A peptide derived from the C-terminal region of acetylcholinesterase modulates extracellular concentrations of acetylcholinesterase in the rat substantia nigra

doi:10.1016/j.neulet.2003.12.078

Neuroscience Letters

Volume 358, Issue 3, 1 April 2004, Pages 210-214

https://doi.org/10.1016/j.neulet.2003.12.078 Get rights and content

Abstract

It is well established that acetylcholinesterase (AChE) has ‘non-classical’ functions independent of cholinergic transmission. A region of AChE distinct from the catalytic site may be responsible for these actions via a 14-residue peptide located between residues 586–599 at the C-terminus of human AChE. This AChE-peptide possesses a high amino acid sequence homology with a region of amyloid precursor protein and shares many biophysical and physiological characteristics. In this study, the effect of AChE-peptide (AEFHRWSSYMVHWK) on the extracellular levels of endogenous AChE was examined in rat substantia nigra in vitro. A chemiluminescent assay was used to continuously measure the soluble AChE concentration from tissue punches of the substantia nigra. Application of NMDA evoked an increase in extracellular AChE levels consistent with previous results obtained from in vivo models. AChE-peptide, when applied alone, had no effect on AChE release: however, when co-applied with NMDA, AChE-peptide reduced the effectiveness of NMDA to evoke release of AChE. These results indicate, in a region of the brain central to the aetiology of Parkinson's disease, that an AChE-peptide fragment derived from AChE displays a bioactivity that could involve regulation of Ca²⁺ availability and hence the release of AChE.

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Acknowledgements

SRE and SAG wish to thank Synaptica Ltd, UK for the funding of this work carried out within the University of Oxford, UK. We would also like to acknowledge the contributions of Maureen Pitkeithley at OCMS, Carolyn Carr at The Dyson Perrins laboratory, Oxford and Kevin Prior at the Department of Pharmacology, Oxford.

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Copper has an important role in nervous system function, as a cofactor of many enzymes and in the synthesis of neurotransmitters. Both the dose and the chemical form of copper can determine the impact of this element on metabolism, the neurological system and the immune system.
The aim of the study was to determine whether and in what form the addition of copper changes the level of amyloid beta and acetylcholinesterase level in selected rat tissues.
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Acetylcholinesterase (AChE) is now well-established widely as a signalling molecule with non-hydrolytic functions including trophic activity in a diverse variety of situations in both neural and non-neural tissues. We have focussed on the observation that AChE, operating as a trophic agent independent of its enzymatic action, does indeed trigger calcium entry into neurons. It is possible that AChE has a dual non-classical action that ranges along a trophic-toxic axis, depending on amount, duration of availability and, most significantly, age. The neurodegenerative diseases could therefore be viewed as aberrant activation of developmental mechanisms with ‘non-cholinergic’ AChE as a, perhaps the, pivotal molecule. We have identified two peptides that could be cleaved from the C-terminus of T-AChE, one (T14), within the other (T30), and which have a strong sequence homology to the comparable region of beta-amyloid whilst the inert residue within the T30 sequence (‘T15’) acts as a control, and is without effect. We have subsequently been able to ascribe the trophic-toxic actions of the both T14 and T30 peptides to the modulation of calcium influx via an allosteric site on the alpha-7 nicotinic acetylcholine receptor (α7-nAChR). If the scenario described here is indeed the primary mechanism of neurodegeneration, then interception of the actions of the ‘non-cholinergic’ AChE-peptides T14 and T30 at the α7-nAChR, would be a promising novel therapy for arresting and stabilising cell loss in Alzheimer’s disease, whereas detection of the peptides ideally in the blood, could provide a sensitive surrogate marker. If the marker was sensitive enough to be detected pre-symptomatically in a routine blood test, then the medication for arresting further cell loss could be initiated at that time, and the symptoms would never appear. This dual approach of identifying the marker and then intercepting its further action, could thus amount to an effective treatment for Alzheimer’s and other neurodegenerative diseases.
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Thereby presenting a new therapeutic possibility: intercepting the actions of C-terminus AChE peptides at α7-nAChRs before sufficient neurodegeneration has taken place to trigger the emergence of symptoms. Although two early electrophysiological studies suggested a bioactivity of the peptide (Bon and Greenfield, 2003; Greenfield et al., 2004), most of the more recent subsequent research has been undertaken in cell cultures (Bond and Greenfield, 2007; Bond et al., 2006; Onganer et al., 2006) and tissue cultures (Day and Greenfield, 2004; Emmett and Greenfield, 2004; Zbarsky et al., 2004). Moreover, an additional drawback of these approaches has been the protracted time course of the effect studied.
Acetylcholinesterase (AChE) is now well known to have a secondary, non-enzymatic function independent of cholinergic transmission. In the last decade, the part of the molecule responsible for this action has been identified, i.e. a 14 amino acid peptide fragment (‘T14’), deriving from the C-terminus of AChE: this peptide has been shown to be bioactive in a range of preparations and to act at an allosteric site on α₇ nicotinic acetylcholine receptors (α₇-nAChRs). Of particular significance is the finding that AChE-related peptides trigger calcium-induced neurotoxicity that may be pivotal in the process of neurodegenerative diseases, such as Alzheimer's. However to date all studies have been performed on isolated cell preparations. The aim of this study was therefore to characterise the bioactivity of T14 on meso-scale in vitro cortical networks (‘neuronal assemblies’) from rat brain slices containing orbitofrontal cortex. Local field potential (LFP) recordings showed that the T14 peptide has a selective, holistic action on cortical networks in a modulatory biphasic manner i.e. predisposing excitation at concentrations of up to 1 μM, after which the trend is reversed in favour of inhibition at higher doses (>1 μM). By contrast, a scrambled variant of the T14 peptide sequence (S14), showed no significant changes in neuronal activation. Optical imaging using voltage-sensitive dyes (VSDI) corroborated the electrophysiological findings and also provided further insight into the spatial dynamics of the effects of the peptide: T14 application had a facilitatory effect i.e. increased the time-course of activation at sub-micromolar concentrations only (700 nM) without significantly affecting the spread of evoked assemblies. Moreover: co-applying T14 with the α₇-nAChR competitive antagonist methyllycaconitine (MLA) produced inhibition in activation synchrony not seen with either agent on their own, suggesting an additive inhibitory effect. In conclusion, the T14 peptide derived from AChE produced a dose-dependent biphasic modulation of cortical networks activity dependent on the α₇-nAChR: these findings should thus provide a more comprehensive insight into the immediate actions of a novel bioactive agent of high potential relevance to neurodegenerative disorders such as Alzheimer's disease.
Evaluation of a technique to identify acetylcholinesterase C-terminal peptides in human serum samples
2010, Chemico-Biological Interactions
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Moreover, AChE is now well-established generally as a signalling molecule that has trophic activity in a wide variety of tissues and situations [20,23–26,36]. The Greenfield group identified the salient region of the AChE molecule, a C-terminal domain peptide [10], which could mediate such non-classical actions and also demonstrated toxic effects including cell death due to increased calcium influx [5,9,13]. More recently, they obtained direct evidence that the respective target for this peptide is an allosteric site on the alpha 7-nicotinic acetylcholine receptor (α7-nAChR), interaction with which causes calcium influx and up-regulation of receptor expression [6,39].
A novel theory for neurodegeneration is that non-cholinergic functions of acetylcholinesterase (AChE) are responsible for the progressive death of global neurons. The C-terminal region of AChE has been shown to be responsible for non-cholinergic actions of AChE by binding to an allosteric site on the alpha 7-nicotinic acetylcholine receptor, thereby causing calcium influx; the resultant signal has trophic effects in immature neurons, but toxic effects in mature neurons. Although there is strong in vitro and in vivo evidence for the involvement of this C-terminal region of AChE in neurodegeneration, a cleaved C-terminal peptide has not yet been identified in human brains. This preliminary study aimed to identify the cleaved AChE C-terminal peptide in serum from human Alzheimer's disease patients using immunoaffinity purification. A number of antibodies were tested for sensitivity and specificity towards peptide sequences from the C-terminus. Although the antibodies were able to identify peptide in vitro, peptide was not detected using immunoaffinity purification of human serum, possibly due to insufficient detection limits of the antibody. Therefore more sensitive techniques are required to identify cleaved AChE C-terminal peptides in human samples. None the less, C-terminal AChE peptide might act as a signalling molecule in an as yet unexplored system.

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A peptide derived from the C-terminal region of acetylcholinesterase modulates extracellular concentrations of acetylcholinesterase in the rat substantia nigra

Abstract

Section snippets

Acknowledgements

Neuroscience

Neuroscience

Biochem. Pharmacol.

Neuroscience

Neuron

Brain Res.

Neuroscience

Mechanisms and effects of intracellular calcium buffering on neuronal survival in organotypic hippocampal cultures exposed to anoxia/aglycemia or to excitotoxins

J. Neurosci.

Bioactivity of a peptide derived from acetylcholinesterase:electrophysiological characterisation in guinea-pig hippocampus

Eur. J. Neurosci.

Origin of various enzymes released from the substantia nigra and caudate nucleus effects of 6-hydroxydopamine lesions of the nigro-striatal pathway

J. Comp. Neurol.