*4.1. Oleuropein and NDDs*

In our previous study performed in neuroblastoma cell lines, we found that OleA induced autophagy by activation of the Ca<sup>2</sup>+/Calmodulin Protein Kinase Kinase β (CaMKKβ)/AMPK/mTOR signalling axis. We proposed that OleA might induce autophagy through a Ca2+ increase in the cytoplasma from the endoplasmic reticulum that, in turn, activated Ca<sup>2</sup>+/CaMKKβ, and subsequently AMPK signaling. This complex facilitates mTORC1 inhibition and ULK1 activation to generate autophagic vacuole induction. We demonstrated that SH-SY5Y cells treated with 50 μM OleA showed an increased level of Beclin-1, critical for inducing autophagy, correlated with a biphasic elevation of the phosphorylation of residue Thr172 of AMPK [103]. The e ffects of OleA as an autophagy inducer have also been investigated in animal transgenic models. Grossi et al. [104], using a wildtype and TgCRND8 transgenic mouse model for human Aβ pathology, demonstrated that a diet supplemented with OleA restored the defective autophagic flux through an improvement of the fusion of lysosomes to autophagic vesicles, resulting in a remarkable cortex plaque reduction, and a recovery of the mice cognitive performance. They suggested that autophagy might be activated by inhibition of the mTOR pathway, reflected by the phosphorylation decrease of its target p70S6 protein kinase, shown in cell culture. These data indicate that autophagy may be considered as a strategic anti-amyloid mechanism, and sugges<sup>t</sup> that OleA and/or its derivatives may exert their neuroprotective function in the brain, crossing the blood-brain barrier, acting as autophagy–related anti-amyolid agents, enhancing the clearance of Aβ plaque deposition. The cognitive recovery showed in these Alzheimer's disease (AlzD) animal models supports the hypothesis that a diet supplementated with these polyphenols may have beneficial e ffects in slowing cognitive decline in patients with clinical signs of this disease.

Activation of NAD-dependent deacetylase sirtuin-1 (SIRT-1) is another mechanism through which OleA may modulate autophagy. SIRT1, a class III HDAC involved in the pathogenesis of several NCDs, deacetylates histones and non-histone proteins, such as transcription factors like p53, NF-κB, and FOXO, by transferring the acetyl group to NAD+. SIRT-1 influences autophagy directly (but also oxidative stress and apoptosis), via deacetylation of key components of this pathway. It showed a functional crosstalk with Poly (ADP-ribose) polymerase-1 (PARP-1) through NAD+ cofactor availability, and so any changes in levels of intracellular NAD+ and/or PARP-1 activity may influence SIRT-1 activity [105]. Luccarini et al. [106] showed that PARP-1 activation matched with a significant accumulation of PAR polymers in the cortex of TgCRND8 mice at the early (3.5 month) and intermediate (six month) stages of Aβ deposition. The same TgCRND8 mice fed with a supplementation of OleA showed a rescue of both PARP-1 activation, the accumulation of its product, and increased SIRT-1 expression. Moreover, OleA was able to reduce the rise of the apoptotic mediators phospho-NF-κ<sup>B</sup> and phospho-p53.
