*2.5.* α*-synuclein Induced Damages and* α*-synuclein Accumulation was Targeted by Olive Oil Polyphenols In Vivo*

The advantage of the C. *elegans* OW13 strain is the yellow fluorescent labelling of synthesized α-synuclein driven by the muscle specific *unc-54*-promoter. Therefore, the potential polyphenolic inhibition of the pathological α-synuclein accumulation in muscles can be observed via fluorescence microscopy. Treatment of this model with either polyphenol resulted in a progressive reduction of α-synuclein accumulation in the body wall muscle cells compared to the control groups: The reduction of α-synuclein accumulation was about 5% at day 3 and 8% at day 7 and 12 of adulthood in OLE-treated groups (Figure 9). Even more pronounced effects were monitored by using HT, with a reduction of α-synuclein accumulation by 6% at day 3, 7% at day 7, and 14% at day 12 of adulthood. Overall, the fluorescence intensity declined with age, a finding in line with the ageing-dependent decline of *unc-54* expression observed by Adamla and Ignatova [66].

**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).

In addition to the OW13 strain, the UA44 transgenic animals were also monitored under fluorescent conditions. The α-synuclein accumulation in dopaminergic neurons causes neurodegeneration in the UA44 transgenic strain [63]. Moreover, GFP linked to the dopamine transporter in dopaminergic nerve cells allows to visualize the quantity and quality of the six anterior and two posterior dopaminergic neurons after α-synuclein-induced damage [67].

Counting and description of each dopaminergic neuron was performed as proposed by Harrington, et al. [68]. We counted six anterior dopaminergic neurons (four CEP neurons and two ADE neurons) and two posterior DA neurons (PDE neurons) [69]. Every type of alteration, such as the loss of uniformity of the neuronal body and the reduction of the fluorescence up to neuronal shutdown, were noted in order to classify the neurons as degenerated (as shown in Figure 10B) or intact (Figure 10C). The fraction of worms with damaged dopaminergic neurons was growing with age (Figure 10A), however, HT was able to minimize neuronal damages especially in older nematodes (Figure 10A). A smaller and non-significant neuroprotective effect on dopaminergic neurons was also obtained with OLE treatment (Figure 10A).

**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 P*dat-1*::GFP + P*dat-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.
