-immunoprecipitation assays using an HA antibody. The α_2AAR^HA/HA allele harbors an HA tag at the N terminus of the endogenous α_2AAR locus. APP/PS1 mouse brains were used as a negative control. Representative blots from multiple experiments are shown.

Fig. 3.. Aβ_O redirects α_2AAR 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 α_2AAR with treatment as indicated. NE (400nM) was applied with prazosin and propranolol to selectively activate α_2AAR. Ctrl, positive controls for array blotting. *, p<0.05 by one-way ANOVA. (B) Primary cortical neurons (14 DIV) were stimulated as indicated for 30 min. V, vehicle; Clon, clonidine (1 μM). Representative Western blots of phospho-GSK3β (pGSK3β), total GSK3β and 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 co-transfected with WT α_2AAR and a siRNA against GSK3β or scrambled (scbd) siRNA. Representative blots of tau phosphorylation are shown. (F-H) Mice that received bilateral intrahippocampal injection of Aβ_O (100 pmole, monomer equivalent, each side) or vehicle were treated with saline, idazoxan (3mg/kg, i.p.), lithium (300mg/kg, i.p.) or idazoxan plus lithium. 24 hrs later, hippocampal lysates were analyzed by Western. 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 mean±SEM.

Fig. 4.. Allosteric binding to the 3eL is required for Aβ_O to induce activation of the GSK3β/tau cascade through α_2AAR.

Neuro2A cells expressing WT or 3eL-9A mutant α_2AAR were treated with vehicle, NE (400nM), Aβ_O (100nM, monomer equivalent) or NE plus Aβ_O. (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) α_2AAR-mediated G protein activation was measured by GTPγS binding assays using membrane homogenates prepared from cells expressing the WT or indicated mutant α_2AAR.

Fig. 5.. Blocking α_2AAR in AD model mice with profound Aβ pathology reduces GSK3β activity, amyloid pathology and tau hyperphosphorylation.

8 month-old APP/PS1 and non-transgenic (nTg) littermate mice were treated with saline or idazoxan for 8 weeks, followed by a one-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 bar, 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 indicated. Scale bar, 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 following the indicated treatment. Scale bar, 20μm. (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 α_2AAR 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 vs. nTg mice; **, p<0.01, saline-treated vs. 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; ****, p<0.0001 by two-way ANOVA Tukey’s multiple comparisons test. All data are shown as mean±SEM.