GluA1 subunit ubiquitination mediates amyloid-β-induced loss of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors

Abstract

The accumulation of soluble amyloid-β (Aβ) peptides produces profound neuronal changes in the brain during the pathogenesis of Alzheimer's disease. Excessive levels of Aβ disrupt excitatory synaptic transmission by promoting the removal of synaptic AMPA receptors (AMPARs), dendritic spine loss, and synaptic depression. Recently, activity-dependent ubiquitination of the GluA1 subunit has been shown to regulate the intracellular sorting of AMPARs toward late endosomes for degradation. However, whether this ubiquitin signaling pathway mediates Aβ-induced loss of surface AMPARs is unknown. In this study, we demonstrate that acute exposure of cultured neurons to soluble Aβ oligomers induces AMPAR ubiquitination concomitant with the removal of AMPARs from the plasma membrane. Importantly, expression of the GluA1 ubiquitin-deficient mutants inhibited the adverse effects of Aβ on the surface expression of AMPARs in neurons. Furthermore, we revealed the cross-talk between GluA1 ubiquitination and phosphorylation, in particular phosphorylation at Ser-845, which is crucial for AMPAR recycling and is known to be dephosphorylated in the presence of Aβ. Our data showed that the GluA1 ubiquitin-deficient mutant enhances GluA1 phosphorylation on Ser-845. Conversely, the GluA1 S845D phosphomimetic mutant reduced binding with Nedd4-1 and hence the ubiquitination of AMPARs. Importantly, the GluA1 S845D mutant also prevented Aβ-induced removal of surface AMPARs. Taken together, these findings provide the first demonstration of the dynamic cross-modulation of GluA1 ubiquitination and phosphorylation, a process that is perturbed by Aβ, in regulating the membrane sorting decision that ultimately determines the number of AMPARs on the cell surface.

Keywords: amyloid-β (Aβ); phosphorylation; trafficking; ubiquitylation (ubiquitination); α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA receptor, AMPAR).

Figure 1.

Aβ induces the ubiquitination of the GluA1 subunit of AMPARs in primary neurons. A, cultured neurons were incubated with DMSO (Veh, vehicle control) or 5 μm Aβ for 1 h and immediately lysed in 1% SDS. Diluted lysates were immunoprecipitated with anti-GluA1 antibodies (IP: GluA1). Eluted proteins were subjected to Western blot analysis and probed with anti-ubiquitin and anti-GluA1 antibodies. Ubiquitinated GluA1 is indicated by the vertical bar (Ub-GluA1). The two right-hand lanes represent biological replicates. B, the effect of Aβ treatment on GluA1 ubiquitination was quantified and normalized to a DMSO control (Ctrl). Data are represented as the mean of five independent experiments (Mann-Whitney test; **, p < 0.01; n = 5). C, cultured neurons were incubated with increasing concentrations of Aβ for 1 h and subjected to the ubiquitination assay. D, a dose-response curve of Aβ effects on the ubiquitination of GluA1. Data are represented as the mean of four independent experiments (one-way ANOVA; *, p < 0.05; ***, p < 0.001; n = 4). E, representative immunoblots depicting Aβ-induced ubiquitination of the GluA1 subunit of AMPARs following immunoprecipitation assays using anti-ubiquitin antibodies (IP: Ub) (from two independent experiments). Error bars represent S.E.

Figure 2.

GluA1 ubiquitination is necessary for the Aβ-induced reduction of surface AMPARs. A, cortical neurons were electroporated with pH-GluA1 constructs, either WT or ubiquitin-deficient mutants as indicated, prior to plating. qKR contains quadruple mutations of Lys-813, Lys-819, Lys-822, and Lys-868 in the GluA1 C-terminal tail into arginines. At DIV14, neurons were treated with 5 μm Aβ or DMSO for 1 h and subjected to a surface (Surf.) biotinylation assay prior to cell lysis. Neuronal lysates were then incubated with NeutrAvidin beads to purify surface receptors. Eluted proteins and total cell lysates were subjected to Western blot analysis and probed with anti-GluA1 and anti-β-actin antibodies. The effects of Aβ on the levels of surface (B) and total (C) receptor expression were quantified as surface/total receptor ratios and as total receptor/β-actin ratios, respectively, and normalized to DMSO controls (Ctrl). Data represent the mean of three independent experiments (Mann-Whitney test; **, p < 0.01; n.s., not significant; n = 5). Error bars represent S.E. Veh, vehicle.

Figure 3.

GluA1 ubiquitination is necessary for the Aβ-induced reduction of surface AMPARs. A, cortical neurons were transfected with pH-GluA1 constructs, either WT or ubiquitin-deficient mutants as indicated, at DIV12. qKR contains quadruple mutations of Lys-813, Lys-819, Lys-822, and Lys-868 in the GluA1 C-terminal tail into arginines. At DIV14, neurons were treated with 5 μm Aβ or DMSO (Veh) for 1 h and incubated with anti-GFP antibodies prior to fixation to visualize surface pH-GluA1 expression (red). Total pH-GluA1 expression was determined with the endogenous GFP signal (green), whereas the level of synaptophysin was visualized by immunostaining with anti-synaptophysin antibodies (blue). Scale bar, 10 μm. The effects of Aβ on the levels of surface receptor expression (B) and synaptophysin (C) were quantified as surface/total receptor ratios and as total synaptophysin staining per dendritic area, respectively, and normalized to DMSO controls (Ctrl). Data represent the mean of two independent experiments (Mann-Whitney test; ***, p < 0.001; n.s., not significant; n = 30). Error bars represent S.E.

Figure 4.

Ubiquitination negatively regulates GluA1 phosphorylation at Ser-845. Cortical neurons were electroporated with pH-GluA1 constructs, either WT or ubiquitin-deficient mutants as indicated, prior to plating. qKR contains quadruple mutations of Lys-813, Lys-819, Lys-822, and Lys-868 in the GluA1 C-terminal tail into arginines. At DIV14, neurons were treated with 20 μm forskolin (A), 0.1 μm PMA (C), or DMSO for 10 min and immediately lysed with 1× SDS sample buffer. Total cell lysates were subjected to Western blot analysis and probed with anti-GluA1 phospho-Ser-845, anti-GluA1 phospho-Ser-831, and anti-GluA1 antibodies. The effects of GluA1 ubiquitin-deficient mutants on the phosphorylation levels at Ser-845 (B) and Ser-831 (D) were quantified as phospho-/total receptor ratios and normalized to wild-type controls. Data represent the mean of five independent experiments (one-way ANOVA; *, p < 0.05; n.s., not significant; n = 12). Error bars represent S.E.

Figure 5.

The S845D phosphomimetic mutant blocks GluA1 ubiquitination by reducing its interaction with Nedd4-1. A, cortical neurons were electroporated with pH-GluA1 constructs, either WT, phosphodeficient (alanine mutations), or phosphomimetic (aspartate mutations) as indicated, prior to plating. At DIV14, neurons were treated with 100 μm AMPA in the presence of 1 μm tetrodotoxin and 50 μm d-2-amino-5-phosphonovaleric acid for 10 min and immediately lysed in 1% SDS. Diluted lysates were immunoprecipitated with anti-GFP antibodies (IP: GFP). Eluted proteins were subjected to Western blot analysis and probed with anti-ubiquitin (Ub) and anti-GluA1 antibodies. B, the effects of GluA1 phosphorylation mutants on agonist-induced ubiquitination were quantified and normalized to the wild-type control. Data are represented as the mean of three independent experiments (one-way ANOVA; **, p < 0.01; n = 4). C, HEK293 cells were transfected with HA-Nedd4-1 alone (−; lane 1) or together with pH-GluA1 constructs, either wild type, phosphodeficient (alanine mutations), or phosphomimetic (aspartate mutations) as indicated. Cells were lysed 48 h later and subjected to immunoprecipitation assays with anti-GFP antibodies (IP: GFP). Total cell lysates and eluted proteins were subjected to Western blot analysis and probed with anti-HA and anti-GluA1 antibodies. D, the effects of GluA1 phosphorylation mutants on HA-Nedd4-1 binding were quantified and normalized to the wild-type control. Data are represented as the mean of three independent experiments (one-way ANOVA; *, p < 0.05; n = 3). Error bars represent S.E.

Figure 6.

The GluA1 S845D phosphomimetic mutant prevents the Aβ-induced reduction of surface AMPARs. A, cortical neurons were electroporated with pH-GluA1 constructs, either wild type, phosphodeficient (alanine mutations), or phosphomimetic (aspartate mutations) as indicated, prior to plating. At DIV14, neurons were treated with 5 μm Aβ or DMSO for 1 h and subjected to the surface (Surf.) biotinylation assay prior to cell lysis. Neuronal lysates were then incubated with NeutrAvidin beads to purify surface receptors. Eluted proteins and total cell lysates were subjected to Western blot analysis and probed with anti-GluA1 and anti-β-actin antibodies. The effects of Aβ on the levels of surface (B) and total (C) receptor expression were quantified as surface/total receptor ratios and as total receptor/β-actin ratios, respectively, and normalized to DMSO controls (Ctrl). Data represent the mean of three independent experiments (Mann-Whitney test; **, p < 0.01; ***, p < 0.001; n.s., not significant; n = 5). Error bars represent S.E. Veh, vehicle.

Figure 7.

Proposed model for the role of protein ubiquitination in mediating Aβ-induced down-regulation of surface AMPARs. A, under normal conditions, the majority of internalized AMPARs are recycled back to the plasma membrane, a process that is facilitated by PKA phosphorylation of the GluA1 subunit at Ser-845. The phosphorylation of GluA1 prevents the ubiquitination of AMPARs as a result of which receptor degradation is maintained at a low level for normal protein homeostasis. B, however, in the presence of high concentrations of soluble oligomeric Aβ, AMPARs are constantly endocytosed due to overactivation of the protein phosphatase calcineurin (CaN) and the E3 ubiquitin ligase Nedd4-1. This in turn leads to an increase in the level of GluA1 ubiquitination in part due to the down-regulation of Ser-845 phosphorylation. As a consequence, the recycling of AMPARs back to the plasma membrane (and therefore their expression on the cell surface) decreases, leading to synaptic depression. Ctrl, control; Ub, ubiquitin.