Oligomeric amyloid-{beta} inhibits the proteolytic conversion of brain-derived neurotrophic factor (BDNF), AMPA receptor trafficking, and classical conditioning

Abstract

Amyloid-β (Aβ) peptide is thought to have a significant role in the progressive memory loss observed in patients with Alzheimer disease and inhibits synaptic plasticity in animal models of learning. We previously demonstrated that brain-derived neurotrophic factor (BDNF) is critical for synaptic AMPA receptor delivery in an in vitro model of eyeblink classical conditioning. Here, we report that acquisition of conditioned responses was significantly attenuated by bath application of oligomeric (200 nm), but not fibrillar, Aβ peptide. Western blotting revealed that BDNF protein expression during conditioning is significantly reduced by treatment with oligomeric Aβ, as were phosphorylation levels of cAMP-response element-binding protein (CREB), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV), and ERK. However, levels of PKA and PKCζ/λ were unaffected, as was PDK-1. Protein localization studies using confocal imaging indicate that oligomeric Aβ, but not fibrillar or scrambled forms, suppresses colocalization of GluR1 and GluR4 AMPA receptor subunits with synaptophysin, indicating that trafficking of these subunits to synapses during the conditioning procedure is blocked. In contrast, coapplication of BDNF with oligomeric Aβ significantly reversed these findings. Interestingly, a tolloid-like metalloproteinase in turtle, tTLLs (turtle tolloid-like protein), which normally processes the precursor proBDNF into mature BDNF, was found to degrade oligomeric Aβ into small fragments. These data suggest that an Aβ-induced reduction in BDNF, perhaps due to interference in the proteolytic conversion of proBDNF to BDNF, results in inhibition of synaptic AMPA receptor delivery and suppression of the acquisition of conditioning.

FIGURE 1.

Monomeric and oligomeric Aβ preparation. Western blots displaying the formation of Aβ_1–42 monomers, oligomers, and fibrils in the preparations of Aβ used to test conditioning are shown. Oligomeric Aβ_1–42 and fibrillar Aβ_1–42 were prepared as described.

FIGURE 2.

Oligomeric but not fibrillar Aβ attenuates the acquisition of in vitro classical conditioning. A, physiological records of abducens nerve recordings taken from experiments in which brainstems were treated with oligomeric or fibrillar Aβ. Both traces show an abducens nerve unconditioned response recorded in the second pairing session after Aβ application. A CR is indicated (arrow) in the record from a preparation treated with fibrillar Aβ. The CS and US are indicated at bottom. B, acquisition curves of the percentage of CRs during the acquisition phase of conditioning. Oligomeric (Oligo), but not fibrillar or scrambled Aβ peptides, significantly suppressed acquisition of CRs as compared with normal conditioning. Asterisks indicate significant differences from pseudoconditioning (p values under “Results”).

FIGURE 3.

Oligomeric Aβ attenuates BDNF protein expression during conditioning. Western blots demonstrating that preparations treated by oligomeric Aβ (AβO; 200 nm) for two pairing sessions resulted in suppression of BDNF protein expression are shown, whereas treatment with fibrillar (AβF) or scrambled (AβS) Aβ showed levels of BDNF expression comparable with normal conditioning (C2). Actin loading controls are also shown. % Ps, percentage of pseudoconditioning. Asterisks indicate significant differences from pseudoconditioning (p values under “Results”).

FIGURE 4.

Oligomeric Aβ blocks the conditioning-related phosphorylation of some protein kinases but not others. Western blots show that treatment with the oligomeric Aβ for two pairing sessions inhibited the phosphorylation levels of CaMKII (B), CaMKIV (C), CREB (D), and ERK (E) to pseudoconditioned values, whereas activation of PKA (A) and PKCζ/λ (F) was not significantly affected. Treatment with fibrillar or scrambled Aβ resulted in little change in the elevated state of protein kinase phosphorylation as compared with normal untreated conditioning. % Ps, percentage of pseudoconditioning. Asterisks indicate significant differences from pseudoconditioning (p values under “Results”).

FIGURE 5.

Real-time RT-PCR analysis of the level of the total tolloid-like metalloproteinase (tTLL), cytosolic tTLL (tTLLc), and secreted tTLL (tTLLs) mRNA shows that oligomeric Aβ treatment does not reduce expression of tTLLs. Preparations were conditioned (C1) or pseudoconditioned (Ps1) for one pairing session in normal saline or treated with oligomeric Aβ for the same period of time and not conditioned (AβO) or conditioned in the presence of Aβ (C1+AβO). Error bars are S.D. Asterisks indicate significant differences from pseudoconditioning (p values under “Results”).

FIGURE 6.

Synaptic localization of GluR1- and GluR4-containing AMPA receptors is inhibited by oligomeric Aβ treatment during conditioning and rescued by BDNF. A, confocal images of GluR1 or GluR4 AMPA receptor subunits (red) and colocalization with synaptophysin (Syn; green) punctate staining. Overlapping (yellow) or adjacent puncta indicate colocalization. C2, normal conditioning. B, quantitative analysis of colocalized staining for GluR1+Syn or GluR4+Syn for the different treatment groups. Treatment with oligomeric Aβ during conditioning resulted in inhibition of CRs and synaptic delivery of GluR1 and GluR4 subunits, whereas application of fibrillar or scrambled Aβ resulted in CR expression and levels of colocalized staining similar to untreated conditioned preparations. The inhibition of colocalized staining observed after oligomeric Aβ treatment was reversed by coapplication of oligomeric Aβ with BDNF. Scale bar = 2 μm. Asterisks indicate significant differences from pseudoconditioning (Ps2) (p values under “Results”).

FIGURE 7.

The tolloid-like metalloproteinase, tTLLs, cleaves oligomeric Aβ at lower concentrations than proBDNF in in vitro cleavage assays. Synthetic human oligomeric Aβ was incubated either alone or with recombinant turtle proBDNF and increasing concentrations of tTLLs for 1 h at 25 °C. Soluble Aβ, particularly the oligomeric forms, was likely cleaved into small fragments by tTLLs, which was complete at 50 nm. In the same assays, tTLLs began to cleave proBDNF into BDNF at about 100 nm. IB, immunoblot.

FIGURE 8.

Incubation of in vitro brainstem preparations in tTLLs resulted in expression of mature BDNF protein that was inhibited by oligomeric Aβ. Western blots of proBDNF and mature BDNF show expression of BDNF protein after incubation in tTLLs alone. Incubation in tTLLs and oligomeric Aβ (200 nm) resulted in significant attenuation of BDNF expression. Asterisks indicate significant differences from pseudoconditioning (p values under “Results”).