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The extensive use of agricultural pesticides to improve crop quality and yield significantly increased the risk to the public of exposure to small but repeated doses of pesticides over time through various routes, including skin, by increasing the risk of disease outbreaks. Although much work was conducted to reduce the use of pesticides in agriculture, little attention was paid to prevention, which could reduce the toxicity of pesticide exposure by reducing its impact on human health. Extra virgin olive oil (EVOO), a major component of the Mediterranean diet, exerts numerous health-promoting properties, many of which are attributed to oleuropein aglycone (OleA), the deglycosylated form of oleuropein, which is the main polyphenolic component of EVOO. In this work, three pesticides with different physicochemical and biological properties, namely oxadiazon (OXA), imidacloprid (IMID), and glyphosate (GLYPHO), were compared in terms of metabolic activity, mitochondrial function and epigenetic modulation in an in vitro cellular model of human HaCaT keratinocytes to mimic the pathway of dermal exposure. The potential protective effect of OleA against pesticide-induced cellular toxicity was then evaluated in a cell pre-treatment condition. This study showed that sub-lethal doses of OXA and IMID reduced the metabolic activity and mitochondrial functionality of HaCaT cells by inducing oxidative stress and altering intracellular calcium flux and caused epigenetic modification by reducing histone acetylation H3 and H4. GLYPHO, on the other hand, showed no evidence of cellular toxicity at the doses tested. Pretreatment of cells with OleA was able to protect cells from the damaging effects of the pesticides OXA and IMID by maintaining metabolic activity and mitochondrial function at a controlled level and preventing acetylation reduction, particularly of histone H3. In conclusion, the bioactive properties of OleA reported here could be of great pharmaceutical and health interest, as they could be further studied to design new formulations for the prevention of toxicity from exposure to pesticide use.

The term neuroinflammation defines the reactions of astrocytes and microglia to alterations in homeostasis in the diseased central nervous system (CNS), the exacerbation of which contributes to the neurodegenerative effects of Alzheimer's disease (AD). Local environmental conditions, such as the presence of proinflammatory molecules, mechanical properties of the extracellular matrix (ECM), and local cell-cell interactions, are determinants of glial cell phenotypes. In AD, the load of the cytotoxic/proinflammatory amyloid β (Aβ) peptide is a microenvironmental component increasingly growing in the CNS, imposing time-evolving challenges on resident cells. This study aimed to investigate the temporal and spatial variations of the effects produced by this process on astrocytes and microglia, either directly or by interfering in their interactions. Ex vivo confocal analyses of hippocampal sections from the mouse model TgCRND8 at different ages have shown that overproduction of Aβ peptide induced early and time-persistent disassembly of functional astroglial syncytium and promoted a senile phenotype of reactive microglia, hindering Aβ clearance. In the late stages of the disease, these patterns were altered in the presence of Aβ-plaques, surrounded by typically reactive astrocytes and microglia. Morphofunctional characterization of peri-plaque gliosis revealed a direct contribution of astrocytes in plaque buildup that might result in shielding Aβ-peptide cytotoxicity and, as a side effect, in exacerbating neuroinflammation.

In Alzheimer's disease (AD), microglia, brain resident immune cells, become chronically inflammatory and neurotoxic. In recent years, neuroinflammation has attracted particular interest in the scientific community. The genetic variants of molecules associated with ''microgliopathies'', including the triggering receptor expressed in myeloid cells-2 (TREM2), result in increased risk of developing AD and cognitive decline. We performed a set of in vitro assays using human neuronal (SH-SY5Y) and microglial (BV2 and C13NJ) cell models. Cells were differentially treated with extra virgin olive oil (EVOO) polyphenols, oleuropein aglycone (OleA) and hydroxytyrosol (HT) before adding LPS. We evaluated the protective effects of these EVOO products by a set of biochemical and cell biology assays, including ELISA, MTT, ROS detection, Western blotting and immunofluorescence. Our results provide an integrated understanding of the neuroprotection exerted by polyphenols in terms of: (i) reduction of pro-inflammatory cytokines release (IL-6, IL-8, IP-10 and RANTES); (ii) activation of the TREM2-dependent anti-inflammatory pathway; (iii) enhancement of protective microglial activity favoring the M2 polarization phenotype. Such findings provide new and important insights into the mechanisms by which the dietary olive polyphenols exert beneficial properties against neuroinflammation and neuronal impairment.

The release of monomers from the homotetrameric protein transthyretin (TTR) is the first event of a cascade, eventually leading to sporadic or familial TTR amyloidoses. Thus, ligands able to stabilize TTR and inhibit monomer release are subject of intense scrutiny as potential treatments against these pathologies. Here, we investigated the interaction between TTR and a non-glycated derivative of the main olive polyphenol, oleuropein (OleA), known to interfere with TTR aggregation. We coupled fluorescence studies with molecular docking to investigate the OleA/TTR interaction using wild-type TTR, a monomeric variant, and the L55P cardiotoxic mutant. We characterized a fluorescence band emitted by OleA upon formation of the OleA/TTR complex. Exploiting this signal, we found that a poorly specific non-stoichiometric interaction occurs on the surface of the protein and a more specific stabilizing interaction takes place in the ligand binding pocket of TTR, exhibiting a K D of 3.23 ± 0.32 µM, with two distinct binding sites. OleA interacts with TTR in different modes, stabilizing it and preventing its dissociation into monomers, with subsequent misfolding. This result paves the way to the possible use of OleA to prevent degenerative diseases associated with TTR misfolding.

The interaction between the cell membrane and misfolded protein species plays a crucial role in the development of neurodegeneration. This study was designed to clarify the relationship between plasma membrane composition in terms of the differently linked sialic acid (Sia) content and cell susceptibility to toxic and misfolded Aβ-42 peptides. The sialylation status in different cell lines was investigated by lectin histochemistry and confocal immunofluorescence and then correlated with the different propensities to bind amyloid fibrils and with the relative cell susceptibility to amyloid damage. This study reveals that expressions of Sias α2,3 and α2,6 linked to galactose/N-acetyl-galactosamine, and PolySia are positively correlated with Aβ-42-induced cell toxicity. PolySia shows an early strong interaction with amyloid fibrils, favoring their binding to GM1 ganglioside containing α2,3 galactose-linked Sia and a loss of cell viability. Our findings demonstrate that cell lines with a prevailing plastic neuron-like phenotype and high monoSia and PolySia contents are highly susceptible to amyloid Aβ-42 toxicity. This toxicity may involve a change in neuron metabolism and promote a compensative/protective increase in PolySia, which, in turn, could favor amyloid binding to GM1, thus exacerbating cell dysmetabolism and further amyloid aggregation.

(1) Background: Autophagy, the major cytoplasmic process of substrate turnover, declines with age, contributing to proteostasis decline, accumulation of harmful protein aggregates, damaged mitochondria and to ROS production. Accordingly, abnormalities in the autophagic flux may contribute to many different pathophysiological conditions associated with ageing, including neurodegeneration. Recent data have shown that extra-virgin olive oil (EVOO) polyphenols stimulate cell defenses against plaque-induced neurodegeneration, mainly, through autophagy induction. (2) Methods: We carried out a set of in vitro experiments on SH-SY5Y human neuroblastoma cells exposed to toxic Aβ 1-42 oligomers to investigate the molecular mechanisms involved in autophagy activation by two olive oil polyphenols, oleuropein aglycone (OleA), arising from the hydrolysis of oleuropein (Ole), the main polyphenol found in olive leaves and drupes and its main metabolite, hydroxytyrosol (HT). (3) Results: Our data show that the mixture of the two polyphenols activates synergistically the autophagic flux preventing cell damage by Aβ 1-42 oligomers., in terms of ROS production, and impairment of mitochondria. (4) Conclusion: Our results support the idea that EVOO polyphenols act synergistically in autophagy modulation against neurodegeneration. These data confirm and provide the rationale to consider these molecules, alone or in combination, as promising candidates to contrast ageing-associated neurodegeneration.

The interplay between α-synuclein and dopamine derivatives is associated with oxidative stress-dependent neurodegeneration in Parkinson's disease (PD). The formation in the dopaminergic neurons of intraneuronal inclusions containing aggregates of α-synuclein is a typical hallmark of PD. Even though the biochemical events underlying the aberrant aggregation of α-synuclein are not completely understood, strong evidence correlates this process with the levels of dopamine metabolites. In vitro, 3,4-dihydroxyphenylacetaldehyde (DOPAL) and the other two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and 3,4-dihydroxyphenylethanol (DOPET), share the property to inhibit the growth of mature amyloid fibrils of α-synuclein. Although this effect occurs with the formation of differently toxic products, the molecular basis of this inhibition is still unclear. Here, we provide information on the effect of DOPAC on the aggregation properties of α-synuclein and its ability to interact with membranes. DOPAC inhibits α-synuclein aggregation, stabilizing monomer and inducing the formation of dimers and trimers. DOPAC-induced oligomers did not undergo conformational transition in the presence of membranes, and penetrated the cell, where they triggered autophagic processes. Cellular assays showed that DOPAC reduced cytotoxicity and ROS production induced by α-synuclein aggregates. Our findings show that the early radicals resulting from DOPAC autoxidation produced covalent modifications of the protein, which were not by themselves a primary cause of either fibrillation or membrane binding inhibition. These findings are discussed in the light of the potential mechanism of DOPAC protection against the toxicity of α-synuclein aggregates to better understand protein and catecholamine biology and to eventually suggest a scaffold that can help in the design of candidate molecules able to interfere in α-synuclein aggregation.

The increasing extension in life expectancy of human beings in developed countries is accompanied by a progressively greater rate of degenerative diseases associated with lifestyle and aging, most of which are still waiting for effective, not merely symptomatic, therapies. Accordingly, at present, the recommendations aimed at reducing the prevalence of these conditions in the population are limited to a safer lifestyle including physical/mental exercise, a reduced caloric intake, and a proper diet in a convivial environment. The claimed health benefits of the Mediterranean and Asian diets have been confirmed in many clinical trials and epidemiological surveys. These diets are characterized by several features, including low meat consumption, the intake of oils instead of fats as lipid sources, moderate amounts of red wine, and significant amounts of fresh fruit and vegetables. In particular, the latter have attracted popular and scientific attention for their content, though in reduced amounts, of a number of molecules increasingly investigated for their healthy properties. Among the latter, plant polyphenols have raised remarkable interest in the scientific community; in fact, several clinical trials have confirmed that many health benefits of the Mediterranean/Asian diets can be traced back to the presence of significant amounts of these molecules, even though, in some cases, contradictory results have been reported, which highlights the need for further investigation. In light of the results of these trials, recent research has sought to provide information on the biochemical, molecular, epigenetic, and cell biology modifications by plant polyphenols in cell, organismal, animal, and human models of cancer, metabolic, and neurodegenerative pathologies, notably Alzheimer's and Parkinson disease. The findings reported in the last decade are starting to help to decipher the complex relations between plant polyphenols and cell homeostatic systems including metabolic and redox equilibrium, proteostasis, and the inflammatory response, establishing an increasingly solid molecular basis for the healthy effects of these molecules. Taken together, the data currently available, though still incomplete, are providing a rationale for the possible use of natural polyphenols, or their molecular scaffolds, as nutraceuticals to contrast aging and to combat many associated pathologies., Competing Interests: The authors declare no conflict of interest