*2.4.* C. elegans *Parkinsonian Models Profit from Olive Oil Polyphenol Treatments*

Exposure to the pesticide rotenone or the transgenic expression of human α-synuclein induces the Parkinsonian-like syndrome in C. *elegans*, which manifests in impaired movement [62,63]. In order to assess whether the polyphenols are able to reduce this symptom, the swimming analysis was performed with nematodes suffering from Parkinson's-like symptoms induced by exposure to rotenone of wild type worms. Furthermore, transgenic Parkinson's models expressing human α-synuclein in muscle cells (strain OW13) and dopaminergic neurons (strain UA44), respectively, were treated with polyphenols as well.

Treatment with 10 μM rotenone led to dramatically decreased movement abilities. Given that untreated nematodes exhibit more than 50 thrashes per minute at A3, the thrashing rate was reduced by more than 80% to less than 10 thrashes per minute in rotenone-exposed C. *elegans* (Figure 6A). Similar proportions could be monitored for the activity index of young control nematodes with or without rotenone treatment (Figure 6C). Furthermore, the body wave number was more than doubled due to exposure to rotenone compared to untreated nematodes (Figure 6B). Interestingly and in contrast to rotenone-untreated nematodes, the locomotion abilities after exposure to rotenone did not decline after the 3rd day of adulthood (Figure 6). OLE was able to partly inhibit the rotenone-induced movement decline in both tested ages and all swim traits by more than doubling the measured values (Figure 6A,C) or by reducing them by at least 42% (Figure 6B). Quite strong effects were also visible by using HT in all tested ages and endpoints: HT increased the thrashing rate by at least 56% and the activity index by a minimum of 116%. The body wave number was decreased by at least 23%.

**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 3rd and 7th 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.

To check the anti-Parkinson's effect of olive oil polyphenols, the motor activity was also assessed in the transgenic C. *elegans* OW13 and UA44 strains, both models of synucleinopathies. The presence of α-synuclein in the body wall muscle cells (strain OW13) leads to movement deficits, as previously described by Van Ham, et al. [64]. Indeed, the thrashing rate clearly deteriorated from about 50 thrashes/min in young untreated wild type adults to about 35 and 23 thrashes per minute, respectively, in young untreated OW13 worms (Figure 7A). This effect, albeit weaker, was also seen, in part, in older nematodes as well as for the activity index (Figure 7C). The body wave number was very stable between the 3rd and 7th day of adulthood (Figure 7B). Interestingly, control DMSO treatment already led to a mild beneficial effect for all three swim parameters in the OW13 strain.

**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.

Treatment with either polyphenol improved several swim performance features in this strain over control treatments (Figure 7A–C). OLE administration provoked a 27% increase of thrashes per minute at day 3 and a 40% increase at day 7 (Figure 7A), whereas the increase of the activity index (27%) was detected only at day 7 (Figure 7C) and no significant change was seen in the body wave number (Figure 7B). HT displayed its advantageous effects in both tested age groups and in all swim parameters (Figure 7A–C), whereas HT remarkably increased the thrashing rate by 71% in A3.

Interestingly, the strain UA44, which is characterized by the presence of α-synuclein in dopaminergic neurons, did only show a negligible decline in locomotion from day 3 to day 7 of adulthood (Figure 8). Furthermore, the difference in swim performance between young wild type and young UA44 nematodes was hardly recognizable, a finding that agrees with data from a previous study, showing that α-synuclein controlled by a dopaminergic promotor did not disturb the thrashing speed [65]. Surprisingly, treatment with either polyphenol resulted only in a limited improvement in swim behaviour of the UA44 strain. OLE treatment increased the activity index in young nematodes (Figure 8C) but resulted only in minor and non-significant changes of the thrashing rate and body wave number for UA44 (Figure 8A,B). On the other hand, HT supplementation showed beneficial effects only on the number of thrashes per minute in older worms (Figure 8A), but not on the magnitude of movement or the waviness (Figure 8B,C).

**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.

To summarize, HT and OLE treatments resulted in enhanced swim performance in nematodes suffering from rotenone exposure or α-synuclein expression in muscle cells (strain OW13). However, the strain UA44, characterized by α-synuclein expression in dopaminergic neurons, only weakly profited from the polyphenol treatments.
