The wild type worm (Bristol N2) and transgenic line of NL5901 (pkIs2386, unc-54p::alphasynuclein::YFP) were obtained from the Caenorhabditis Genetics Center (CGC). Worms were grown on the nematode growth medium (NGM) agar plate and fed with Escherichia coli OP50 at 20°C (Brenner, 1974). Synchronization of nematode culture was achieved using treatment with sodium hypochlorite and 1 M NaOH (1:1). The synchronized eggs were cultured in M9 buffer for 24 h at 20°C to hatch L1 larvae. Then, the L1 stage worms were transferred to NGM plate.

RNAi feeding experiments were performed on synchronized L1 to L4 larvae at 20°C. E. coli HT115 (DE3) containing empty vector (pL4440) or target genes strains were cultured overnight in LB medium containing 100 μg/mL ampicillin and 12.5 μg/mL tetracycline at 37°C. Then spread to NGM plates containing 25 μg/mL carbenicillin and 1 mM isopropyl 1-thio-b-β-galactopyranoside (IPTG) at 37°C overnight. The synchronized NL5901 worms were transferred to NGM plates seeded with RNAi bacteria and allowed to grow until mature. RNAi efficiency was evaluated prior to starting the experiment.

Effect of MD6a on the α-syn aggregation was evaluated using NL5901 strain. Briefly, age-synchronized worms were washed three times with M9 buffer to get rid of remaining bacteria and mounted onto 2% agarose pads. Then, worms were immobilized with 20 mM sodium azide. To monitor the α-syn aggregation, YFP protein was visualized and photographed with a fluorescence microscope (Olympus BX63). The fluorescence intensity was quantified using ImageJ.

Briefly, the collected worms added extract liquid were grinded by freeze-thawing with liquid nitrogen. Centrifugation was performed at 4°C for 10 min at 12,000 rmp and 30 µL of the supernatant was taken and then assayed with the ATP Content Assay Kit (Solarbio). And protein quantification was performed with BCA Protein Assay kit (Biosharp).

Oxygen consumption rates were measured using the Oxytherm (Hansatech, United Kingdom), a Clark-type oxygen electrode as described previously (Schulz et al., 2007). The collected nematodes were resuspended in 1 mL of M9 and transferred to the chamber. Oxygen concentration was monitored with a Clark electrode in a closed chamber for 10 min. The nematodes were subsequently collected. Protein concentration was measured using the BCA Protein Assay kit.

At least 15 worms on day 0 of adulthood were randomly selected and transferred to a glass slide containing 10 µL M9 buffer. After allowing the worms to acclimate to the liquid medium for 30 s, their movement was continuously recorded by a microscope (MZ62) for 10 s.

To determine mitochondrial ROS levels, nematodes were stained with MitoSOX™ Red for 20 min. Then, the worms were anaesthetised with 20 mM sodium azide solution and mounted on microscopic slides. The fluorescence was examined under a fluorescence microscope (Olympus BX63). The Fluorescence intensity was quantified using ImageJ.

Nematodes were cultured to the L3 stage placed into 100 nM TMRE dishes stained for 24 h, and worms were transferred to plates without dye for 1 h prior to imaging to clear the gut of residual dye (Berry et al., 2023). Nematodes were mounted on 2% agarose pads under 20 mM sodium azide anesthesia. Changes in mitochondrial membrane potential fluorescence intensity were observed using a confocal (Leica TCS SP8). Fluorescence intensity was quantified using ImageJ.

Nematodes were treated with MD6a to incubate the L4 stage, and total RNA was extracted from nematodes of each treatment group using TRIzol reagent (Invitrogen), as previously described (Lapierre et al., 2013). The RNA of worms was reverse transcribed into cDNA by using PrimeScript™ RT reagent Kit with gDNA Eraser. Real-time PCR was performed according to the primers designed in Table 1.

The accumulation and aggregation of α-syn is known as one of the pathological features in PD patients. Previous studies have demonstrated that pathological α-syn can activate PARP-1, thus leading to the loss of dopaminergic neurons in PD (Kam et al., 2018). We used the C. elegans strain NL590, which expresses human α-syn fused with YFP under an unc-54 promoter, to elucidate the effects of MD6a on α-syn aggregation. The parp-1 mRNA levels in wild-type N2 and NL5901 worms were examined by qPCR. The result showed parp-1 expression in NL5901 nematodes was significantly higher than that in N2 worms, treatment with 10 μM MD6a significantly reduced parp-1 mRNA levels in NL5901 nematodes (Figure 1A). To confirm the role of parp-1 in the neuroprotective effects of MD6a, we genetically knocked down parp-1 expression using RNAi. Reduced α-syn accumulation was observed in NL5901 worms treated with both 10 μM MD6a and parp-1 RNAi (Figures 1B,C). However, under parp-1 RNAi, 10 μM MD6a failed to further reduce α-syn aggregation in NL5901 nematodes compared with parp-1 RNAi group (Figures 1B,C). Together, these results indicate that the MD6a-induced reduction of α-syn aggregation in C. elegans is dependent on parp-1.

Mitochondrial dysfunction is widely considered as a main contributor to neurodegeneration in PD (Park et al., 2018). The complex interplay between mitochondrial dynamics and bioenergetics is especially important for neuronal function. In general, neurons have high energy demands that require many functional mitochondria. The activation of pathological parp-1 causes mitochondrial dysfunction, leading to ATP depletion and mitochondrial membrane potential decline (Cipriani et al., 2005; Luo and Kraus, 2011). To elucidate whether MD6a acts as a PARP-1 inhibitor to contribute to protecting mitochondrial function in PD, we measured the oxygen consumption rate (OCR), ATP levels, movement ability, mitochondrial ROS production and mitochondrial membrane potential. The results demonstrated that MD6a treatment significantly affected mitochondrial biogenesis in NL5901 worms, with markedly increased OCR levels (Figure 2A), ATP production (Figure 2B), movement ability (Figure 2C) and mitochondrial membrane potential (Figures 2D,E), while observably decreased mitochondrial ROS levels (Figures 2F,G). Furthermore, parp-1 RNAi could mimic all these beneficial effects of MD6a which suggested that MD6a improve mitochondrial dysfunction through parp-1. However, MD6a could not further increased OCR, ATP, movement, mitochondrial membrane potential levels (Figures 2A–E), and decreased mitochondrial ROS levels (Figures 2F,G) under parp-1 RNAi. Taken together, these results indicate that MD6a significantly enhances mitochondrial function in the PD nematode model through parp-1.

The mammalian target of rapamycin (mTOR) signaling pathway plays an important homeostatic function in the regulation of energy metabolism, cell survival, senescence, and neurodegeneration (Wullschleger et al., 2006). Moreover, increasing evidence indicates that mTOR is critical for the pathogenesis of PD. A previous study reported that mTOR protein expression levels are increased in the temporal cortex of patients with α-syn accumulation (Crews et al., 2010). We explored the effects of MD6a on the TOR signaling pathway in C. elegans, and revealed that the expression of let-363, an ortholog of TORC1, was downregulated by 10 μM MD6a (Figure 3A). Furthermore, although 10 μM MD6a did not decrease let-363 expression in parp-1 RNAi-treated worms (Figure 3A), it decreased parp-1 expression in let-363 RNAi-treated worms (Figure 3B). These results suggest that the MD6a-mediated regulation of let-363 is dependent on parp-1. Additionally, the fluorescence intensity of α-syn was reduced by let-363 RNAi treatment in NL5901 nematodes; however, 10 μM MD6a did not reduce α-syn fluorescence intensity in let-363 RNAi-treated nematodes compared with let-363 RNAi group (Figure 3D). These findings indicate that MD6a downregulates let-363 in a parp-1-dependent manner to reduce α-syn aggregation.

Previous studies have identified a number of transcription factors downstream of TOR, including DAF-16/FOXO, SKN-1/NRF, HSF-1/HSFs, PHA-4/FOXA, HLH-30/TFEB, and RPC-1/POL III (Blackwell et al., 2019). In this study, we found hsf-1 mRNA levels were downregulated by treatment with 10 μM MD6a, and parp-1 RNAi blocked this MD6a-mediated reduction (Figure 4A). Previous reports have demonstrated that α-syn aggregation is related to dysfunction of the protein degradation pathway, including of heat shock proteins (HSPs) (Jones et al., 2014). HSP-4 and HSP-6 are involved in regulating intracellular protein degradation processes by binding to damaged or obsolete proteins and removing unstable proteins via endoplasmic reticulum-associated protein degradation (ERAD) (Shin et al., 2022) and mitochondria-associated degradation pathway (Rolland et al., 2019). We thus evaluated the mRNA levels of hsp-4 and hsp-6, which are associated with HSPs in NL5901 worms. The results revealed that MD6a significantly reduced degradation-regulated genes in NL5901 nematodes (Figures 4B,C). Furthermore, parp-1 RNAi reduced the expression levels of hsp-4 and hsp-6 in NL5901 worms. However, 10 μM MD6a treatment could not further reduce the mRNA levels of hsp-4 and hsp-6 under parp-1 RNAi (Figures 4B,C). Together, these results indicate that MD6a inhibits PARP-1 downregulation of the let-363/hsf-1 signaling pathway by targeting the reduction of hsp-4 and hsp-6 mRNA levels.