ABSTRACT Mitochonic acid-5 ameliorates the pathophysiology of human mitochondrial-disease fibroblasts and _Caenorhabditis elegans_ Duchenne muscular dystrophy and Parkinson’s disease models.

Here, we found that 10 μM MA-5 attenuates the age-related decline in motor performance, loss of muscle mitochondria, and degeneration of dopaminergic neurons associated with mitochondrial

Ca2+ overload in _C. elegans_. These findings suggest that MA-5 may act as an anti-aging agent against a wide range of neuromuscular dysfunctions in metazoans. SIMILAR CONTENT BEING VIEWED

BY OTHERS NRF2 ACTIVATION INDUCES MITOPHAGY AND REVERSES PARKIN/PINK1 KNOCK DOWN-MEDIATED NEURONAL AND MUSCLE DEGENERATION PHENOTYPES Article Open access 03 July 2021 MITOCHONDRIA-AFFECTING

to these aging processes is the accumulation of dysfunctional mitochondria1,2. Maintaining normal mitochondrial function is therefore crucial in overcoming the effects of aging.

Indole-3-acetic acid (IAA), a plant hormone, is also found in animals, synthesized by liver, kidney3 and gut microbes4,5. It also accumulates in human renal failure6. Additionally, IAA also

promotes fibroblast proliferation in both mice and humans7. Recent research has even demonstrated that microbiota-derived IAA enhances the effectiveness of chemotherapy in pancreatic ductal

adenocarcinoma8. Through screening in-house chemical library of IAA derivatives, a compound called Mitochonic Acid 5 (MA-5) was developed. MA-5 exhibits ameliorative effects on fibroblasts

from patients with mitochondrial disease9. MA-5 enhances ATP production without increasing mitochondrial reactive oxygen species (ROS) generation9,10. Furthermore, it has been shown to

prolong the survival of a mouse model for mitochondrial disease, known as the “Mitomouse”11. The nematode _C. elegans_, with its relatively short lifespan and molecular similarity to

vertebrate systems, offers a valuable model for studying aging. We recently found that MA-5 (final concentration 10 μM) ameliorates the pathogenesis of Duchenne muscular dystrophy (DMD) and

Parkinson’s disease (PD) in a nematode model12. Thus, one of the next studies aims to investigate whether MA-5 can impede the progression of aging in _C. elegans_. First, in this study, we

found that the administration of 10 μM MA-5 tended to increase endogenous ATP levels in one-day-old young (D1) and mature D4 adults (no significant difference) and markedly ameliorated the

age-related decline in ATP levels in D7 and D14 adults (Fig. 1a). At the same time, MA-5 significantly suppressed the age-related decline in locomotor performance as evidenced by higher

thrashing rate in liquids and crawling velocity on agar plates compared to the control (Fig. 1b, c). In D1 adults, MA-5 administration also significantly increased thrashing rate, suggesting

that MA-5 is a general enhancer of muscle function. Age-related mitochondria fragmentation and volume loss are known to occur in body wall muscular cells (BWMC), and these impairments

correlate well with decreased motor performance13,14. Indeed, the administration of MA-5 ameliorated age-related mitochondrial fragmentation and volume loss in elderly D14 (Fig. 1d, e). We

recently found that mitochondrial Ca2+ ([Ca2+]mito) levels in BWMC increase with age by using the _aceIs1_ transgene of mitochondrial Ca2+ sensor mtLAR-GECO (strain ATU3301)15. Remarkably,

the administration of MA-5 was able to suppress the age-related elevation in [Ca2+]mito levels (Fig. 1d, f). Interestingly, MA-5 was also found to maintain low [Ca2+]mito levels even in D1

adults (Fig. 1d, f). Furthermore, when compared to the mitochondrial calcium uniporter (MCU) inhibitor Ru36015, MA-5 did not inhibit mitochondrial Ca2+ oscillations synchronized with

cytoplasmic Ca2+ oscillations during the muscular contraction and relaxation cycle in _C. elegans_ BWMC (Supplementary Fig. 1). This indicates that MA-5 maintains mitochondrial Ca2+

homeostasis through an action distinct from MCU inhibition. To investigate the effects of MA-5 on age-related neurodegeneration, we utilized _vtIs1 dat-1p::_GFP (strain TG2435)16 to observe

four anterior cephalic dopaminergic neurons (CEPs) that function to sense mechanosensory stimuli17. In the mock control, GFP fluorescent puncta increased and became apparent on the dendrites

of elderly D16 animals (Fig. 2a, b). These puncta are the formation of axonal spheroids or inclusion bodies commonly observed in degenerating neurons18. However, administration of MA-5

significantly reduced the age-related puncta formation (Fig. 2a, b). Furthermore, to evaluate the effect of MA-5 on neuronal function decline during aging, harsh touch responses on agar

plates19 were also observed. In D7 animals, the number of backward body bend responses to head harsh touch decreased by approximately half due to aging, but by about one-third after MA-5

treatment (Fig. 2c). In D16 animals, no backward bending due to harsh touch was observed in either group, but the touch-induced head deflection was observed in half of the control group (_n_

 = 20/40 worms tested) and in 85% (_n_ = 34/40 worms tested) of the MA-5 treated group. The number of mitochondria in CEPs is much lower than in muscle cells, and age-related changes such as

further reduction and fragmentation have not been successfully observed. We therefore constructed and utilized the _aceIs2_ [_dat-1p::mitochondrial LAR-GECO+myo-2p::GFP_] transgene (strain

ATU5301) in this study to investigate age-related changes in mitochondrial Ca2+ levels in CEPs. Based on previous observations, puncta in CEPs increased not only during the aging process but

also in young gravid animals 24 h after administration of 2 μM-rotenone, a mitochondrial complex I inhibitor, and these increases were effectively suppressed by MA-5 treatment12. Therefore,

we aimed to examine whether low-dose rotenone induces an elevation in mitochondrial Ca2+ levels in CEPs and whether MA-5 can counteract this effect. The result showed that treatment of

ATU5301 D2 animals with 2 μM rotenone for 24 h increased mtLAR-GECO signals in CEPs, which was markedly suppressed by MA-5 administration (Fig. 2d, e). Furthermore, similar to its ability to

reduce [Ca2+]mito levels in BWMC of D1 and D14 animals (Fig. 1), MA-5 also reduced [Ca2+]mito levels in CEPs of D2 animals under control conditions without rotenone treatment. These

findings indicate that MA-5 maintains low [Ca2+]mito levels not only in muscular cells but also in neurons. Compared to long-lived _C. elegans_ mutants with reduced insulin/insulin-like

growth factor-1 signaling (IIS), such as _daf-2_ and _age-1_ deficiency20, MA-5 had no effect on maximum lifespan (Supplementary Fig. 2a). A slight increase in median lifespan was observed,

although it was not statistically significant (control: 13.6 ± 0.6 days, MA-5: 15.3 ± 0.5 at 5 μM, 14.8 ± 0.4 at 10 μM and 14.8 ± 0.3 at 20 μM after D1 adults). Additionally, MA-5 at

concentrations of 5–20 μM also ameliorated age-related decreases in locomotion (thrashing) in D10 animals and mitochondrial mass in D14 animals (Supplementary Fig. 2b–d). Since MA-5

significantly increased intracellular ATP levels at 3 and 10 µM in a previous study with Hep3B cells9, it is likely that similar dose effects are conserved in _C. elegans_. Taken together,

MA-5 functions as an anti-aging agent that can significantly improve age-related neuromuscular decline and extend a healthy lifespan. The suppression of mitochondrial Ca2+ overload is a key

challenge for ameliorating brain aging and the progress of neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s diseases and heart failure21,22. Ca2+ plays a crucial

role in maintaining optimal mitochondrial function. In contrast, an excessive influx of Ca2+ can have detrimental effects on mitochondria, resulting in impaired functionality. This overload

of Ca2+ leads to a decrease in mitochondrial inner membrane potential (ΔΨm) and a reduction in ATP production. Additionally, the increased release of reactive oxygen species (ROS) further

exacerbates the damage to mitochondria. Ultimately, these dysfunctions contribute to cellular demise and cell death23. Moreover, our recent findings indicate that Ca2+ overload promotes

mitophagy and results in mitochondrial volume loss in _C. elegans_ BWMC15. Both genetic (_mcu-1_ mutation) and pharmacological (Ru360 administration) suppression of MCU can ameliorate muscle

weakness induced by _C. elegans_ aging and _dys-1 (eg33)_ mutation of DMD model15. However, MA-5 administration reduced mitochondrial Ca2+ overload induced by aging, rotenone treatment

(Figs. 1, 2), and the _dys-1 (eg33)_ mutation12, while maintaining MCU activity. These results highlight the role of MA-5 in maintaining mitochondrial homeostasis by inhibiting excessive

Ca2+ accumulation, rather than inhibiting normal Ca2+ uptake into mitochondria. MA-5 stabilizes mitochondrial cristae structures through binding to Mitofilin/Mic60 in cultured mammalian

cells and facilitates ATP synthetase oligomerization10. Here we also examined whether Mitofilin/Mic60 is the target of MA-5 in _C. elegans_ as in mammalian cells. Our previous data showed

that MA-5 fluorescently labeled with BODIPY efficiently translocated into mitochondria of live wild-type _C. elegans_12. Using this experimental system, BODIPY-MA-5 signal was markedly

reduced in mitochondria of gonadal and oocyte cells of deletion mutants of each of the two Mitofilin genes _immt-1_ and _immt-2_24 in _C. elegans_ genome (Supplementary Fig. 3). In the

_immt-1_ and _immt-2_ deletion double mutants, the mitochondrial fluorescence signal of BODIPY-MA-5 was almost completely lost (Supplementary Fig. 3). These results strongly suggest that

MA-5 binds in common with Mitofilin/Mic60/IMMT-1 and IMMT-2, which are conserved in metazoans. MA-5 also improves distended cristae in _C. elegans immt-1_ single mutants12. Mitofilin/Mic60

depletion led to a loss of cristae junctions (CJs)25. Intriguingly, MICU-1, one of the components of the MCU complex, was also recently reported to be important not only for Ca2+ transport

but also for maintenance of CJs width26. MICU-1 depletion widened the CJs, increased the release of cytochrome _c_, and loss of ΔΨm26. Overall, our study suggests that stabilization of

mitochondrial CJs by MA-5 causes not only enhanced ATP production but also maintenance of mitochondrial Ca2+ homeostasis. This homeostatic effect of MA-5 maintains mitochondrial quality and

extends a healthy life span. METHODS _C. ELEGANS_ STRAINS AND CULTURE CONDITIONS The strains used in this study are as follows: wild-type N2, ATU2301: _goeIs3_

[_myo-3p::SL1::GCamP3.35::SL2::unc54 3’UTR+unc-119(_+_)_] _V_15_, acels1_[_myo-3p::mitochondrial LAR-GECO+myo-2p::RFP_] _II_, ATU3301: _ccIs4251_ [_(pSAK2) myo-3p::GFP::LacZ::NLS_ + 

_(pSAK4)myo-3p::mitochondrialGFP+dpy-20_+_)_] _I, acels1_ [_myo-3p::mitochondrial LAR-GECO+myo-2p::RFP_] _II_15, ATU3307: _ccIs4251, aceIs1, immt-1_ (_tm1730_), ATU3308: _ccIs4251, aceIs1,

immt-2_ (_tm2366_), ATU3310: _ccIs4251, aceIs1, immt-1_ (_tm1730_), _immt-2_ (_tm2366_), TG2435: _vtIs1_ [_dat-1p::GFP + rol-6(su1006)_] _V_, and ATU5301: _aceIs2_ [_dat-1p::mitochondrial

LAR-GECO+myo-2p::GFP_]. The nematodes were synchronously cultured from the eggs on _Escherichia coli_ OP50 NGM agar plates (60 mm diameter, 8 ml volume) at 20 °C. MA-5 (Hayashi K-I, Okayama

University of Science) and the ETC inhibitor rotenone (Millipore Sigma, Burlington, MA, USA) were applied to the OP50 seeded plates at a final concentration of 5, 10, 20, and 2 μM,

respectively. These plates were allowed to permeabilize for 24 h and used for nematode culture. ATP DETECTION ATU3301 worms on desired days were collected in 100 µM M9 buffer for further ATP

assays. Endogenous ATP was extracted by 3 cycles of sonication (15”, 60” resting at 20 kHz, Ultrasonic Homogenizer Smurt NR-50M, Microtec Co. Ltd, Cheshire, CT, USA) and centrifugation for

1 min at 5000 g. An ATP determination kit (Molecular Probes, Eugene, OR, USA) was used to measure endogenous ATP levels. LIFESPAN AND MOTOR ACTIVITY ANALYSES A total of 300 worms at the L4

stage were set up on three replicate solid media with or without MA-5 treatment under 20 °C. Worms were gently touched with a worm picker to record the number of worms alive, dead, or

censored for each day. The Kaplan–Meier survival curves were performed using Microsoft Excel. To analyze the motor activity, the thrashing frequency of synchronized adult worms was measured

in 1 ml of M9 buffer for 30 s. The maximum velocity was determined by transferring ATU3301 animals to new NGM-agar plates without bacterial lawn, irradiating them with blue light (GFP-B

mode: Excitation wavelength 480 nm and Emission bandwidth 40 nm) using a fluorescence stereomicroscope (SMZ18; Nikon, Tokyo, Japan), video recording their movement behavior using a

microscope camera (DP74; Olympus, Tokyo, Japan), and calculating by ImageJ software. MITOCHONDRIA AND MITOCHONDRIAL CA2+ LEVELS MEASUREMENT _C. elegans_ BWMC and their mitochondrial images

were obtained using confocal laser-scanning microscopy (FluoView Olympus FV10i; Olympus, Tokyo, Japan). Synchronized worms were washed with M9 buffer, 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 NaN3 solution, and immediately observed. Muscular mitochondrial volume and

length of mitochondrial networks were analyzed by Image J software (National Institutes of Health, Bethesda, MD, USA). For live imaging of the cytoplasmic and mitochondrial Ca2+ oscillation

in BWMC using GCaMP fluorescence (_goeIs3_ transgene) and mtLAR-GECO (_aceIs1_ transgene), the synchronized ATU2301 worms were washed and mounted with 2.5% polystyrene microspheres (0.10 μm,

Polysciences Inc. Warrington, PA, USA). The [Ca2+]mito was calculated using the following equation: [Ca2+]mito = Kd ∙ (R − Rmin) / (Rmax − R), where Kd (12 μM) indicates the dissociation

constant between Ca2+ and the LAR-GECO probe, and R indicates the ratio of fluorescence intensity of mtLAR-GECO to that of mtGFP15. Time-lapse confocal images of cytosolic GCaMP fluorescence

were acquired at room temperature (20~22 °C) by FV10i. In dopaminergic cephalic (CEP) neurons, mitochondrial Ca2+ levels were monitored by the mitoLAR-GECO fluorescent intensities of

ATU5301 carrying _aceIs2_ [_dat-1p::mitochondrial LAR-GECO+myo-2p::GFP_]. Day 1 adults of ATU5301 were treated with MA-5 and rotenone for 24 h and the mitoLAR-GECO signal levels were

observed and measured by Fv10i z-stack images. DOPAMINERGIC NEURONAL DEGENERATION MEASUREMENT Age-synchronized adult day 1 and day 16 worms with _dat-1p::GFP_ (TG2435) were used in this

experiment. Approximately 12 worms were analyzed for each condition. Images were obtained using confocal laser-scanning microscopy, and ImageJ software was used to calculate the number of

beads in all four CEP neurons. HARSH TOUCH RESPONSE A total of 40 wild-type N2 (each D1, D7, and D16 synchronized adults) were analyzed under each experimental condition. The head of the

forward-moving worm was touched with a platinum wire, and the number of backward body bends was counted using a stereomicroscope (SZ61; Olympus, Tokyo, Japan)19. BODIPY-BASED

FLUORESCENT-CONJUGATED MA-5 STAINING ATU3301 and its derivatives with _immt-1_ and _immt-2_ deletion mutants were stained with 2 μM BODIPY-MA-511 for 2 h. After washing with M9 buffer and

fixing with 100 mM NaN3, the fluorescent images of BODIPY-MA-5 were immediately observed using a confocal laser-scanning microscope (Olympus, Tokyo, Japan) at a constant laser power of Ex

490 / Em 504 nm. 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. REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary linked to this article. DATA AVAILABILITY Data sets generated from

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ACKNOWLEDGEMENTS _C. elegans_ strains were provided by the Caenorhabditis Genetics Center funded by the U.S. National Institutes of Health (NIH) Office of Research Infrastructure Program

(P40 OD010440) and the National Bioresource Project, Tokyo, Japan. _immt-1_ (_tm1730_) and _immt-2_ (_tm2366_) knockout mutants were generated by the National Bioresource Project, Tokyo,

Japan, which is part of the International C. elegans Gene Knockout Consortium. We also thank Dr. Mika Teranishi for the construction of ATU5301 _aceIs2_ [_dat-1p::mitochondrial

LAR-GECO+myo-2p::GFP_] and the critical reading of the manuscript. This work was funded in part by the Advanced Research and Development Programs for Medical Innovation, AMED-Moonshot

(JP22zf0127001), and AMED-CREST (16814305). XT.W. is grateful to the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) Scholarship. AUTHOR INFORMATION AUTHORS AND

AFFILIATIONS * Graduate School of Life Sciences, Tohoku University, Sendai, 980-8577, Japan XinTong Wu, Miku Seida & Atsushi Higashitani * Division of Medical Science, Tohoku University

Graduate School of Biomedical Engineering, Sendai, 980-0872, Japan Takaaki Abe * Department of Clinical Biology and Hormonal Regulation, Tohoku University Graduate School of Medicine,

Sendai, 980-0872, Japan Takaaki Abe Authors * XinTong Wu View author publications You can also search for this author inPubMed Google Scholar * Miku Seida View author publications You can

also search for this author inPubMed Google Scholar * Takaaki Abe View author publications You can also search for this author inPubMed Google Scholar * Atsushi Higashitani View author

publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS XT.W., T.A., and A.H. conceived and designed the study. XT.W., M.S., and A.H. conducted experiments and

analyzed the data. XT.W. and A.H. wrote the manuscript. All authors have read and agreed to the published version of the manuscript. CORRESPONDING AUTHOR Correspondence to Atsushi

Higashitani. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to

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attenuates age-related neuromuscular dysfunction associated with mitochondrial Ca2+ overload in _Caenorhabditis elegans_. _npj Aging_ 9, 20 (2023). https://doi.org/10.1038/s41514-023-00116-2

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