Astrocytes associated with senile plaques possess muscarinic acetylcholine receptors.

AUTOPSY sections of the temporal and parahippocampal gyri were incubated with the monoclonal antibody M35 to label muscarinic acetylcholine receptors. Senile plaques were visualized either by counterstaining with Congo red or by Bielschowsky silver-stain of adjacent sections. The antibody labelled pyramidal and non-pyramidal cells in the grey matter, as well as fibrous astrocytes in the white matter and first cortical layer, of elderly control brains. In AD brains however, the grey matter was characterized by prominently labelled fibrous astrocytes and few or poorly labelled neurones. M35-immunoreactive astrocytes were often associated with senile plaques. These findings suggest that plaque-associated astrocytes may be responsive to acetylcholine or cholinergic drugs.

Human neutrophil phagocytic granules contain a truncated soluble form of the Alzheimer beta/A4 amyloid precursor protein (APP).

We have characterized the molecular species and subcellular distribution of Alzheimer beta/A4 amyloid precursor protein (APP) in neutrophilic granulocytes purified from human peripheral blood. APP was readily detectable in these cells. Immunochemical analysis with a panel of antibodies revealed that this APP species lacked the transmembrane and cytoplasmic domains previously demonstrated in cell-associated APP. However, it contained a protease inhibitor domain of the Kunitz type, indicating that neutrophil APP is a potent inhibitor of certain serine proteases. Upon subcellular fractionation, APP was primarily localized to azurophilic granules, which are neutrophil-specific phagocytic organelles assigned to enzymatic digestion of invading microbes and dead or injured tissue. Apparently, in the neutrophil, a nonsecretory organelle stores truncated, soluble APP, a species previously found only in blood plasma and cerebrospinal fluid or in conditioned medium of cultured cells. Soluble APP in neutrophils may therefore have intracellular functions in addition to its previously described extracellular functions. These findings also indicate that there are previously uncharacterized cell-specific differences in processing, trafficking, and storage of the APP molecule. Finally, the precise subcellular localization of APP to neutrophil-specific phagocytic organelles is suggestive of a role for APP in the nonimmunological host defense.

beta-Amyloid related peptides exert differential effects on [3H]MK-801 binding to rat cortical membranes.

The effects of beta-amyloid protein 1-40 (beta AP 1-40), substance P (SP), and the amidated and carboxylic acid C-terminated forms of the SP homologous beta AP fragment 25-35 (beta AP 25-35-NH2 and beta AP 25-35-COOH) were studied on [3H]MK-801 binding to the rat brain NMDA receptor cation channel. All peptides gave dose-dependent enhancements of [3H]MK-801 binding stimulated by low glycine. beta AP 25-35-COOH, but not beta AP 25-35-NH2 produced an inhibition of [3H]MK-801 binding stimulated by high glycine in the presence of either low or high glutamate. Low glutamate-stimulated [3H]MK-801 binding was also inhibited by SP but not by beta AP 1-40. It is concluded that beta AP related peptides exert differential effects on the NMDA receptor complex at the glycine and possibly also the glutamate recognition sites.

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.

Cholinesterase inhibitor effects on extracellular acetylcholine in rat striatum.

The effects of inhibition of cholinesterase on levels of extracellular acetylcholine in the striatum of freely moving rats, were investigated with a microdialysis technique. Acetylcholine could not be detected under basal conditions. However, local administration of the cholinesterase inhibitors neostigmine, physostigmine or heptyl-physostigmine through the dialysis probe elevated acetylcholine above the detection limit. Complex interactions between the effects of local and systemic cholinesterase inhibitors were observed. Whereas systemic administration of physostigmine or heptylphysostigmine alone increased the extracellular concentration of acetylcholine, they also caused acetylcholine to fall below baseline levels, which had been established by perfusing physostigmine through the microdialysis probe. These studies demonstrate the ability of local inhibition of cholinesterase to affect the observation of effects of systemically-administered drugs.

Biochemical pathology and treatment strategies in Alzheimer's disease: emphasis on the cholinergic system.

The neurochemical pathology of Alzheimer's disease (AD) has been consistently shown to involve cholinergic degeneration in the cerebral cortex. This together with evidence from experimental animal studies showing that cholinergic neurones play a role in learning and memory processes has formed the basis of the cholinergic hypothesis of Alzheimer's dementia and the major rationale for neurotransmitter replacement therapy of the disorder.

Preferential inhibition of acetylcholinesterase molecular forms in rat brain.

The effect of eight different acetylcholinesterase inhibitors (AChEIs) on the activity of acetylcholinesterase (AChE) molecular forms was investigated. Aqueous-soluble and detergent-soluble AChE molecular forms were separated from rat brain homogenate by sucrose density sedimentation. The bulk of soluble AChE corresponds to globular tetrameric (G4), and monomeric (G1) forms. Heptylphysostigmine (HEP) and diisopropylfluorophosphate were more selective for the G1 than for the G4 form in aqueous-soluble extract. Neostigmine showed slightly more selectivity for the G1 form both in aqueous- and detergent-soluble extracts. Other drugs such as physostigmine, echothiophate, BW284C51, tetrahydroaminoacridine, and metrifonate inhibited both aqueous- and detergent-soluble AChE molecular forms with similar potency. Inhibition of aqueous-soluble AChE by HEP was highly competitive with Triton X-100 in a gradient, indicating that HEP may bind to a detergent-sensitive non-catalytic site of AChE. These results suggest a differential sensitivity among AChE molecular forms to inhibition by drugs through an allosteric mechanism. The application of these properties in developing AChEIs for treatment of Alzheimer disease is considered.

Cholinesterase inhibitor therapy for Alzheimer dementia: what do animal models tell us?

Despite a strong rationale for the use of cholinesterase (ChE) inhibitors and related drugs to augment cholinergic function as palliative treatment for Alzheimer dementia, this approach met with limited and variable success until the striking results recently reported with tacrine (THA). Our previous studies have shown the potential utility of blood ChE inhibition to define the optimum dose of such drugs. In the present studies, THA was reinvestigated in rats because several literature reports of weak inhibition of blood and brain acetylcholinesterase (AChE) after systemic treatment leave unresolved the mechanism of action for THA. Consistent with previous reports, we find an in vitro IC50 of 1 microM THA or less for brain or red blood cell AChE, dependent on the substrate concentration. Results were independent of tissue dilution in vitro. However, after in vivo THA, the inhibition of plasma ChE or brain AChE declined as a log function of tissue dilution. These results indicate that the degree of inhibition is underestimated as a result of dilution of tissue for enzyme assay. We therefore used minimal tissue dilution to establish the dose-response and time course functions after s.c. administration of THA and to compare the effect of THA in various brain regions with that on blood enzymes. Pons-medulla AChE was much less sensitive to the effects of THA than hippocampus, cortex, cerebellum or plasma ChE, particularly at doses of 2.5 mg/kg or less. In these other brain regions, AChE was inhibited 22-44% after doses of 1.25 to 2.5 mg/kg THA, the dose range which maximally improved retention performance in mice. Peak inhibition occurred rapidly in plasma, but was delayed in brain (30-60 min after THA). Inhibition was long-lasting in both plasma and cortex (greater than or equal to 5 hr). It is concluded that long-lasting inhibition of the metabolism of acetylcholine is the most heuristic explanation of THA's pharmacological activity.