β‐Amyloid activates presynaptic α7 nicotinic acetylcholine receptors reconstituted into a model nerve cell system: involvement of lipid rafts

Abstract

Beta amyloid (Abeta) plays a central role in the pathogenesis of Alzheimer's disease. Abeta is the major constituent of senile plaques, but there is a significant presence of Abeta in the brain in soluble forms. The results of functional studies indicate that soluble Abeta interacts with the alpha7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Abeta-alpha7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist-like and antagonist-like effects have been reported. In particular, agonist-like effects have been observed for presynaptic nAChRs. Here, we demonstrate Abeta(1-42)-evoked stimulatory changes in presynaptic Ca(2+) level via exogenous alpha7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108-15 cells as a model, presynaptic system. The Abeta(1-42)-evoked responses were concentration-dependent and were sensitive to the highly selective alpha7 nAChR antagonist alpha-bungarotoxin. Voltage-gated Ca(2+) channels and internal Ca(2+) stores were both involved in Abeta(1-42)-evoked increases in presynaptic Ca(2+) following activation of alpha7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Abeta(1-42), whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist-like action of pico- to nanomolar concentrations of soluble Abeta(1-42) on the presynaptic nerve terminal, including the possible involvement of receptor-associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.

Role of Key Aromatic Residues in the Ligand-binding Domain of α7 Nicotinic Receptors in the Agonist Action of β-Amyloid

“…Mock cells were treated with FuGENE 6 alone. Successful expression of ␣7-nAChRs required another 48 h, and functional responses of ␣7-nAChRs in varicosities were monitored by confocal imaging (23). Moderate expression levels were typically obtained, allowing for detection of negative or positive changes following receptor mutation.…”

“…Confocal Imaging-Changes in Ca 2ϩ level in individual varicosities of differentiated NG108-15 cells were monitored by the specific Ca 2ϩ fluorescent dye Fluo-4 as described previously (23). In brief, Fluo-4 was loaded into the cells cultured on CellTak-coated coverslips as the acetoxymethylester derivative (Invitrogen).…”

“…Cell Cultures and Transfection-NG108-15 cells were cultured in DMEM containing 15% FBS and differentiated in 1% FBS-containing DMEM on exposure to dibutyryl cyclic AMP (1 mM, Sigma Aldrich) for 48 h until the presence of neurites and varicosities were observed (typically 2-3 days) as described (23). Under these conditions, the neurites have a predominantly axonal character, and the varicosities display all of the necessary features of a presynaptic element (26 -31).…”

“…A␤ applied to NG108-15 varicosities expressing ␣7-nAChRs induced pronounced and sustained increases in Ca 2ϩ , reconstituting the agonist-like effect of A␤ in presynaptic-like structures, and the response could be fully blocked by the selective ␣7-nAChR antagonist ␣-bungarotoxin (23). To define the basis for A␤ activation of ␣7-nAChRs, this study examined candidate residues in the ligand binding domain of ␣7-nAChRs.…”

“…To demonstrate that the agonist-like action of A␤ involved direct activation of nAChRs, ␣7-nAChRs were expressed in the axonal varicosities of NG108-15 cells, which normally do not express functional nAChRs (23). A␤ applied to NG108-15 varicosities expressing ␣7-nAChRs induced pronounced and sustained increases in Ca 2ϩ , reconstituting the agonist-like effect of A␤ in presynaptic-like structures, and the response could be fully blocked by the selective ␣7-nAChR antagonist ␣-bungarotoxin (23).…”