Healthspan Maintenance and Prevention of Parkinson's-like Phenotypes with Hydroxytyrosol and Oleuropein Aglycone in C. elegans

Abstract

Numerous studies highlighted the beneficial effects of the Mediterranean diet (MD) in maintaining health, especially during ageing. Even neurodegeneration, which is part of the natural ageing process, as well as the foundation of ageing-related neurodegenerative disorders like Alzheimer's and Parkinson's disease (PD), was successfully targeted by MD. In this regard, olive oil and its polyphenolic constituents have received increasing attention in the last years. Thus, this study focuses on two main olive oil polyphenols, hydroxytyrosol (HT) and oleuropein aglycone (OLE), and their effects on ageing symptoms with special attention to PD. In order to avoid long-lasting, expensive, and ethically controversial experiments, the established invertebrate model organism Caenorhabditis elegans was used to test HT and OLE treatments. Interestingly, both polyphenols were able to increase the survival after heat stress, but only HT could prolong the lifespan in unstressed conditions. Furthermore, in aged worms, HT and OLE caused improvements of locomotive behavior and the attenuation of autofluorescence as a marker for ageing. In addition, by using three different C. elegans PD models, HT and OLE were shown i) to enhance locomotion in worms suffering from α-synuclein-expression in muscles or rotenone exposure, ii) to reduce α-synuclein accumulation in muscles cells, and iii) to prevent neurodegeneration in α-synuclein-containing dopaminergic neurons. Hormesis, antioxidative capacities and an activity-boost of the proteasome & phase II detoxifying enzymes are discussed as potential underlying causes for these beneficial effects. Further biological and medical trials are indicated to assess the full potential of HT and OLE and to uncover their mode of action.

Keywords: C. elegans; Parkinson’s disease; ageing; healthspan; lifespan; olive oil; polyphenols.

Figure 1

Chemical structure of (A) oleuropein aglycone and (B) hydroxytyrosol.

Figure 2

Heat stress survival in presence of different OLE and HT concentrations. Survival is expressed as a percentage of the initial population combined with three biological replications. At the third day of adulthood (day 0), wild type nematodes were exposed to heat stress at 37 °C for 3 h prior monitoring survival. (A) Survival curves during OLE treatment with n (control) = 70, n (30 µg/mL) = 74, n (100 µg/mL) = 44, n (250 µg/mL) = 68, and n (500 µg/mL) = 64; (B) Survival curves during HT treatment with n (control) = 90, n (30 µg/mL) = 50, n (100 µg/mL) = 52, n (250 µg/mL) = 67, and n (500 µg/mL) = 79. Statistical significance was calculated by log-rank test including Bonferroni correction. Differences compared to control were considered significant at p < 0.05 (*) and p < 0.001 (**). n: number of tested nematodes.

Figure 3

Effect of OLE and HT on lifespan in C. elegans. The survival curves of controls and polyphenol-treated nematodes are shown. Survival is expressed as a percentage of the initial population per day. (A) The curve represents two independent experiments (n control = 184, n OLE = 111); (B) Representative survival curve of three independent experiments with control and HT-treated worms (n control = 250, n HT = 286). Statistical significance was calculated by log-rank test; differences compared to control were considered significant at p < 0.05 (*) and p < 0.001 (**). n: number of tested nematodes.

Figure 4

Age pigment quantification after OLE and HT treatment in C. elegans. Nematodes were observed by fluorescence microscopy in red spectrum at day 3, 7, and 12 of adulthood in two biological repeats. The bar charts (left) show the mean red fluorescence intensity of OLE (n A3 = 34; n A7 = 38; n A12 = 88) and DMSO (n A3 = 39; n A7 = 43; n A12 =84)-treated nematodes as well as HT (n A3 = 38; n A7 = 45; n A12 = 94)-treated nematodes and their respective water control (n A3 = 38; n A7 =44; n A12 = 95). Data are represented as mean ± SEM, and statistical differences compared to control were considered significant at p < 0.05 (*). n: number of tested nematodes; A3, A7, A12: 3^rd, 7^th and 12^th day of adulthood. In addition, example pictures (right) representing bright field and red fluorescence shots at the 12^th day of adulthood in the control (DMSO and water) and polyphenol-treated (OLE and HT) groups are shown (all scale bars = 200 µm).

Figure 5

Swim behaviour characteristics in wild type C. elegans treated with OLE and HT. Locomotory performances were determined at day 3, 7, and 12 of adulthood in two independent repeats. The determination of locomotion differences comprises three parameters: (A) the thrashing rate, (B) the body wave number, and (C) the activity index. The number of analysed animals accounts for: DMSO control = 67 (A3), 70 (A7) and 117 (A12); OLE = 63 (A3), 70 (A7) and 106 (A12); water control = 76 (A3), 70 (A7) and 111 (A12); HT = 70 (A3), 70 (A7) and 104 (A12). Data are presented as mean ± SEM and differences compared to control were considered significant at p < 0.05 (*) and p < 0.001 (**). A3, A7, A12: day 3, 7, 12 of adulthood.

Figure 6

Effect of OLE and HT on rotenone-induced locomotion deficits. The administration of 10 µM rotenone, starting at the fourth larval stage, led to Parkinsonian-like phenotype exhibiting movement impairments at the 3^rd and 7^th day of adulthood. (A) The thrashing rate, (B) the body wave number, and (C) the activity index are shown with and without simultaneous OLE or HT administration. The number of tested nematodes was: DMSO control = 58 (A3) and 69 (A7); OLE = 60 (A3) and 63 (A7); water control = 62 (A3) and 46 (A7); HT = 44 (A3) and 61 (A7). A3, A7: day 3 and 7 of adulthood. Data are pooled from two biological repeats and presented as mean ± SEM, and differences compared to control were considered significant at p < 0.05 (*) and p < 0.001 (**). To enable direct comparisons, data from nematodes without rotenone and polyphenol exposures (see Figure 5) are shown in addition.

Figure 7

OLE and HT effects on the swim performance in the OW13 strain. (A) The thrashing rate, (B) the body wave number, and (C) the activity index were determined at day 3 and day 7 of adulthood with and without OLE or HT treatment. Here, the nematode strain OW13, characterized by α-synuclein in the body wall muscle cells, was used. The number of tested nematodes was: DMSO control = 65 (A3) and 71 (A7); OLE = 61 (A3) and 60 (A7); water control = 51 (A3) and 71 (A7); HT = 61 (A3) and 66 (A7). A3, A7: day 3 and 7 of adulthood. Data are collected in two independent trials and are presented as mean ± SEM. Differences compared to control were considered significant at p < 0.05 (*) and p < 0.001 (**). To enable direct comparisons, data from wild type nematodes without polyphenol exposures (see Figure 5) are shown in addition.

Figure 8

Effect of OLE and HT on the swim performance in the C. elegans UA44 strain. (A) The number of thrashes per minute, (B) the number of waves running through the body, and (C) the area covered by the body per minute were observed in presence and absence of OLE and HT at day 3 and 7 of adulthood in two biological repeats. The UA44 strain used in this study is characterized by α-synuclein in dopaminergic neurons. The bar charts represent the following number of individuals: DMSO control (n A3 = 63, n A7 = 50), OLE (n A3 = 72; n A7 = 55), water control (n A3 = 57, n A7 = 55) and HT (n A3 = 48, n A7 = 65). n: number of tested nematodes. A3, A7: day 3, 7 of adulthood. Data are represented as mean ± SEM and differences compared to control were considered significant at p < 0.05 (*). To enable direct comparisons, data from wild type nematodes without polyphenol exposures (see Figure 5) are shown in addition.

Figure 9

Changed α-synuclein accumulation in muscle cells of the OW13 strain after OLE and HT treatment. Nematodes from the OW13 strain were subjected to polyphenolic treatments starting from L4 and analysed by fluorescence microscopy using a yellow filter at day 3, 7, and 12 of adulthood. The bars represent the mean fluorescent intensity ± SEM from two biological repeats and the number of tested nematodes were: DMSO control (n A3 = 32, n A7 = 48, n A12 = 38), OLE (n A3 = 28; n A7 = 43, n A12 = 30), water control (n A3 = 47, n A7 = 75, n A12 = 66) and HT (n A3 = 35, n A7 = 39, n A12 = 44). A3, A7, A12: day 3, 7, 12 of adulthood. Differences compared to control were considered significant at p < 0.05 (*) and p < 0.001 (**). In addition, three example pictures from untreated OW13 nematodes visualising the age-dependent fluorescent change are shown (all scale bars = 200 µm).

Figure 10

The impact of OLE and HT on dopaminergic neurodegeneration caused by α-synuclein in the C. elegans UA44 strain. (A) Shown are the percentages of nematodes with degenerated dopaminergic anterior neurons with and without polyphenolic treatment. The determination of the type and frequency of aberrations of dopaminergic neuronal viability was performed at day 3, 7, and 12 of adulthood. The number of tested nematodes in three biological repeats was: DMSO control = 43 (A3), 31 (A7) and 45 (A12); OLE = 38 (A3), 47 (A7) and 39 (A12); water control = 41 (A3), 44 (A7) and 71 (A12); HT = 39 (A3), 44 (A7) and 54 (A12). A3, A7, A12: day 3, 7, 12 of adulthood. Data were analysed using chi-square test with *p < 0.05 and **p < 0.001. In addition, an example of α-synuclein-induced degeneration in the anterior DA neurons of the C. elegans UA44 strain, expressing both Pdat-1::GFP + Pdat-1::α-syn is shown (B,C). The DA neurons are sub-classified as four CEP neurons, which are superimposed in most pictures, and two ADE neurons. (B) Degeneration of CEP and ADE; (C) intact DA neurons.