Peptide Blockers of the Inhibition of Neuronal Nicotinic Acetylcholine Receptors by Amyloid β

Margaret H. Magdesian, Arthur A. Nery, A. Henrique B. Martins, Maria Aparecida Juliano, Luiz Juliano, Henning Ulrich, Sérgio T. Ferreira
2005 Journal of Biological Chemistry  
Alzheimer disease (AD) is characterized by accumulation of the neurotoxic amyloid ␤ peptide (A␤) and by the loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs) throughout the brain. Direct inhibition of nAChRs by A␤ has also been suggested to contribute to cholinergic dysfunction in AD. In an effort to find ligands capable of blocking A␤-induced inhibition of nAChRs, we have screened a phage display library to identify peptides that bind to A␤. Using this approach, we
more » ... tified a heptapeptide denoted IQ, which binds with nanomolar affinity to A␤ and is homologous to the acetylcholine-binding protein and to most subtypes of nAChRs. Rapid kinetic whole-cell current-recording measurements showed that A␤ inhibits nAChR function in a dose-dependent manner in neuronal differentiated PC12 cells and that nanomolar concentrations of IQ completely block the inhibition by A␤. These results indicate that the A␤ binding site in nAChRs is homologous to the IQ peptide and that this is a relevant target for A␤ neurotoxicity in AD and, more generally, for the regulation of nAChR function by soluble A␤ in a physiological context. Furthermore, the results suggest that the IQ peptide may be a lead for the development of novel drugs to block the inhibition of nAChRs in AD. Alzheimer disease (AD) 1 is the most common age-related neurodegenerative disorder. This irreversible disease is caused by extensive synaptic dysfunction, resulting in the impairment of cognitive and other cerebral functions. Clinical investigations as well as studies on animal models suggest that the primary agent of neurodegeneration is a peptide of 39 -43 amino acid residues known as amyloid ␤ peptide (A␤) (1, 2). Although the majority of secreted A␤ is 40 amino acids long (A␤40), the smaller fraction of longer, 42-amino acid species (A␤42) has received greater attention because of its high propensity to nucleate and drive the formation of amyloid aggregates and fibrils (3). Amyloid fibrils found in the senile plaques present in the brains of AD patients were originally considered to be primarily responsible for the clinical progression of AD. However, more recent evidence indicates that the cognitive decline correlates better with the amount of soluble A␤ and with the loss of synaptic proteins than with the formation and deposition of amyloid plaques (e.g. Refs. 4 and 5). Multiple neural systems are affected in AD, and a key feature of the neurodegenerative process is the loss of cholinergic neurons and nicotinic ACh receptors (nAChRs) throughout the brain (6). High affinity association of A␤42 with ␣7 and ␣4␤2 nAChRs has recently been observed in amyloid plaques and neurons of AD patients (7, 8). Partial functional inhibition of ␣4␤2 and ␣7 nAChRs by A␤ has also been demonstrated (9 -12). This, however, is still a somewhat controversial issue, as a recent report has described that picomolar concentrations of A␤ activate, rather than inhibit, whole-cell current responses of ␣7-nAChRs (13). In this study, we have used a phage display approach to identify peptides that bind to A␤40 with high affinity. The display of random peptides on filamentous bacteriophage has allowed the identification of peptides that bind specifically to a variety of targets, including fibrillar A␤ (14, 15). Peptides thus identified have been shown to act as agonists and antagonists of various receptors and to mimic the binding sites of physiological receptors (16). Using this approach, we have now isolated a cysteine-linked heptapeptide (denoted IQ) that binds to A␤40 and is homologous to AChBP and to several subtypes of nAChRs. Remarkably, nanomolar concentrations of IQ completely block the inhibition of nAChRs by A␤ in differentiated PC12 cells. These results indicate that the A␤ binding site in nAChRs is homologous to the IQ peptide and suggest that IQ may be a lead for the development of novel drugs to alleviate the inhibition of cholinergic function by A␤ in AD. EXPERIMENTAL PROCEDURES Phage Display-A␤40 (Bachem) was diluted to 0.1 g/l in either PBS or in a 50% trifluoroethanol solution in PBS, and 50 l of each solution were dried in the wells of a 96-well plate at 37°C under constant shaking for 16 h. To check the state of aggregation of the samples, the material from one well of each solution was extracted with 1% SDS and submitted to SDS-PAGE. Although all of the material from the PBS solution sample was found to be aggregated, silver staining of the gel indicated that A␤40 dried from the trifluoroethanol solution did not aggregate. The wells were washed with PBS and blocked with 0.5% bovine serum albumin in PBS for 1 h. After washing, 10 l of a phage display library (PhD-C7C, New England Biolabs) was added to each well and incubated according to the manufacturer's instructions. After
doi:10.1074/jbc.m502406200 pmid:15987688 fatcat:l4ejgoqq3jah7a2qjmmuvigwn4