Regulation of aging by balancing mitochondrial function and antioxidant levels.

doi:10.21203/rs.3.rs-1322686/v1

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Regulation of aging by balancing mitochondrial function and antioxidant levels.

https://doi.org/10.21203/rs.3.rs-1322686/v1

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Aging is the deterioration of physiological mechanisms that is associated with getting old. There is a link between aging and mitochondrial function. In C. elegans, it has been reported that inhibiting the electron transfer system decreases ATP production and extends lifespan. However, it has also been suggested that energy loss due to mitochondrial dysfunction or impairment is a mechanism of diseases and aging. Thus, there is an unresolved relationship between ATP levels and aging. To address this issue, we administered febuxostat (FBX), an inhibitor of human xanthine oxidase (XO)/xanthine dehydrogenase (XDH), to C. elegans. XO/XDH is an enzyme that participates in the in vivo synthesis of uric acid. When FBX is added to cells, the salvage pathway of purine metabolism becomes dominant, and the ATP concentration increases. Since humans and C. elegans but few mammalian species lack uricase, which degrades uric acid, we used C. elegans as a model to evaluate the effects of FBX and to challenge the enigma of the relationship between ATP and lifespan.

In this study, we showed that FBX protects mitochondria and prevents age-related muscle deterioration in C. elegans. In addition, we showed that FBX administration could increase ATP levels without overloading the mitochondria while extending the lifespan. We also showed that the combination of FBX and an antioxidant as a protection against ROS prolongs lifespan more. We have shown that the antioxidant effects and increased ATP levels may lead to antiaging effects.

Aging is the deterioration of physiological mechanisms that is associated with getting old. In humans, aging is accompanied by hearing impairment, vision deterioration, muscle mass reduction, respiratory capacity reduction, and organ weight decrease. The accumulation of DNA damage, mitochondrial damage, and damaged proteins are believed to cause aging ¹, ², ³. Radiation, ultraviolet rays, and reactive oxygen species are known to cause the accumulation of DNA damage ⁴. The mitochondrial electron transfer system produces ROS. The accumulation of mitochondrial damage is also believed to involve ROS. The antioxidant defense system normally works to protect target molecules from ROS in the mitochondria of normally functioning young cells. However, the activity and expression of antioxidant enzymes are decreased in the mitochondria of aged cells, and these decreases are believed to lead to the accumulation of oxidative damage ⁵, ⁶. It has been suggested that reducing ROS can improve aging and age-related diseases in model organisms ⁷, ⁸, ⁹. In humans, uric acid, vitamin C, and vitamin E exert antioxidant effects on ROS. Uric acid is the end product of purine metabolism in humans and is one of the most abundant antioxidant molecules ¹⁰. Uric acid is known to extend lifespan by activating DAF-16/FOXO and SKN-1/NRF2 in C. elegans ¹¹.

In C. elegans, it was reported that inhibiting the electron transfer system decreases ATP production and extends lifespan and that suppressing oxygen consumption extends lifespan ¹², ¹³, ¹⁴, ¹⁵, ¹⁶. However, it has also been suggested that energy loss due to mitochondrial dysfunction or impairment is a mechanism of diseases and aging ¹⁷, ¹⁸, ¹⁹. In C. elegans, it was reported that mutants of clk-1, 3, 5, 6, and 10 demonstrated increased ATP levels and longer lifespans ⁷. Thus, there are apparent contradictory hypotheses regarding mitochondrial function, ATP levels, and lifespan.

Additionally, calorie restriction can inhibit aging in laboratory animals ²⁰. In mice, C. elegans, and flies, calorie restriction extends lifespan. Calorie restriction suppresses insulin signaling and activates FOXO, which is suggested to inhibit aging ²¹. The inhibition of insulin receptor/daf-2 expression in C. elegans has been reported to increase ATP levels and prolong lifespan ¹⁴. Although there are reports on genes involved in lifespan as described above, no treatment has been found to reliably suppress aging. Furthermore, the relationship between ATP levels and lifespan is still elusive.

In this study, we administered febuxostat (FBX), an inhibitor of human xanthine oxidase (XO)/xanthine dehydrogenase (XDH), to C. elegans. XO/XDH is an enzyme that participates in the in vivo synthesis of uric acid. When FBX is added to cells, the salvage pathway of purine metabolism becomes dominant, and the ATP concentration increases ²², ¹⁷. It is also known that inhibitors of XO/XDH prevent the onset of heart disorders and dementia, similar to aging ²³, ²⁴. Additionally, inhibitors of XO/XDH were reported to partially prevent disuse muscle atrophy in mice and humans ²⁵. Most mammals, including mice, have uricase, which breaks down uric acid. However, humans cannot breakdown uric acid because of inactive urate oxidase gene. Since C. elegans also lacks uricase ²⁶, ²⁷, we used C. elegans as a model to evaluate the inhibitory effect of XO/XDH and challenged the enigma of ATP-lifespan relationships.

In this paper, we have shown that balancing mitochondrial function, ATP levels, and antioxidant levels can prevent aging and extend lifespan.

・Febuxostat (FBX) ameliorates age-dependent damage to body wall muscle cells in Caenorhabditis elegans.

FBX, an inhibitor of XO/XDH, has been shown to increase intracellular adenosine triphosphate (ATP) ²², ²⁸. In addition, energy loss due to mitochondrial dysfunction or impairment is a mechanism of aging ¹⁸, ¹⁹. In particular, muscle aging is correlated with increased mortality and an increased risk of developing an aging-related disease ²⁹, ³⁰. The efficacy of FBX was tested in C. elegans, and the effects of FBX on muscle was examined. Evidence for age-related muscle deterioration was monitored by green fluorescent protein (GFP)-tagged proteins localized to the body wall muscle nuclei (Pmyo-3::GFP/nuclear localization signal [NLS]). Pmyo-3::GFP/NLS-expressing ccIs4251 were grown on FBX-supplemented medium, and the number of detectable GFP-labeled body wall muscle nuclei was counted on Days 14, 16, and 18. On Day 14, FBX had no effect (Fig. 1A). On Day 16, the numbers of detectable GFP-labeled body wall muscle nuclei were approximately 10% higher in C. elegans reared on a medium supplemented with FBX at 5, 10, and 20 µg/ml (Fig. 1B). On Day 18, more detectable GFP-labeled nuclei of the body wall muscle were found in C. elegans reared on a medium supplemented with FBX at 10 µg/ml (approximately 7% higher) than in those reared without FBX (Fig. 1C).

Mitochondrial defects, such as increased fragmentation and reduced mitochondrial volume, are said to be observed in the body wall muscles of aged C. elegans. In aged C. elegans, neuronal mitochondria exhibit ultrastructural changes, including loss of visible membrane and cristae structures ³¹. A strain expressing mitochondrial matrix-targeted GFP (mitoGFP) under the control of the myo-3 promoter (Pmyo-3::mitoGFP) ³² was used to monitor the mitochondrial morphology in aging body wall muscle cells. A qualitative analysis of the changes to mitochondrial morphology with age was performed. Mitochondrial fragmentation tended to be suppressed in FBX-treated C. elegans (Sup. Figure 1A–C). Mitochondria in the body wall muscle cells of aged C. elegans were observed by transmission electron microscopy. Broken mitochondrial cristae and membrane structures were observed in the animals not treated with FBX. In contrast, the mitochondrial cristae and membrane structures were intact in the FBX-treated worms. These findings indicate that the addition of FBX protects against age-related muscle deterioration.

Remarkably, there was a bell-shaped dose–response of this effect of FBX.

・FBX increases resistance to the mitochondrial inhibitor NaN₃.

Because FBX effectively protected muscle cells in C. elegans, we considered that FBX could also inhibit the XDH-1 activity in C. elegans. FBX is known to be an inhibitor of XO/XDH in humans (Fig. 2A). The amount of uric acid (UA) in the body of worms treated with FBX (Sup. Figure 2) was measured to investigate the effect of FBX on C. elegans XDH-1, which metabolizes xanthine to give rise to UA. FBX at concentrations of 5 and 10 µg/ml did not change the amount of UA in C. elegans, but 20 µg/ml FBX decreased the amount of UA by approximately 50%. This finding indicates that FBX acts as an inhibitor of XDH-1 in C. elegans, as it does in humans. Since 5 and 10 µg/ml FBX had effects on muscle cell survival above, we speculated that the unchanged UA concentration was caused by the kinetics of UA excretion and not the inhibition of XDH-1. The protective effect of FBX on the mitochondria (Fig. 1D) was hypothesized since FBX increased undegraded hypoxanthine, increased the salvage pathway of nucleic acid metabolism, and increased ATP synthesis (Fig. 2A). To test this hypothesis, NaN₃ was administered to wild-type C. elegans to inhibit mitochondrial function, and the effects of FBX were observed. When NaN₃ was added at 500 µM, all worms died after 32 h. When FBX (5 and 10 µg/ml) was added with NaN₃, the survival rate of the worms increased. However, the addition of FBX at 20 and 40 µg/ml did not increase the survival rate (Fig. 2B). In the mutant lacking the enzyme that acts in the salvage pathway (hprt-1), FBX did not restore the survival rate upon NaN₃ addition (Fig. 2C, Sup. Figure 3A). The same experiment was performed using xdh-1 mutants (tm9919 and tm9909) (Sup. Figure 3B). Unexpectedly, the effect of NaN₃ was stronger in the xdh-1 mutant than in the wild-type (Fig. 2D, Sup. Figure 5A, B), and the same tendency was observed when NaN₃ was added at 400 µM (Sup. Figure 4A–C). In the xdh-1 mutant (ok3234) that lacks the NAD-binding domain but restores the xanthine dehydrogenase domain, the effect of NaN₃ was not strong (Sup. Figure 5C, D).

The survival rate of worms with xdh-1 mutations (tm9911 and tm9909) was lower than that of the wild-type worms when NaN₃ was added (Fig. 2D). Because deletions in the xdh-1 alleles (tm9911 and tm9909) almost completely span the whole gene, the mutants are likely null mutants. This suggests that tm9911 and tm9909 have almost no UA (Sup. Figure 2). To test this possibility, 2 mM UA was added to the medium, and the viability of wild-type and xdh-1 mutant animals (tm9911 and tm9909) upon the addition of NaN₃ was examined. UA increased the resistance to NaN₃ in both wild-type and xdh-1 mutant animals (tm9911 and tm9909) (Fig. 3A, B, Sup. Figure 6). Since UA is known to be an antioxidant that exists in vivo ¹⁰, another antioxidant (sodium ascorbate, vitamin C) was added, and resistance to NaN₃ was examined. In wild-type animals, resistance to NaN₃ increased when vitamin C was added. This was similar to the effects of UA, although the effects of vitamin C were smaller (Fig. 3A). The xdh-1 mutant (tm9911) and hprt-1 mutant (tm6318) animals showed no changes in resistance to NaN₃ upon vitamin C treatment (Fig. 3B, Sup. Figure 6). This indicates that UA acts as an antioxidant, and to some extent, this action can be substituted by vitamin C. When FBX was added to wild-type animals at 20 µg/ml, resistance to NaN₃ was not observed (Fig. 2B, Fig. 3C), and when 4 mM vitamin C was added together with FBX, resistance to NaN₃ increased (Fig. 3C). The increased resistance to NaN₃ by FBX appears to be due to the activation of the salvage pathway and the increased ATP. We measured the amount of ATP in wild-type animals when FBX and/or vitamin C were added. The amount of ATP per mitochondria increased when both FBX and vitamin C were added (Fig. 4A–C).

・The addition of FBX and vitamin C extends the lifespan of C. elegans.

Treatment of wild-type C. elegans with UA, an antioxidant, is known to prolong its lifespan ¹¹. Energy loss due to mitochondrial dysfunction or impairment is suggested to be a mechanism of aging ¹⁸, ¹⁹. We have shown that the mitochondria were protected by supplementation with FBX. Therefore, FBX and vitamin C were added to wild-type animals, and the lifespan was measured. Treatment with vitamin C alone did not improve the lifespan (Fig. 5A). FBX at 5 µg/ml alone had an effect on lifespan extension compared to FBX-free conditions (Fig. 5B). FBX at 10 µg/ml also had a similar or slightly weaker effect than FBX at 5 µg/ml (Fig. 5C). FBX at 20 µg/ml alone did not differ significantly from FBX-free conditions (Fig. 5D). In addition, the coadministration of FBX and 4 mM vitamin C prolonged the lifespan of the animals compared to FBX alone (Fig. 5B–D), and the effect was not attenuated even when the FBX concentration was increased to 20 µg/ml (Fig. 5D).

・Coadministration of FBX and vitamin C suppresses the phenotype of a familial Parkinson’s disease nematode model.

Treatment of wild-type C. elegans with FBX slowed the rate of nuclear change (Fig. 1A–C). The degree of nuclear change was reported to correlate with locomotor performance ³³. FBX (0, 5, 10, 20 µg/ml) and vitamin C (0, 4 mM) were administered to transgenic animals expressing αSyn (S129A) (tmIs913 [Punc-51::αSynS129A, Punc-51::EGFP]) and tmIs907 [Punc-51::EGFP] as a control, and the body size was measured 96 h after bleaching. S129A-expressing nematodes showed abnormal locomotion and growth retardation. Administration of vitamin C alone had no growth-promoting effect. The growth-promoting effect of FBX alone was also weak. In addition, growth was promoted in the presence of FBX (10 and 20 µg/ml) and vitamin C (4 mM) (Fig. 6A, B). These results indicate that the synergistic effect of FBX and vitamin C reduces the toxicity of α-synuclein.

・FBX administration suppresses the phenotype of tauopathies.

XO/XDH inhibitors have been reported to suppress the onset of dementia ²⁴, ³⁴. The effect of FBX was tested using transgenic animals expressing Tau (WT4R) in the nerve (tmIs390 [Punc-119::WT4R, Pges-1::EGFP]) as a model for Alzheimer’s disease, which is said to account for approximately 70% of dementia. tmIs390 has been reported to produce the phenotype of Unc ³⁵. tmIs388[Pges-1::EGFP] was used as a control, and tmIs390 was supplemented with FBX (0, 5, 10, and 20 µg/ml). The area of nematode movement over 30 min was examined at 11 days after bleaching. The migration distance of the nematodes was prolonged in the presence of FBX (5 and 10 µg/ml) and was more prolonged in FBX (20 µg/ml) (Sup. Figure 7).

In this study, we investigated the effects of FBX in C. elegans. Low concentrations of FBX elicited protective effects on age-related muscle deterioration and led to NaN₃ resistance and longer survival. However, these effects were hardly observed when FBX was added at high concentrations or to xdh-1 mutant animals (tm9911). When FBX was added to C. elegans at a high concentration (20 µg/ml), the concentration of UA in the body was reduced by approximately half, and the xdh-1 mutant animals (tm9911) had almost no UA in the body (Sup. Figure 2). We hypothesized that this may be the reason for the decreased drug efficacy of FBX at higher concentrations because UA is known to be an antioxidant that exists in vivo. In support of this hypothesis, the addition of antioxidants with a high concentration of FBX led to NaN₃ resistance and longer survival (Fig. 2, Fig. 3, Fig. 5).

It has been suggested that energy loss due to mitochondrial dysfunction or impairment is a mechanism of diseases and aging ¹⁷, ¹⁸, ¹⁹. However, it was also reported that inhibiting the electron transfer system decreases ATP production and extends lifespan and that suppressing oxygen consumption extends lifespan ¹², ¹³, ¹⁵, ¹⁶. Thus, there are conflicting reports on the relationship between mitochondrial function, ATP contents and lifespan.

In this study, we showed that FBX administration could increase ATP levels while extending the lifespan, suggesting that an increase in ATP content alone does not shorten the lifespan. We also showed that the combination of FBX and antioxidants protect against ROS to prolong the lifespan.

When xdh-1 mutant animals (ok3234) were treated with NaN₃ (500 µM) and FBX (10 µg/ml), the resistance to NaN₃ was increased compared with only NaN₃ treatment in xdh-1 (tm9911) (sup. Figure 5D, Fig. 2D). XDH-1 contains FAD and molybdopterin domains ³⁶. The FAD domain is the NAD binding site, and the molybdopterin domain is the redox center. XDH is an enzyme that metabolizes hypoxanthine to xanthine by hydroxylation and metabolizes xanthine to uric acid by hydroxylation downstream of the purine metabolic pathway. The fact that the xdh-1 mutant animals (ok3234) lacking only the NAD binding site show a normal concentration of ROS ³⁶ also supports this possibility.

In the present study, we showed that FBX protects mitochondria (Fig. 1). Simultaneous administration of FBX (20 µg/ml) and vitamin C in the presence of NaN₃ did not decrease ATP production in mitochondria (Fig. 4). Together with the high sensitivity to NaN3 of hprt-1 (tm6318) mutant animals, which cannot use the salvage pathway, these results suggest that FBX activated the salvage pathway of purine metabolism and contributed to ATP production. Thus, it reduced mitochondrial overuse and led to mitochondrial protection.

We demonstrated that FBX and vitamin C have synergistic effects on reducing α-synuclein toxicity (Fig. 6A, B), and FBX administration represses the tauopathy phenotypes (Sup. Figure 7). Mitochondrial or bioenergetic dysfunction may be related to the etiology of Alzheimer’s disease (AD) and Parkinson’s disease (PD). Additionally, gout patients treated with UA-lowering drugs have a lower risk of both vascular and nonvascular dementia ³⁴. Furthermore, impaired removal of defective mitochondria is a pivotal event in AD pathogenesis, suggesting that targeting the maintenance of mitochondrial quality may be a promising therapeutic strategy ³⁷. Based on the protective effects of FBX on mitochondria in C. elegans (Fig. 1), FBX may be a potential therapeutic or preventive agent for the treatment of AD and PD. The therapeutic effects of FBX may be enhanced by coadministration of FBX and vitamin C. A recent study identified FBX as a potential new treatment for AD using artificial intelligence approaches ³⁸.

In humans, vitamin C intake is inversely associated with serum uric acid concentrations ³⁹. Vitamin C decreases free radical-induced damage to somatic cells and likely modulates serum uric acid concentration via its uricosuric effect ³⁹, ⁴⁰, ⁴¹. Furthermore, vitamin C intake may improve renal function and increase the glomerular filtration rate ⁴¹, ⁴², ⁴³.

We hypothesize that the combination of FBX and vitamin C would not only increase the therapeutic treatment of gout but also have a preventive effect on other diseases and prolong lifespan. We also hypothesize that it is important to maintain ATP levels for lifespan extension and that protection from ROS generated during ATP production is also important.

Nematode strains

C. elegans strain N2 worms were used as wild-type animals. Worms were grown at 20 °C under well-fed conditions using standard methods ⁴⁴. The strain carrying tm6318 was obtained from a UV/TMP-mutagenized library as described previously ⁴⁵. These were identified via PCR amplification with primers spanning the deletion region of hprt-1(tm6318), as described previously ⁴⁵, ⁴⁶. The primers used for PCR genotyping were as follows: 5’-CAATCGCGCTGCTCTGCGTA-3’ and 5’-CTATACTGGCAAAACGCGGT-3’ (tm6318 1^st round); 5’-GCGTACTCAAAGGATCCTAT-3’ and 5’-GACGGTCATAATACACCGAA-3’ (tm6318 2^nd round). The mutant was backcrossed twice with N2 before use.

Strains carrying the xdh-1 mutation (tm9909 and tm9911) were obtained from a CRISPR-cas9 system as described previously ⁴⁷ and identified via PCR amplification with primers spanning the deletion region of tm9909 and tm9911. The primers used for PCR genotyping were 5’-GAGTGCAAGACTAATAGGGAG-3’ and 5’-GTGTTTCACCCCTTCTCTAG-3’. The mutants were backcrossed twice with N2 before use.

The Caenorhabditis Genetics Center provided the xdh-1(ok3234) sample.

All assays were performed on kanamycin (Km)-supplemented NGM plates with UV-irradiated OP50 as the food, unless otherwise indicated. UV treatment of bacteria was prepared as described previously ⁴⁸.

Plasmid construction

We used site-directed mutagenesis to insert the guide sequences into a Cas9-sgRNA (single guide RNA) expression vector (pDD162) containing both sgRNA and Cas9 protein expression units, which were obtained through Addgene ⁴⁹. Then, xdh-1 was targeted for Cas9 cleavage using the guide sequences (GAATACGTTCAGGAGTTGC and GATGCAATGAGGGAGGATG) that were inserted into pDD162 individually.

Movement analyses

Eggs were collected by bleaching Tg animals (tmIs388 or tmIs390) reared at 20 °C on OP50 normally-seeded NGM plates (Day 0). After 36 h, bleached Tg animals were transferred to NGM plates with FBX. OP50 was irradiated with UV and treated with Km. When the Tg animals reached the young adult age, FUdR (15 μM) was added to the NGM plate. Eleven days after bleaching, a Tg animal expressing Tau was placed on a new NGM plate with one animal each. After 30 min, we photographed the traces of worm movement using a stereomicroscope (Olympus). The areas with worm movement were quantified using ImageJ (NIH, Bethesda, MD). At least 30 animals were observed per condition at a time. The experiments were repeated four times.

Growth analysis of S129A-α-Synuclein Tg Worms

We used C. elegans overexpressing Ser-129 mutant α-synuclein and EGFP (α-Syn [S129A], tmIs913) or only EGFP (tmIs907) as a control. α-Syn (S129A) and EGFP were expressed under the unc-51 promoter, which drives the pan-neuronal expression. α-Syn (S129A) Tg worms exhibit growth retardation ³⁵.

Eggs were collected by bleaching Tg animals (tmIs913 or tmIs907) reared at 20 °C on OP50 normally-seeded NGM plates. Then, 36 hours after bleaching, Tg animals were transferred to NGM plates with FBX. OP50 was irradiated with UV and treated with Km. Then, 96 hours after bleaching, we individually photographed the fluorescence of the worms using a fluorescence stereomicroscope (Olympus). The glowing area was measured using ImageJ (NIH, Bethesda, MD). At least 30 animals were observed per condition at a time. The experiments were repeated four times.

Transmission Electron Microscopy

TEM analyses of C. elegans were performed as previously described ⁵⁰.

For synchronized worms, eggs were obtained using alkaline hypochlorite treatment of gravid N2 adult hermaphrodites (Day 0). These eggs were reared at 20 °C on OP50 normally-seeded NGM plates. Then, 36 hours after bleaching, worms were transferred to NGM medium with FBX (0, 5, 10, or 20 μg/ml). OP50 was irradiated with UV and treated with Km. After Day 4, we replanted every day until the worm stopped laying eggs. On Day 18, we fixed the worm. In brief, the worms were anesthetized in M9 buffer with 8% ethanol for 5 min and then cut into 2–3 pieces in the primary fixative solution (2% glutaraldehyde, 2% paraformaldehyde in 100 mM phosphate buffer, pH 7.4). Then, they were postfixed in osmium tetroxide at 4 °C, dehydrated, and embedded in EPON812. The posterior side of the vulva was used as a sample. Ultrathin sections were analyzed using a transmission electron microscope (HITACHI H-7600) at 100 KeV. TEM analyses were performed at the Hanaichi Ultrastructure Research Institute Co. (Okazaki, Japan).

Lifespan Analysis

For synchronized worms, eggs were obtained using alkaline hypochlorite treatment of gravid adult hermaphrodites (Day 0). These eggs were reared at 20 °C on OP50 normally-seeded NGM plates. Then, 36 hours after bleaching, the Tg animals were transferred to NGM plates with FBX (0, 5, 10, or 20 μg/ml) and vitamin C (0 or 4 mM). OP50 was irradiated with UV and treated with Km (20 µg/mL). The UV treatment of bacteria was prepared as described previously ⁴⁸.

Upon reaching young adult age, worms were supplied with FUdR (15 μM). Lifespan measurements were initiated by the transfer of adult-stage worms (Day 5) to new NGM plates containing 5-fluoro-2’-deoxyuridine (FUdR, 15 μM), FBX (0, 5, 10, or 20 μg/ml) and vitamin C (0 or 4 mM). OP50 was irradiated with UV and treated with Km (20 µg/mL). Worms were transferred to fresh plates every 7 days. To prevent progeny development, plates were supplemented with FUdR (15 μM) on the day before use. Survival was monitored every other day; worms were considered dead if they showed no movement when prodded with a platinum wire. Lifespan analysis via log-rank tests was performed using GraphPad Prism 6.

ATP detection

The assessment of the ATP levels of C. elegans was performed as previously described ⁵¹. In brief, N2 worms (approximately 50 animals each) in the L4 stage were placed on NGM plates supplemented with 500 µM NaN3, FBX (0 or 20 µg/ml), and sodium ascorbate (Vit. C, 0 or 4 mM). After 14 h, only adult nematodes were collected and washed 5 times with M9 buffer. The samples were frozen using liquid nitrogen, and then the frozen worm was boiled for 15 min. The samples were centrifuged at 14,800 g for 10 min at 4 °C, and then the supernatants were transferred into new 1.5-ml tubes. ATP was determined using the ATP Determination Kit (A22066, Molecular Probes) according to the manufacturer’s protocol.

mtDNA content quantification

The mitochondrial DNA (mtDNA) copy number was quantified using real-time quantitative PCR (qPCR). N2 worms (approximately 50 animals each) in the L4 stage were placed on NGM plates supplemented with 500 µM NaN3, FBX (0 or 20 μg/ml), and sodium ascorbate (0 or 4 mM). After 14 h, only adult nematodes were collected and lysed using proteinase K. Then, qPCR was performed using SYBR GREEN PCR Master Mix (Applied Biosystems) with the nd-1 forward (5-AGCGTCATTTATTGGGAAGAAGAC-3) and reverse (5-AAGCTTGTGCTAATCCCATAAATGT-3) primers, as well as the ama-1 forward (5-AGATGGACCTCACCGACAAC-3) and reverse (5-CTGCAGATTACACGGAAGCA-3) primers. The mtDNA copy number was calculated as the amplification of the mitochondrial gene relative to the amplification of the nuclear gene. The experiments were repeated nine times.

Assay for NaN3 resistance

C. elegans of the L4 stage were placed on NGM plates, which were supplemented with NaN3 (400 or 500 μM), FBX (0, 5, 10, or 20 μg/ml), sodium ascorbate (0 or 4 mM), uric acid (0 or 2 mM), and Km. UV-irradiated OP50 was fed to the animals. After 8, 14, 20, and 32 h, we observed the survival of C. elegans.

Uric acid quantity measurement

For synchronized worms, eggs were obtained using alkaline hypochlorite treatment of gravid N2 or tm9911 worms (Day 0). After 36 h, the worms were transferred to NGM plates with FBX (0, 5, 10, or 20 μg/ml). UV-irradiated OP50 was used as bait. Worms were supplied with FUdR (15 μM) on Day 4. On Day 6, adult worms were collected for the preparation of urate samples as described previously ²⁶. In brief, ~500 worms in the experimental or control groups were collected, washed with M9 buffer three times, and ultrasonicated. Protein was precipitated by centrifugation (4000 rpm for 20 min), and then the supernatant was analyzed using the Uric Acid Assay Kit (CBL, STA-375) according to the manufacturer’s protocol.

Mitochondrial imaging and nuclei imaging

ccIs4251 (Pmyo-3::Ngfp-lacZ; Pmyo-3::Mtgfp), which has GFP fusion proteins localized to the body wall muscle mitochondria and nuclei, was used for this study. For synchronized worms, eggs were collected by bleaching Tg animals (ccIs4251) and reared at 20 °C on OP50 normally-seeded NGM plates (Day 0). After 36 h, the worms were transferred to NGM plates with FBX (0, 5, 10, or 20 µg/ml) and Km. UV-irradiated OP50 was used as food. After Day 4, we replanted every day until the worm stopped laying eggs. On Days 14, 16, and 18 after bleaching, worms were anesthetized by placing M9 buffer with a drop of 50 mM sodium azide on the solidified pads of 5% agarose laid on the slides. After adding a coverslip, worms were observed using a BX-51 microscope (Olympus). Mitochondrial morphology was classified in accordance with previous studies ³².

Statistical analysis

Data are presented as mean ± SEM for all data. All the tests were performed GraphPad Prism version 6. All assays for drug effects were done by double-blinded experiments.

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Competing interest reported. We have a pending application of patent: PCT/JP2020/001551

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Regulation of aging by balancing mitochondrial function and antioxidant levels.

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