Alzheimer's disease is a chronic progressive neurodegenerative disease with symptoms of dementia and from which in Europe suffer approximately 7.3 million people. Cholinergic hypothesis represents the first attempt to interpret the pathogenesis of Alzheimer’s disease and has led to the development of acetylcholinesterase inhibitors. The role of cholinesterases in the pathogenesis of Alzheimer's disease has led to intensive research of functions of cholinesterases in the central nervous system. Despite all the research, however, some key questions about the role of cholinesterases remain unclear. One of the still unexplained phenomena is also that in mammals besides acetylcholine esterase, also related enzyme butyrylcholinesterase can be found. Activity of butyrylcholnesterase may increases from 1.2 to up to 11-fold in sever Alzheimer’s disease, which suggests that in the brains of patients with Alzheimer's disease butyrylcholinesterase gradually takes over the role of acetylcholinesterase in hydrolysis of acetylcholine. Selective inhibitors of butyrylcholinesterase can be clinically useful for the restoration of cholinergic activity, which decline in late stages of disease. In this research, we successfully optimized Koelle’s method for displaying the activity of cholinesterases on rat brain slices, by reducing the incubation time and the quantity of reagents used in relation to the original protocol. It has also been showen that the modified method is useful for qualitative evaluation of the potency of cholinesterase inhibitors. With the modified method, we have first determined areas of the brain with highest butyrylcholinesterase activity and then on those areas, we have evaluated the inhibitory potency of novel selective inhibitor of this enzyme. We also tried to develop a new method for detecting butyrylcholinesterase on brain slices by means of fluorescence-labeled analogue of the novel inhibitor, that would allow quick and easy display of butyrylcholinesterase on brain slices and would also confirmed the binding capacity of the inhibitor in the active site of enzyme on the brain slices - an environment that is more complex than a test system in vitro, and represent an important step for the advancement of the compounds in experiments in vivo. Method development was disabled mainly due to excessive lipophilicity of fluorescent analogue and its non-specific binding across all of the brain slice, as well as strong autofluorescence of lipofuscin. However we successfully proved affinity of analog to enzyme, confirmed the detection of fluorescent signal of bounded analog and thus laid the foundations and highlighted the problems for further experiments in the development of this method.
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