Abstract
Alzheimer's Disease (AD) is a progressive, age-related disease that is positively
characterised by the post-mortem presence of neurofibrillary tangles and amyloid plaques.
At present ~ 5 % of those aged over 65 years are diagnosed with AD, with this figure
increasing to ~ 20 % for those aged over 75 years. It is nearly 100 years since AD was first
characterised in 1907, however the exact cause has yet to be determined with the cholinergic
and beta-amyloid (Ap) hypotheses remaining central to the aetiology. The cholingergic
hypothesis suggests that loss of the cholinergic pathways precedes that of any other
neurotransmitter making it a primary target in AD and predating the amyloid hypothesis
which itself postulates that deposition of Ap is primarily responsible for the
neurodegeneration in AD. Until recently, these two hypotheses have been almost
exclusively studied in separation. However, recent studies suggest that the two major CNS
cholinergic nicotinic acetylcholine receptor subtypes a4p2 and a7 may interact directly with
Ap and therefore, a multidisciplinary approach has been used to evaluate the reported
interaction.
Radioligand binding was used to characterise the α₄ß₂ nAChR ([³H]-epibatidine
and [³H]-cytisine) and α₇ nAChR ([³H]-methyllycaconitine ([³H]-MLA) and [³H]-
αBungarotoxin ([³H]-αBgTx)) in rat and mouse brain tissue and in SH-EP1 cell lines
overexpressing human forms of the α₄ß₂ or α₇ nAChRs. No species difference in ligand
affinities were observed for the α₄ß₂ nAChR. In contrast, nicotinic agonists exhibited
significantly higher affinity (~100 fold) for human α₇ nAChRs compared to their rat
counterparts with no change in antagonist affinity. Interestingly, evaluation of [³H]-MLA
and [³H]-aBgTx binding indicated the latter ligand bound to a restricted number of sites on
the α₇ nAChR. Furthermore, neither human nor rat αß₁₋₄₂ inhibited [³H]-cytisine or [³H]-
MLA binding to nAChRs. In parallel to behavioural studies, these binding assays were also
employed to assess nAChR pharmacology in transgenic α₇ knockout mice. With no
alteration in α₄ß₂ nAChR pharmacology, the deficit in sustained attention exhibited by these
α₇-KO mice is probably due to loss of the α₇ nAChR.
Finally, a series of studies was performed to examine the functional interaction
between αß₁₋₄₂ and nAChRs. (-)Nicotine evoked changes in calcium flux or membrane
potential in SH-EPl-hα₄ß₂ cells were not inhibited by soluble or insoluble human αß₁₋₄₂.
Even whole cell patch clamp analysis of single cells showed no direct interaction between
the αß₁₋₄₂ nAChR and αß₁₋₄₂. The examination of the functional interaction between α₇
nAChRs and αß₁₋₄₂ using whole cell patch clamp or fluorescence based assays was
compromised by a lack of consistent expression of functional human α₇ nAChRs in the SHEP1 cell line. In addition, neither human α₄ß₂ nor α₇ nAChRs co-immunoprecipitated with
human αß₁₋₄₂.
In conclusion, using a systematic multidisciplinary approach incorporating
functional and non-functional assays the following major observations were made (1)
species differences in α₇ but not α₄ß₂ nAChR pharmacology, (2) the two α₇ nAChR
antagonists MLA and αBgTx differ in their ability to bind the α₇ homopentamer, (3)
removal of the α₇ nAChR does not alter a4p2 nAChR pharmacology in transgenic mice (4)
neither soluble or insoluble αß₁₋₄₂ interacted in pharmacological, functional, or biochemical
assays with the rodent or human α₄ß₂ or α₇ nAChRs.
