Description

Parkinson’s disease (PD) patients have been shown to benefit greatly from intense physical activity (Schenkman et al. 2018). Recent studies have demonstrated that exercise causes changes in the levels of ⍺-synuclein aggregate species, a hallmark of PD, in different mammalian animal models (Koo and Cho 2017; Shin et al. 2017; Zhou et al. 2017; Minakaki et al. 2019). However, questions still remain about how exercise affects specifically native ⍺-synuclein protein species directly after the cessation of exercise and the longer-term downstream effects which exercise may have on organismal health. It was recently discovered that periods of thrashing in liquid solution, otherwise called swimming exercise, in C. elegans worms, induces many mechanisms invoked during mammalian exercise (Laranjeiro et al. 2017). This has provided an avenue for studying exercise conditions in various C. elegans models of neurodegeneration (Laranjeiro et al. 2019). In order to study the effect of exercise on native human ⍺-synuclein protein species, we utilized the NL5901- pkIs2386 worm model of Parkinson’s which contains human ⍺-synuclein tagged to a yellow fluorescent protein (YFP) in the muscle cells (van Ham et al. 2008). We performed tissue analysis via Blue Native (BN) page westerns and confocal microscopy. In addition, because pharyngeal pumping is decreased while worms are swimming, we controlled for this effect by exposing worms in parallel to a period of food restriction (FR) conditions (Vidal-Gadea et al. 2012). We also performed thrashing assays to assess longer term downstream behavioral effects on the animals after either exercise or food restriction conditions.

It has been previously reported that NL5901 worms exhibit a lower thrashing ability than N2 worms due to the presence of human ⍺-synuclein species in the muscle cells (Anand et al. 2020). In our experiments, we subjected Day 1 NL5901 worms to a period of swimming exercise — 15-20 mins — and observed a dramatic effect upon the quantity of native human ⍺-synuclein proteins via BN Page westerns using an anti-GFP antibody to detect ⍺-synuclein::YFP proteins as previously described (Figure 1 A-D) (van Ham et al. 2008). As worms continue to swim while they are being collected for Western Blot assays, and because the speed of this collection can vary, we have indicated the 15-20 mins time frame in order to account for this variation. Three different protein groups were analyzed — 66-<480 kDa, 720 kDa, and proteins trapped within the wells (well proteins), which may be large molecular weight protein species. In the exercised worms, 66-<480 kDa and well proteins were found to be significantly decreased compared to control (un-treated) and food restricted worms. Interestingly, we also observed that the protein profile in the 66-<480 kDa range was different for exercised versus food restricted worms — while in exercised worms there appeared only a single band for ⍺-synuclein at ~90 kD, in food restricted worms we detected a range of protein similar to, but less abundant than those proteins present in control (un-treated) worms (Figure 1B). It has been shown that ⍺-synuclein (~14 kDa) runs at a higher molecular weight (~50 kDa) in BN page gels (Bartels et al. 2011). Therefore, we hypothesize that ⍺-synuclein tagged to YFP (~41 kDa in SDS gels) is the ~90 kDa band visible in the BN page gel in Figure 1B. For FR worms, of the three protein groups analyzed, only the 66-<480 kDa range of proteins and the well proteins show a statistically significant reduction when compared to control (un-treated) worms (Figure 1A and D, respectively), while 720 kDa proteins do not show a significant decrease (Figure 1C). It has been previously reported that exercise increases autophagy and plays a role in attenuating levels of ⍺-synuclein in an MPTP/P mouse model of Parkinson’s disease (Koo and Cho 2017). We therefore hypothesize that the decrease in human ⍺-synuclein protein species we have observed due to 15-20 min of swimming exercise in the NL5901 C. elegans model of PD may be attributed to protein degradation through an increase in autophagy.

We also utilized thrashing assays in order to determine the physiological effects of exercise and food restriction on ⍺-synuclein expressing worms. This method is commonly used to quantify worm neuromuscular function and is performed by quantifying the rate with which the worms swim, or thrash, during a 30 second interval. Since our results have shown that placing NL5901 worms into liquid and allowing them to thrash/swim causes ⍺-synuclein protein species to decrease, we performed the thrashing assay at 3 mins, a time point which does not induce a strong effect upon ⍺-synuclein protein species. In addition, in order to observe the effect of a full bout of swimming exercise upon ⍺-synuclein protein species in real time, we allowed worms to continue swimming undisturbed until 20 mins and recorded their thrashing rates at this time as well. On Day 2, we found that at the 3 min time point, previously exercised worms showed a significantly higher thrashing rate than control (untreated) or FR worms (Figure 1E). By 20 mins (i.e. after a period of exercise), control (untreated) worms had improved their thrashing rate and attained the speed of previously exercised worms (Figure 1F), suggesting a reduction in ⍺-synuclein aggregation due to swimming, as supported by the BN page results (Figure 1 A-D). In contrast, FR worms did not improve their thrashing speed over the 20 min period, indicating that prior food restriction may have negative effects on either ⍺-synuclein aggregation and/or the health of the worms.

Thrashing assays were also performed on Day 4. At the 3 min mark, the worms did not exhibit any differences between the three conditions, albeit all displayed lower thrashing rates than on Day 2 (Figure 1G). The phenomenon of reduced thrashing rates with age has also been previously observed in N2 worms (Hahm et al. 2015). After 20 mins however, previously exercised worms achieved a significantly higher thrashing rate than control (un-treated) worms and a trend towards a significantly higher rate than FR worms (p≤0.0673) (Figure 1H). In addition, previously exercised worms achieved a thrashing rate similar to the control (un-treated) worms on Day 2, suggesting that exercise exerts a restorative effect upon older NL5901 worms. We also noticed that unlike on Day 2, FR worms were able to improve their thrashing frequency between 3 mins and 20 mins, indicating that any negative effect which food restriction may have had at Day 2 was reduced by Day 4.

Using confocal microscopy, we assessed the effect of exercise and FR on the surface area (SA) size of ⍺-synuclein puncta located in the head region of the NL5901 worms as previously reported (Goya et al. 2020) (Figure 1 I-N). Here, we observed that exercised and FR worms have less puncta overall than control (un-treated) worms. However, we also observed that the average surface area values for the puncta in each worm did not reflect significant differences between Ex and FR conditions (calculations not shown.) Therefore, we investigated if differences may be more apparent between these conditions if instead, we observed the individual sizes of each puncta rather than the average values calculated per worm. As shown in the heat map in Figure 1O, exercised worms have a greater concentration of smaller puncta (dark blue) than control (un-treated) or FR worms. All values were normalized to their respective controls and correspond to values from 0 to 3. We hypothesize that the preparation of samples in PFA solution may have resulted in a window of time where worms continued to swim in solution, and ultimately, may have caused smaller differences across the three separate conditions, as observed in panels I-N, particularly between exercised and FR worms.

We observed that 15-20 mins of swimming exercise on Day 1 of adulthood caused a dramatic effect upon ⍺-synuclein protein quantity and species, as well as improved the thrashing rate of worms on Day 2 and Day 4 (Figure 1 A-H). Currently, we hypothesize that autophagy mechanisms may be upregulated during this brief period of exercise leading to the dramatic differences in ⍺-synuclein protein species observed. Exercise on Day 1 also allowed worms on Day 4 to reach the thrashing frequency of Day 2 control (un-treated) worms after 20 mins of swimming (Figure 1H). In contrast, FR on Day 1 appeared to hinder the swimming abilities of worms on Day 2 (Figure 1E, F.) By Day 4 however, food restricted worms appeared to be rehabilitated, although they still showed a trend towards a slower swimming pattern than exercised worms after 20 mins of swimming (Figure 1H). In support of this data, our confocal analysis shows that exercised worms have a higher concentration of smaller puncta (Figure 1O.) However, we observed that while both exercised and FR worms have less puncta than control (un-treated) worms, the differences between exercised and FR worms are not as dramatic as in the results observed in the BN page and thrashing assays. We are currently exploring differences between exercised and food restricted worms using alternate confocal methods and/or sample preparation. In the future, we are also interested in exploring further the mechanistic basis for the observed divergent effects of Ex versus FR on ⍺-synuclein aggregates. However, our results indicate the presence of a set of unique and separate relationships between ⍺-synuclein aggregation in the context of exercise versus food restriction.