Elsevier

Neurobiology of Aging

Volume 36, Issue 2, February 2015, Pages 648-663
Neurobiology of Aging

Regular article
Oleuropein aglycone protects against pyroglutamylated-3 amyloid-ß toxicity: biochemical, epigenetic and functional correlates

https://doi.org/10.1016/j.neurobiolaging.2014.08.029Get rights and content

Abstract

Amyloid-ß (Aß) fragments, oligomeric Aß aggregates, and pyroglutamylated-Aß peptides, as well as epigenetic mechanisms and autophagy dysfunction all appear to contribute in various ways to Alzheimer's disease progression. We previously showed that dietary supplementation of oleuropein aglycone, a natural phenol abundant in the extra virgin olive oil, can be protective by reducing Aß42 deposits in the brain of young and middle-aged TgCRND8 mice. Here, we extended our study to aged TgCRND8 mice showing increased pE3-Aß in the brain deposits. We report that oleuropein aglycone is active against glutaminylcyclase-catalyzed pE3-Aß generation reducing enzyme expression and interferes both with Aß42 and pE3-Aß aggregation. Moreover, the phenol astonishingly activates neuronal autophagy even in mice at advanced stage of pathology, where it increases histone 3 and 4 acetylation, which matches both a decrease of histone deacetylase 2 expression and a significant improvement of synaptic function. The occurrence of these functional, epigenetic, and histopathologic beneficial effects even at a late stage of the pathology suggests that the phenol could be beneficial at the therapeutic, in addition to the prevention, level.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the occurrence of 2 main histopathologic signs in the affected brain regions: extracellular amyloid plaques, composed of deposits of the amyloid-ß (Aß) peptides, and intracellular neurofibrillary tangles, composed of fibrillar polymers of the hyperphosphorylated tau protein (Selkoe and Schenk, 2003). The main component of the extracellular plaque deposits is a polymeric fibrillar form of the 42 amino acid Aß peptide (Aß42) generated by sequential proteolytic cleavage by ß- and γ-secretases of the amyloid precursor protein. Although the plaques are widely considered as the main contributors to the functional alterations and behavioral deficits that characterize AD (Haass, 2004), several lines of evidence point to a key role played by the early oligomeric Aß aggregates preceding the appearance of mature fibrils and/or possibly leaked from the latter (Hayden and Teplow, 2013). Oligomeric tau aggregates and hyperphosphorylated tau tangles (Barten et al., 2012, Gendron and Petrucelli, 2009) together with the effects of Aß peptides on tau phosphorylation state and degradation are also increasingly considered (Giacobini and Gold, 2013), leading to include AD among the tauopathies (Wang and Liu, 2008).

Besides full-length Aß peptides, a variety of different N-truncated Aβ peptides have been identified within the cored and diffuse amyloid plaques in the AD brain starting with amino residue Ala-2, pyroglutamylated Glu-3, Phe-4, Arg-5, His-6, Asp-7, Ser-8, Gly-9, Tyr-10, and pyroglutamylated Glu-11 (Antonios et al., 2013, Bayer and Wirths, 2014, Lemere et al., 1996, Saido et al., 1995).

Aß peptides can undergo N-terminal truncation by 2 or by 10 amino acids and subsequent cyclization of resulting N-terminal glutamate (E) to pyroglutamate (pE) which generates pE3-Aß(3-40/42) and pE11-Aß(11-40/42) peptides that display a loss of 3 charges and 6 charges, respectively with ensuing increase of their hydrophobicity (Gunn et al., 2010, Mori et al., 1992, Saido et al., 1995). These shortened pyroglutamylated peptides are reported to be more neurotoxic and to aggregate more rapidly than the full-length isoforms and to seed further Aß aggregation (He and Barrow, 1999, Nussbaum et al., 2012, Schilling et al., 2008). It is unclear whether pE3-Aß is present in early plaque deposits or it accrue later, and some authors have proposed that pyroglutamylated-Aß (pE-Aß) (40/42) peptides could be initiators of AD pathogenesis and others report that Aß4-42 precedes pE3-Aß (Antonios et al., 2013, Jawhar et al., 2011a, Schilling et al., 2008, Wirths et al., 2010).

The modification which originates the pE-Aß peptides is catalyzed by glutaminyl cyclase (QC, also known as QPCT) both in vitro (Schilling et al., 2004) and in vivo (Cynis et al., 2006, Cynis et al., 2008, Nussbaum et al., 2012, Schilling et al., 2008). In the mammalian brain, a physiologically relevant neuronal expression of QC was described in the hypothalamus, and enzyme involvement in neuropeptide and hormone maturation was shown (Bockers et al., 1995, Fischer and Spiess, 1987). In the AD brain, QC activity and pE-Aß levels are increased by several folds (Schilling et al., 2008) and distinct types of pE-Aß deposits, where pE3-Aß(3-42) predominates, have been identified at sites of QC-immunoreactive neurons and in target fields of QC-rich projection neurons (Hartlage-Rubsamen et al., 2011). Chronic pharmacologic inhibition of QC (Schilling et al., 2008) or suppression of its encoding gene (Alexandru et al., 2011, Wirths et al., 2009) in transgenic mouse models of AD resulted in reduced pE-Aß peptide generation and improved performance in cognitive tasks, whereas QC overexpression worsened neuropathology and cognitive dysfunction (Jawhar et al., 2011b).

We have recently reported that 8-week dietary supplementation of oleuropein aglycone (OLE), a natural phenol (secoiridoid) abundant in the extra virgin olive oil, improves learning and memory, reduces de novo Aß42 deposition, favors preformed Aß42 plaque disassembly and greatly stimulates the autophagic response in the young and middle-aged TgCRND8 (Tg) mouse model of Aß deposition (Grossi et al., 2013). Our aim was to provide more information on the molecular and biological basis of these effects by investigating whether the same dietary supplementation of OLE resulted, directly or indirectly, in a reduction of the toxic pE3-Aß peptide deposits in the brain of the same Tg mouse model at different ages corresponding to early, intermediate, and late stage of Aß deposition. We also checked the level of histone acetylation in the oldest treated and untreated animals to ascertain whether the protective effects of OLE supplementation also involved epigenetic modifications. In fact, recent data indicate that epigenetic changes are implicated in learning and memory (Graff et al., 2012) and these modifications have been suggested as potential targets for AD therapeutics (Adwan and Zawia, 2013). Accordingly, we investigated the functional effects of OLE in aged animals by assessing in brain slices whether its administration in vitro was able to counteract dysfunctions of transgene-induced long-term potentiation (LTP) in the CA1 hippocampal area.

Our study provides evidence that the massive age-dependent deposition of toxic pE3-Aß is strongly reduced in the TgCRND8 mice fed with a normal diet supplemented with OLE and that such a decrease likely reflects the parallel reduction of QC expression. Furthermore, we show that, in our mouse model of Aβ deposition, autophagy activation, histone acetylation, and LTP facilitation underlie the OLE-promoted benefits at the histopathologic and functional level, suggesting new perspectives for AD not only at the prevention but also at the therapeutic level.

Section snippets

Ethics statement

Transgenic hemizygous CRND8 male and female mice, harboring a double-mutant gene of APP695 (Chishti et al., 2001) with a (C57)/(C57/C3H) genetic background, and non-Tg hybrid (C57)/(C57/C3H) wild type (wt) control littermate mice were used following the ECC (DL 116/92, Directive 86/609/EEC) and National guidelines for animal care. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Italian Ministry of Health (Permit Number: 283/2012-B).

Animals

The following 3 age

OLE modifies pE3-Aß aggregation in vitro

Our previous data showed that OLE interferes with the aggregation in vitro of Aß42 by skipping the formation of toxic species (Rigacci et al., 2011). Therefore, initially we sought to assess whether, and to what extent, OLE modified pE3-Aß aggregation as well with respect to Aß42. To this purpose, 25 μM pE3-Aß was incubated under aggregation conditions in the presence or in the absence of 100 μM OLE, and its secondary structure was analyzed at different aggregation times by CD. Immediately

Discussion

The TgCRND8 mouse strain is widely used as a model of Aß deposition in AD studies. In this model, Aß deposits increase over time and become robust by 7–9 months of age (Bellucci et al., 2007, Rosi et al., 2010). We have previously reported that OLE administration in young and middle-aged TgCRND8 mice results in remarkably beneficial behavioral, molecular, and histopathologic effects, suggesting that it could be useful to treat patients with presymptomatic or early AD (Grossi et al., 2013).

Conclusion

In conclusion, our previous (Grossi et al., 2013) and present data do indicate that OLE or its main metabolites produced after OLE administration, as shown by our LC-MS/MS data, improve memory and behavioral performance; they do this by acting as multifunctional drugs that restore, at least in part, in the brain of a mouse model of AD different dysfunctional pathways by interfering with Aß aggregation as well as with signaling and epigenetic pathways. At present, the identified cellular targets

Disclosure statement

The authors declare that they have no conflicts of interest.

Acknowledgements

The authors thank Dr P.St.G. Hyslop and Dr D. Westaway for supplying the TgCRND8 mouse strain. This work was supported by the Regione Toscana: “Programma per la Ricerca Regionale in Materia di Salute 2009,” by the ECRF 2010-2011 and the Università degli Studi di Firenze.

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