Cholinesterase inhibitors in Alzheimer's disease : an experimental study on mechanisms of interatcion with muscarinic and nicotinic receptors and neuroprotection

Abstract

The overall aim of this thesis was to investigate the mechanisms beyond the interaction of cholinesterase inhibitors (ChEIs) with the cholinergic system in brain, with emphasis on their regulatory and mechanistic effects on subtypes of muscarinic and nicotinic receptors and neuroprotective properties.

To investigate muscarinic receptor subtypes in frontal cortices of AD brains, the muscarinic antagonists pirenzepine, AF-DX 116, HHSiD, p-F-HHSiD and himbacine were used in receptor binding assays. The number of muscarinic receptors was found to be preserved in frontal cortices of AD brains, with a relative increase in the number of M 1 receptor subtype compared to controls. The presence of the APOE e4 allele was not found to be related to deficits in ChAT activity and number of nicotinic receptors (nAChRs) in the temporal cortex of AD brains.

The ChEI tacrine was found to both increase and decrease the acetylcholine (ACh) release in a concentration-dependent manner. The enhanced release induced by tacrine was observed at concentrations (,uM range) that are expected to be relevant for clinical situations. Pirenzepine (M1 selective antagonist) could prevent the increase in ACh release induced by tacrine, indicating that the effect was mediated via M1 muscarinic receptors. These findings together with the finding of preserved Ml receptors in AD brains, support a favourable effect of tacrine in the treatment of AD.

In order to investigate the mechanism by which ChEIs interact with nAChRs in brain, a transfected fibroblast cell line, stably expressing the major neuronal nAChR subtype a4B2, was utilized as a model system. Chronic treatment with tacrine, galanthamine, donepezil and NXX-066 increased the number of a4B2 nAChRs in the concentration range of 10 - 10-5 M, probably via post-transcriptional mechanisms. In addition, tacrine and galanthamine at higher concentrations decreased the number of nAChRs. These results demonstrate that donepezil and NXX-066 interact solely with an allosteric activator site on the nAChR, while tacrine and galanthamine interact with both an allosteric activator site as well as the ACh binding site.

Tacrine (10-10 - 10-5 M) and donepezil (10-8 and 10-7 M) were able to reduce the neurotoxicity induced by B-amyloid(25-35) (AB) in PC12 cells. The neuroprotective effect of tacrine and donepezil may be mediated through nAChRs, at least for tacrine since mecamylamine blocked the effect of tacrine. Estrogen potentiated the neuroprotective effect of tacrine and donepezil. Since estrogen might sensitize the nAChR, it is plausible that the effect may be via the nAChR.

In conclusion, the present study demonstrates that ChEIs, in clinical relevant concentrations (uM range), increases the release of ACh via Ml receptors, upregulates the a4B2 nAChR subtype by interacting with an allosteric site on the nAChR and prevent AB-induced toxicity in vitro.