Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

Bárbara Rienda,Tarson Tolentino-Cortez,Ane Elexpe,Gabriel Barreda-Gómez,Cristina Bruzos-Cidón,María Torrecilla,Marina Gulak,Egoitz Astigarraga

doi:10.3390/analytica2010003

Abstract

Acetylcholinesterase (AChE) is responsible for hydrolyzing the acetylcholine neurotransmitter, bringing an end point to cholinergic neurotransmission. Thus, AChE is the primary target of a wide spectrum of compounds used as pesticides, nerve agents or therapeutic drugs for neurodegenerative diseases such as Alzheimer’s disease (AD). This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. Therefore, the aim of this work is to report a novel technology that enables the simultaneous determination of tissue specific AChE activity, as well as the analysis and screening of specific inhibitors, by using cell membrane microarrays. These microarrays were composed of cell membranes, isolated from 41 tissues, organs and brain areas, that were immobilized over a slide, maintaining the functionality of membrane proteins. To validate this platform, demonstrating its usefulness in drug discovery as a high throughput screening tool, a colorimetric protocol to detect the membrane-bound AChE activity was optimized. Thus, rat cortical and striatal AChE activities were estimated in presence of increased concentrations of AChE inhibitors, and the donepezil effect was assessed simultaneously in 41 tissues and organs, demonstrating the major potential of this microarray’s technology.

Highlights

Acetylcholinesterase (AChE) is responsible for degrading the acetylcholine neurotransmitter into choline and acetic acid, bringing an end point to cholinergic neurotransmission
We modified and optimized the traditional histochemical methodology implemented by Karnovsky and Roots for AChE localization in tissue sections [24] to detect membranebound ChE and AChE activity in the cell membrane microarrays (CMMs)
We propose a novel platform based on CMMs obtained from a tissue collection to estimate membrane-associated AChE activity and to screen compounds that act through membrane-bound cholinesterases, in a single miniaturized assay

Summary

Introduction

Acetylcholinesterase (AChE) is responsible for degrading the acetylcholine neurotransmitter into choline and acetic acid, bringing an end point to cholinergic neurotransmission This enzyme is heterogeneously distributed in the brain showing different activity depending on the nervous region. The most commonly used technologies are based on the photometric Ellman’s method, which consists of measuring the increase in yellow color produced from thiocholine, generated from the hydrolysis of the acetylthiocholine by cholinesterases when it reacts with dithiobisnitrobenzoate ion [6] This method has been adapted to microtiter plates for inhibitor screening, but the amount of sample required limits their use with specific brain areas involved in the AChE physiological effects. Regional distribution of cholinesterase and specific lipids has been described using CMMs [22]

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Conclusion