Biochimica et Biophysica Acta (BBA) - General Subjects
Oleuropein aglycone: A polyphenol with different targets against amyloid toxicity
Graphical abstract
Introduction
Systemic amyloidosis include severe, potentially fatal, diseases characterized by multiorgan deposition of insoluble fibrillar polymeric material grown from misfolded proteins. The rarity and the variable involvement of different organs and tissues are often responsible for missed or delayed diagnosis, which makes amyloid diseases a severe clinical challenge. A large wealth of data support the idea that the pathological features of systemic amyloidosis stem primarily from the concurrent presence of amyloid deposits arising from the ordered polymerization of a number of specific proteins and their circulating amyloidogenic precursors [1,2]. Consequently, at the present, the research for new compounds, either synthetic, natural or derived from natural molecular scaffolds, able to interfere with the aggregation of proteins involved in amyloid diseases, is considered a rewarding approach to develop new therapeutic strategies. Plant polyphenols have been extensively studied for their multiple biological activities, including anti-oxidant, anti-inflammatory, anti-diabetic, anti-aggregation and anti-cancer power [3]. These effects have raised the interest in plant polyphenols for their possible use, among others, to prevent tissue deposits of amyloid aggregates and to contrast pathological tissue reactions to the latter [4]. In particular, it has repeatedly been reported that the Mediterranean diet (MD), naturally enriched in plant polyphenols such as resveratrol (contained in red wine) and oleuropein (the main polyphenol in the olive leaves, drupes and oil) and many others is effective against age-related deterioration and improves aging-associated degenerative diseases and neurological deficits [5].
The glycated oleuropein molecule consists of the polyphenol hydroxytyrosol (4-(2-hydroxyethyl)benzene-1,2-diol) esterified to both elenolic acid and glucose. Recent research on oleuropein aglycone (OleA), a metabolic product resulting from the action of endogenous β-glucosidases, has focused on its general ability to remodel and inactivate toxic amyloid oligomers [6]. In particular, OleA interferes with amyloid aggregation of human islet amyloid polypeptide (hIAPP) [7], Aβ42 [8] and transthyretin [9] skipping the appearance of toxic oligomers and/or favoring aggregation into off-pathway aggregates. The behaviour of OleA matches that of many other polyphenols, even though the mechanism of their anti-aggregation effect can differ in different molecules [10]. In vivo studies have confirmed that OleA protects against Aβ42 aggregation and plaque formation in tissue. These studies were carried out using transgenic animal models of Aβ42 aggregation and plaque deposition such as C. elegans [11] and the TgCRND8 mouse model [12]. In particular, in the latter, dietary supplementation of OleA strongly improved the cognitive performance with respect to untreated littermates. Such a positive effect matched reduced Aβ42 levels and plaque deposits in the animal brain together with reduced inflammatory response, increased synaptic function (LTP), and neurogenesis, microglia migration to the plaques for phagocytosis and an intense activation of the autophagic flux. These biochemical modifications in OleA-fed mice were traced back, at least in part, to the induction of epigenetic modifications [13]. Finally, a recent study reports that OleA is able to locate at the surface of the cell membrane interacting specifically with negatively charged phospholipids of synthetic lipid bilayers [14], suggesting its effective ability to interact with the cell membrane. This finding is of interest considering that the interaction of OleA with the cell membrane can either interfere with bilayer-binding properties of amyloid species and/or increase the local concentration of the polyphenol itself; the latter can be a particularly relevant effect considering the low amounts of polyphenol intake with a normal diet and its limited bioavailability.
An increasing body of data considers amyloid cytotoxicity as the overall result of a complex interplay between the biochemical and biophysical properties of both amyloid aggregates and the cell membrane they interact with [[15], [16], [17], [18]]. The biochemical, biophysical and mechanistic aspects of this interaction are widely considered an early event of amyloid cytotoxicity and have been the focus of intense research in recent years [[15], [16], [17], [18], [19]]. However, the development of therapeutic strategies has been largely directed to search substances able to interfere with the process of amyloid aggregation [19], whereas the investigation of molecules able to interfere with membrane activity and lipid-binding properties of amyloid species has been addressed only marginally.
In this study, we extended and implemented our previous data on the effects of OleA against the first described naturally occurring structural variant, Asp76Asn (D76N), of human β2-microglobulin (b2m) [[20], [21], [22]], a 99 residue protein subunit of the MHC-I produced by all nucleated cells as a 119 residue precursor. b2m displays a β-sandwich fold typical of the immunoglobulin superfamily and contains seven β-strands. Normally, once released from the quaternary structure of MHC-I, b2m is cleared from the circulation in the proximal tubules of the kidneys. However, in patients undergoing long-term dialysis, the serum concentration of b2m rises from normal levels (1–2 mg/L) to as much as 50–70 mg/L. Such a dramatic increase of concentration is believed to be the main cause of b2m aggregation in these patients [23]. The pathogenic naturally occurring D76N variant is aggressively fibrillogenic in vitro, forcing a re-evaluation of previously hypothesized mechanisms of b2m fibrillogenesis. In contrast to patients with dialysis-related amyloidosis, all members of the family carrying such mutation had normal circulating concentrations of b2m and normal renal function. Despite the misfolding propensity of the D76N variant and the essential contribution of b2m to the structure of the MHC-I complex, none of the heterozygote subjects displayed clinical evidence of immunodeficiency [20]. On the contrary, extensive amyloid deposits of D76N b2m were found in the spleen, liver, heart, salivary glands, and nerves of the homozygotes where, in the absence of adequate therapy, the disease resulted in a fatal outcome [20].
We have recently reported that amyloid aggregates of D76N b2m interact specifically with the plasma membrane of the exposed cells at the level of GM1, a major lipid rafts component, and this findings was assumed to at the basis, at least in part, of their toxic effects [21]. These data led us to hypothesize that, particularly for D76N b2m amyloidosis, inhibiting aggregate-membrane interaction could be a very important way to protect cells against aggregate cytotoxicity thus reducing at least some symptoms of the disease. Accordingly, we extended our previous findings in SH-SY5Y cells by investigating the ability of OleA to interfere with D76N b2m aggregation and aggregate cytotoxicity. We found that OleA reduces D76N b2m cytotoxicity (i.) by favoring the appearance of non-toxic aggregates; (ii.) by decreasing aggregate interaction with the plasma membrane at GM1-enriched sites; (iii.) by enhancing cell resistance against the toxic effects of the aggregates. Our data offer the possibility to validate and optimize the use of OleA to rationally design novel and promising pharmacological tools to be used for prevention and therapy of systemic amyloidosis.
Section snippets
In vitro aggregation of D76N b2m
D76N b2M was expressed and purified as previously reported [22]. The aggregation reaction was initiated starting from the freeze-dried protein. Briefly, the protein was dissolved in PBS (Sigma Aldrich, Saint Louis, MO, USA) and centrifuged for 5 min at 13200 ×g. To achieve aggregation, the resulting supernatant was diluted to 20 μM concentration; and 300 μL of the solution was incubated at 37 °C in a 1.5 mL eppendorf tube lying in horizontal position under vigorous shaking. Protein aggregation
OleA interferes with amyloid aggregation of D76N b2m
We initially used different biophysical techniques to investigate whether OleA interfered with the aggregation path of D76N b2m previously described (see under Materials and methods) [22]. DLS analysis and negatively stained TEM showed that the general size and morphology of D76N b2m species obtained until 144 h of incubation under aggregating conditions were affected by the presence of the polyphenol (Fig. 1A, B). We chose the 144 h time since we previously showed that at that time the sample
Discussion
In systemic amyloidosis the accumulation in tissues and organs of large amounts of proteinaceous deposits may in itself be the primary origin of the clinical symptoms. Anyway, tissue damage and organ dysfunction move forward through mechanisms that are complexes and incompletely understood. The simple explanation of a physical mechanical replacement of parenchymal tissue by amyloid deposits seems to be insufficient. For instance, clinical observations of patients with amyloid light-chain (AL)
Conclusion
Our data add to, extend and confirm, many others obtained with different amyloidogenic proteins/peptides including amylin, Aβ peptides, transthyretin and wild type b2m, highlighting the interference of OleA with protein aggregation and aggregate cytotoxicity. Taken together, this body of coherent knowledge allows to further generalize the effects of OleA as a molecule with important protective effects against protein amyloid aggregation and some of the molecular and cellular derangements
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Acknowledgments
This project was supported by Ente Cassa di Risparmio di Firenze [N° 2015.1002A2202.3931]. M.L. was supported by grant from AIRAlzh Onlus financed by ANCC-COOP/Airalzh ONLUS (Reg.N°0043966.30-10-2014). S.R. was supported by grants from the Cariplo Foundation [Projects 2013 0964 and 2014 0700], the Telethon Foundation [Grant GG14127], the Italian Ministry of health [Ricerca Finalizzata RF 2013 02355259].
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