Acetylcholinesterase promotes beta-amyloid plaques in cerebral cortex.

Studies in vitro have suggested that acetylcholinesterase (AChE) may interact with beta-amyloid to promote deposition of amyloid plaques in the brain of patients with Alzheimer's disease. To test that hypothesis in vivo, we crossed Tg2576 mice, which express human amyloid precursor protein and develop plaques at 9 months, with transgenic mice expressing human AChE. The resulting F1 hybrids (FVB/N x [C57B6 x SJL/J]) expressed both transgenes in brain. By 6 months of age, their cerebral cortex showed authentic plaques that stained both by thioflavin S and by beta-amyloid 1-40 and 1-42 immunohistochemistry. The plaques also stained positively for other components including Cd11b, GFAP, and AChE. Plaque onset in the hybrids occurred 30-50% sooner than in the parental lines. Plaque numbers increased with age and plaques remained more numerous in the doubly transgenic animals at 9 and 12 months. Quantitative immunoassay via ELISA also showed an increase of total amyloid content in brain at 9-12 months. These histological and biochemical results support the conclusion that AChE may play a role in pathogenesis of Alzheimer's disease

Cholinesterase reactivation in vivo with a novel bis-oxime optimized by computer-aided design.

Recently, several bis-pyridiniumaldoximes linked by a variable-length alkylene chain were rationally designed in our laboratories as cholinesterase reactivators. Extensive in vitro tests of these oximes with acetylcholinesterase inhibited by two different organophosphate agents, echothiophate and diisopropylfluorophosphate, revealed one compound with particularly good reactivation kinetics and affinity for phosphorylated acetylcholinesterase (AChE). This compound, designated "ortho-7", with a heptylene chain bridging two aldoximes ortho to a pyridinium ring nitrogen, was chosen for detailed comparison with the classic reactivator pyridine-2-aldoxime methochloride (2-PAM). In vitro, ortho-7 reactivated AChE selectively, without restoring activity of the related enzyme butyrylcholinesterase (BChE). For in vivo studies, rats were injected with ortho-7 or 2-PAM before or after organophosphate exposure, and the activities of AChE and BChE were determined at multiple intervals in blood and solid tissues. Ortho-7 behaved nearly as well in the animal as in vitro, reactivating AChE to the same extent as 2-PAM in all peripheral tissues studied (serum, red blood cell, and diaphragm), but at doses up to 100-fold smaller. Like other oxime reactivators, ortho-7 did not reactivate brain AChE after systemic administration. Nonetheless, this agent could be useful in combination therapy for organophosphate exposure, and it may provide a platform for development of additional, even more effective reactivators.

Effect of extrinsic denervation on muscarinic neurotransmission in the canine ileocolonic region.

To explore the hypothesis that denervation hypersensitivity increases ileocolonic motor activity after extrinsic denervation, we compared muscarinic neurotransmission in canine ileocolonic loops that were isolated and either extrinsically innervated or extrinsically denervated. We recorded ileal, ileocolonic sphincter (ICS) and colonic pressures, and colonic tone, compliance and relaxation during ileal distention. Muscarinic effects were probed by neostigmine, and minimally effective doses of muscarinic receptor antagonists. Denervation augmented ileal, ICS and colonic contractile activity; colonic high-amplitude propagating contractions (HAPCs) were also augmented; colonic relaxation during ileal distention was abolished. Neostigmine induced HAPCs in both loop preparations. Pirenzipine (M1 antagonist) reduced ileal contractile activity in all loops and reduced colonic relaxation during ileal distention in innervated loops. Pirenzipine also reduced colonic tone and colonic HAPCs, more in denervated loops. Darifenacin (M3 antagonist) reduced ileocolonic contractile activity and tone more than did AF-DX 116 (M2 antagonist) in all loops. Cholinergic receptor subtypes modulate different facets of ileocolonic motor activity in the canine ileocolonic region. Increased sensitivity at M1 muscarinic receptors may partly account for the effects of extrinsic denervation.

Selective disruption of neurotransmission by acetylcholinesterase antibodies in sympathetic ganglia examined with intracellular microelectrodes.

Antibodies to acetylcholinesterase (AChE) induce adrenergic dysfunction in rats by selective, complement-mediated destruction of preganglionic sympathetic nerve terminals. To analyze this phenomenon at the neuronal level, monoclonal antibodies to AChE (1.6 mg) were injected via the tail vein, and superior cervical ganglia (SCG) or inferior mesenteric ganglia (IMG) were studied in vitro. In control SCG, all impaled neurons generated action potentials during direct injection of depolarizing current or indirect stimulation through the preganglionic nerve. Current injection remained effective in ganglia from treated rats, but preganglionic stimulation was greatly impaired: at 12 h and 3 d, less than 10% of the neurons responded, even to a maximal stimulus (150 V); at 9 d, only 25% responded. By contrast, in IMG, synaptic transmission was much less affected by antibody exposure: 60% or more of examined neurons responded to preganglionic stimulation. Differences in antibody access did not explain differing sensitivities of SCG and IMG since immunohistochemistry showed rapid accumulation of IgG deposits in both ganglia. These results are believed to reflect widespread but subtotal preganglionic sympathectomy by AChE antibodies. Current information indicates that paravertebral ganglia are all antibody-sensitive, but some prevertebral ganglia are resistant, suggesting immunochemical differences between them.

Quantitative, video-based histochemistry to measure regional effects of anticholinesterase pesticides in rat brain.

Acetylcholinesterase histochemistry was coupled to inexpensive and widely available apparatus for video microscopy and densitometry to study enzyme activity and inhibition in different parts of the rat brain. Quantitative histochemistry, under properly defined conditions, yielded an output that increased linearly with incubation time and section thickness and was a smooth hyperbolic function of substrate concentration. The time-course of staining after in vivo exposure to eserine revealed no sign that carbamate-induced cholinesterase inhibition was readily reversed in vitro. Brains from rats treated either with a carbamate or an organophosphate anticholinesterase pesticide showed significant regional variation in cholinesterase inhibition. The histochemical data corresponded well with data from biochemical assays of acetylcholinesterase activity (overall correlation coefficient of absolute values, r = 0.95). Also, a comparison of assay types by two-way analysis of variance showed no significant main effect. These results support the conclusion that video-based histochemistry is suitable for detailed studies of developmental and toxicological influences on cholinesterases in multiple microscopic regions of the rat brain.

Pathology of the senescent heart: anatomic observations on 237 autopsy studies of patients 90 to 105 years old.

Persons older than 85 years of age will constitute the fastest growing segment of the US population in the next decade, and despite the recent decline in mortality from heart disease, cardiovascular disease remains the single greatest cause of death in these elderly persons. We studied the pathologic changes in the hearts of 237 patients (93 men and 144 women) who had lived to the age of 90 years or older. The degree and extent of coronary atherosclerosis in these senescent hearts were similar to findings reported in younger patients who died of coronary heart disease, but calcification of the coronary arteries, mitral annulus, and aortic valve was more prevalent, as was cardiac amyloidosis. Multiple cardiac disorders were common in these very elderly hearts, but they seemed to play a lesser role in precipitating heart failure or shortening the life-span of the patients. Important factors in attaining longevity seem to be protection from the development of severe coronary artery disease by an unexplained mechanism and an innate resistance to cardiac dysfunction from a multitude of structural changes that occur with advancing age.

Paroxysmal nocturnal hemoglobinuria: erythrocyte acetylcholinesterase deficit analyzed by immunoassay and fluorescence-activated sorting.

An immunodisplacement assay based on a specific, solid-phase monoclonal antibody was designed to measure acetylcholinesterase in tissue extracts. Sample enzyme content was determined from the competitive reduction of binding of a purified acetylcholinesterase standard, with a detection limit of 5 ng or less. Washed erythrocyte membranes from six normal subjects averaged 1.8 units of acetylcholinesterase activity and 0.45 microgram of acetylcholinesterase content per milligram of total protein. Enzyme activity and content in samples from three patients with paroxysmal nocturnal hemoglobinuria (PNH) were reduced approximately in parallel, by as much as 70%. The residual cholinesterase had almost the same homospecific activity as the normal enzyme and was bound with equivalent affinity by six different antibodies. Therefore, the cholinesterase defect was dominated by enzyme loss rather than by structural abnormalities affecting enzyme function. Fluorescence-activated sorting of antibody-labeled erythrocytes revealed a bimodal population distribution. Up to 66% of the PNH cells lacked cholinesterase, and the rest had a near-normal enzyme content. Inasmuch as enzyme-deficient cells represent the complement-sensitive population, cell sorting may help in assessing clinical status and, perhaps, in developing new therapeutic modalities for PNH.

Acrylamide neuropathy in the rat: effects on energy metabolism in sciatic nerve.

Energy metabolism was examined in rat sciatic nerve before and after induction of neuropathy by treatment with acrylamide monomer. The in vivo activities of two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase and nerve-specific enolase, were resistant to acrylamide. The levels of adenosine 5'-triphosphate and creatine phosphate were also unaffected by acrylamide after either short-term or long-term treatment. The turnover of high-energy phosphate was somewhat reduced in the nerves of severely intoxicated animals. These findings cast doubt on the hypothesis that acrylamide neuropathy begins with an attack on the means of generating metabolic energy, although the eventual failure of one or more energy-consuming processes in peripheral nerve remains likely.