Soluble oligomers of amyloid-β peptide disrupt membrane trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor contributing to early synapse dysfunction

Abstract

β-Amyloid (Aβ), a peptide generated from the amyloid precursor protein, is widely believed to underlie the pathophysiology of Alzheimer disease (AD). Emerging evidences suggest that soluble Aβ oligomers adversely affect synaptic function, leading to cognitive failure associated with AD. The Aβ-induced synaptic dysfunction has been attributed to the synaptic removal of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors (AMPARs). However, the molecular mechanisms underlying the loss of AMPAR induced by Aβ at synapses are largely unknown. In this study we have examined the effect of Aβ oligomers on phosphorylated GluA1 at serine 845, a residue that plays an essential role in the trafficking of AMPARs toward extrasynaptic sites and the subsequent delivery to synapses during synaptic plasticity events. We found that Aβ oligomers reduce basal levels of Ser-845 phosphorylation and surface expression of AMPARs affecting AMPAR subunit composition. Aβ-induced GluA1 dephosphorylation and reduced receptor surface levels are mediated by an increase in calcium influx into neurons through ionotropic glutamate receptors and activation of the calcium-dependent phosphatase calcineurin. Moreover, Aβ oligomers block the extrasynaptic delivery of AMPARs induced by chemical synaptic potentiation. In addition, reduced levels of total and phosphorylated GluA1 are associated with initial spatial memory deficits in a transgenic mouse model of AD. These findings indicate that Aβ oligomers could act as a synaptic depressor affecting the mechanisms involved in the targeting of AMPARs to the synapses during early stages of the disease.

FIGURE 1.

oAβ induces Ser-845 dephosphorylation in GluA1 and a decrease in cell surface AMPARs. A, Western blots showing phosphorylated levels of Ser-845 (upper blot), total GluA1 (middle blot), and GAPDH protein levels (lower blot) as loading control. B, Western blots showing phosphorylated levels of Ser-831 and Ser-880 (upper blot), total GluA1 and GluA2 (middle blot), and GAPDH protein levels (lower blot) as loading control. C, graph represents quantification of phosphorylated AMPA subunits in response to oAβ at different times after stimulation compared with baseline. Represented values are the ratio of the levels of phosphorylated AMPA subunits versus normalized total levels (related to GAPDH) of the corresponding AMPA subunit. **, p < 0.01; ***, p < 0.001 (n = 8); error bars indicate ± S.E. D, surface proteins were analyzed by biotinylation. Blots show surface GluA1 (upper blot) and total GluA1 (lower blot). E, quantified changes in surface fraction of GluA1 and GluA2 in response to oAβ at different times after stimulation. *, p < 0.05; **, p < 0.01; ***, p < 0.001. F, Western blot and percentage of GluA1 (left) and GluA2 (middle) and GluA1/GluA2 ratio (right) in basal and oAβ at 60 min. Surface values are the ratio between surface and total amounts of each subunit. oAβ alters the GluA1/GluA2 ratio (n = 3–6). Error bars indicate ± S.E. *, p < 0.05.

FIGURE 2.

oAβ affects the interaction between GluA1 and GluA2. A, representative immunoblots for GluA1 immunoprecipitation (IP) experiments. Almost all GluA1 subunits were pulled down by the GluA1 C-terminal antibody, as observed with no GluA1 signal in the unbound lane. GluA2 subunits in the unbound lane are likely GluA2/3 heteromers. B–D, graphs represent quantified changes in GluA1/2 heteromers. oAβ affects interaction between GluA1 and GluA2, inducing a decrease of GluA1/2 heteromers. *, p < 0.05; **, p < 0.01; ***,p < 0.001. (n = 6). Error bars indicate ± S.E.

FIGURE 3.

oAβ increases intracellular calcium into primary neurons and reduces surface expression of AMPAR through ionotropic glutamate receptors. A, oAβ induces a rapid and sustained increase in [Ca²⁺]_i in primary neurons. Cells were loaded with fura2/AM and subjected to calcium imaging. Images show a phase-contrast image and [Ca²⁺]_i levels before (resting calcium) and after treatment with oAβ (oligomers). B, traces correspond to 52 representative neurons (n ≥ 150 cells in three experiments) for oAβ treatment. In vehicle control, cells were treated with DMEM/F-12. C, effect of calcium chelator BAPTA-AM on GluA1 internalization. Neurons were pre-treated with 20 μm BAPTA-AM followed by treatment with 5 μm oAβ for 30 min. The amounts of surface GluA1 were assessed by surface biotinylation. Values indicate mean ± S.E. normalized to basal.*, p < 0.05 basal versus oAβ; ^#, p < 0.05 oAβ versus oAβ plus BAPTA-AM (n = 4). D, ionotropic glutamate receptor antagonists (MK-801 for NMDAR and CNQX for AMPAR) prevent oAβ-induced surface GluA1-containing AMPAR loss. Primary cultures were treated with oAβ for 30 min in the presence or absence of MK-801 (10 μm) and CNQX (50 μm). Changes in surface GluA1 subunit were examined by surface biotinylation. Values indicate mean ± S.E. normalized to basal. *, p < 0.05 basal versus oAβ; ^#, p < 0.05 oAβ versus oAβ plus MK-801 and oAβ plus CNQX (n = 5).

FIGURE 4.

oAβ reduces surface expression and dephosphorylation at Ser-845 of GluA1 in a calcineurin-dependent manner. A, increased calcineurin activity in cultured neurons in presence of oAβ (5 μm). Neurons were treated with oAβ at indicated times and calcineurin activity was determined (see “Experimental Procedures” for details). B, immunoblots showing expression of calcineurin in total lysates from cultured neurons treated with oAβ (5 μm). Neither catalytic subunit (calcineurin A; upper panel) nor regulatory subunit (calcineurin B; lower panel) amounts were affected by oAβ treatment. Values indicate mean ± S.E. normalized to basal (n = 4). C and D, effect of a calcineurin-selective inhibitor (FK-506) on GluA1 internalization. Neurons were pre-treated with 10 μm FK-506 followed by treatment with 5 μm oAβ for 30 min. C, representative blot showing phosphorylation levels of Ser-845 (upper blot) and total GluA1 (lower blot). D, representative blots showing surface GluA1 (upper blot) and total GluA1 (lower blot). Note that FK-506 alone is affecting basal levels of Ser-845 phosphorylation but has no effect on surface GluA1 compared with basal levels. E, graph represents quantified changes in phosphorylated Ser-845 (left axis) and surface GluA1 (right axis) in response to oAβ. *, p < 0.05 basal versus oAβ; ^#, p < 0.05 oAβ versus oAβ plus FK-506. (n = 5); bars represent mean ± S.E. normalized to basal.

FIGURE 5.

oAβ-mediated block of phosphorylation at Ser-845 impairs AMPAR priming for synaptic incorporation. Neurons (pre-treated or not with 5 μm oAβ during 30 min) were stimulated with forskolin/rolipram (F/R; 50 μm/0.1 μm) for 30 or 60 min. A, sample blots showing phosphorylation levels of Ser-845 (upper blot), total GluA1 (middle blot), and GAPDH protein level (lower blot) as loading control. The graph represents quantified changes in phosphorylated subunit in response to F/R stimulation compared with baseline. B, sample blots showing surface GluA1 (upper blot) and total GluA1 (lower blot). The graph represents quantified changes in surface GluA1 in response to F/R stimulation compared with baseline. oAβ treatment during 30 min impair Ser-845 phosphorylation induced by F/R stimulation affecting GluA1 surface delivery. *, basal versus oAβ or F/R; ^# and ^§, F/R versus oAβ plus F/R. *, p < 0.05; **, p < 0.01; ^#, p < 0.05; ^§, p < 0.001 (n = 6); bars represent mean ± S.E. normalized to basal.

FIGURE 6.

Reduced cell surface AMPARs in APP_Sw,Ind neurons. Primary neurons were prepared from APP_Sw,Ind mice or wild-type littermates in the presence or absence of DAPT (1 μm during 72 h). Surface expression of GluA1 subunit was analyzed by biotinylation at 12 days in vitro. A, surface expression of GluA1 was reduced in neurons from APP_Sw,Ind mice but was restored partially by DAPT (upper blot). The total amount of GluA1 was not affected by APP_Sw,Ind expression (middle blot). DAPT treatment shows light reduction in protein expression (lower blot, GAPDH from total extract). B, quantification of treated primary neurons. Bars represent mean ± S.E. (n = 3 embryos/genotype). ***, p < 0.001 WT versus APP_Sw,Ind; ^#, p < 0.01 APP_Sw,Ind versus APP_Sw,Ind plus DAPT.

FIGURE 7.

APP_Sw,Ind mice show lower levels of phosphorylated GluA1 at Ser-845 at 6 months of age. A, APP_Sw,Ind transgenic mice display learning deficits in the Morris water maze. Six-month-old littermate APP_Sw,Ind and non-transgenic control mice (n = 4 WT/n = 6 APP_Sw,Ind genotype) were trained in the Morris water maze for 5 days. APP_Sw,Ind mice learned the task at 5 days, but they required significantly longer latencies to locate the platform after 2 days of training (two-way analysis of variance; latencies: genotype effect, F₍₁₎ = 10.71; day effect, F₍₄₎ = 24.38; p < 0.0001). Data represent the mean ± S.E. **, p < 0.01. B, representative immunoblots of hippocampal total protein extract from naive, 2 days and 5 days after Morris water maze task mice. Extracts were probed with the indicated antibodies. C, densitometric quantification of changes expressed as mean ± S.E. (naive wild type is indicated as 100%). *, p < 0.05.