Neuronal nicotinic receptor deficits in Alzheimer patients with the Swedish amyloid precursor protein 670/671 mutation.

The influence of beta-amyloid on cholinergic neurotransmission was studied by measuring alterations in nicotinic acetylcholine receptors (nAChRs) in autopsy brain tissue from subjects carrying the Swedish amyloid precursor protein (APP) 670/671 mutation. Significant reductions in numbers of nAChRs were observed in various cortical regions of the Swedish 670/671 APP mutation family subjects (-73 to -87%) as well as in sporadic Alzheimer's disease (AD) cases (-37 to -57%) using the nicotinic agonists [3H]epibatidine and [3H]nicotine, which bind with high affinity to both alpha3 and alpha4 and to alpha4 nAChR subtypes, respectively. Saturation binding studies with [3H]epibatidine revealed two binding sites in the parietal cortex of AD subjects and controls. A significant decrease in Bmax (-82%) for the high-affinity site was observed in APP 670/671 subjects with no change in K(D) compared with controls (0.018 nM APP 670/671; 0.036 nM control). The highest load of neuronal plaques (NPs) was observed in the parietal cortex of APP 670/671 brains, whereas the number of [3H]nicotine binding sites was less impaired compared with other cortical brain regions. Except for a positive significant correlation between the number of [3H]nicotine binding sites and number of NPs in the parietal cortex, no strict correlation was observed between nAChR deficits and the presence of NPs and neurofibrillary tangles, suggesting that these different processes may be closely related but not strictly dependent on each other.

Regional distribution of subtypes of nicotinic receptors in human brain and effect of aging studied by (+/-)-[3H]epibatidine.

Epibatidine, a potent nicotinic agonist, was used to study the regional distribution of nicotinic acetylcholine receptor binding sites in the human brain. Saturation studies performed in the human temporal cortex with (+/-)-[3H]epibatidine revealed binding to two binding sites with Kd and Bmax values of 0.018 and 4.2 nM, 12.7 and 15.4 fmol/mg protein, respectively. Competition studies with (+/-)-[3H]epibatidine/unlabelled nicotine or [3H]nicotine/unlabelled (+/-)-epibatidine showed binding to two binding sites in the human temporal cortex (Ki=0.16 and 12.6 nM; 0.007 and 0.3 nM, respectively). Similarly, when unlabelled nicotine was used to displace (+/-)-[3H]epibatidine, two binding sites were also revealed in the thalamus and the cerebellum of human brain (Ki=0.065 and 7.7 nM; 0.07 and 12.5 nM, respectively). The regional binding of (+/-)-[3H]epibatidine binding in human brain was somewhat different from that of [3H]nicotine. A proportionally higher binding was observed for (+/-)-[3H]epibatidine in the cerebellum and the thalamus compared to [3H]nicotine, probably reflecting different selectivity to nicotinic receptor subtypes. A marked significant age-related decrease in (+/-)-[3H]epibatidine binding was observed in the frontal and the temporal cortices (-79%, -84%, respectively) of human subjects between 56-85 years of age, which was similar to that of [3H]nicotine (-82%, -79%, respectively). The (+/-)-[3H]epibatidine binding in the cerebellum decreased significantly with age (-77%), while [3H]nicotine binding showed no significant age-related changes in this brain region. The findings indicate that a specifically modulate regional nicotinic receptors in human brain.

Regulation of nicotinic receptor subtypes following chronic nicotinic agonist exposure in M10 and SH-SY5Y neuroblastoma cells.

The present study further investigated whether nicotinic acetylcholine receptor (nAChR) subtypes differ in their ability to up-regulate following chronic exposure to nicotinic agonists. Seven nicotinic agonists were studied for their ability to influence the number of chick alpha4beta2 nAChR binding sites stably transfected in fibroblasts (M10 cells) following 3 days of exposure. The result showed a positive correlation between the Ki values for binding inhibition and EC50 values for agonist-induced alpha4beta2 nAChR up-regulation. The effects of epibatidine and nicotine were further investigated in human neuroblastoma SH-SY5Y cells (expressing alpha3, alpha5, beta2, and beta4 nAChR subunits). Nicotine exhibited a 14 times lower affinity for the nAChRs in SH-SY5Y cells as compared with M10 cells, whereas epibatidine showed similar affinities for the nAChRs expressed in the two cell lines. The nicotine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was shifted to the right by two orders of magnitude as compared with that in M10 cells. The epibatidine-induced up-regulation of nAChR binding sites in SH-SY5Y cells was one-fourth that in M10 cells. The levels of mRNA of the various nAChR subunits were measured following the nicotinic agonist exposure. In summary, the various nAChR subtypes show different properties in their response to chronic stimulation.

Nicotinic receptors, muscarinic receptors and choline acetyltransferase activity in the temporal cortex of Alzheimer patients with differing apolipoprotein E genotypes.

The number of nicotinic and muscarinic receptors and choline acetyltransferase (ChAT) activity were investigated in the temporal cortex of patients with Alzheimer's disease (AD) with different apolipoprotein E (APOE) genotypes. A significant reduction in the ChAT activity (P < 0.001) and in the number of nicotinic receptors (P < 0.001) was observed in the temporal cortex of AD brains independent of APOE genotype. The number of muscarinic receptors were unchanged in AD brains compared to control in both epsilon 4 and epsilon 3 carriers. A significant negative correlation (P < 0.001) was observed in AD brains between the histopathological dementia score and ChAT activity, which was independent of the APOE genotype. In this study the presence of the APOE epsilon 4 allele was not related to specific deficits in cholinergic activity in the temporal cortex of AD brains.

Effect of tacrine on in vivo release of dopamine and its metabolites in the striatum of freely moving rats.

The effects of tacrine (THA) on extracellular concentrations of dopamine (DA), 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid were investigated in the striatum of freely moving rats, using a microdialysis technique in which tacrine was administered locally via the microdialysis membrane. THA in concentrations of 10(-8) to 10(-5) M, significantly elevated the levels of the DA metabolites 3,4-dihydroxyphenylacetic acid and homovanillic acid, whereas a significantly increased content of extracellular DA was observed at higher concentrations of THA (10(-3) to 10(-2) M). Local administration of the muscarinic antagonist atropine (10(-6) M) or the nicotinic antagonist mecamylamine (10(-5) M) both prevented the effects of THA on DA and its metabolites. In vitro receptor binding studies showed that THA displaced the binding of muscarinic antagonists [3H]pirenzepine (IC50, 2.1 +/- 0.4 microM) and [3H]AFDX 384 (IC50, 3.4 +/- 0.2 microM) equally in striatal tissue, suggesting that THA binds with equal affinity to M1 and M2 muscarinic receptor subtypes. THA showed a 20-fold lower affinity to high-affinity nicotinic receptors compared with muscarinic receptors when determined by [3H]cytisine competition curves. The study indicates that THA enhances monoamine neurotransmission in the rat striatum, probably via an interaction with both muscarinic and nicotinic heteroreceptors.

Epibatidine and ABT 418 reveal selective losses of alpha 4 beta 2 nicotinic receptors in Alzheimer brains.

Subtypes of nicotinic acetylcholine receptors (nAChRs) were characterized in human temporal cortex and in a alpha 4 beta 2 nAChR-transfected M10 cell line using the nicotinic agonists epibatidine, ABT 418 and (-)nicotine in competition studies with [3H]nicotine and [3H]cytisine. The three agonists best fitted to an one-site model in M10 cells with the rank of potencies: epibatidine > > (-)-nicotine > ABT 418. Heterogeneous nAChRs were revealed for epibatidine and ABT 418 in the human temporal cortex. Both compounds labelled a major binding site (Ki 0.35 nM and 68.6 nM respectively) and an additional minor binding site with higher affinity (Ki 0.002 nM and 0.86 nM respectively). Corresponding data for (-)nicotine were a major site (Ki 2.80 nM) and an additional minor site with lower affinity (Ki 2150 nM). Selective losses in the major population of nAChR were observed in the temporal cortex of brains from individuals with Alzheimer's disease with all three agonists. This suggested that the alpha 4 beta 2 nAChR subunit might be the most vulnerable in Alzheimer's disease (AD) and a possible target for therapeutic strategies.

Muscarinic receptors mediate attenuation of extracellular acetylcholine levels in rat cerebral cortex after cholinesterase inhibition.

Muscarinic autoregulation of extracellular acetylcholine levels was investigated by microdialysis in the cerebral cortex of freely moving rats under basal conditions as well as following systemic administration of a reversible cholinesterase inhibitor. Atropine (2.2 mg/kg s.c. or 0.2 microM via the dialysis probe) did not affect basal extracellular acetylcholine levels in the cerebral cortex. However, it did potentiate the elevation of extracellular acetylcholine levels produced by a dose of systemic heptylphysostigmine which inhibited 25% of cortical and 40% of plasma cholinesterase activity. These observations suggest that the extracellular concentration of acetylcholine following moderate acetylcholinesterase inhibition is regulated through muscarinic receptors.

Cholinesterase inhibitor effects on extracellular acetylcholine in rat cortex.

A microdialysis technique was used to sample acetylcholine (ACh) from the cerebral cortex of conscious rats. We thus investigated the effects of systemically administered cholinesterase inhibitors (ChEI) such as physostigmine (300 micrograms/kg), heptylphysostigmine (5 mg/kg) and tetrahydroaminoacridine (tacrine, 5 mg/kg) on extracellular ACh levels. Baseline quantities of extracellular ACh could be detected, even in the absence of ChEI. Acetylcholine levels increased to 1100% over baseline within 30 min of physostigmine administration and returned to control levels after 1.25 hr. Heptylphysostigmine elicited a maximal increase of 1000% within 1.5 hr, and the effect persisted up to 9.5 hr. A 500% increase was observed 1.5 hr after tacrine administration, and ACh returned to control levels after 4 hr. Although the ACh effects observed in this study correlated with previously determined levels of acetylcholinesterase (AChE) inhibition, we conclude that measures of cortical AChE activity alone are not sufficient to predict extracellular ACh levels following systemic ChEI administration.

Coupling of muscarinic receptors to GTP proteins in postmortem human brain--alterations in Alzheimer's disease.

The coupling of muscarinic agonist receptors to guanine nucleotide-binding (G) proteins was investigated in the frontal, temporal cortices and thalamus of control and Alzheimer brains by using carbachol in competition experiments with [3H]QNB. In the presence of GppNHp, the carbachol/[3H]QNB competition binding data showed a 6-fold increase in the high-affinity muscarinic agonist coefficient (Ki high) in the thalami of control brains and a significantly increased proportion of low-affinity agonist binding sites (Bmax low) in the temporal cortices of control brains, while no significant effect of GppNHp was observed in Alzheimer brains. The results suggest a disturbance of the muscarinic receptor-G protein coupling in Alzheimer's disease.

Tacrine restores cholinergic nicotinic receptors and glucose metabolism in Alzheimer patients as visualized by positron emission tomography.

Three patients with Alzheimer's disease, a 68-year-old woman with mild dementia and 2 men (aged 64 and 72 years) with moderate dementia were treated orally with the cholinesterase inhibitor tacrine (tetrahydroaminoacridine), 80 mg daily, for several months. The patients were investigated using positron emission tomography (PET) prior to, and after 3 weeks and 3 months of treatment. The PET studies involved a multi-tracer system consisting of [18F]-fluoro-deoxy-glucose (18F-FDG) (tracer for glucose metabolism); 11C-butanol (cerebral blood flow) and (S)(-)- and (R)(+)-[N-11C-methyl]-nicotine (nicotinic receptors; cholinergic neural activity). Tacrine treatment increased the uptake of 11C-nicotine to the brain. Significant reduced difference in uptake between the two enantiomers (S)(-)- and (R)(+)11C-nicotine was observed in the frontal and temporal cortices after tacrine treatment in all three patients. The kinetic analysis indicated increased binding of (S)(-)11C-nicotine in brain compatible with a restoration of nicotinic cholinergic receptors. The most pronounced effect was observed after 3 weeks and 3 months treatment in the patient with mild dementia. An increase in cerebral glucose utilization was found in the 68-year-old patient with mild dementia but also slightly in the 64-year-old man with moderate dementia when treated with tacrine for 3 months. Tacrine administration did not affect cerebral blood flow. The PET data obtained after 3 weeks of tacrine treatment was paralleled by improvement in neuropsychological performance. This study shows in vivo by PET neurochemical effects induced in brain by treatment with tacrine to Alzheimer patients. Intervention with tacrine in the early course of the disease might be necessary for clinical improvement.