Oleuropein aglycone induces protective autophagy: molecular mechanisms and therapeutic targets in pathological models of autophagy dysfunction.

Macroautophagy, also referred to as autophagy, is an intracellular process aimed to degrade and recycle cytoplasmic components, including long-lived proteins and damaged organelles. Due to the pivotal role of autophagy in maintaining cellular proteostasis, its dysfunction is associated with a wide number of human diseases such as cancer, cardiomyopathies and neurodegenerative disorders. In these pathological conditions, autophagy is initially activated as a survival mechanism but subsequently becomes defective, leading to cell damage. Many recent studies aim to better understand why autophagy is compromised in pathological conditions. Consequently restoration of defective autophagy now appears as an important therapeutic strategy in disease contexts. Several small molecules acting as autophagy modulators, such as plant polyphenols, or natural compounds present in fruit and vegetables, have been proposed as potential therapeutic applications. Plant polyphenols are able to regulate autophagy through different pathways. In particular, resveratrol and epigallocatechin-3 gallate (EGCG) stimulate the autophagic pathway via CamKK-AMPK-mTOR signalling. Polyphenols can also activate the sirtuins (SIRT), family of class III histone deacetylases, which are also involved in autophagy modulation. SIRT-induction results in many cellular outcomes and is considered responsible for the epigenetic effects of polyphenols. Oleuropein is the main polyphenol found in the olive tree and its main product, olive oil. Our previous studies have highlighted the beneficial effects of oleuropein aglycone (OLE), both in neuroblastoma cell lines (N2a) and in TgCRND8 mice, a model of Aβ deposition. In the latter, food supplementation with OLE resulted in remarkable plaque reduction and in the reduction of cognitive impairment when compared to non-OLE fed littermates. These protective effects were strongly correlated to an increased activation of autophagy in OLE fed mice. In light of the benefits associated with the upregulation in autophagy, the aim of this thesis was to investigate the cellular and molecular effectors of OLE-induced autophagy in vitro, by use of cultured human neuroblastoma cells (SH-SY5Y) and in vivo, using our TgCRND8 mice. Our in vitro results showed that OLE supplementation induces a rapid release of Ca2+ from the endoplasmic reticulum stores which, in turn, activates CAMKKβ with subsequent phosphorylation and activation of AMPK. The interplay between AMPK activation and mTOR inhibition shown in the OLE-fed animal model supports the idea that autophagy activation by OLE proceeds through mTOR inhibition. SIRT1 activation, another mechanism that synergizes with OLE-induced Ca2+-CaMKKβ-AMPK-mTOR signalling was also found in N2a cells. Given our findings for OLE dependent promotion of autophagy in our in vitro and in vivo neurodegeneration models, we aimed to determine whether OLE promotion of autophagy is ubiquitous and could protect against other pathological conditions displaying autophagy dysfunction. We selected an in vitro model of cardiomyopathy characterized by overexpression of monoamine oxidase-A (MAO-A). It is well established that catecholamine and serotonin degradation by MAO-A produces H2O2, which then disrupts nuclear translocation of TFEB, a master regulator of autophagy, causing autophagosome accumulation and ultimately cell death. Using this model we have shown that OLE treatment counteracts the effects of the MAO-A/H2O2 axis by improving mitochondrial function and decreasing cell necrosis. We demonstrate that these protective outcomes are, at least in part, related to the activation in autophagy. Indeed, increased autophagy observed in cardiac cells treated with OLE was a measure of the increase in autophagic vacuoles and autophagy-specific marker (LC3II) expression. Double immunofluorescence imaging of RFP-GFP-LC3 after 6 h of OLE treatment showed an increase of the autophagic flux; in addition, nuclear translocation of TFEB in OLE-treated cells was also observed. Together these data suggest that OLE treatment evokes transcriptional regulation of autophagy. In conclusion, our findings demonstrate that the underlying molecular mechanism of OLE stimulated autophagy includes the activation of the Ca2+-CaMKK-AMPK-mTOR signalling pathway. The identified molecular underpinnings of OLE treatment are indeed similar to other plant polyphenols such as resveratrol and EGCG. We show further that SIRT1-activation could synergize to maintain OLE-induced autophagy. TFEB translocation to the nucleus supports the importance of the transcriptional regulation of autophagy, findings that warrent further investigation. The results of this thesis add to the growing knowledge base of the molecular mechanisms of OLE-induced autophagy and provide strong evidence that similar to other plant polyphenols OLE can be a potential therapeutic against age-related diseases associated with autophagy dysfunction, including neurodegeneration and cardiovascular diseases.