Release of acetylcholinesterase (AChE) from β-amyloid plaques assemblies improves the spatial memory impairments in APP-transgenic mice

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Abstract

The major protein constituent of amyloid deposits in Alzheimer's disease (AD) is the amyloid-β-peptide (Aβ). Amyloid deposits contain “chaperone molecules” which play critical roles in amyloid formation and toxicity. In the present work, we test an analog of hyperforin (IDN 5706) which releases the AChE from both the Aβ fibrils and the AChE-Aβ burdens in transgenic mice. Hyperforin is an acylphloroglucinol compound isolated from Hypericum perforatum (St. John's Wort), which is able to prevent the Aβ-induced spatial memory impairments and Aβ neurotoxicity. Altogether this gathered evidence indicates the important role of AChE in the neurotoxicity of Aβ plaques and finding new compounds which decrease the AChE-Aβ interaction may be a putative therapeutic agent to fight the disease.

Introduction

Alzheimer's disease (AD) is one of the most common neurodegenerative dementias, characterized by a progressive deterioration of cognitive functions [1]. Although a small percentage of AD cases involve mutations in some known genes, referred as familial AD, the large majority of AD cases occur sporadically with unknown etiology [2]. Therefore, there is a need to search for the responsible mechanisms of the progressive cognitive decline observed in AD patients. The behavioral impairment observed in AD patients has been attributed to the accumulation, conformation, quaternary structure and morphology of the amyloid-β-peptide (Aβ) bundles formed [3], [4], [5], [6]. The Aβ accumulation and deposition has been causally implicated in the neuronal dysfunction and neuronal loss that underlies the clinical manifestations, however, the synaptic damage observed prior to the amyloid plaque formation has been attributed to the neurotoxic effect of Aβ oligomers [7]. One of the several proteins associated with amyloid plaque deposits is the enzyme acetylcholinesterase (AChE), which is predominantly associated with the amyloid core of mature senile plaques, pre-amyloid diffuse deposits, and cerebral blood vessels in AD brain [8], [9]. AChE has been described in cholinergic and non-cholinergic processes in both the central and peripheral nervous system [10], [11]. The enzyme is secreted and becomes associated with extracellular structures, namely the synaptic basal lamina at the neuromuscular junction and, as above mentioned, the amyloid plaques of brain [12], [13]. In vitro studies indicated that AChE has the ability to enhance Aβ aggregation and amyloid fibril formation. In fact, when AChE was infused stereotaxically into the CA1 region of the rat hippocampus, novel plaque-like structures were formed [14], [15]. As Aβ is toxic to neurons [2], the main targets for therapeutic intervention of the Aβ cascade include the inhibition of Aβ production, the inhibition of Aβ aggregation and fibril formation, in addition to the inhibition of the consequent inflammatory responses caused by the Aβ deposition. In this context, several substances are known to inhibit Aβ fibrillogenesis in vitro, including laminin [16], polyphenols [17], site-directed monoclonal antibodies [18], Ginkgo biloba extract [19], β-sheet breaker peptides [20] and hyperforin [21]. Nevertheless, an effective therapeutic approach that interferes directly with the neurodegenerative process in AD is eagerly awaited. Hyperforin (HYP) is an acylphloroglucinol, a natural derivative from Hypericum perforatum, also known as the St. John's Wort. The natural product is a complex mixture of compounds comprising several natural derivatives and HYP was identified as the molecule responsible for the antidepressant activity by a mechanism involving the inhibition of uptake of monoamines and other neurotransmitters [22]. It was suggested that HYP possesses memory-enhancing properties in rodents [23]. In recent studies we determined that HYP decreases amyloid deposit formation, Aβ-induced neuropathological changes and behavioral impairments in a rat model of amyloidosis; prevents Aβ-induced neurotoxicity in hippocampal neurons, avoiding the increase in reactive oxidative species associated with amyloid toxicity; disaggregates amyloid fibrils in a dose and time-dependent manner [21]. These antecedents drove us to investigate whether the HYP derivate, IDN 5706, would reduce β-amyloid deposition and improve spatial learning acquisition, and how these compounds could be able to destabilize Aβ-AChE interaction in an attempt to find a putative therapeutic agent for the disease. We report here that IDN 5706 inhibits the aggregation, depolymerizes the fibrils and removes the AChE from the complex. We also show that IDN 5706 induces a decrease in the number of mature AChE-positive plaques and that this effect improves behavior of the APP-transgenic mice.

Section snippets

Synthetic peptides and reagents

1–42 peptide corresponding to the human sequence (Bachem Inc., Torrance, CA., lot no. T-20964amd and Genemed Synthesis Inc., South San Francisco, CA) were dissolved in dimethyl sulphoxide (DMSO) at a concentration of 15 μg/μl and immediately stored in aliquots at −20 °C before assaying. Tetrahydrohyperforin (IDN 5706) is a semi synthetic derivative of hyperforin (WO 03/091194 A1; WO 2004/106275 A2) and was provided by Indena SpA, Milano, Italy. Recombinant human acetylcholinesterase (rhAChE)

Results

The hyperforin derivative (IDN 5706) inhibits Aβ aggregates formation, disaggregates Aβ fibrils and releases the AChE from AChE-Aβ complexes.

A major issue in AD research is to find some new therapeutic drugs which decrease the Aβ aggregation and inhibit the AChE with dual specificity, being directed to both the active and ‘peripheral’ sites. For these reasons, we investigate the effect of the hyperforin derivative IDN 5706 in those activities. Hyperforin was described to prevent Aβ

Discussion

In vitro and in vivo studies indicated that AChE has the ability to enhance Aβ aggregation and amyloid fibril formation. In fact, when AChE was infused stereotaxically into the CA1 region of the rat hippocampus novel plaque-like structures were formed [14]. More recently, studies confirm that AChE accelerates Aβ deposition: a double transgenic mouse overexpressing both the human APP containing the Swedish mutation and the human AChE has been developed. Such double transgenic mouse starts to

Acknowledgments

This research was supported by grants from FONDAP (no. 13980001) and the Millennium Institute for Fundamental and Basic Biology (MIFAB). We thank to Christopher Adams for a critical review of the manuscript. Margarita C. Dinamarca and Waldo Cerpa are predoctoral fellows from CONICYT.

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