β-Amyloid Directly Inhibits Human α4β2-Nicotinic Acetylcholine Receptors Heterologously Expressed in Human SH-EP1 Cells

Abstract

Amyloid-␤ (A␤) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human ␣4 and ␤2 subunits (h␣4␤2-nAChR). However, the relationships between these phenomena are uncertain, and effects of A␤ on h␣4␤2-nAChR function have not been investigated in detail. We first confirmed expression of h␣4 and h␤2 subunits as messenger RNA in transfected, human SH-EP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed ␣4 and ␤2 subunit protein expression and coassembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-h␣4␤2 cells of functional h␣4␤2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by A␤ 1-42 in a dosedependent manner. Functional inhibition was selective for A␤ 1-42 compared with the functionally inactive, control peptide A␤ 40 -1 . A␤ 1-42 -mediated inhibition of h␣4␤2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5-yl thiophosphate failed to prevent A␤ 1-42 -induced inhibition, suggesting that down-regulation of h␣4␤2-nAChR function by A␤ 1-42 is not mediated by nAChR internalization. Sensitivity to A␤ 1-42 antagonism at 1 nM was evident for h␣4␤2-nAChRs, but not for heterologously expressed human ␣7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nM A␤ 1-42 , with the magnitude of functional block being higher for 100 nM A␤ 1-42 acting on h␣7-nAChRs. These findings suggest that h␣4␤2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for A␤ neurotoxicity in AD.Alzheimer's disease (AD) 1 is a progressive, neurodegenerative disorder manifest as a severe impairment of learning and memory. Pathophysiological hallmarks of AD include extracellular deposits of ␤-amyloid peptide (A␤) in senile plaques, formation of intraneuronal neurofibrillary tangles, and cholinergic neuron death (1). Although the precise mechanisms of AD pathogenesis are only partially understood, it is now widely accepted that the accumulation and aggregation of A␤ 1-42 plays a key role in the disease (2). Evidence has indicated an interaction between A␤ and the cholinergic system (3). For example, very low concentrations (pico to nanomolar) of A␤ can directly induce cholinergic hypofunction (4 -6). It has been reported that solubilized A␤ inhibits several steps of acetylcholine synthesis and release (4, 7), inhibits cholinergic enzyme activity (6), impairs cholinergic metabolism and neurotransmission (8 -10), and depresses hippocampal synaptic function (11).Recent evidence suggests possible roles for nicotinic acetylcholine receptors (nAChRs) as central targets for A␤-induced neurotoxicity manifest as...

Nicotinic Acetylcholine Receptor Signalling: Roles in Alzheimer's Disease and Amyloid Neuroprotection

“…A␤ action on nAChRs depends on subunit composition; it has been reported to block ␣7, transiently potentiate ␣4␤2 before blocking, and to have no action on ␣3␤4 (Pym et al, 2005). However, in contrast to its reported transient enhancement when expressed in oocytes, an inhibition of ␣4␤2 has been reported when expressed in human SH-EP1 cells (Wu et al, 2004).…”

Amyloid β Protein Modulates Glutamate-Mediated Neurotransmission in the Rat Basal Forebrain: Involvement of Presynaptic Neuronal Nicotinic Acetylcholine and Metabotropic Glutamate Receptors

“…A number of electrophysiological studies, in a variety of in vitro preparations, have demonstrated that A␤ 1-42 can exert its effects through both ␣7 and ␣4␤2 nAChRs (Pettit et al, 2001;Dineley et al, 2002;Fu and Jhamandas, 2003;Wu et al, 2004;Lamb et al, 2005). However, the precise nature of A␤ 1-42 interactions with nAChRs is controversial because some studies have reported that A␤ 1-42 activates nAChRs (Dineley et al, 2002;Fu and Jhamandas, 2003), whereas other studies suggested that A␤ 1-42 inhibits nAChRs (Pettit et al, 2001;Wu et al, 2004;Lamb et al, 2005). Both ␣7 and non-␣7 subtypes of nAChRs were reported to be involved in such A␤-mediated interactions.…”

Dopamine release in prefrontal cortex in response to β-amyloid activation of α7∗ nicotinic receptors

“…Previous work has demonstrated a potent action of soluble Aβ on nicotinic acetylcholine receptors (nAChRs), including both antagonist (Pettit et al, 2001;Liu et al, 2001;Grassi et al, 2003;Wu et al, 2004) and agonist (Dineley et al, 2001;Dougherty et al, 2003;Fu et al, 2003) effects. We have shown that pM-nM Aβ 1-42 induces increased [Ca 2+ ]i in isolated presynaptic terminals from rat cortex and hippocampus in a manner susceptible to partial antagonism by classical nAChR antagonists, such as α-bungarotoxin and dihydro-β-erythroidine.…”