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Acetylcholinesterase (AChE) catalyses the hydrolysis of the neurotransmitter acetylcholine and it has been implicated in several non-cholinergic actions, including neurite outgrowth and amyloid formation. We have studied the trophic function of brain AChE on neuronal cell metabolism and proliferation as well as the enzyme domain involved in such effects. Low AChE concentrations (0.1-2.5 nM) stimulated neurite outgrowth and induced cell proliferation as measured by MTT reduction and [3H]thymidine incorporation. The action of AChE was not affected by edrophonium and tacrine both active site inhibitors, but it was abolished by propidium and gallamine, two peripheral anionic binding site (PAS) ligands. We conclude that the PAS domain of AChE is involved in the neurotrophic activity of the enzyme.

This work addresses the differential effects of several oxidative insults on two neuronal cell lines, PC12 and Neuro 2a cells, extensively used as neuronal models in vitro. We measured cellular damage using the cytotoxic assays for MTT reduction and LDH release and found that acetylcholinesterase (AChE)-amyloid-beta-peptide (Abeta) complexes, Abeta25-35 fragment, glutamate and H2O2 were over 200-fold more toxic to PC12 than to Neuro 2a cells. 17alpha and 17beta estradiol were able to protect both cell types from damage caused by H2O2 or glutamate. By contrast, other insults not related to oxidative stress, such as those caused by the nonionic detergent Triton X-100 and serum deprivation, induced a similar level of damage in both PC12 and Neuro 2a cells. Considering that the Abeta peptide, H2O2 and glutamate are cellular insults that cause an increase in reactive oxygen species (ROS), the intracellular levels of the antioxidant compound, glutathione were verified. Neuro 2a cells were found to have 4- to 5-fold more glutathione than PC12 cells. Our results suggest that Neuro 2a cells are less susceptible to exposure to AChE-Abeta complexes, Abeta25-35 fragment, glutamate and H2O2 than PC12 cells, due to higher intracellular levels of antioxidant defense factors.

Alzheimer's disease (AD) is a neurodegenerative disorder whose hallmark is the presence of senile plaques and neurofibrillary tangles. Senile plaques are mainly composed of amyloid beta-peptide (Abeta) fibrils and several proteins including acetylcholinesterase (AChE). AChE has been previously shown to stimulate the aggregation of Abeta1-40 into amyloid fibrils. In the present work, the neurotoxicity of different amyloid aggregates formed in the absence or presence of AChE was evaluated in rat pheochromocytoma PC12 cells. Stable AChE-Abeta complexes were found to be more toxic than those formed without the enzyme, for Abeta1-40 and Abeta1-42, but not for amyloid fibrils formed with AbetaVal18-Ala, a synthetic variant of the Abeta1-40 peptide. Of all the AChE-Abeta complexes tested the one containing the Abeta1-40 peptide was the most toxic. When increasing concentrations of AChE were used to aggregate the Abeta1-40 peptide, the neurotoxicity of the complexes increased as a function of the amount of enzyme bound to each complex. Our results show that AChE-Abeta1-40 aggregates are more toxic than those of AChE-Abeta1-42 and that the neurotoxicity depends on the amount of AChE bound to the complexes, suggesting that AChE may play a key role in the neurodegeneration observed in Alzheimer brain.

The senile plaques present in Alzheimer's disease (AD) are composed of a core of amyloid beta-peptide (Abeta) plus several proteins including acetylcholinesterase (AChE). Recently we found that AChE forms complexes with the Abeta peptide in vitro and that these are more cytotoxic than Abeta fibrils alone. Considering that estrogen has been reported to act as a protective agent against Abeta-induced cytotoxicity, the effect of 17beta-estradiol was studied in rat pheochromocytoma (PC12) and mouse neuroblastoma (Neuro 2a) cells exposed to either Abeta alone or AChE-Abeta complexes. Estrogen showed a powerful protective effect in response to the challenge of AChE-Abeta complexes as well as with Abeta fibrils. This was also the case for other cytotoxic agents such as glutamate and H2O2. Our results suggest a common mechanism for cellular protection by estrogen against the toxicity of both Abeta fibrils and AChE-Abeta complexes, likely avoiding the free radical apoptotic pathway.

Brain acetylcholinesterase (AChE) forms stable complexes with amyloid-beta peptide (Abeta) during its assembly into filaments, in agreement with its colocalization with the Abeta deposits of Alzheimer's brain. The association of the enzyme with nascent Abeta aggregates occurs as early as after 30 min of incubation. Analysis of the catalytic activity of the AChE incorporated into these complexes shows an anomalous behavior reminiscent of the AChE associated with senile plaques, which includes a resistance to low pH, high substrate concentrations, and lower sensitivity to AChE inhibitors. Furthermore, the toxicity of the AChE-amyloid complexes is higher than that of the Abeta aggregates alone. Thus, in addition to its possible role as a heterogeneous nucleator during amyloid formation, AChE, by forming such stable complexes, may increase the neurotoxicity of Abeta fibrils and thus may determine the selective neuronal loss observed in Alzheimer's brain.