Mitochonic Acid 5 Improves Duchenne Muscular Dystrophy and Parkinson’s Disease Model of Caenorhabditis elegans

Mitochonic Acid 5 (MA-5) enhances mitochondrial ATP production, restores fibroblasts from mitochondrial disease patients and extends the lifespan of the disease model “Mitomouse”. Additionally, MA-5 interacts with mitofilin and modulates the mitochondrial inner membrane organizing system (MINOS) in mammalian cultured cells. Here, we used the nematode Caenorhabditis elegans to investigate whether MA-5 improves the Duchenne muscular dystrophy (DMD) model. Firstly, we confirmed the efficient penetration of MA-5 in the mitochondria of C. elegans. MA-5 also alleviated symptoms such as movement decline, muscular tone, mitochondrial fragmentation and Ca2+ accumulation of the DMD model. To assess the effect of MA-5 on mitochondria perturbation, we employed a low concentration of rotenone with or without MA-5. MA-5 significantly suppressed rotenone-induced mitochondria reactive oxygen species (ROS) increase, mitochondrial network fragmentation and nuclear destruction in body wall muscles as well as endogenous ATP levels decline. In addition, MA-5 suppressed rotenone-induced degeneration of dopaminergic cephalic (CEP) neurons seen in the Parkinson’s disease (PD) model. Furthermore, the application of MA-5 reduced mitochondrial swelling due to the immt-1 null mutation. These results indicate that MA-5 has broad mitochondrial homing and MINOS stabilizing activity in metazoans and may be a therapeutic agent for these by ameliorating mitochondrial dysfunction in DMD and PD.

The most severe and common muscular dystrophy, Duchenne muscular dystrophy (DMD), is a serious progressive muscle disease caused by mutations in the DMD (dystrophin-encoding) gene. In the meantime, Parkinson's disease (PD) is the second most prevalent neurodegenerative disease in the world. There is increasing evidence that mitochondrial dysfunction leads to progressive deterioration in patients with DMD or PD [8,9]. The underlying pathophysiologies of DMD and PD are complex, but mitochondrial dysfunction is a prominent early consequence established in both [10,11]. At Int. J. Mol. Sci. 2022, 23, 9572 2 of 15 present, treatments of these diseases are largely targeted at controlling the symptoms and focus on maximizing quality of life. Current standard pharmaceutical treatments such as the corticosteroid prednisone for DMD, and L-DOPA and dopamine agonists for PD are also associated with several undesirable side effects [12,13]. Therefore, although there are still no satisfactory therapies available for mitochondrial disorders, pharmaceutical treatments that enhance mitochondrial function or treat the consequences of mitochondrial dysfunction are considered to be effective not only for mitochondrial diseases but also for DMD and PD. Therefore, the novel MA-5, which is effective against mitochondrial disease models [1,[5][6][7], is also expected to be effective against skeletal muscle dysfunction in DMD and dopaminergic neurodegeneration in PD.
The nematode Caenorhabditis elegans (C. elegans) provides the advantage of conducting aging and disease model studies due to the homology it shares with human genome [14]. C. elegans has approximately 20,000 protein-coding genes, about the same number as humans, and most proteins involved in basic cell function and metabolism are mammalian homologues [15]. In particular, many genes associated with human diseases such as dys-1 (an ortholog of human DMD) and pdr-1 (an ortholog of human PRKN) are highly conserved in nematodes at the molecular level [16,17]. Furthermore, green fluorescent protein (GFP)-based live-imaging techniques can visualize neuromuscular dysfunctions in C. elegans. As an example, in studying PD, the administration of the neurotoxins 6-hydroxydopamine (6-OHDA) leads to the visualization of dopaminergic neurodegeneration in the dat-1p::GFP transgenic C. elegans TG2435 [18,19]. Similarly, chronic exposure to low concentration (2-4 µM) of rotenone, an inhibitor of mitochondrial electron transport chain (ETC) complex I, causes dopaminergic neurodegeneration not only in rodents but also in C. elegans [19][20][21][22][23][24]. Moreover, in C. elegans upon overexpression of human mutant αsynuclein, similar dopaminergic neurodegeneration can be visualized in an age-dependent manner [25]. Recently, using Cas-CRISPR technology, if there is in sufficient conservation in the C. elegans homologue, the target protein can be replaced by one encoded by the human gene [26,27]. These disease and aging models have been used to develop novel drugs [15,[28][29][30][31][32]. In this study, we evaluated whether MA-5 could alleviate the manifestations in body wall muscle (BWM) cells in a C. elegans DMD model and alleviate the changes in dopaminergic neurons in a C. elegans PD model.

Penetration and Homing Activity of MA-5 into Intact C. elegans Mitochondria
In normal human fibroblasts, fluorescence-labeled MA-5 (BODIPY-MA-5) efficiently penetrates mitochondria as mitochondrial homing activity [5]. To confirm the penetration of MA-5 into C. elegans, wild-type adult hermaphrodites were treated with BODIPY-MA-5 solution for 30 min. The green fluorescent signals of BODIPY-MA-5 were observed on mitochondria in BWM cells, germ cells and intestine and closely matched the blue fluorescent signals of AIE™ Mitochondria Blue ( Figure 1A,B). In contrast, BODIPY on its own effectively stained intestinal lipid droplets and germ cell membranes [33] ( Figure 1A). These clearly show that MA-5 can penetrate almost all mitochondria in intact living C. elegans.
Administration of MA-5 at a final concentration of at least 20 µM even increased the median lifespan of ATU3301 animals by about 1 day, but the change was not significant (Table 1). Subsequent experiments were treated at a final concentration of 10 µM, as used in human patient fibroblasts [1]. Administration of MA-5 at a final concentration of at least 20 μM even increased the median lifespan of ATU3301 animals by about 1 day, but the change was not significant (Table 1). Subsequent experiments were treated at a final concentration of 10 μM, as used in human patient fibroblasts [1].

Alleviation of C. elegans DMD Model Symptom by MA-5
Similar to the human DMD, the C. elegans dys-1 (eg33) mutation synthesizes a C-terminal truncated dystrophin protein that loses scaffolding function [35]. In the dys-1 (eg33) mutant adults synchronized on day 2, a decrease in motility (thrashing rate) was significantly attenuated by treatment with MA-5 ( Figure 2A). In addition, the movement of wildtype (WT) worms was slightly but significantly increased by MA-5 treatment. Next, using the goeIs GCaMP sensor, we studied muscular cytoplasmic Ca 2+ ([Ca 2+ ]cyto) cycling with contraction and relaxation. In the BWM cells of a WT worm immobilized with  I, acels1 [myo-3p::mitochondrial LAR-GECO + myo-2p::RFP]II) in N2 wild-type background [34]. b Median life expectancy is the age at which half of the population died. One test plot was approximately n = 50, and each experiment was performed in triplicate. There is no significant difference between control and every MA-5 treatment.

Alleviation of C. elegans DMD Model Symptom by MA-5
Similar to the human DMD, the C. elegans dys-1 (eg33) mutation synthesizes a Cterminal truncated dystrophin protein that loses scaffolding function [35]. In the dys-1 (eg33) mutant adults synchronized on day 2, a decrease in motility (thrashing rate) was significantly attenuated by treatment with MA-5 ( Figure 2A). In addition, the movement of wild-type (WT) worms was slightly but significantly increased by MA-5 treatment. Next, using the goeIs GCaMP sensor, we studied muscular cytoplasmic Ca 2+ ([Ca 2+ ] cyto ) cycling with contraction and relaxation. In the BWM cells of a WT worm immobilized with microspheres, the full-width half-maximum (FWHM) time of delta [Ca 2+ ] cyto was around 10 s for each cycle ( Figure 2B,C, Supplementary Movie S1). On the other hand, the FWHM of dys-1 mutants was broadened to more than 20 s, indicating that the dys-1 mutant had a longer accumulation of [Ca 2+ ] cyto , similar to typical human muscular dystrophy [36,37]. Intriguingly, the MA-5 treatment significantly improved the expanded FWHM ( Figure 2C). This suggests that in the DMD model, the administration of MA-5 smoothed the movement of muscle contraction and relaxation cycle and increased the thrash rate.

MA-5 Ameliorates Muscular Mitochondrial Perturbations with Rotenone Treatment
To evaluate the effect of MA-5 on mitochondrial perturbations in BWM cells, WT adults synchronized on day 1 (D1) were treated with a low concentration of 2 μM rotenone, an inhibitor of ETC complex I. The fluorescent signals of the mitochondria-specific ROS generation with MitoTracker Red CMXRos significantly increased after exposure to rotenone for 6 h (Figure 4). In contrast, the addition of MA-5 at the same time as rotenone  Continuous treatment of the SD1347 wild-type strain carrying the ccIs4251 transge mtGFP and nuclear targeted GFP (nucGFP), with 2 μM rotenone from synchronous adulthood for 48 h significantly suppressed body growth associated with increased m cle mass ( Figure 5A). Using confocal fluorescent microscopy, mitochondrial network fr mentation and nuclear destruction were also observed in BWM cells ( Figure 5C-E). Ho ever, the administration of MA-5 significantly improved not only body growth retar tion but also mitochondrial and nuclear damage ( Figure 5A,C-E). In addition, roten reduced the total amount of endogenous ATP to one-third of WT, whereas MA-5 tr ment was able to rescue it to two-thirds of WT ( Figure 5B). Continuous treatment of the SD1347 wild-type strain carrying the ccIs4251 transgene, mtGFP and nuclear targeted GFP (nucGFP), with 2 µM rotenone from synchronous D1 adulthood for 48 h significantly suppressed body growth associated with increased muscle mass ( Figure 5A). Using confocal fluorescent microscopy, mitochondrial network fragmentation and nuclear destruction were also observed in BWM cells ( Figure 5C-E). However, the administration of MA-5 significantly improved not only body growth retardation but also mitochondrial and nuclear damage ( Figure 5A,C-E). In addition, rotenone reduced the total amount of endogenous ATP to one-third of WT, whereas MA-5 treatment was able to rescue it to two-thirds of WT ( Figure 5B).

Alleviation of PD Model Progression by MA-5
Low concentrations of rotenone and 6-OHDA are known to be the PD stressors in mammals [21][22][23][24]. Rotenone and 6-OHDA have also been reported to induce C. elegans dopaminergic neurodegeneration visualized in the TG2435 strain carrying dat-1p::GFP as PD models [18][19][20]. Here, we confirmed the appearance of GFP fluorescence puncta in dopaminergic CEP neurons 24 and 48 h after treatment of synchronized L4 larvae with 2 µM rotenone ( Figure 6). The number of puncta increased with the exposure time, indicating that 2 µM rotenone exposure caused the progression of dopaminergic neurodegeneration in C. elegans as a PD model ( Figure 6B). On the other hand, rotenone-induced neurodegeneration was significantly suppressed by the administration of MA-5, suggesting that MA-5 alleviates not only muscle damage but also neuronal dysfunction that arises with chronic mitochondrial perturbation ( Figure 6A,B).

Alleviation of PD Model Progression by MA-5
Low concentrations of rotenone and 6-OHDA are known to be the PD stressors in mammals [21][22][23][24]. Rotenone and 6-OHDA have also been reported to induce C. elegans dopaminergic neurodegeneration visualized in the TG2435 strain carrying dat-1p::GFP as PD models [18][19][20]. Here, we confirmed the appearance of GFP fluorescence puncta in dopaminergic CEP neurons 24 and 48 h after treatment of synchronized L4 larvae with 2 μM rotenone ( Figure 6). The number of puncta increased with the exposure time, indicating that 2 M rotenone exposure caused the progression of dopaminergic neurodegeneration in C. elegans as a PD model ( Figure 6B). On the other hand, rotenone-induced neurodegeneration was significantly suppressed by the administration of MA-5, suggesting that MA-5 alleviates not only muscle damage but also neuronal dysfunction that arises with chronic mitochondrial perturbation ( Figure 6A,B).   C. elegans. has two homologs of mitofilin, IMMT-1 and IMMT-2. These are not redundant, as each mutation affects the mitochondrial morphologies of the same cell and the effects of double mutations are additive [39,40]. In particular, immt-1 defects cause severe local swelling of the mitochondria by disrupting the cristae morphology. In mammalian cells, MA-5 interacts with mitofilin and modulates the mitochondrial inner membrane organizing system (MINOS) [5]. We, therefore, investigated whether MA-5 could suppress the mitochondrial swelling caused by immt-1 mutant in C. elegans. Muscle mitochondria visualized with the ccIs4251 transgene mtGFP showed that irregularly enlarged mitochondria were increased in the immt-1 mutant compared to the wild type ( Figure 7A,B). When the immt-1 mutants were cultured on NGM-plates containing MA-5 from L4 larvae to adulthood, the number of enlarged mitochondria were significantly reduced ( Figure 7A,B). In addition, transmission electron microscopy showed that not only was the mitochondrial diameter suppressed but the deformation of the cristae morphology in the immt-1 mutant was also suppressed by MA-5 treatment ( Figure 7C). These results suggest that MA-5 functions stabilize and repair MINOS in C. elegans. muscles.

Suppression of Mitochondrial Swelling in C. elegans Mitofilin immt-1 Mutant with MA-5
C. elegans. has two homologs of mitofilin, IMMT-1 and IMMT-2. These are not redundant, as each mutation affects the mitochondrial morphologies of the same cell and the effects of double mutations are additive [39,40]. In particular, immt-1 defects cause severe local swelling of the mitochondria by disrupting the cristae morphology. In mammalian cells, MA-5 interacts with mitofilin and modulates the mitochondrial inner membrane organizing system (MINOS) [5]. We, therefore, investigated whether MA-5 could suppress the mitochondrial swelling caused by immt-1 mutant in C. elegans. Muscle mitochondria visualized with the ccIs4251 transgene mtGFP showed that irregularly enlarged mitochondria were increased in the immt-1 mutant compared to the wild type ( Figure 7A,B). When the immt-1 mutants were cultured on NGM-plates containing MA-5 from L4 larvae to adulthood, the number of enlarged mitochondria were significantly reduced ( Figure  7A,B). In addition, transmission electron microscopy showed that not only was the mitochondrial diameter suppressed but the deformation of the cristae morphology in the immt-1 mutant was also suppressed by MA-5 treatment ( Figure 7C). These results suggest that MA-5 functions stabilize and repair MINOS in C. elegans. muscles.

Discussion
Human DMD is an X-linked muscle-wasting disease caused by the loss of dystrophin protein, a rod-shaped cytoskeletal protein that is primarily expressed in muscles. In the absence of dystrophin, the structural integrity of the sarcolemma is lost and leads to disruption in skeletal muscle signaling, such as nitric oxide, ROS production pathways and Ca 2+ cycles [41]. In particular, Ca 2+ cycling between the sarcoplasmic reticulum (SR) and the cytoplasm is essential for the normal muscle contraction and relaxation cycle. It is also reported that in cardiomyocytes from mdx mice, an animal model of DMD, elevated [Ca 2+ ] mito is associated with the excessive opening of the mitochondrial permeability transition pore (mPTP), loss of mitochondrial membrane potential and mitochondrial depolarization [42]. It leads to substantial structural damage to the mitochondria and ultimately promotes cell death. Similar to human muscular dystrophy, an abnormal increase in [Ca 2+ ] cyto is observed in the C. elegans. dys-1 mutants, which in turn activates various matrix metalloproteinase-mediated extracellular matrix degradation [30,43]. First of all, the fluorescent MA-5, BODIPY-MA-5, was used to evaluate the efficiency and predominant penetration into the mitochondria of the C. elegans. tissues (muscle, germline, intestine and mammalian cells) [5] (Figure 1). Here, we also confirmed a muscle tone disorder in which [Ca 2+ ] cyto accumulates longer in the dys-1 (eg33) mutants immobilized with microspheres ( Figure 2, Supplementary Movie S1). In addition, the over-accumulation of [Ca 2+ ] mito levels in BWM cells of the dys-1 mutant was observed with mitochondrial fragmentation (Figure 3).
Intriguingly, we found that the mitochondrial homing drug MA-5 significantly improved elevated [Ca 2+ ] mito and mitochondrial fragmentation in the BWM cells of dys-1 mutants (Figure 3). In addition, prolonged [Ca 2+ ] cyto cycles and decreased thrash movements were recovered by the administration of MA-5 ( Figure 2). These suggest that MA-5 is a potential therapeutic agent that works by ameliorating mitochondrial dysfunction in DMD, as demonstrated using the DMD model of C. elegans. Ellwood et al. [31] have identified that the use of hydrogen sulfide (H 2 S) supplementation (GY4137 and AP39) acts similar to prednisone and improves neuromuscular health using the C. elegans DMD model. They also find a decline in total sulfide and H 2 S-producing enzymes in dystrophin/utrophin knockout mice, suggesting the deficit with H 2 S may contribute to DMD pathology. On the other hand, the loss of Ca 2+ homeostasis in C. elegans DMD model does not appear to be corrected by either prednisone or H 2 S supplementation [31]. Therefore, MA-5 may act by a different mechanism than H 2 S supplementation and prednisone. Interestingly, pharmacological activation of Sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) with CDN1163 ameliorates dystrophic phenotypes in mdx mice [44]. The administration of CDN1163 reduced [Ca 2+ ] cyto levels in vitro and ex vivo, reversed the mitochondrial swelling, increased OCR and reduced ROS production in isolated mitochondria of mdx mice. Taking together, controlling Ca 2+ homeostasis in muscular mitochondria and cytoplasm by MA-5 and CND1163 is effective in treating and alleviating muscular dystrophy.
The mitochondria generate ROS as an intrinsic by-product of ATP synthesis. The generation of ATP and ROS in healthy mitochondria is generally coupled [45,46]. On the other hand, mitochondrial dysfunction causes two detrimental consequences, decreased ATP synthesis and increased ROS production. To artificially cause such disorders, low concentrations of rotenone chronic exposure were applied in this study. MA-5 significantly suppressed the increase in mitochondrial ROS and the decrease in endogenous ATP levels (Figures 4 and 5B). In addition, rotenone-induced the fragmentation of the mitochondrial network and the nuclear destruction of BWM cells was suppressed ( Figure 5C-E). Moreover, chronic exposure of rotenone causes dopaminergic neurodegeneration in C. elegans as a PD model [19,20]. MA-5 alleviated dopaminergic neurodegeneration in the rotenone-treated PD model ( Figure 6). In cultured mammalian neuronal cells, a recent mechanism has reported that rotenone (as one of the PD stressors) promotes the translocation of Parkin to mitochondria and increases the interaction between Parkin and mitofilin [24]. It finally causes ubiquitination-induced mitofilin degradation. Previously, we have shown that MA-5 binds directly to mitofilin and stabilizes the cristae structure. This promotes the oligomerization of ATP synthase and supercomplex formation, thereby increasing local ATP production as a mitochondrial-homing activity [6]. Thus, this pharmacological effect of MA-5 may be widely conserved in metazoans, from nematodes to humans. MA-5 may bind to another C. elegans mitofilin, IMMT-2 molecules, because the immt-1-deficient mutation also improved mitochondrial hypertrophy and cristae deformity (Figure 7). Taken together, MA-5 mitochondrial homing activity increases ATP production and reduces ROS levels, and MA-5 mitofilin binding may suppress the ubiquitination or degradation of mitofilin by Parkin even in the presence of PD stressors. Further work on the verification of this working hypothesis is needed in the future.
In conclusion, it is becoming increasingly clear that mitochondrial dysfunction plays a causal role in a number of neuromuscular diseases including DMD, PD, mitochondrial myopathy, amyotrophic lateral sclerosis and Alzheimer's disease. This work demonstrates the beneficial effect of MA-5 on C. elegans DMD and PD models. We also found that the mitochondrial homing drug MA-5 significantly improves mitochondria Ca 2+ homeostasis, in addition to the previously known ATP production and ROS reduction. MA-5 may act by a unique mechanism through its interaction with mitofilin and may be more beneficial when used in combination with other agents, such as H 2 S donors and prednisone.

C. elegans Strains and Culture Conditions
The standard procedures for C. elegans maintenance were followed using nematode growth media (NGM) agar plates with Escherichia coli OP50 as a food source and incubator at 20 • C [47]. MA-5 (Hayashi K-I, Okayama University of Science) and the ETC inhibitor rotenone (Millipore Sigma, Burlington, MA, USA) were applied to plates at a final concentration of 10 and 2 µM, respectively. Worms were age-synchronized from the eggs and allowed to grow to the designated day. The strains used in this study are as follows: wild-type N2, TG2435: vtIs1 [dat-1p::GFP + rol-6(su1006)] V, ATU2301: goeIs3

Measurement of Body Length
The worms were synchronized, and their movement was recorded using stereomicroscopy (SMZ18; Nikon, Tokyo, Japan), a device camera (DP74; Olympus, Tokyo, Japan) and an imaging software (cellSens Standard 2.2; Olympus, Tokyo, Japan). Body length was measured from images captured by the software.

Thrashing Speed
To determine the body bending of the worms in the liquid, the thrashing speed of synchronized adult worms was measured in 1 mL of M9 buffer for 30 s. In total, 10 worms were measured for each treatment.

Microscopic Imaging
C. elegans BWM cells and their mitochondrial images were obtained using confocal laser-scanning microscopy (FluoView Olympus FV10i; Olympus, Tokyo, Japan). For observation, synchronized worms were washed with M9 buffer and mounted on a microscope slide (6.5 mm square, 20 µm deep well made with a water-repellent coating (Matsunami Glass Ind., Ltd., Osaka, Japan)) with 100 mM NaN 3 solution. Muscular mitochondrial volume and length of mitochondrial networks were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA).
For the live imaging of the cytoplasmic Ca 2+ cycling in BWM cells using GCaMP fluorescence (goeIs3 transgene), the synchronized worms were washed and mounted with 2.5% polystyrene microspheres (0.10 µm, Polysciences Inc. Warrington, PA, USA). Timelapse confocal images of cytosolic GCaMP fluorescence were acquired at room temperature (20~22 • C) by FV10i.
To examine BWM mitochondrial structures by transmission electron microscopy (TEM), four-day-old adult hermaphrodites of wild-type and immt-1 mutants treated with or without MA-5 were used. Worms were fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at 4 • C, then washed 2 times in 0.1 M cacodylate buffer for 15 min each and fixed in 1% osmium tetroxide on ice for 90 min. They were dehydrated through a 50-95% ethanol series for 10 min each and 100% ethanol for 20 min three times, rinsed twice for 10 min in propylene oxide and embedded in resin containing TAAB Epon 812 (TAAB, Reading, UK) for 48 h at 60 • C. Ultra-thin sections (70 nm) cut with a diamond knife on an ultramicrotome (Leica Microsystems, Wetzlar, Germany) were mounted on copper grids, stained with 2% uranyl acetate at room temperature for 15 min, secondary-stained with lead stain solution (Sigma-Aldrich, Burlington, MA, United States) at room temperature for 3 min and then examined by TEM (H-7600, Hitachi, Ltd., Tokyo, Japan).

Measurement of Mitochondrial Ca 2+ Levels
Mitochondrial Ca 2+ and cytoplasmic Ca 2+ in BWM cells were, respectively, observed by measuring the expression of the transgenes aceIs1 and goeIs3 as described recently from our group [34]. The Ca 2+ concentration in muscle mitochondria ([Ca 2+ ] mito ) was calculated using the following equation [48].
[Ca 2+ ] mito = Kd·(R−Rmin)/(Rmax−R) where Kd (12 µM) indicates the dissociation constant between Ca 2+ and the LAR-GECO probe [49] and R indicates the ratio of fluorescence intensity of mtGECO to that of mtGFP of ccIs4251 transgene in a constant area.

Dopamine Neuron Degeneration Measurement
Age-synchronized two-day-old adult worms with dat-1p::GFP (TG2435) were used in this experiment. Approximately 12 worms were analyzed for each condition. Images were obtained using confocal microscopy and ImageJ software was used to calculate the number of beads in all four cephalic (CEP) neurons [50].

Mitochondrial ROS Measurement
Wild-type (N2) one-day-old adult nematodes were incubated with rotenone for 6 h in the presence or absence of MA-5. Subsequently, worms were treated with 0.5 µM MitoTracker ® Red CMXRos (Fisher Scientific, Schwerte, Germany) and 25 µM AIE™ Mito-chondria Blue (AIEgen Biotech Co., Limited, Hong Kong, China) for 3 h. Nematodes were then transferred to fresh NGM agar plates and incubated overnight. Images were obtained using confocal microscopy and fluorescence intensity was analyzed using FV10-ASW Viewer software (Olympus, Tokyo, Japan).

ATP Detection
Wild-type (N2) worms on two-day-old adults were collected in 100 µM M9 buffer for further ATP assays. An ATP determination kit (Molecular Probes, Eugene, OR, USA) was used to measure endogenous ATP levels, as previously reported for rotenone treatment [51].

Analysis of mitofilin/immt-1gene Mutation Mitochondrial Morphology
The synchronized immt-1 mutant (ATU3307) on a four-day-old adult was used in this experiment. Approximately 50 muscle cell images were taken from 10 worms for each treatment. Images of mitochondrial morphology were observed using confocal microscopy and analyzed using ImageJ software.

Statistical Analysis
The one-way ANOVA with post hoc Tukey's HSD and Dunn's test were used for comparisons between groups as appropriate (R or Origin software). All data points including outliers were used for means and statistical significance. A p value of < 0.05 was considered significant. Different letters indicate significant differences between the groups.