*2.3. Benefits by HT and HD in* α*-Synuclein-Induced Parkinsonian Models*

The transgenic *C. elegans* synucleinopathies-model 'OW13 features α-synuclein expression in the body wall muscle cells driven by the muscle specific *unc-54*-promoter. The resulting movement deficits were already described by Van Ham et al. [51] and were also visible in the current study. The wave initiation rate, for instance, deteriorated by more than 50% when comparing untreated wild type animals (Figure 2A) with OW13 nematodes (Figure 5A) at the 3rd day of adulthood. Both polyphenols were able to mitigate the α-synuclein-induced locomotion impairments, whereas HD showed slightly higher capacities in aged (A7) nematodes (Figure 5A–C): At the 7th day of adulthood, the wave initiation rate (Figure 5A), the body wave number (Figure 5B), and the activity index (Figure 5C) were improved by HD-treatment by 96%, 42%, and 70%, respectively, whereas HT led to an enhancement by 47%, 25 %, and 34%, respectively.

**Figure 5.** Benefits from HD & HT treatment in the OW13 strain. The nematode strain OW13 is characterized by α-synuclein expression in the body wall muscle cells. After polyphenol treatment, the wave initiation rate (**A**), the body wave number (**B**) and the activity index (**C**) were determined in two independent trials with ≥51 nematodes per treatment and age. Furthermore, the α-synuclein accumulation in muscle cells was quantified in two trials with ≥35 nematodes per treatment and age by fluorescence microscopy using a yellow filter (**D**). Data are presented as mean ± SEM. Differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). A3, A7, A12: 3rd, 7th and 12th day of adulthood. Finally, examples for the appearance of α-synuclein accumulation with increasing fluorescence intensities are shown (**E**).

The accumulation of α-synuclein in the muscle cells can be directly observed and quantified in the OW13-strain (Figure 5E). This is possible due to the transparent nature of *C. elegans* and due to the linkage of the yellow fluorescent protein (YFP) to the synthesized α-synuclein. Thus, fluorescence microscopy enabled the detection of potential α-synuclein-inhibiting abilities of the tested polyphenol treatments. Indeed, both treatments led to a reduction of accumulated YFP (Figure 5D), which is a direct indication for the decrease of the α-synuclein amount. The enhancement by the polyphenols is quite similar at A3 (5–6%), whereas HD showed clearly stronger effects at A7 and A12 with a decrease of 17% and 18%, respectively, compared to HT-treated nematodes with a decrease of 7% and 14% (Figure 5D). The overall reduction of the fluorescence intensities with age is based on the aging-dependent decline of *unc-54* expression [52].

Furthermore, the Parkinson's model 'UA44 was used to investigate the anti-Parkinsonian capacities of the polyphenol treatments. This strain is characterized by the expression of α-synuclein in dopaminergic neurons, which does not lead to distinct movement deficits [53] but to accelerated neurodegeneration [54]. Interestingly, only the wave initiation rate could profit from HT and HD treatment in this model (Figure 6A): HT increased the rate by 11% (A3) and 26% (A7) and HD by 45% (A3) and 28% (A7). No enhancement could be observed in the body wave number (Figure 6B) or the activity index (Figure 6C) by either polyphenol treatment.

**Figure 6.** Benefits from HD & HT treatment in the UA44 strain. The nematode strain UA44 is characterized by α-synuclein as well as GFP expression in dopaminergic neurons. After polyphenol treatment, the wave initiation rate (**A**), the body wave number (**B**) and the activity index (**C**) were determined in two independent trials with ≥48 nematodes per treatment and age. Furthermore, the neuronal viability was analysed in three trials with ≥39 nematodes per treatment and age by fluorescence microscopy using a green filter. Shown are the percentages of nematodes with degenerated dopaminergic anterior neurons with and without polyphenolic treatment (**D**). Data are presented as mean ± SEM. Differences compared to control were considered significant at *p* < 0.05 (\*) and *p* < 0.001 (\*\*). A3, A7, A12: 3rd, 7th and 12th day of adulthood. Two nematodes with neurodegeneration characterized by missing or weak fluorescence in the DA neurons (**E**) as well as two nematodes with intact neuronal appearance (**F**) are shown. The DA neurons are sub-classified as four CEP neurons (red arrows), which are superimposed in most pictures and two ADE neurons (yellow arrows).

In the UA44 strain, the green fluorescent protein (GFP) is linked to the dopamine transporter in dopaminergic neurons, thus, the vitality of the six anterior and two posterior dopaminergic (DA) neurons can be observed with a fluorescent microscope. The α-synuclein-induced damage of the nerve cells manifests as lowered or missing fluorescence in single neurons, whereas the classification of intact and degenerated anterior DA neurons were performed as described in Harrington et al. [55] and as illustrated in Figure 6E,F. The increase of degenerated neurons with age could be completely blocked by 250 μg/mL HD (Figure 6D). In all age classes, the quantity of degenerated anterior DA neurons constantly amounts to about 26% in the HD-treated group, whereas the quantity of degenerated neurons in the control group increased by 22% from 34% (A3) to 56% (A12). HT-treated nematodes also featured a neuroprotective effect, however, an increase of neurodegeneration with age is still visible.
