Endogenous chondroitin extends the lifespan and healthspan in C. elegans

Chondroitin, a class of glycosaminoglycan polysaccharides, is found as proteoglycans in the extracellular matrix, plays a crucial role in tissue morphogenesis during development and axonal regeneration. Ingestion of chondroitin prolongs the lifespan of C. elegans. However, the roles of endogenous chondroitin in regulating lifespan and healthspan mostly remain to be investigated. Here, we demonstrate that a gain-of-function mutation in MIG-22, the chondroitin polymerizing factor (ChPF), results in elevated chondroitin levels and a significant extension of both the lifespan and healthspan in C. elegans. Importantly, the remarkable longevity observed in mig-22(gf) mutants is dependent on SQV-5/chondroitin synthase (ChSy), highlighting the pivotal role of chondroitin in controlling both lifespan and healthspan. Additionally, the mig-22(gf) mutation effectively suppresses the reduced healthspan associated with the loss of MIG-17/ADAMTS metalloprotease, a crucial for factor in basement membrane (BM) remodeling. Our findings suggest that chondroitin functions in the control of healthspan downstream of MIG-17, while regulating lifespan through a pathway independent of MIG-17.

glucuronyltransferase and acetylgalactosamine-transferase activities.The length of the chondroitin chains varies depending on the specific combination of subunits of the chondroitin polymerizing enzyme complex 9 .Chondroitin proteoglycans not only fill the gaps in the ECM, but are also believed to play a role in signal transduction.Dysfunction of chondroitin synthase has been associated with abnormal development and impaired nerve regeneration 10,11 .Longevity associated with chondroitin ingestion has been observed in both humans and C. elegans 12,13 .Nevertheless, the roles of endogenous chondroitin in aging mostly remain to be elucidated.
The BM is a specialized sheet-like ECM that envelops tissues.The ADAMTS protease MIG-17 is secreted from the body wall muscle cells and localizes to the BMs of various tissues, and regulates cell migration and organ size in C. elegans depending through its protease activity [14][15][16] .MIG-17 is involved in the recruitment and modulation of BM molecules, including collagen IV and fibulin to regulate organogenesis [17][18][19] .While the function of MIG-17 in organogenesis has been investigated, its role in organismal aging remains to be elucidated.
In this study, we found that mig-17 mutants exhibited accelerated senescence and that a gain-of-function mutation in MIG-22/ChPF, mig- 22(k185gf), suppressed this phenotype.We also demonstrated that mig- 22(k185gf) mutants had an increased level of chondroitin and extended the lifespan and healthspan than wildtype animals.Genetic analyses suggest that MIG-17 regulates healthspan through chondroitin proteoglycans (CPGs), while lifespan is influenced by CPGs through mechanisms independent of MIG-17.

Dominant mutation k185 in MIG-22/ChPF
During development of the C. elegans gonad, the gonadal leader cells, called distal tip cells (DTCs), migrate in a U-shaped pattern to form the U-shaped gonad arms.The mig-17 mutants exhibit a misshapen gonad phenotype due to the meandering DTC migration 20 (Fig. 1A,B).To elucidate the mechanism of DTC migration regulated by MIG-17, we screened and isolated a mutation k185 as a genetic suppressor of the DTC migration defect in mig-17(k174) null mutants (Fig. 1A-C,E).The causative gene for k185 was identified as mig-22, a gene previously known to be essential for DTC migration 10 .MIG-22 is a homologue of the human chondroitin polymerizing factor ChPF, which forms a complex with SQV-5/chondroitin synthase (ChSy) 8 .The k185 mutation corresponds to a single nucleotide substitution from C to T, resulting in the L325P amino acid change.(Fig. S1).The amino acid Leucine corresponding to L325 of C. elegans MIG-22 is highly conserved among different species and is likely critical for protein function.
It has been reported that mig-22 reduction-of-function (rf) alleles k141 and tk24, as well as the deletion allele tk69, display meandering DTC migration defects similar to those observed in mig-17 mutants 10 .In contrast, mig-22(k185) alone exhibited DTC migration phenotypes comparable to the wild type (Fig. 1D,E).While mig-22(k185) strongly suppressed the DTC migration defect of mig-17(k174) mutants, mig-22(k185)/ + ; mig-17(k174) showed weak defects, suggesting that k185 exhibits a semi-dominant effect (Fig. 1E).Since k185 heterozygous with a wild-type allele showed a stronger suppressor activity compared to k185 heterozygous with the deletion allele tk69, k185 is a gain-of-function (gf) allele that enhances the function of the wild-type mig-22 gene.mig-22 reduction-of-function (rf) alleles k141 and tk24, as well as the deletion allele tk69, failed to suppress the gonadal defects observed in mig-17 mutants.We examined whether the overexpression of wild-type mig-22 can suppress the mig-17 defect.The extrachromosomal array carrying multicopy mig-22 genes partially but significantly suppressed the gonadal defect in mig-17 mutants (Fig. 1E).These results indicate that MIG-22 functions downstream of MIG-17.
The levels of chondroitin are known to decrease in the sqv-5(k175) mutant 10 , and we also observed a substantial reduction in chondroitin in the sqv-5(k175) mutant (Figs.2, S2).The k175 mutation represents a reductionof-function mutation, and the enzyme activity may not be completely lost 10 .Therefore, we investigated whether the increase in chondroitin levels resulting from the mig-22(k185gf) mutation also occurs in the sqv-5(k175) mutant background.We found a slight but statistically significant increase in chondroitin levels in sqv-5(k175); mig-22(k185gf) when compared to sqv-5(k175) (Figs. 2, S2).

mig-22(k185gf) extends lifespan and healthspan
Since chondroitin sulfate intake has been shown to extend lifespan in C. elegans 13 , we investigated whether an increase in endogenous chondroitin affects lifespan.Interestingly, we observed that mig-22(k185gf) exhibited a longer lifespan compared to the wild type (p < 0.005 by logrank test) (Figs.3A, S3).The lifespan of mig-22(k185gf) was 5.9 days (30.6%) longer on average than that of wild type, with a median increase of 9 days and a maximum lifespan extension of 7 days.
We also assessed whether mig-22(k185gf) had an impact on healthspan during adulthood in addition to lifespan.Specifically, we examined body length, pumping rate, and defecation cycle.In wild-type animals, the body length increased until day 4 and remained relatively stable through day 5.However, beyond day 7, a gradual decrease in body length was observed (Fig. 3B).Although the body length of mig-22(k185gf) animals increased as in the wild type, the shortening was significantly slower than the wild type.Notably, mig-22(k185gf) animals exhibited larger body sizes compared to the wild type at days 7, 10, and 14 (Figs.3B, S4A,B).
The pharyngeal pumping rate is known to decrease with aging 21 .We examined changes in pumping rates along aging.In wild-type adults, the pumping rate was 137.3 ± 9.1 per 30 s on day 1, and this rate remained relatively consistent until day 3. Subsequently, at day 5, the pumping rate began to decline, reaching 59.0 ± 36.4 per 30 s by day 9 (Figs.3C, S5A).However, in mig-22(k185gf) animals, no significant decrease in pumping rate was observed until day7.Consequently, mig-22(k185gf) maintained a faster pumping rate for a longer duration when compared to the wild type (Figs.3C, S5A,B).
The defecation cycle elongates with aging 22 .In the wild type, the defecation cycle was 1.67 times longer in day 5 adults compared to day 1 adults (Fig. 3D).In mig-22(k185gf), the defecation cycle also lengthened with age.However, mig-22(k185gf) exhibited a significantly shorter cycle compared to the wild type at day 5 and 7. www.nature.com/scientificreports/ In C. elegans, the mobility of animals decreases with aging.In day 9 wild-type adults, mobility after tapping was almost half that of day 1 adults.The mobility of mig-22(k185) at day 1 adult was slower than that of wildtype animals (Fig S6).Therefore, we used the ratio to day 1 adult to compare the effect of aging on mobility (Fig. 3E).The downregulation of mobility with aging tended to be less in the mig-22(k185) background at day 9 adult than in the wild-type background.Taken together, these results indicate that the mig-22(k185gf) mutation, which increases endogenous chondroitin, slows aging by extending both lifespan and healthspan in the wild-type background.
If slower pumping in sqv-5(k175) mutants is caused by the reduction of chondroitin, supplementation of chondroitin can rescue the slower pumping in sqv-5(k175) background.On day1 of adulthood, supplementation of chondroitin had no effect in the sqv-5(k175) background, although it caused a slight reduction in the pumping rate in the wild-type background (Fig. 5I).On day 5 of adulthood, a significant rescue of slower pumping was observed by the supplementation of chondroitin in the sqv-5(k175) background (Fig. 5J).
We also measured brood size and found no correlation between aging and brood size (Fig S9).For example, mig-22(k185gf) mutation suppressed small brood size of sqv-5(k175), but not affect the short lifespan of sqv-5(k175).
Although the body length of mig-17 mutants was slightly longer than that of the wild type at day 1, it became significantly shorter than wild type at days 3 to 5 (Figs.6C-F,I, S4A,E).mig-22(k185gf) suppressed the shortening phenotype of mig-17 (Figs.6E-I, S4B,E,F).Although a fraction of mig-17 animals showed very short body sizes of less than 1 mm at day 3 to 5, mig-22(k185gf) also suppressed this phenotype (Figs.6I, S4F).
The pumping rate, defecation cycle, and mobility of mig-17(k174) mutants were similar to those of the wildtype animals in day 1 adults, suggesting that mig-17 mutants can form fully functional pharynx, intestine, and locomotive system (Figs.6J-L, S5A,E, S6C).However, mig-17 mutants showed significantly slower pumping at day 9 and altered defecation behaviors at day 3 compared to wild-type animals.Downregulation of mobility at the day 7 adult was larger in mig-17(k174) mutants than that of wild type animals.These phenotypes of mig-17 were suppressed by mig-22(k185gf) (Figs.6J-L, S5A,B,E,F, S6C,D).These results indicate that mig-22(k185gf) suppresses multiple phenotypes observed in aged mig-17 mutants, including alterations in the body length and periodic behaviors.

Discussion
In this study, we have isolated a gain-of-function mutation of the mig-22 gene, which encodes a chondroitin polymerizing factor.The mig-22(k185gf) mutation suppressed the gonadal defects of the mig-17 mutant.Furthermore, we observed that the mig-22(k185gf) mutation extended lifespan and healthspan when compared to the wild type.Conversely, the reduction-of-function mutation k175 in the sqv-5 gene, encoding ChSy, led to premature aging and a shortened lifespan.Both MIG-22 and SQV-5 are homologs of mammalian ChPF and ChSy, which form a complex to synthesize chondroitin chains 23 .The chondroitin levels in mig- 22(k185gf) were increased twofold compared to those of the wild type, whereas the levels in sqv-5(k175rf) were markedly reduced compared to the wild type.We also previously reported a significant reduction in chondroitin levels in mig-22(k141rf) 10 .In this study, we observed a slight but significant increase in chondroitin levels in the presence of mig-22(k185gf), even in the sqv-5(k175rf) background, suggesting that the residual ChSy activity contributes to this increase.
Mammalian ChSy binds to ChPF, and the chondroitin polymerization activity of ChSy is higher when ChSy and ChPF are co-expressed 23 .While ChPF is homologous to ChSy in its primary structure, but ChPF itself has a weak chondroitin chain polymerizing activity.In mammals, multiple ChPF and ChSy complexes result in varying chondroitin chain lengths depending on the specific combination of these complexes 9 .Notably, the knockout of ChPF and ChPF2 in cancer cells has been reported to shorten the length of chondroitin chains 25 .Therefore, it is plausible that the altered chondroitin chain length, rather than the quantity of chondroitin chains attaching to the core proteins, contributes to the increased levels in mig-22(k185gf) and the decreased levels in mig-22(rf) and sqv-5(rf) mutants.
mig-22(k185gf) was identified as a strong suppressor of the DTC migration defect associated with the mig-17(k174) null allele.This suggests that the elevated chondroitin levels may serve to compensate for the loss of MIG-17-dependent proteolysis within the BM.Given that MIG-17 plays a role in ensuring proper attachment of the DTCs to the body wall 14 , it is conceivable that the increased chondroitin levels in proteoglycans residing within the body wall BM or in the gonadal BM could potentially restore the appropriate adhesion between these tissues in the absence of MIG-17.
Since mig-17 mutants did not affect the lifespan in an otherwise wild-type background, it is reasonable to conclude that mig-17 does not play a role in the regulation of lifespan.However, phenotypes observed in aged adults, including shortening of body length and the decline in the pumping rate and defecation cycle, were observed earlier in mig-17 mutants than in wild-type animals.These observations suggest that MIG-17 likely governs the BM physiology in multiple tissues to support normal aging.Based on our genetic analyses, we propose that CPGs act downstream of MIG-17, promoting DTC migration and contributing to healthy aging in a manner dependent on the amount of chondroitin attached (Fig. 7).Given that ADAMTS proteases in mammals often degrade proteoglycans, it is plausible that the substrates of MIG-17 might include CPGs 26 .
mig-22(k185gf) exhibited a longer lifespan than the wild type, whereas the lifespan of sqv-5(k175rf) was significantly shorter compared to the wild type.Notably, mig-22(k185gf) failed to suppress the shortened lifespan and shorter body length of sqv-5(k175rf).However, mig-22(k185gf) could suppress the accelerated aging phenotypes of periodic behaviors in the sqv-5(k175rf) background.These results suggest that chondroitin levels play a crucial role in the regulation of both lifespan and healthspan.It is possible that different thresholds of chondroitin levels may exist to control distinct outputs for lifespan and healthspan.It is also noteworthy that an increase in chondroitin levels beyond wild-type levels extends both lifespan and healthspan.MIG-17 appears to be required for lifespan extension only when excessive amount of chondroitin is produced.In summary, these findings indicate that the regulation of healthspan is under the MIG-17 pathway, while a separate regulatory pathway independent of MIG-17 is implicated in the regulation of lifespan (Fig. 7).
CPGs are known to be involved in various critical processes, including ECM assembly, cell adhesion, cell migration, proliferation, and nerve regeneration 10,11,24,27 .There is existing evidence that chondroitin levels decrease with aging 28 .Moreover, chondroitin has been reported to prevent the induction of aging-related phenomena.For instance, in the nervous system of mice, reduced chondroitin synthesis is associated with accelerated MIG-17 www.nature.com/scientificreports/age-related memory decline 29 .In human studies, a cohort ranging from 50 to 76 years old showed that chondroitin intake was linked to reduced mortality, implying a potential lifespan-extending effect 12 .Furthermore, in C. elegans, feeding chondroitin sulfate has been shown to extend lifespan 13 .This indicates a consistent correlation between chondroitin intake and extended lifespan extension is observed across different species.Nevertheless, the precise role of endogenous chondroitin in the regulation of aging is remains enigmatic.
In this study, we demonstrated a correlation between chondroitin levels in vivo and lifespan and healthspan.Chondroitin modifies over 20 core proteins in C. elegans 30 .Different chondroitin proteoglycans may be involved in various aging-related processes.The mig-22(k185gf) mutation represents a senescence-suppressing genetic polymorphism.The Leucine residue at position 325, corresponding to the mig-22(k185gf) mutation site is conserved across different species.It would be intriguing to investigate whether genetic polymorphisms in ChPF proteins, similar to mig-22(k185gf), have lifespan-extending effects in mammals, including humans.Further research is essential to elucidate the precise mechanisms underlying aging in relation to chondroitin levels of CPGs.

Strains and genetic analysis
Culture, handling and ethyl methanesulfonate (EMS) mutagenesis of C. elegans were conducted as described 31 .

Microscopy
Gonad migration phenotypes were scored using a Nomarski microscope (Axioplan 2; Zeiss).Analysis of gonadal phenotypes was performed in the young-adult stage, as previously described 14 .
Measure body length, photographs of animals on an agar plate were taken using a 5 × objective lens.The measurements were conducted using ImageJ software.Each strain was sampled with 30 individuals per day.

Analysis of chondroitin levels
Wild-type, mig-22(k185), sqv-5(k175), and sqv-5(k175); mig-22(k185) animals were grown on NGM plates at 20 °C and collected as mixed-stage animals using M9 buffer.The animals were washed twice with M9 buffer and cultured for 1 h at room temperature.After removing the supernatant, 10 ml of acetone was added.The analysis of the CS chain analysis was conducted by enzymatic treatment and HPLC-based quantification as described previously 11,27 .

Behavioral analyses
Defecation cycle and pumping rate were analyzed as previously described 33 .To synchronize the animals for the analysis of defecation cycle, pumping rate or body size, we collected young adults with no fertilized eggs.To distinguish them from their progeny, they were transferred them to a new plate every two days while they were laying eggs.The pumping rate was measured for 30 s, and the average of three consecutive measurements was calculated.If the defecation cycle exceeded 3 min, further measurements were discontinued.The sample size was 20 for pumping and 30 for the defecation cycle.
Mobility assays were conducted by measuring the speed of animals after tapping plates with E. coli.Movies were recorded for 90 s after tapping, and images were analyzed using the MTrack2 plugin in ImageJ.
Supplementation of chondroitin was performed as described 13 .Animals were maintained for two generations before measuring pumping rate.

Analysis of lifespan
Animals were synchronized for lifespan analysis by collecting L4 stage individuals.Lifespan was analyzed as previously described 34 .

Analysis of brood size
Brood size is measured as described 35 .

Figure 2 .
Figure 2. Amounts of chondroitin in mutants relative to wild type.Amounts of chondroitin were quantified by four independent sets of assays.The quantified values were averaged and shown as ratios relative to that of wild type.Error bars indicate standard error.p-values for t test are indicated: ***p < 0.005, **p < 0.01, *p < 0.05.

Figure 3 .
Figure 3. Suppression of aging by mig-22(k185gf) mutation.(A) The lifespan of wild-type and mig-22(k185gf).The x axis shows days of adulthood.The y axis shows survival probability.The table displays the average, standard deviation, median, and maximum of lifespan.p-values for the logrank test are indicated: ***p < 0.005, -Not determined.(B-E) Body length (B), pumping rate (C), defecation cycIe (D), and mobility on E.coli (E) of wild-type and mig-22(k185) animals.Black and white bars indicate wild-type and mig-22(k185), respectively.The x axis shows days in adulthood.The y axis shows body length (B), pumping rate in 30 s (C), defecation cycle (D), and ratio to average speed of day1 animals.p-values for t test against wild type are indicated: ***p < 0.005, **p < 0.01, *p < 0.05.