Insights into antiamyloidogenic properties of the green tea extract (−)-epigallocatechin-3-gallate toward metal-associated amyloid-β species

Abstract

Despite the significance of Alzheimer's disease, the link between metal-associated amyloid-β (metal-Aβ) and disease etiology remains unclear. To elucidate this relationship, chemical tools capable of specifically targeting and modulating metal-Aβ species are necessary, along with a fundamental understanding of their mechanism at the molecular level. Herein, we investigated and compared the interactions and reactivities of the green tea extract, (−)-epigallocatechin-3-gallate [(2R,3R)-5,7-dihydroxy-2-(3,4,5-trihydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-3-yl 3,4,5-trihydroxybenzoate; EGCG], with metal [Cu(II) and Zn(II)]-Aβ and metal-free Aβ species. We found that EGCG interacted with metal-Aβ species and formed small, unstructured Aβ aggregates more noticeably than in metal-free conditions in vitro. In addition, upon incubation with EGCG, the toxicity presented by metalfree Aβ and metal-Aβ was mitigated in living cells. To understand this reactivity at the molecular level, structural insights were obtained by ion mobility-mass spectrometry (IM-MS), 2D NMR spectroscopy, and computational methods. These studies indicated that (i) EGCG was bound to Aβ monomers and dimers, generating more compact peptide conformations than those from EGCGuntreated Aβ species; and (ii) ternary EGCG-metal-Aβ complexes were produced. Thus, we demonstrate the distinct antiamyloidogenic reactivity of EGCG toward metal-Aβ species with a structurebased mechanism.amyloid-β peptide | metal ions | natural products | amyloidogenesis T he brain of individuals with Alzheimer's disease (AD) has protein aggregates composed of misfolded amyloid-β (Aβ) peptides (1-4). The Aβ peptides are produced endogenously through enzymatic cleavage of amyloid precursor protein. Aβ monomers can misfold and oligomerize into various intermediates before the formation and elongation of fibrils that exhibit a characteristic cross-β-sheet structure (1-4). The accumulation of aggregated Aβ species has been a key feature of the amyloid cascade hypothesis, which cites that these aggregates are possible causative agents in AD. In addition, transition metals, such as Cu and Zn, whose misregulation leads to aberrant neuronal function, have a suggested link to AD pathology (1,(3)(4)(5)(6)(7)(8). In vitro and in vivo studies have provided evidence for the direct interactions of metal ions with Aβ and their presence within Aβ plaques, indicating the formation of metal-associated Aβ (metal-Aβ) species. These metal-Aβ species have been implicated in processes that could lead to neurotoxicity (e.g., metal-induced Aβ aggregation and metal-Aβ-mediated reactive oxygen species generation) (1, 3-8). The involvement of metal-Aβ species in AD pathogenesis, however, has not been clearly elucidated. To advance our understanding of the potential neurotoxicity of metal-Aβ species, efforts to develop chemical tools capable of interacting directly with metal-Aβ species and modulating their reactivity in vitro and in biological systems are under way (1,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)...

Modulators of amyloid protein aggregation and toxicity: EGCG and CLR01

“…and NMR experiments suggested that EGCG interaction with metal-Aβ species caused formation of more compact configurations than those formed by metal-free Aβ [106]. These observations suggested that EGCG binds to Aβ oligomers but not monomers, in agreement with the study by Palhano et al [78].…”

“…Similarly, EGCG disaggregated Aβ fibrils formed in the presence of metals after short incubation periods of 2-4 h, leading to formation of low-mass Aβ species [106]. In the presence of EGCG, regardless of the presence of metal ions, Aβ preparations were not toxic to murine Neuro-2a neuroblastoma cells [106].…”

“…[106]. Similarly, EGCG disaggregated Aβ fibrils formed in the presence of metals after short incubation periods of 2-4 h, leading to formation of low-mass Aβ species [106].…”

Brain Food for Alzheimer-Free Ageing: Focus on Herbal Medicines

“…Formed more compact peptide conformations compared to EGCG-untreated A" species; ternary EGCG-metal-A" complexes were produced. This illustrates the selective modulation of the anti-amyloidogenic reactivity of EGCG towards metal-A" species (Hyung et al 2013) Effect of the addition of EGCG in drinking water (1.5, 3 mg/kg for 3 weeks) intake in mice Prevented lipopolysaccharide-induced A" production by the inhibition of "-secretase activity, and improved effects on memory deficiency in liposaccharide-induced AD mice models (Lee et al 2009) Isothermal titration calorimetry studies on the interactions between EGCG and A" EGCG-A" binding was enhanced by increasing temperature, salt concentration and at pH values away from the pI of A" EGCG encapsulated in nanoparticles Improved in vivo efficacy, doubled bioavailability; improved chemical stability and enhanced its biological activity Hu et al 2013;Smith et al 2010) Protonation of EGCG at low pH Resulted in aggregation and reduced oral bioavailability of EGCG-dispersed selenium nanoparticles (Wu et al 2013b) Modulation of A"-induced tau hyperphosphorylation by curcumin (Cur) in human neuroblastoma SH-SY5Y cells Cur inhibits phosphorylation of tau at Thr231 and Ser396 by modulating the phosphatase and tensin homolog (PTEN) PTEN/Akt/GSK-3" pathway. Involves down-regulation of phosphorylation of Akt and of PTEN, a negative regulator of PIP3 induced by A" (Huang et al 2014a) Effects of Cur after 3-month administration to APPswe/PS1dE9 double transgenic mice, an AD model…”

Targeting Assembly and Disassembly of Protein Aggregates

“…Binding of EGCG with amyloidogenic proteins is supposed to happen via its oxidized 6.8b form, which should establish covalent bonds with free NH 2 (represented as a 4:1 Ab:6.8b adduct in Figure 6.4, real stoichiometry unknown) and/or SH groups of amyloidogenic proteins, contributing to neurotoxicity-reducing protein remodeling [137]. The metal chelation ability of EGCG supports its remodeling activity on naturally occurring, neurotoxicity-involved metalAb species [138].…”