Intracellular oligomeric amyloid-beta rapidly regulates GluA1 subunit of AMPA receptor in the hippocampus

Abstract

The acute neurotoxicity of oligomeric forms of amyloid-β 1-42 (Aβ) is implicated in the pathogenesis of Alzheimer's disease (AD). However, how these oligomers might first impair neuronal function at the onset of pathology is poorly understood. Here we have examined the underlying toxic effects caused by an increase in levels of intracellular Aβ, an event that could be important during the early stages of the disease. We show that oligomerised Aβ induces a rapid enhancement of AMPA receptor-mediated synaptic transmission (EPSC(A)) when applied intracellularly. This effect is dependent on postsynaptic Ca(2+) and PKA. Knockdown of GluA1, but not GluA2, prevents the effect, as does expression of a S845-phosphomutant of GluA1. Significantly, an inhibitor of Ca(2+)-permeable AMPARs (CP-AMPARs), IEM 1460, reverses the increase in the amplitude of EPSC(A). These results suggest that a primary neuronal response to intracellular Aβ oligomers is the rapid synaptic insertion of CP-AMPARs.

Figure 1. Generation of lower-n oligomers of Aβ1-42 (Aβ)

(a) A schematic of the principle of single molecule two-colour fluorescence coincidence detection and analysis of oligomers. The protein is labeled with a red or blue fluorophore and aggregated. The sample is then diluted to picomolar concentrations and analysed using single molecule fluorescence. Monomers passing through the probe volume give rise to non-coincident bursts of fluorescence while oligomers give rise to coincident fluorescent bursts, enabling the fraction of oligomers present in the sample to be determined. The intensity of a coincident burst relative to average monomer bursts was determined, allowing the oligomer size to be estimated. (b) Histogram depicting the proportion of monomers and oligomers. (c) Histogram depicting the size distribution of oligomers present in the preparation of Aβ oligomers.

Figure 2. Intracellular infusion of Aβ causes a rapid increase in the AMPAR-mediated EPSC (EPSCA).

(a) The infusion of 1–5 nM oligomeric Aβ into post-synaptic neurons induces a rapid increase in EPSC_A (n = 7). (b) Monomeric Aβ did not induce an increase in EPSC_A (n = 6). (c) Clusterin (500 nM) prevented the Aβ oligomer-induced facilitation of EPSC_A (n = 6). (d) The increase in EPSC_A is independent of synaptic activity (n = 6). Filled circles depict Aβ infused neurons and open circles depict control neurons. (e) An NMDAR-antagonist, D-AP5 (50 M) has no effect on the Aβ oligomer-induced facilitation of EPSC_A (n = 6). (f) The NMDAR mediated EPSC (EPSC_N) is unaffected by infusion of Aβ oligomers (n = 6). In this (and subsequent figures) graphs plot the mean ± S.E.M. of n experiments.

Figure 3. Aβ oligomer-induced increase in EPSCA is dependent on Ca²⁺ and PKA.

(a) Neurons were infused with Aβ oligomer in the presence of BAPTA (10 mM) in the filling solution. This prevented the Aβ oligomer facilitated increase in EPSC_A (n = 7). (b) Ryanodine infusion via the pipette prevented the Aβ facilitated increase in EPSC_A (n = 7). (c) There was no increase in EPSC_A following preincubation (30 min) with RP-cAMPS (100 μM) (n = 6). (d) H89 infusion via the pipette prevented the Aβ facilitated increase in EPSC_A (n = 6). (e) Ro 32-0432 (10 μM) infusion via the pipette had no effect on the Aβ-mediated increase of EPSC_A (n = 6). (f) PKC 19-31 infusion via the pipette did not prevent the Aβ facilitated increase in EPSC_A (n = 6). (g) KN-62 (10 μM) preincubation (45 min) and infusion via the pipette prevented the sustained Aβ facilitation of EPSC_A (n = 7).

Figure 4. Aβ oligomer-induces expression of CP-AMPARs

(a) Aβ failed to increase EPSCA in GluA1-shRNA transfected cells (n = 7). (b) Aβ oligomers infusion increases EPSC_A in GluA2-shRNA transfected cells (n = 8). (c) Aβ failed to increase EPSC_A in GluA1-S845 phosphomutant transfected cells (n = 7). (d) Bath application of IEM 1460 (100 μM) has no effect on basal transmission EPSC_A (n = 6). (e) The Aβ oligomer-mediated increase in EPSC_A is reduced by bath application of IEM (n = 8).

Figure 5. Exogenous application of Aβ induces GluA1 surface expression.

(a) Aβ treatment caused an increase in the surface expression of GluA1, but not GluA2/3 as shown through a biotinylation assay. (b) Exogenous application of Aβ caused an increase in EPSC_A (n = 6), (c) which was prevented when slices were perfused with IEM (n = 6).