Tetrapeptide Ac-HAEE-NH2 Protects α4β2 nAChR from Inhibition by Aβ

Abstract

The cholinergic deficit in Alzheimer's disease (AD) may arise from selective loss of cholinergic neurons caused by the binding of Aβ peptide to nicotinic acetylcholine receptors (nAChRs). Thus, compounds preventing such an interaction are needed to address the cholinergic dysfunction. Recent findings suggest that the ¹¹EVHH¹⁴ site in Aβ peptide mediates its interaction with α4β2 nAChR. This site contains several charged amino acid residues, hence we hypothesized that the formation of Aβ-α4β2 nAChR complex is based on the interaction of ¹¹EVHH¹⁴ with its charge-complementary counterpart in α4β2 nAChR. Indeed, we discovered a ³⁵HAEE³⁸ site in α4β2 nAChR, which is charge-complementary to ¹¹EVHH¹⁴, and molecular modeling showed that a stable Aβ₄₂-α4β2 nAChR complex could be formed via the ¹¹EVHH¹⁴:³⁵HAEE³⁸ interface. Using surface plasmon resonance and bioinformatics approaches, we further showed that a corresponding tetrapeptide Ac-HAEE-NH₂ can bind to Aβ via ¹¹EVHH¹⁴ site. Finally, using two-electrode voltage clamp in Xenopus laevis oocytes, we showed that Ac-HAEE-NH₂ tetrapeptide completely abolishes the Aβ₄₂-induced inhibition of α4β2 nAChR. Thus, we suggest that ³⁵HAEE³⁸ is a potential binding site for Aβ on α4β2 nAChR and Ac-HAEE-NH₂ tetrapeptide corresponding to this site is a potential therapeutic for the treatment of α4β2 nAChR-dependent cholinergic dysfunction in AD.

Keywords: Alzheimer’s disease; cholinergic deficit; molecular modeling; nicotinic acetylcholine receptor; peptide drugs; β-amyloid.

Figure 1

Model of the interaction of the α4β2 nAChR site ³⁵HAEE³⁸ with Aβ₄₂ after 100 ns of molecular dynamics structure equilibration. (A) Model of the α4β2 structure with bound Aβ₄₂ peptide, viewed from the extracellular side. (B) Detailed view of the interaction interface. The Aβ₄₂ peptide is colored green with the ¹¹EVHH¹⁴ site shown in cyan. The ³⁵HAEE³⁸ site is colored magenta. The N-terminal α-helix of both α4 and β2 subunits is colored red.

Figure 2

Global docking of Ac-HAEE-NH₂ to Aβ₄₂. (A) A histogram of Aβ₄₂ atomic contacts to the Ac-HAEE-NH₂ tetrapeptide for the data from six docking servers. The position of the ¹¹EVHH¹⁴ site is highlighted in red. Calculated by QASDOM [20] metaserver. (B,C) Examples of the docked Ac-HAEE-NH₂ peptide. The Aβ₄₂ peptide is colored green, with the ¹¹EVHH¹⁴ site shown in cyan, and the Ac-HAEE-NH₂ tetrapeptide is colored magenta.

Figure 3

Sensorgrams showing direct binding of Ac-HAEE-NH₂ (1 mM–2 mM) to immobilized Aβ₁₆. Spikes at the start and end of Ac-HAEE-NH₂ injections are due to a slight time delay in the reference cell and appear when reference subtraction is carried out.

Figure 4

Global docking of Ac-HAEE-NH₂ to Aβ₁₆ (A,B) Examples of the docked Ac-HAEE-NH₂ peptide. The Aβ₁₆ peptide is colored green with the ¹¹EVHH¹⁴ site shown in cyan, and the Ac-HAEE-NH₂ tetrapeptide is colored magenta. (C) The proposed interface of HAEE-EVHH interaction based on a docking model.

Figure 5

(A) Representative ion current traces and (B) normalized amplitudes of ACh (100 μM)-induced ion currents in α4β2 nAChR-expressing Xenopus laevis oocytes in control and after 3 min pre-incubation with 10 µM Aβ₄₂ (“Aβ₄₂”), 10 µM Aβ₄₂ and 100 µM Ac-HAEE-NH₂ (“HAEE + Aβ₄₂”), or 10 µM Aβ₄₂ followed by washout with Barth’s solution containing 100 µM of Ac-HAEE-NH₂ (“HAEE after Aβ₄₂”). (B) Individual current amplitude values are depicted as black dots. (C) Normalized ACh (100 μM)-induced current amplitudes in α4β2 nAChR-expressing Xenopus laevis oocytes in control and after 3 min pre-incubation with 10 µM Aβ₄₂, followed by 3 min washout with Barths’ solution in the absence (“Aβ₄₂”) or presence (“HAEE”) of Ac-HAEE-NH₂. (A,C) Data are presented as mean ± SD, n ≥ 3. *—p < 0.05, **—p < 0.005, ***—p < 0.001, ****—p < 0.0001, ns—nonsignificant. (D) The leakage current in α4β2 nAChR-expressing Xenopus laevis oocytes was measured after 3 min consecutive incubations in Barth’s solution (“Barth”), in Barth’s solution containing 10 µM Aβ₄₂ (“Aβ₄₂”), and in Barth’s solution in the absence (“Barth”) and presence of 100 µM Ac-HAEE-NH₂ (“HAEE”).

Figure 6

The possible role of ¹¹EVHH¹⁴:³⁵HAEE³⁸ interface in cholinergic deficit associated with Alzheimer’s disease. In brains of Alzheimer’s disease patients, interaction of Aβ with nAChRs causes transition of the receptor from functional state (green) to dysfunctional state (violet), which may lead to selective loss of cholinergic neurons (top left). Our results suggest that the interaction of Aβ with α4β2 nAChR is mediated by charge complementary interface ¹¹EVHH¹⁴:³⁵HAEE³⁸ (bottom left and middle) and that Ac-HAEE-NH₂ peptide corresponding to this interface can competitively displace Aβ from the complex and restore the functionality of α4β2 nAChR (top right).