*4.2. Polyphenol and Rotenone Treatment*

The treatment plates were prepared with the polyphenols oleuropein aglycone (Extrasynthese) and hydroxytyrosol (Sigma-Aldrich, St. Louis, MO, USA). Glycated oleuropein was de-glycosylated by treatment with almond β-glucosidase (EC 3.2.1.21, Fluka, Sigma Aldrich, St. Louis, MO, USA) as previously described [121], with minor modifications. Briefly, 10 mM oleuropein in 0.1 M phosphate buffer (pH 7.0) was incubated overnight with β-glucosidase (8.9 I.U.) at RT. Then, the reaction mixture was centrifuged, the precipitate re-suspended in 50% (*v*/*v*) dimethyl sulfoxide (DMSO) and the solution kept frozen. Complete deglycosylation was assessed by assaying the released glucose. HT powder was dissolved in bidest water at 60 mg/mL and the solution stored at −20 ◦C. OLE and HT were added to the bacteria and agar at a final concentration of 30 μg/mL, 100 μg/mL, 250 μg/mL, or 500 μg/mL, respectively. A final concentration of 0.05% DMSO (for OLE assays) or equal amounts of bidest water (for HT assays) were applied in all treatment and control plates as well as feeding bacteria.

To trigger the Parkinsonian-related phenotype, wild type nematodes were treated with rotenone (Sigma-Aldrich, St. Louis, MO, USA). A stock solution of 0.5 mg/mL rotenone was prepared in DMSO and added with a final concentration of 10 μM to the control and polyphenol plates. After distribution with a spatula and drying for 24 h in the dark, OP50, including 10 μM rotenone and the respective polyphenol, was spread on the plates. L4 nematodes were transferred to the rotenone plates until they were used for bioassays.
