Characterization of the interaction of [beta]-amyloid with the agonist binding domain of [alpha]7 nicotinic acetylcholine receptors
Authors:   , Tong Mei , Nichols Robert A 
Published: July 2024

Alzheimer's Disease (AD) is a degenerative neurological disorder characterized by neuronal loss, visible extracellular senile plaques, and intracellular neurofibrillary tangles. Plaques result largely from dense accumulations of the peptide beta amyloid (A[beta]). On the other hand, at the earliest stage of the disease, A[beta] in a soluble, oligomeric state increases significantly in the brain. It has been proposed that this early accumulation of A[beta] triggers progression to AD. Though targets for A[beta] have yet to be clearly identified, one of the possibilities is the homomeric [alpha]7 nicotinic acetylcholine receptor ([alpha]7-nAChR) present on presynaptic nerve terminals. We have previously shown that picomolar to nanomolar soluble A[beta] induced increases in presynaptic Ca²⁺ via [alpha]7-nAChRs reconstituted in a model nerve cell system, the NG108-15 neuroblastoma cell line, indicating a potent agonist-like action of A[beta]. Here, I first demonstrated that A13's activation requires the extracellular domain of the [alpha]7-nAChR. To delineate the specific interaction sites for A[beta]'s agonist-like action on [alpha]7-nAChRs, I focused on the "aromatic cluster" in the ligand-binding domain of the receptor. This cluster includes tyrosines(Y) at positions 188, 195 and 93 and tryptophan(W) at position 149, and all of these residues have been shown to be important in agonist binding. These amino acids were mutated either to phenylalanine, serine or alanine. By recording increases in presynaptic [Ca²⁺]i upon [alpha]7-nAChR activation, responses of Y188 mutants of [alpha]7-nAChR to A[beta] or ACh stimulation were nearly completely abolished. In contrast, responses of Y188 mutants to nicotine were unaffected. Mutation of Y195 had the opposite effect: responses to nicotine were abolished, while responses to Al3 or ACh were unaffected. Mutations of the other aromatic residues, Y93 or W149, had little or no effect. A[beta] was found to bind to the Y188 mutant, as assessed via co-immunoprecipitation. Together, these results indicate a critical role for Y188 in the activation of [alpha]7-nAChR by A[beta] in the absence of a significant change in binding. This tyrosine in the snail acetylcholine binding protein (AChBP) as previously found to interact with the quaternary amine of the acetylcholine derivative carbamylcholine in X-ray crystallographic analysis, suggesting a possible cation-[pi] interaction for its interaction in [alpha]7-nAChRs with A[beta]. In complementary experiments, the roles of the hydrophilic and hydrophobic domains in A[beta] in the activation of presynaptic [alpha]7-nAChRs were investigated using various A[beta] fragments. The hydrophilic A[beta] peptides, A[beta]12-28, A[beta]1-15 and A[beta] 17-28, evoked larger [Ca²⁺]i responses of [alpha]7- nAChR in comparison to full-length A[beta]1-42. These fragments did not show any fibrillogenesis, but did form oligomers as assessed with gel electrophoretic analysis. A hydrophobic fragment of the peptide, A[beta]33-42, had little effect over background. In summary, the aromatic residue Y188 was shown to be a key component in the ligandbinding domain of the [alpha]7-nAChR in the regulation of the receptor on presynaptic terminals by A[beta]. The activation by A[beta] of the [alpha]7-nAChR correlated with the hydrophilic domain of the A[beta] peptide. As A[beta] is produced at the synapse, regulation of presynaptic [alpha]7-nAChRs by picomolar to nanomolar soluble A[beta] may control synaptic activity and plasticity.