Caenorhabditis elegans, the FOXO ortholog DAF-16A and D/F isoforms extend lifespan in daf-2 insulin-like receptor mutants. Here we identify the DAF-21/Hsp90 chaperone as a longevity regulator. We find that reducing DAF-21 capacity by daf-21(RNAi) initiated either at the beginning or at the end of larval development shortens wild-type lifespan. daf-21 knockdown employed from the beginning of larval development also decreases longevity of daf-2 mutant and daf-2 silenced nematodes. daf-16 loss-of-function mitigates the lifespan shortening effect of daf-21 silencing. We demonstrate that DAF-21 specifically promotes daf-2 and heat-shock induced nuclear translocation of DAF-16A as well as the induction of DAF-16A-specific mRNAs, without affecting DAF-16D/F localization and transcriptional function. DAF-21 is dispensable for the stability and nuclear import of DAF-16A, excluding a chaperone-client interaction and suggesting that DAF-21 regulates DAF-16A activation upstream of its cellular traffic. Finally, we show a selective requirement for DAF-21 to extend lifespan of DAF-16A, but not DAF-16D/F, transgenic daf-2 mutant strains. Our findings indicate a spatiotemporal determination of multiple DAF-21 roles in fertility, development and longevity and reveal an isoform-specific regulation of DAF-16 activity.The FOXO transcription factor family is a conserved regulator of longevity and the downstream target of insulin/insulin-like signaling. In  The Forkhead box O (FOXO) transcription factor family is the critical downstream mediator of the ILS pathway3. Activation of a Forkhead transcription factor consistently extends lifespan from yeast4 through worms6 to flies7. In mice, FOXO3 is required for dietary restriction induced longevity8 and loss of FOXO function appears to play a role in various age-related diseases including diabetes, cancer and atherosclerosis9. A potential role of FOXO in human longevity is corroborated by independent studies reporting the enrichment of certain FOXO1 and FOXO3a polymorphisms in nonagenarians11.A conserved endocrine insulin/insulin-like nutrient signaling pathway (ILS) modulates longevity from yeast to mammalsCaenorhabditis elegans2. Here, under nutrient-rich conditions insulin-like peptides (ILPs) bind to the DAF-2 insulin-like receptor which in turn initiates a signal cascade going through the C. elegans phosphoinositide 3-kinase AGE-1, the serine-threonine kinases PDK-1, AKT-1 and AKT-212. Phosphorylation of the single FOXO ortholog DAF-16 by AKT kinases in three conserved serine/threonine residues leads to its exclusion from the nucleus thereby preventing its transcriptional function. In low nutrient conditions DAF-16 translocates into the nucleus and activates DAF-16 dependent gene expression13. Besides the AKT-mediated inhibitory phosphorylations there are a number of mechanisms regulating DAF-16/FOXO function including activatory phosphorylations, redox modifications, ubiquitinylation and proteasomal degradation revealing a highly complex integration of various regulatory inputs at the level of DAF-16/FOXO protein which is not fully understood14.Much of our knowledge on the regulation of ILS and FOXO function are obtained through studies in the roundworm 15, indicating a conserved general longevity promoting “core” gene set. Indeed, DAF-16/FOXO has an intimate connection to stress. It is both activated by, and confers tolerance to, various stresses16. Likewise, reduced ILS mutants not only exhibit increased lifespan but also extended healthspan with more robust cross-tolerance to stresses17. Moreover, the proposed role of DAF-16/FOXO in proteostasis is illustrated by the facts that it upregulates genes of both autophagy and proteasomal protein degradation, while downregulates those of protein synthesis18. Finally, DAF-16 acts in concert with the heat shock transcription factor HSF-1 to extend nematode lifespan in response to reduced ILS19, in part by a cooperative induction of the longevity promoting small heat shock protein genes19. The above-mentioned findings both illustrate an important role of DAF-16 in proteostasis and imply further crosstalks to finely tune the DAF-16 and HSF-1 dependent responses.The transcriptional output of activated DAF-16/FOXO is similarly abundant. Targets shared across species and tissues throughout evolution encompass genes involved in growth factor signaling, metabolism, resistance to various stresses, and proteostasisC. elegans DAF-16 gene gives rise to three groups of transcripts that are transcribed from different promoters20. Isoforms A, B and D/F/H share the same Forkhead domain but have distinct N-terminal domains2. Further studies consistently revealed that DAF-16A and DAF-16D/F are responsible for lifespan extension21, however, their relative contribution remains controversial. These findings add another layer and a yet unknown complexity to DAF-16 regulation.In contrast to the four mammalian FOXO genes originating from gene duplication, the single 22. It accounts for 1–2% of the total cellular proteins. In yeast, Hsp90 is the most connected protein, affecting the function of ~20% of the proteome22. In mammalian cells Hsp90 is essential for the folding and activity of several hundred thermodynamically unstable ‘client’ proteins, predominantly kinases and transcription factors mainly involved in cell signaling and proliferation23. A comprehensive list of Hsp90 clients can be found at the Picard laboratory homepage (www.picard.ch/downloads/Hsp90interactors.pdf). Although Hsp90 is a promising tumor target24 and is implicated in the evolution of new traits22, several pieces of evidence suggest it might play a role in aging. For example, others and we reported that Hsp90 also chaperones antiproliferative, differentiation promoting clients, such as the TGF-β receptor25 and PPARγ26. Moreover, Hsp90 also regulates the heat-shock response by releasing the heat shock factor HSF-1 upon binding to denatured proteins27 which may be utilized in age-related diseases of proteostasis and metabolism, such as diabetes28. Yet, the impact of Hsp90 on aging is largely unexplored.The 90-kDa heat-shock protein (Hsp90) is an essential, evolutionarily conserved eukaryotic molecular chaperoneC. elegans Hsp90 ortholog encoded by the daf-21 gene. Previous work using a weak gain-of-function point mutant daf-21(p673) unraveled its role in chemosensory perception and dauer development through DAF-1130, and muscle homeostasis via UNC-4531. The loss of DAF-21 in embryos either by large deletion mutation in the daf-21(nr2081) strain30 or in the F1 progeny of daf-21(RNAi) treated worms32 induced embryonic and early larval lethality and revealed a conserved function of Hsp90 in cell division through chaperoning the WEE-1 kinase34. Perhaps due to the essential role of daf-21 in embryogenesis, its role in C. elegans lifespan regulation is largely unexplored. In this study, using gene silencing from larval development we investigated the involvement of DAF-21 in wild-type and daf-2 induced longevity and found its specific requirement for DAF-16 isoform A activity.Despite the plethora of information in yeast and mammals, little is known about the biological function and interactions of the sole daf-21(RNAi) construct described earlier35. In agreement with an essential role of Hsp90 in cell proliferation and development22daf-21(RNAi) treatment of the parental F0 generation induced embryonic and early larval lethality in F1 in one32, and sterile F1 worms exhibiting developmental phenotypes in another study35. To investigate the impact of DAF-21 during larval development we started daf-21 silencing from hatching. daf-21(RNAi) efficiently reduced daf-21 mRNA and protein levels in young adults compared to empty vector (EV) control  fed worms exhibited a protruding vulva in ~90% of the population and a mild hypomotility, but no other developmental phenotypes  from the midst of L4 stage. As expected, no protruding vulva phenotype was detected. Reflecting an essential role in oogenesis and embryonic development, daf-21 silencing from hatching caused sterility accompanied by a lack of oocytes in the gonad, whereas that from the L4 stage caused reduced brood size and sometimes embryonic lethality  from hatching , employed either during or after larval development is a safe approach to reduce DAF-21 capacity without compromising develoment and health.To differentiate between the effects exerted by DAF-21 during larval development and adulthood, respectively, we also treated worms with lts Fig. , consisting Fig. , reflectdaf-21(RNAi) from hatching throughout the entire life. Our results show that daf-21(RNAi) shortened lifespan by ~27% compared to EV  was less pronounced, but still significant, if RNAi treatment was administered from the L4 stage throughout adulthood  gain-of-function allele appears to decrease lifespan mainly via bagging 31. We did not observe bagging and premature death in the daf-21 silenced population. The comparable lifespan reduction by daf-21 knockdown during and after development, respectively, suggests that daf-21 affects longevity independent of egg laying and fertility  impaired in the absence of an optimal DAF-21 capacity.Next, we addressed how reducing DAF-21 capacity starting at the beginning or at the end of larval development, respectively, might affect the natural lifespan of wild-type nematodes. First, we measured the lifespan of N2 worms fed by either empty vector or  EV Fig. . The redood Fig. . The daf5. Therefore we compared the lifespan of daf-2(e1370) and daf-2(e1370); daf-16(mgDf50) single and double mutant animals, respectively, grown on plates with bacteria harboring empty vector or daf-21(RNAi). In accordance with the literature daf-2 mutation caused an increase in lifespan compared to wild type which was entirely abrogated by daf-16 loss of function  treatment from hatching significantly reduced the lifespan of daf-2(e1370) worms showing a requirement for DAF-21 to fully manifest the increased longevity conferred by reduced ILS. The partial inhibitory effect of daf-21(RNAi) might be due to the unsuccessful penetration of dsRNA into neurons which do not express SID-1 protein43. To address this possibility, we used RNA interference to silence the expression of daf-2 combined with daf-21 knockdown. The lifespan of wild-type animals upon daf-2(RNAi)/EV showed a ~100% increase compared to empty vector  mutant background. Again, we found a 20–50%, partial decrease of the lifespan of daf-2 silenced nematodes upon daf-21(RNAi) treatment comparable to that observed in daf-2 mutants, indicating a longevity supporting effect of DAF-21 in non-neuronal tissues.Increased longevity of animals with reduced ILS has been established as one of the most robust effects that prolong lifespanion Fig. 20,41,42.daf-21(RNAi) employed from the L4 stage on the lifespan of daf-2 mutants. In all 4 trials it extended the longevity of daf-2(e1370) animals, however, in two of them the effect was non-significant  employed either from hatching or from the L4 stage  treatment on daf-2 lifespan appears to be independent of fertility.Next, we determined the effect of ant Fig. . These odaf-21(RNAi) further shortened the lifespan of daf-16 and daf-2; daf-16 mutants, however, it was significantly less than its lifespan shortening effect in the strains harboring wild-type daf-16  in early adulthood of daf-2 strains  point mutant also promotes dauer formation in C. elegans29 by inhibiting the DAF-11 guanylcyclase in chemosensory neurons30. Hence, we investigated the interference between daf-2 and daf-21 in dauer development. As shown before45, daf-2(e1370) mutants grown on 25 °C almost exclusively turned into dauer larvae, while having a secondary mutation in daf-16 abrogated this effect  L1. The dauer arrest by neuronal daf-21(RNAi) and the embryonic/early larval lethality caused by the embryonic loss of DAF-2132 prevented us to test its impact on daf-2 induced dauer formation. However, our findings together with the lifespan data indicate that DAF-21 in peripheral tissues affects wild-type and daf-2 longevity that is spatiotemporally separated from its effect on development.Nematodes with lowered ILS have a high tendency to initiate an alternative developmental pathway and form dauer larvae under stressful conditions. Likewise, the ect Fig. . In agreted Fig. . Therefot30 Fig. . Dauer ddaf-2 lifespan is dependent on DAF-16 suggested a functional link between DAF-21 and DAF-16. Hence, we asked how daf-21 knockdown affects DAF-16 function. An important step in DAF-16 activity in response to lowered ILS, as well as stresses including heat shock, is its nuclear translocation49. First, we monitored the intracellular localization of DAF-16 in the TJ356 strain containing a daf-16a/b::GFP transgene16 in response to daf-2 and daf-21 knockdown employed from hatching. To ensure similar RNAi dosages, animals were fed with bacteria containing empty vector (EV), daf-2(RNAi)/EV, daf-21(RNAi)/EV and daf-2(RNAi)/daf-21(RNAi) in 1:1 ratios. In daf-2 silenced nematodes a large proportion of DAF-16A/B was localized in the nuclei of both intestinal as well as muscle cells. We found that silencing daf-21 partially inhibited the daf-2 knockdown-induced nuclear translocation of DAF-16A/B::GFP  was confirmed under these conditions as well as daf-21(RNAi)‘s ability to inhibit such translocation  null mutant background: daf-16a::rfp and daf-16d/f::gfp and their respective daf-2(e1370) variants20. The transcripts and proteins structures of the two isoforms are depicted in Fig. daf-2(e1370) compared to the respective strains with intact daf-2 alleles  DAF-16D/F::GFP was still nuclear, while the nuclear localization of both DAF-16A/B::GFP and DAF-16A::RFP were abolished , some that are reportedly DAF-16A-regulated (scl-20 and gst-20) and genes that are the targets of DAF-16 isoform F . In the first series of experiments, in order to clearly isolate the undesired cross-talk from the other DAF-16 isoforms, we took use of the above daf-16a::rfp and daf-16d/f::gfp strains. The induction of sod-3 and old-1 mRNA expression – caused by the daf-2(e1370) mutation – were both diminished by daf-21(RNAi) in the daf-16a::rfp strain  on wild-type animals  mutant variant of the daf-16a::rfp strain  allele in the daf-16a::rfp strain and showed an efficient inhibition of gst-20 expression by daf-21(RNAi)  family54. In contrast to DAF-16A target genes, these two transcriptional targets in a daf-16d/f::GFP transgenic background were not inhibited by daf-21(RNAi)  Fig. . To addrAi) Fig. .Figure 6daf-16 background in response to daf-21 silencing from the L4 stage. Comparing the mRNA expression in a daf-2(e1370) and daf-16(mgDf50); daf-2(e1370) strains, respectively, showed that the DAF-16A specific sod-3, old-1, gst-20 and scl-20 were induced by the daf-2 mutation in a daf-16 dependent manner and their expression was inhibited by silencing daf-21 with the exception of scl-20  mutant but not inhibited by daf-21(RNAi)  sequence35  Fig. . Some of22. If Hsp90 function is compromised, destabilized clients are polyubiquitinylated by ubiquitin conjugating enzyme complexes and subsequently degraded by the proteasome57. Several E3 ubiquitin ligases target mammalian FOXOs to degradation18. In C. elegans, the null mutation of the RLE-1 E3 ubiquitin ligase has been shown to lead to DAF-16 protein stabilization and DAF-16 dependent lifespan extension58. We reasoned if DAF-21 stabilized DAF-16 conformation, then reduced DAF-21 capacity would result in DAF-16 aggregation and disrupt lifespan extension in rle-1 mutants. Although we did not detect a substantial increase of DAF-16A::RFP in rle-1 background, daf-21(RNAi) neither interfered with DAF-16 distribution nor caused DAF-16 aggregation . Thus, DAF-16 is unlikely to be a DAF-21 client.Mammalian Hsp90 is a specific chaperone that stabilizes the conformation of a large number of client proteins including various transcription factors49. In response to reduced ILS, DAF-16 translocates into the nucleus. To assess if DAF-21 acts upstream or at the level of DAF-16 nuclear traffic, we employed a strain harboring the daf-16aAM::gfp (AM: “AKT site mutant”) transgene, in which serines and threonines in the AKT phosphorylation sites were changed to alanines49. In agreement with previous work49, DAF-16AAM::GFP was nuclearly localized in animals possessing wild-type daf-2   mutant , suggesting that nuclear DAF-16 is able to exert a partial transcriptional activity which is independent of DAF-21. These findings corroborate the idea that DAF-21 acts upstream of DAF-16A nuclear traffic.Similarly to mammalian cells, when ILS signaling is ample, AKT-1 and AKT-2 kinases phosphorylate DAF-16/FOXO which prevents its accumulation in nuclei by anchoring it to cytosolic 14-3-3 scaffold proteinsdaf-2 induced longevity. Hence, we investigated how daf-21(RNAi) employed from hatching affects longevity specified by individual DAF-16A and D/F isoforms in the context of reduced ILS by using strains expressing single isoforms in a daf-2(e1370); daf-16(mgDf50) mutant background. In concordance with published data21 both daf-16a::rfp and daf-16d/f::gfp transgenic strains exhibited longer lifespan compared to daf-2(e1370); daf-16(mgDf50) background  further shortened the lifespan of daf-2(e1370); daf-16(mgDf50) double mutants suggesting DAF-21 might also target longevity promoting factors other than DAF-16. Importantly, daf-21 knockdown consistently diminished the lifespan of the DAF-16A::RFP expressing strain in all four biological replicates, while in two out of four trials it failed to modify that of the DAF-16D/F::GFP transgenic worms  require further studies, potential candidates might be a less ordered organization of protein-protein and signaling networksdaf-2 mutants60, in which we have observed that daf-21 silencing during adulthood does not shorten, but marginally extend lifespan. We speculate that the reduction of DAF-21 in early adulthood acting in concert with the mild metabolic stressor daf-2 activates a pro-longevity response that antagonizes the daf-21 induced lifespan shortening effect. Potential mechanisms might involve the HSF-1 dependent heat shock response27, the differential regulation of the respective DAF-16 isoforms21 (this study) or a daf-21 independent pathway. However, the validation of this idea and the identification of the real mechanism(s) await further research.All the proteostatic defense, tissue integrity and immunity are more robust in via specifically ensuring DAF-16A function, reflected by the specific requirement of DAF-21 for DAF-16A nuclear translocation, transcriptional function and lifespan extension mediated by DAF-16A as the sole DAF-16 isoform. Importantly, DAF-21 was necessary for the induction of three out of four DAF-16 target genes, each of them extends lifespan: sod-364, old-165, scl-2052 and gst-2021, the latter two identified as DAF-16A specific targets21 21. Based on this study, we propose a model: DAF-21 affecting the DAF-16A-regulated genes is required to elicit daf-2-induced daf-16-mediated longevity. If DAF-21 capacity is scarce, DAF-16A activity is compromised, DAF-16D/F may still evoke a compensatory response resulting in lifespan extension. Perhaps, this might underlie the lifespan extension and partial lifespan decrease, respectively, of daf-2 worms treated by daf-21(RNAi) in adulthood and from hatching. Other lifespan extending signals that selectively activate DAF-16D/F operate independently of DAF-21 availability.This study confirms the involvement of both DAF-16A and D/F in lifespan control when ILS is lowered59. We observed a DAF-21 dependent DAF-16A nuclear translocation in muscle, hypodermis and intestine. Previous work identified that the intestinal, but not neuronal or muscle-specific expression of DAF-16 is responsible for the longevity effect of lowered ILS46. Furthermore, both muscle and intestinal DAF-16 combat reproductive aging67. Based on this, we speculate that the longevity promoting DAF-21-DAF-16A interaction may predominantly occur in the muscle and intestine.What molecular mechanism(s) may be involved in the functional interaction between DAF-21 and DAF-16A? The fact, that all DAF-21, DAF-16A and DAF-16D/F are pleiotropically expressed in almost every tissue excludes the possibility of a merely tissue-specific interaction20 . Thus, the reciprocal regulations through DAF-21 co-ordinating DAF-16 (and HSF-1) activity may ensure an integrated organismal response sensing both the steady state proteostatic milieu and nutrient availability, which promote proper self-maintenance and on the long term, longevity.DAF-21 is not the only heat shock protein that regulates DAF-16 nuclear traffic. HSF-1 and the constitutive HSP70 isoform HSP-1 are required for the nuclear export of DAF-16A/B in the C. elegans longevity which provides a crosstalk between the proteostasis and nutrient signaling networks via an isoform specific regulation of DAF-16 activity. Considering the strong structural and functional conservation of DAF-16/FOXO isoforms18 and DAF-21/Hsp9022, a similar regulatory mechanism might operate in mammals.In summary, our work uncovers a previously unappreciated role of DAF-21 in C. elegans techniques69. Caenorhabditis elegans strains used in this study: N2: wild type, GR1307: daf-16(mgDf50), CF1038: daf-16(mu86), CB1370: daf-2(e1370); e1370/mu86: daf-16(mu86); daf-2(e1370), EFS7: daf-16(mgDf50); daf-2(e1370), TJ356: daf-16p::daf-16a/b::gfp + rol-6, HT1888: daf-16(mgDf50); unc-119(ed3); lpIs12 [daf-16a::rfp + unc-119(+)], HT1889: daf-16(mgDf50); unc-119(ed3); lpIs14 [daf-16d/f::gfp + unc-119(+)], HT1881: daf-16(mgDf50); daf-2(e1370); unc-119(ed3); lpIs12 [daf-16a::rfp + unc-119(+)], HT1883: daf-16(mgDf50); daf-2(e1370); unc-119(ed3); lpIs14 [daf-16d/f::gfp + unc-119(+)], HT1890: daf-16(mgDf50); daf-2(e1370), CF1371: , JT6130: daf-21(p673), LL1009: daf-21(nr2081)/nT1 [unc-?(n754) let-?], KB6: rle-1(cxTi510), HT1888xKB6: daf-16(mgDf50); unc-119(ed3); lpIs12 [daf-16a::rfp + unc-119(+)]; rle-1(cxTi510), TU3335: lin-15B(n744) X; uIs57 [unc-119p::YFP + unc-119p::sid-1 + mec-6p::mec-6].All strains used were obtained from CGC. Animals were kept at 20 °C using standard daf-2(e1370); daf-16(mu86) double mutant strain was generated by crossing the CB1370[daf-2(e1370)] and CF1038[daf-16(mu86)] single mutants. Genotype of the F2 generation was monitored by allele-specific PCR (see Supplementary Table The daf-16a::rfp transgene was monitored by fluorescence microscopy in the F2 generation while the presence of rle-1(cxTi510) mutation was tested using allele-specific PCR.The HT1888 and KB6 strains were crossed together and the E. coli strains producing dsRNA against daf-21 (hsp90)35 were kindly provided by Eileen Devaney  and were created by cloning a 74 bp and a 294 bp region of hsp90 into the L4440 vector. daf-2(RNAi)70 was from the Vellai Lab  created by cloning a 1204 bp fragment of daf-2 into the L4440 vector. All primers used can be found in Supplementary Table 71: RNAi feeding E. coli clones were grown overnight in LB medium containing 100 μg/ml ampicillin. Worms were grown on plates containing 1 mM IPTG, 50 μg/ml ampicillin and 6.25 μg/ml tetracyclin and seeded with E. coli HT115 strains harboring the L4440 empty vector (EV) control and specific RNAi vectors, respectively, from hatching – or they were transferred onto RNAi plates after reaching the L4 larval stage on OP50. Measurements were made after 2 days on RNAi bacteria. To control for proper RNAi dosage in double RNAi treatments, overnight cultures of the RNAi bacterial strains were mixed in a 1:1 ratio, mixing in the empty vector harbouring strain in single RNAi controls.HT115(DE3) All lifespan assays were performed at 20 °C. Animals were synchronized by allowing gravid adults to lay eggs for 4 hours and using the next generation for experiments after they reached young adult stage. Approximately 35 animals were transferred to each of 3 plates containing 5-fluorodeoxyuridine (FUDR) (Sigma-Aldrich) to a final concentration of 51 µM. Day 1 is defined as the day the worms were placed on the FUDR plates. Every second day animals were scored by tapping with a platinum worm pick starting from day 7. Worms that crawled into the agar, onto the wall of the plate or died from vulval bursting were censored.3 in M9 buffer. Pictures were taken by a Leica DMI6000B epifluorescence microscope with a DFC480 camera . The mRNA was then transcribed into cDNA by RevertAid™ Premium Reverse Transcriptase (Thermo Scientific). qPCR measurements were performed in an ABI 7300 Real-time PCR machine using Maxima™ SYBR Green/ROX qPCR Master Mix (Thermo Scientific). Primer sequences are listed in Supplementary Table 30 young adult animals from a synchronized population were transferred to each of 3 plates for every condition. Plates were put in an incubator preheated to 35 °C for 6 hours. Then, plates were placed back into a 20 °C incubator. Following a 5 hour recovery time the animals were scored every 24 hours by tapping with a platinum worm pick. Worms that crawled into the agar, onto the wall of the plate or died from vulval bursting were censored. All thermotolerance measurements were repeated at least three times. Statistical analysis was performed by comparing different conditions and using two-tailed Student’s T-test to determine the level of significance.10 gravid hermaphrodites were allowed to lay eggs on each plate for 4 hours. Then the plates were transferred to 25 °C and animals were allowed to grow until the third day, when adults and dauer larvae were scored from each strain and condition.daf-21(RNAi) plates for 1 hour in order to get a highly synchronous population of animals. The plates were kept at 20 °C and progeny was scored for phenotypic differences after three days. Pictures were taken using Nikon Eclipse E400 microscope with Diagnostic Instruments SPOT model 1.5.0 camera.10 gravid hermaphrodites were allowed to lay eggs on EV and 10 L4 hermaphrodites from each condition of each strain were placed onto treatment plates individually. All plates were incubated at 20 °C. Every 24 hours mothers were transferred onto new plates. Progeny was scored on the plates 48 hours after the removal of the mother. The assay was carried on till the last animal stopped laying eggs.daf-21(RNAi) or empty vector (EV). Worms were washed three times using M9 buffer and frozen at −80 °C. After thawing, 200 µl of lysis buffer ) was added to the samples. After three freeze-thaw cycles samples were sonicated 6 times for 10 seconds and centrifuged for 10 minutes at 10000 g at 4 °C. Supernatant was transferred into new tubes and was stored at −20 °C or at −80 °C for longer storage. Western blotting was carried out as previously described74. Samples were run in 12% poly-acrylamide gel (Bio-Rad) and transferred to nitrocellulose membrane (Bio-Rad). Blocking was done by incubation in TBS-T with 5% skim milk powder for 1 hour at room temperature. Primary antibodies were Actin  and Hsp9075 . Secondary antibodies were HRP-labelled anti-mouse (1:2000 in TBS-T with 5% skim milk powder) and anti-rabbit (1:3000) IgG, respectively (Dako). Membranes were incubated with ECL reagent  for 1 min and developed.Synchronized population of animals were grown on 10 cm NGM plates with IPTG seeded with either Statistical analysis was done by the SPSS 15.0 software . Survival curves were compared using the Kaplan-Meyer log rank test. Pairwise comparisons were done using Student’s t-test. Multiple comparisons were done with ANOVA using the Fisher’s Least Significant Difference (LSD) test. Variables were expressed as mean ± standard error of the mean (SEM). Statistical levels of significance are as follows: *p < 0.05, **p < 0.01, ***p < 0.001.Supplementary Information


Sox8 nor Amh activity in the ovarian tissues is essential for such ectopic appearance of SOX9-positive cells. The transcriptome analyses of the grafted ovaries during this masculinization process showed early downregulation of pro-ovarian genes such as Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 by days 7–10 post-transplantation, and subsequent upregulation of several pro-testis genes, such as Bhlhe40, Egr1/2, Nr4a2, and Zc3h12c by day 20, leading to a partial sex reversal with altered expression profiles in one-third of the total numbers of the sex-dimorphic pre-granulosa and Sertoli cell-specific genes at 12.5 dpc. Our data imply that the paternal testosterone exposure is partially responsible for the sex-reversal expression profiles of certain pro-ovarian and pro-testis genes in the fetal ovaries in a temporally dependent manner.In most of mammalian embryos, gonadal sex differentiation occurs inside the maternal uterus before birth. In several fetal ovarian grafting experiments using male host mice, an experimental switch from the maternal intrauterine to male-host environment gradually induces partial masculinization of the grafted ovaries even under the wild-type genotype. However, either host-derived factors causing or molecular basis underlying this masculinization of the fetal ovaries are not clear. Here, we demonstrate that ectopic appearance of SOX9-positive Sertoli cell-like cells in grafted ovaries was mediated by the testosterone derived from the male host. Neither  Sox) gene, Sox9, in bipotential supporting cells from 11.0 days post-coitum (dpc) [Sox8 expression [Sox9 and Sox8 cooperatively maintain the function of Sertoli cells during the later stages [Sry, bipotential supporting cells differentiate into pre-granulosa cells from approximately 11.5 dpc in mouse XX embryos [Esr1/2 (estrogen receptor-1/-2) double-null females develop ectopic SOX9-positive Sertoli cell-like (Sertoli-like) cells during the initial round of folliculogenesis, leading to ovotestis formation upon reaching the adult stages [In mouse sex differentiation, both testicular Sertoli cells and ovarian granulosa cells develop from common supporting cell precursors in the genital ridges ,2. In XYum (dpc) –7. SOX9 pression –12. After stages –16. In t embryos . After 1 embryos ), which  embryos ,19–22. It stages ,24. In mt stages . This suFreemartin syndrome causes infertility in a female cattle twin born with a male twin ,27. SincSox9 transcription) during 7–10 days post-transplantation [Similar to freemartin ovaries, fetal ovarian grafts under the kidney capsules of adult male mice undergo a partial masculinization ,34, togeantation , showingSox8 and Amh activity in the partial masculinization of fetal ovaries in the male-host environment. We also examined temporal changes in the gene expression profiles of grafted fetal ovaries during the masculinization process in male nude mice and compared these expression profiles with those from XY/XX embryos during the normal testicular/ovarian differentiation process.In the present study, we examined the roles of host-derived testosterone and donor-derived In fetal ovaries grafted under the kidney capsules of adult male mice (XY-host), the ovarian transplants undergo follicular degeneration by day 10 post-transplantation in which cord-like structures with SOX9-positive Sertoli-like cells appear in the gonadal parenchyma on day15–20 post-transplantation ,35.First, to examine the contribution of the male-host environment to the follicular degeneration, we transplanted fetal ovaries  under the kidney capsules of intact female (XX) or male (XY) nude mice, and then conducted immunohistochemical staining with anti-AMH (a marker for both Sertoli cells and mature granulosa cells of growing follicles) and anti-GATA4  antibodies . In ovarNext, we examined the effect of XY-host-derived androgen on the ectopic appearance of SOX9-positive Sertoli-like cell in ovarian transplants. In the ovarian explants grafted into control XY-host, any SOX9-positive signals were not detectable throughout the ovarian parenchyma on day 10 post-transplantation, but ectopic SOX9-positive Sertoli-like cells were found to appear in the testis cord-like structure near the edge of the GATA4-positive gonadal parenchyma by day 20 ; S2 Fig,Rspo1/Sox9-double null XX mice, Sox8 appears to act as a pro-testis gene that induces male differentiation without any actions of SRY and SOX9 [in vivo and in vitro experiments, excessive AMH exposure was able to induce seminiferous cord-like structures in fetal rodent ovaries [Sox8 and Amh, to partial masculinization of fetal ovaries in the present model.In the testis differentiation of and SOX9 ,41,42. Aand SOX9 ; French and SOX9  and refe ovaries ,46. BaseSox8 expression in ovarian grafts, we used serial sections of ovarian grafts on days 10 and 20 post-transplantation for in situ hybridization using a Sox8 antisense probe  and Amh-null (Amh-/-) lines using the CRISPR/Cas9 system , Tgfbr2 , etc.  before and after transplantation into XY-host and examined temporal changes in the expression profiles of ovarian somatic cells from 4 to 20 days post-transplantation. According to hierarchical clustering, the grafted ovaries exhibited gene expression profiles that differed from those of 13.0 dpc ovaries pre-transplantation . GO analI), etc. . This maI), etc. . These rNext, to analyze the genes that were not associated with these side effects, we selected genes shared between the 1,561 pre-granulosa and 1,849 Sertoli cell-specific genes in fetal ovaries and testes at 12.5 dpc . Among tFst, Bmp2, Wnt4, and Rspo1 [Irx3, Axin2, Fst, Mbnl3, Nr0b1, Wnt4, Wnt9a; p = 1.5E-03) and “reproductive structure development” . In clusters II and III, Sertoli cell-specific genes were upregulated starting from days 7–10 and 15–20 post-transplantation, respectively  and “positive regulation of transcription” .Based on gene clustering analyses, the down- and upregulated genes were divided into three clusters . Within nd Rspo1 ,48,49 weectively ; Table 1ectively  includedIrx3 , Emx2  , Nr0b1 ic cells ), and Fen cancer ), exceptning 7a) . All of 2 action . Nr0b1/DSry gene ,54, was Bhlhe40  and Egr1 (early growth response 1) was upregulated on day 5–10 post-transplantation, and its high expression levels were maintained by day 20 post-transplantation. Nr4a1 , Nr4a2, Egr2, Zfp367 (zinc finger protein 367), Sox8 and Zfp148, albeit of no significance in some genes , displayed a transient upregulation pattern around on day 10–15 post-transplantation , we selected 22 transcription/nuclear factors showing differences in expression more than 1.5-fold at each stage  and AR-target genes in supporting cells such as Ube2b , B4galnogenesis –60), andli cells ) among tFinally, the global alteration of the shared transcriptomes with Sertoli cell- or granulosa cell-specific genes in the masculinization of ovarian grafts was schematically shown in Irx3, Nr0b1/Dax1, Emx2, and Fez1/Lzts1 [Foxl2 action [In fetal mouse ovarian explants grafted into male hosts, the masculinization processes are separated into distinct early (follicular degeneration/tubular structure formation) and late stages (subsequent appearance of ectopic SOX9-positive Sertoli-like cells in the ovarian grafts). Our temporal gene expression profiles of 4,747 downregulated and 6,730 upregulated genes in ovarian graft tissue post-transplantation revealed downregulation of 41.2%  of the pre-granulosa cell-specific genes and upregulation of 37.6%  of the Sertoli cell-specific genes Figs  and 7. Iz1/Lzts1 ,62,63, w2 action . Our pre2 action . FurtherNr4a1/2, Egr1/2, and Sox8 on days 10–15 post-transplantation, albeit not statistically significant in Nr4a1 and Sox8  derived from XY-host contribute to the follicular degeneration and subsequent masculinization along the hypothalamo-pituitary-gonadal axis [Ar and AR-target genes) in this masculinization will require further genetic studies using fetal ovarian grafts with corresponding null mutations.It has been demonstrated that li cells , can uprli cells , furtherdynamics ,70, whildynamics . The pre XY-cast , suggestdal axis –75. MoreSox8 is widely expressed in granulosa cells in degenerating follicles during both the AMH-positive and -negative stages in the tubular structures even at day 10 post-transplantation (Sox8-/- and Amh-/- ovarian explants on day 20 post-transplantation (Sox8 or Amh activity resistance and/or delay of follicular degeneration in the ovarian grafts. This is consistent with the recent data showing the association between human primary ovarian insufficiency (POI) and SOX8 variants [Sox8-null or Amh-null explants  in testis development.In the present study, antation , S5 Fig.antation , suggestvariants , togethevariants ). Howeveexplants . From thntiation ,16,42,78Sox9 expression in normal developing ovaries, it is possible that such marginal subpopulation may contribute to this testosterone-dependent masculinization.Finally, in this masculinization model, SOX9-positive Sertoli-like cells are restricted to the presumptive medullary region 15–20 days post-transplantation . One posIn conclusion, the present study reveals the transcriptome patterns of the testosterone-mediated masculinization in ovarian grafts in male nude mice. These gene expression profiles may help elucidate the infertility and masculinization of mammalian fetal ovaries caused by in utero exposure to inadequate hormonal conditions, as in the case of freemartin syndrome in mixed-sex cattle twins and polycystic ovary syndrome in mammals.All animal experiments in this study were carried out in strict accordance with the approved guidelines by the institutional committees for animal and recombinant DNA experiments at the University of Tokyo (UT) and Tokyo Medical and Dental University (TMDU). The procedures were approved by the Institutional Animal Care and Use Committee of UT  and TMDU .Nude mice  were used as host mice for the transplantation of fetal ovaries. 1 week prior to transplantation, some of the 7 weeks old nude mice were castrated and implanted subcutaneously with a 0.5–1.0 cm silicon tube with an internal diameter of 2 mm  containing testosterone , dihydrotestosterone , or sesame oil alone. In the present study, we used the almost same or higher dose of T/DHT than the previous experiments of androgen-treatment with silicon tube into mice –81. The At 13.0 dpc, female embryos were obtained from pregnant wild-type females . For microarray and RT-qPCR analyses, all pregnant females were pretreated with busulfan  to eliminate the influence of germ cells and their transcripts on the expression profiles in the grafts. We confirmed that the fetal ovaries of busulfan-treated mice could induce the appearance of testis cord-like structures with SOX9-positive Sertoli-like cells  in a simSox8-/- and Amh-/- embryos were obtained by mating either heterozygous or homozygous females with the heterozygous male mice that were established by the CRISPR/Cas9 system , as described by Hashimoto and Takemoto [5’-GGGCCGCTTCACGTGTGGCT-3’ (Sox8) and 5’-TGCAGGGCTTACGTGCCGAG-3’ or 5’-CGTTGGGACACAGCGCTAGC-3’ (Amh). The vectors expressing Cas9 mRNA and these guide RNAs targeting Sox8 or Amh were introduced into fertilized eggs collected from C57BL/6 females. Several independent lines with frame-shift mutations in Sox8  or Amh e domain ) were es reports –87: both tubules , and Amhuteruses .Transplantation of fetal ovaries was carried out using a previously reported procedure . In brieThe samples were fixed in 4% PFA-PBS at 4°C for 12 hours, dehydrated, and then embedded in paraffin. Serial sections at the horizontal level of each thin, flat graft (approximately 4 μm in thickness) were used for immunostaining of various markers as described below. The sections were incubated with anti-AMH , anti-DMRT1 , anti-FOXL2 , anti-GATA4 , anti-GDNF , and anti-SOX9  antibodies at 4°C for 12 hours. The reactions were visualized using Elite ABC Kit (Vector Laboratories), TSA Plus Biotin Kit (Perkin Elmer), or Alexa Fluor 488 Tyramide SuperBoost Kit (Thermo Fisher) with 4’, 6-diamidino-2-phenylindore (DAPI).th section from the horizontal sections) were photographed, and the numbers of normal healthy follicles, degenerating follicles derived from secondary or antral follicles, and SOX9-positive cells were counted in the sections with maximum areas. The degenerating follicle was estimated as the follicular structure except for a healthy follicle by the histological criteria such as an atretic oocyte and disarranged granulosa cells using the image data of the immunohistochemical and histological adjacent sections. The gonadal area (mm2) containing GATA4-positive cells was also measured using ImageJ 1.48V software , and the relative numbers of follicles and SOX9-positive cells were estimated.For morphometric analyses, anti-GATA4/AMH/SOX9-stained serial sectioning samples (every 10in situ hybridization experiments were performed using an RNAscope [in situ detection using the RNAscope 2.0 High Definition-RED Kit according to the manufacturer’s instructions (ACDBio).All RNAscope . All samFor microarray analyses, ovarian transplants initiated on 13.0 dpc (approximately 10 grafts at each stage per one microarray sample) were recovered on days 4, 7, 10, 15, and 20 post-transplantation from the host kidneys. Temporal fold changes in gene expression during each stage were examined and compared with those of 13.0 dpc ovaries pre-transplantation  .Gene expression analysis was conducted using Mouse Genome 430 2.0 GeneChip arrays (Affymetrix). Total RNA from all samples was purified using the NucleoSpin RNA XS Kit (Macherey-Nagel); 100 ng purified total RNA were amplified and labelled with biotin, according to the GeneChip 3’ IVT Express Kit and GeneChip 3’ IVT PLUS Reagent Kit manuals (Affymetrix). The amplified biotinylated RNA (11 μg) was fragmented and hybridized to each array for 16 hours at 45°C in a rotating hybridization oven. Chips were washed and stained with streptavidin/phycoerythrin using the Fluidics Station 450 (Affymetrix) following protocol FS450_0001. Chips were scanned using the GeneChip 3000 Scanner (Affymetrix), and the outputs were obtained using the GeneChip Operating Software v. 1.4 (Affymetrix). All data are accessible via GEO Series accession number GSE88797.https://sites.google.com/site/dchipsoft/home). The averaged normalized values were used to derive fold changes in the duplicated raw data obtained from transplants on days 0, 10, and 20 post-transplantation. Among the altered genes (|fold| > 2), 4,747 were downregulated and 6,730 upregulated on day 4, 7, 10, 15, or 20 post-transplantation (http://david.abcc.ncifcrf.gov/).Data were normalized using the robust multiarray algorithm , and furantation . The celantation  from theSox8 and Amh allele, we performed the RT-PCR analysis using the following primers: Sox8 forward primer: 5’-TGT CAC ACG TGG AGG ATT CGG ACT C-3’, Sox8 reverse primer: 5’-AGG GTC TTG CTG AGC TCT GCG TTA TGG AG-3’; Amh forward primer: 5’-ACG GAG AGG GAA CCT ATG CCG CTG-3’, Amh reverse primer: 5’-TGG CAG TTG TTG GCT TGG TAG GTC TC-3’ . In order to confirm the shorter transcripts of mutated Ar (Mm00442688_m1), Bhlhe40 (Mm00478593_m1), B4galnt1/Galgt1 (Mm01135934_g1), Egr1 (Mm00656724_m1), Egr2 (Mm00456650_m1), Emx2 (Mm00550241_ml), Lzts1 (Mm01345507_m1), Irx3 (Mm00500463_m1), Nr0b1/Dax1 (Mm00431729_m1), Nr4a1 (Mm01300401_m1), Nr4a2 (Mm00443060_m1), Sox8 (Mm00803423_m1), Sox9 (Mm00448840_m1), Tubb3 (Mm00727586_s1), Ube2b (Mm00494000_m1), Zbtb7c (Mm02375515_s1), Zc3h12c (Mm01177355_m1), Zfp148 (Mm00711990_m1), and Zfp367 (Mm00615562_m1) were purchased from Applied Biosystems. The relative levels of the transcripts were normalized against Gapdh (4352339E) levels as an endogenous reference.For Quantitative RT-PCR, Taqman gene expression probes for p-value of 0.05 or less.Normality of all quantitative data were analyzed by Shapiro-Wilk test prior to parametric or nonparametric test. As parametric test, the data were analyzed by one-way analysis of variance followed by Dunnett’s test. As non-parametric test, the data were analyzed by Kruskal–Wallis test followed by Steel’s test. Statistical significance was assessed at a threshold S1 FigA bar graph shows the relative seminal vesicle weight  of each host group. Compared to control (intact) male-host , the relative seminal vesicle weight in other groups was approximately 11% in castrated host males (XY-cast-host), 59% in testosterone-treated castrated males (XY-cast-host +T), or 54% in 5α-Dihydrotestosterone-treated castrated males (XY-cast-host +DHT), respectively . The numbers in parentheses indicate the number of the host males examined after the transplants were recovered from their kidney capsule.(TIF)Click here for additional data file.S2 FigAnti-SOX9 immunostaining of the wild-type ovarian tissues grafted into male (XY), female (XX), and castrated male (XY-cast) hosts, showing no ectopic SOX9-positive cells in all grafted ovaries on day 10 post-transplantation. Scale bars, 100 μm.(TIF)Click here for additional data file.S3 Fig Amino acid sequences of wild-type and independent lines with frame-shift mutations of Sox8  and Amh . The HMG box domain and C-terminal TGF-beta-like domain are shown in blue. Predicted amino acid sequences caused by frame-shift mutations are written in red . Red arrowheads show the positions of the RT-PCR primer sets , as shown in C. (C) RT-PCR analyses of the Sox8 (left) or Amh (right) transcripts in the testes of wild-type (wild) and mutant (mut) males (2-month-old) by using the primer set that flanks the deleted mutation site (red arrowheads in a). The RT-PCR analyses confirm the presence of the only short (deleted) transcripts in each mutant testis. All blots are on the same gel. RT+ or RT- in each panel indicates the RT-PCR reaction samples treated with or without reverse transcriptase, respectively.(TIF)Click here for additional data file.S4 Fig(A) Anti-Claudin-3  immunostaining of the testes of wild-type and Sox8-/- male mice (5-month-old), showing a typical Sox8-null phenotype of reduced CLDN3 expression (arrowheads) in the basal compartment of seminiferous tubules [(B) Reproductive tracts of wild-type and Amh-/- male mice at 3 months old, showing the presence of the uterus tubule (yellow arrow) that runs parallel to the vas deference (white arrow) in the Amh-/- male. Scale bars, 20 μm. tubules . (B) Rep(TIF)Click here for additional data file.S5 Fig(A) Anti-AMH immunostaining of wild-type (on day 10 post-transplantation) and Sox8-/- (on day 20 post-transplantation) ovarian grafts, showing AMH-positive signals are properly seen in the follicles of Sox8-/- grafted ovarian tissues. (B)In situ hybridization using a Sox8 antisense probe of wild-type and Amh-/- ovarian grafts on day 20 post-transplantation, showing no appreciable differences of Sox8-positive signals in the degenerating follicles (arrowheads) between wild-type and Amh-/- ovarian grafts. Scale bars, 100 μm.(TIF)Click here for additional data file.S6 FigAnti-AMH or SOX9 immunostaining of ovarian tissues of busulfan-treated wild-type mice grafted into male host mice on days 10 (upper) and 20 (lower) post-transplantation. Typical testis cord-like structures (arrows in insets) and ectopic appearance of SOX9-positive cells in the medullary region were detected in busulfan-treated grafted ovaries, similarly to non-treated grafted ovaries. The low-magnification images of each ovarian tissues are shown in the insets at the upper right corner. Scale bars, 100 μm.(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 TableS2A and S2B Table show the expression fold changes of 695 down/643 upregulated pre-granulosa/Sertoli cell-specific genes in the ovarian grafts at each stage. S2C and S2D Table show the expression fold changes of 1,561 pre-granulosa/1,849 Sertoli cell-specific gene expression in the ovarian grafts.(XLSX)Click here for additional data file.S3 TableS3A Table shows cluster-I genes, which are downregulated pre-granulosa cell-specific genes, and S3B and S3C Table show clusters-II/III genes, which are upregulated Sertoli cell-specific genes in ovarian grafts. Cluster-I is the 189 pre-granulosa cell-specific genes downregulated in grafted ovaries on day 10 post-transplantation (|fold| > 2). Cluster-II is the 141 Sertoli cell-specific genes upregulated in grafted ovaries, especially on day 10 post-transplantation (|fold| > 2). Cluster-III is the other 261 Sertoli cell-specific genes upregulated in grafted ovaries on day 20 post-transplantation (|fold| > 2). S3D and S3E Table show the lists of GO terms of the genes in clusters-I (S3A Table) and clusters-II/III (S3B and S3C Table). S3F Table shows lists of gene content in the selected GO terms from S3D and S3E Table.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.


N-acetylcysteine (NAC) pretreatment. Moreover, sinularin dose-responsively induced oxidative stress and DNA damage according to flow cytometry analyses of reactive oxygen species (ROS), mitochondrial membrane potential (MitoMP), mitochondrial superoxide, and 8-oxo-2′-deoxyguanosine (8-oxodG)). In conclusion, sinularin induces selective killing, G2/M arrest, apoptosis, and oxidative DNA damage of breast cancer cells.The natural compound sinularin, isolated from marine soft corals, is antiproliferative against several cancers, but its possible selective killing effect has rarely been investigated. This study investigates the selective killing potential and mechanisms of sinularin-treated breast cancer cells. In 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H- tetrazolium, inner salt (MTS) assay, sinularin dose-responsively decreased the cell viability of two breast cancer (SKBR3 and MDA-MB-231) cells, but showed less effect on breast normal (M10) cells after a 24 h treatment. According to 7-aminoactinomycin D (7AAD) flow cytometry, sinularin dose-responsively induced the G2/M cycle arrest of SKBR3 cells. Sinularin dose-responsively induced apoptosis on SKBR3 cells in terms of a flow cytometry-based annexin V/7AAD assay and pancaspase activity, as well as Western blotting for cleaved forms of poly(ADP-ribose) polymerase (PARP), caspases 3, 8, and 9. These caspases and PARP activations were suppressed by  Breast cancer is the most common type of cancer to affect women. Reactive oxygen species (ROS) play an important role in breast cancer cell survival and proliferation , i.e., lFor example, 4β-hydroxywithanolide  and withSinularia flexibilis (S. flexibilis)-derived sinulariolide has been reported to suppress the migration and invasion of bladder cancer (TSGH-8301) cells [Nephthea erecta)-derived natural steroid 24-methyl-cholesta-5,24(28)-diene-3beta, 19-diol-7beta-monoacetate was reported to inhibit the proliferation of lung cancer H1688 cells [Sarcophyton glaucum also display cytotoxicity to several types of cancer [Effective drug development relies on identifying more and better bioactive compounds, such as lead molecules for subsequent modeling and synthesis, with selective killing effects against breast cancer cells. Some natural marine products have been found to inhibit proliferation ,9 and in1) cells , and to 1) cells . The wil88 cells . SeveralSarcophyton flexibilis [S. manaarensis [The current study examines the marine natural product sinularin isolated from the soft coral exibilis . The samaarensis . It is oaarensis  and gastaarensis . Howeveraarensis . Here, wTo test this hypothesis, we selected two types of breast cancer (SKBR3 and MDA-MB-231) cells and one type of breast normal (M10) cells to evaluate the potential selective killing effect of sinularin and to explore its antiproliferative mechanism in terms of cell viability, cell cycle distribution, apoptosis, ROS generation, mitochondrial membrane potential (MitoMP), mitochondrial superoxide, and oxidative DNA damage.To examine apoptosis, the annexin V/7AAD patterns of sinularin-treated breast cancer (SKBR3) and normal breast (M10) cells were analyzed using flow cytometry. To further examine the degree of apoptosis for sinularin-treated breast cancer (SKBR3) (top side) and normal breast (M10) (bottom side) cells, the flow cytometry-based pancaspase patterns for generic activity of caspases 1, 3, 4, 5, 6, 7, 8, and 9 ) are proN-acetylcysteine (NAC) pretreatment  polymerase (PARP) and caspases 3, 8, and 9 were performed. As shown in DCFH-DA-based flow cytometry was used to measure the change of sinularin -induced ROS generation in breast cancer (SKBR3) and normal breast (M10) cells. 2(3)-based flow cytometry was used to measure the changes of sinularin -induced MitoMP change in breast cancer (SKBR3) and normal breast (M10) cells. DiOCThe role of oxidative stress in sinularin-treated SKBR3 and normal breast (M10) cells was examined in terms of superoxide detection. 8-Oxo-2′-deoxyguanosine (8-oxodG) is the main product of oxidative DNA damage . We furtThis study investigated the modulating effects of proliferation, cell cycle progression, oxidative stress, and DNA damage in sinularin-treated breast cancer cells. The following discusses a comparison of drug sensitivity, the changes of cell cycle disturbance, possible signal transduction, and the role of oxidative stress in sinularin-treated breast cancer cells.50 values of sinularin with 9.28, 17.73, 23.5, and 33 μM at 24 h treatment. By contrast, sinularin is only moderately cytotoxic to normal breast (M10) cells, and shows at least 80% viability at the highest concentration (60 μM). The results suggest that sinularin has a selective killing effect against breast cancer cells, but low cytotoxicity to normal breast cells.Different cancer cell types display different sensitivities to sinularin. For example, human melanoma A2058) cells , gastric58 cells Several oxidative stress-inducing drugs cause G2/M arrest and delay cell proliferation ,25,26,27Moreover, we found that the levels of both intrinsic apoptotic protein c-Cas9 and extrinsic apoptotic protein c-Cas8 are increased gradually in sinularin-treated breast cancer cells. Similarly, sinularin also induces c-Cas9 and c-Cas 8 expressions in hepatocellular carcinoma cells  and gastAtaxia telangiectasia mutated (ATM)/checkpoint kinase 2 (Chk2) was activated to induce DNA damages in terms of γH2AX [Sinularin was reported to induce anticancer effects against melanoma, gastric, and liver cancer cells. For example, sinularin induced apoptosis in melanoma A2058) cells . In sinuof γH2AX . In sinu58 cells of γH2AX . In addiTo further address the role of oxidative stress in sinularin-treated breast cancer cells, we pretreated them with the free radical scavenger NAC. We found that the sinularin-induced PARP and caspase activations were inhibited by NAC pretreatment C. This sIn conclusion, the soft coral-derived natural compound sinularin induces antiproliferative and apoptotic effects on breast cancer cells and is associated with G2/M arrest and oxidative DNA damage in an oxidative stress-dependent manner. The selective killing effect of sinularin presents benefits for breast cancer therapy with few apparent side effects.2 at 37 °C.Two human breast cancer cell lines (SKBR3 and MDA-MB-231) and a normal human breast cell line (M10) were respectively ordered from the American Type Culture Collection  and Bioresource Collection and Research Center . SKBR3 and MDA-MB-231 cells were maintained in DMEM/F12 (3:2) medium and supplemented with 10% fetal bovine serum (FBS), antibiotics, and 0.03% glutamine . M10 cells were maintained in alpha medium with 10% FBS and regular antibiotics . Cells were kept in a humidified atmosphere containing 5% COS. flexibilis [S. manaarensis [Sinularin was isolated and purified from the soft corals exibilis  and S. maarensis  and dissaarensis ,37,38.Cell viability was detected based on mitochondrial activity. We applied the 3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) kit  as previously described [DNA was stained by 7AAD  for cell cycle analysis as previously described . After dAnnexin V /7AAD was used to detect apoptosis. After drug treatment, cells were treated with 10 μg/mL of annexin V-fluorescein isothiocyanate and 1 μg/mL of 7AAD for 30 min followed by flow cytometry using an Accuri™ C6.Caspase activity for apoptosis   (18C8) rabbit monoclonal antibody (mAb); cleaved form of caspase-9 (Asp330) (D2D4) rabbit mAb; and cleaved form of caspase-3 (Asp175) (5A1E) rabbit mAb . The mAb-β-actin (clone AC-15)  (diluted 1:5000) was used as an internal control. After secondary antibodies were incubated and washed, the signal was generated using ECL substrate .Detailed Western blotting procedures were previously described . BrieflyROS detecting dye, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) , was used to measure the intracellular ROS levels . After dTM DiOC2(3) assay kit  was used to measure the mitochondrial membrane potential (MitoMP), as previously described [2(3) in 2 mL medium/well of 6-well culture plate for 20–30 min. After washing and resuspension, cells were studied by flow cytometry using an Accuri™ C6.A MitoProbeescribed . After dMitoSOX™ Red  reacts with mitochondrial superoxide and becomes a fluorescent molecule  that allA fluorometric OxyDNA assay kit  was used to measure 8-oxodG levels for flow cytometry analysis ,46. Afte® 12 software  with one-way analysis of variance (ANOVA) and the Tukey HSD post hoc test. Treatments without the same small letters differed significantly.Data are presented as mean ± SD. Group differences were determined by JMP


SMN1) gene. Caenorhabditis elegans mutants lacking SMN recapitulate several aspects of the disease including impaired movement and shorted life span. We examined whether genes previously implicated in life span extension conferred benefits to C. elegans lacking SMN. We find that reducing daf-2/insulin receptor signaling activity promotes survival and improves locomotor behavior in this C. elegans model of SMA. The locomotor dysfunction in C. elegans lacking SMN correlated with structural and functional abnormalities in GABAergic neuromuscular junctions (NMJs). Moreover, we demonstrated that reduction in daf-2 signaling reversed these abnormalities. Remarkably, enhancing GABAergic neurotransmission alone was able to correct the locomotor dysfunction. Our work indicated that an imbalance of excitatory/inhibitory activity within motor circuits and underlies motor system dysfunction in this SMA model. Interventions aimed at restoring the balance of excitatory/inhibitory activity in motor circuits could be of benefit to individuals with SMA.Spinal muscular atrophy (SMA) is a neuromuscular disease characterized by degeneration of spinal motor neurons resulting in variable degrees of muscular wasting and weakness. It is caused by a loss-of-function mutation in the survival motor neuron ( Enhancing GABAergic neurotransmission alone can correct the locomotor dysfunction. This work leads to new understanding of disease pathogenesis and opens up new opportunities for therapy.Spinal muscular atrophy (SMA) is a pediatric motor neuron disease resulting from the loss of the survival motor neuron (SMN) protein. While great effort has been expended on interventions aimed at increasing levels of compensatory SMN1, identification of genes that modify the SMA phenotype has lagged. Here, we undertook a targeted genetic screen to identify SMA disease suppressors. We demonstrated that reduced insulin/insulin-like signaling is beneficial for not only longevity but also locomotor activity in SMA worm models. Our results from anatomic, functional, and genetic studies show that the impairment of GABAergic neurotransmission contributes to locomotor dysfunction in SMN) protein  is an autosomal recessive disease in infants and children, which is characterized by progressive degeneration of the motor neurons resulting from a reduction in abundance of functional survival motor neuron  that differ in a C/T nucleotide inside exon 7, which alters its splicing patterns. This critical difference results in an inclusion of exon 7 in the majority of SMN1-derived mRNA but an exclusion of exon 7 in the most of SMN2-derived mRNA. SMN protein lacking sequences encoded by exon 7 is un stable  pathway significantly extends lifespan and improves locomotor phenotypes in this SMA model. These effects are intriguing because reducing other genes previously implicated in lifespan extension did not extend lifespan in smn-1 null animals. Furthermore, we further demonstrate that by loss of daf-2, locomotion is improved due to enhancement of GABAergic transmission. Our data suggest that impaired GABAergic transmission is disrupted normal homeostatic locomotion in SMA disease model. Interventions aims at restoring the balance of excitatory/inhibitor activity in motor circuits could be benefits to individuals with SMA.In an effort to identify new disease modifiers we asked whether genes previously implicated in life span extension of  elegans  would inEscherichia coli OP50 (daf-2(e1370)III, LM99 smn-1(ok355) I/ht2 [bli-4(e937) qIs48] , RM2710 snf-11(ok156) V, TJ356 zIs356 IV [daf-16::daf-16a/b::GFP + rol-6], FY297  oxls22 [UNC-49B::GFP] II, CZ333 1::GFP] IVjuIs1 [Punc-25-SNB. Bruce Bamber and Anne Hart kindly shared FY297 oxls22[UNC-49B-GFP] and smn-1(rt248), respectively. N2 Bristol was used as the wild-type reference strain. Double or triple worms were generated by standard genetic crosses and verified by PCR or fluorescence expression (Worms were maintained at 20°C on Nematode growth media (NGM) plates seeded with oli OP50 . Stains pression .p values were calculated using the Log-rank (Mantel–Cox) method.Strains were grown at 20°C for at least two generations without starvation before lifespan analysis. Eggs were collected by bleaching reproductive adults of the desired genotype and letting them hatch and grow at either 20°C or 25°C. Survivors were monitored and counted every day. In all cases, Each RNAi (RNA Interference) colony was grown overnight in Luria broth (LB) containing ampicillin (50 µg/ml), and 200 µl was seeded onto NGM plates containing isopropylthiogalactoside  to induce dsRNA expression at room temperature. All RNAi clones used in this study were generated as described previously in the Ahringer library  and wereAnimals at L2 or Late (L2 + 3 d) stage were mounted on dried 2% agarose pads with M9 buffer. A stack of fluorescent images was captured from same posterior region of the dorsal nerve cord using Olympus laser scanning confocal microscopy with 100× objective lens. Stacked images were projected into a single plane using Fluoview software. The acquired images were processed and analyzed by an in house-developed PunctaAnalyzer program and R script .For analysis of sensitivity to the drugs pyridostigmine bromide and levamisole, three replicates of at least 20 worms per genotype were placed on NGM plates supplemented with either 100 mM pyridostigmine bromide (Sigma) or 100 µM levamisole (Sigma). Number of worms paralyzed on each plate was counted every 30 min to a total 360-min duration. Paralyzed worms were identified as those failing to response after 5 s of stimulation by plate-tapping and tail-prodding. The percentage paralyzed was averaged at each time point and plotted graphically using Log-rank (Mantel–Cox test) in PRISM.tba-1) as an endogenous control. Primer sequences for amplification of target mRNA were as following: smn-1, 5′-ATACCTCGATGCCATTTCCA-3′ and 5′-ATCCGCTCATGTACCAGCTC-3′; tba-1, 5′-AAGATGCCGCCAACAACTAC-3′ and 5′-CCTCCTCCGAATGAATGAAA-3′.We hand-picked 500 worms of the various genotypes and at the examined ages for extraction of total RNA using TRIzol (Invitrogen) and RNeasy Mini kit (QIAGEN). A total 1 µg of DNase-treated RNA were reverse transcribed using iScript Reverse Transcription Supermix (Bio-Rad) according to the manufacture’s protocol. The cDNAs were subjected to real-time qPCR in a total volume of 25 µl, containing 1× Power SYBR Green PCR Master mix (Applied Biosystems) and 200 nM primer. The qPCRs were amplified and analyzed in triplicate using StepOne Real-Time PCR system (Invitrogen). Relative mRNA level of the genes of interest were normalized against α tubulin  beating cycles. Excluding discarded movies, we obtain an average of 20 movies of unique individuals per group tested, with a minimum of 11.C. elegans genotypes . The modified version of software in this study is freely available via the University of Pennsylvania Complex Fluids Lab website (http://arratia.seas.upenn.edu/software.html). The modifications allow the code to be used for the study of immature rather than adult C. elegans. We have implemented changes that allow us to accurately measure the biomechanical properties of younger and therefore smaller and thinner worms by replacing parameters valid for adult, approximately millimeter-length C. elegans with parameters calculated directly from measured lengths and diameters.Movies of swimming nematodes are analyzed using in-house software with slight modification from the published software which was previously used and validated for a study on biomechanical profiling of enotypes . The oriC. elegans by extracting the body-shape of the swimmer and then computing the nematode's biomechanical properties, including frequency, amplitude, speed, force, and power  output, and more intuitively, as an indirect measure of strength.C. elegans by multiplying the component of the propulsive (or drag) forces for each segment by its local velocity components:Furthermore, we can compute the power, or rate of energy expenditure, of C. elegans for locomotion.A period-averaged calculation of power is therefore an estimate for how much mechanical energy is consumed by post hoc Tukey HSD was applied for multiple comparisons. For survival analysis, a Log-rank (Mantel–Cox) test was used. The threshold for significance was set to p < 0.05. For normalized body curvature, multiple t tests followed by Holm–Sidak method correction were used.Statistical analysis was performed using GraphPad Prism. For data containing more than three groups of non-normally distributed data, a non-parametric Kruskal–Wallis test with Dunnett’s multiple comparisons test was used. For normally distributed data , one-way ANOVA with the C. elegans smn-1 null mutant ok355 (gene name C41G7.1). The null allele is maintained in a heterozygous state using the hT2g balancer chromosome and heterozygous animals (smn-1(ok355)/hT2g) are phenotypically normal. Maternally contributed SMN message to the developing embryo allows for the smn-1(ok355) mutant animals to develop and mature until their premature demise before adulthood. Animals homozygous for the null allele smn-1(ok355) have two prominent phenotypes: (1) impaired locomotion , and (2) death at 7–8 d of life at 20°C  of a variety of single genes that influence caloric intake (smn-1(ok355) mutant animals by RNAi-mediated knock-down. Nine RNAi candidates were selected (Extended Data daf-2 significantly extended the life-span of smn-1(ok355) mutant animals  and this might be due to compensation by the parallel insulin receptor substrate (IST-1) pathway  due to down-regulated daf-2, we placed the smn-1 null allele in the daf-2 (e1370) background. The e1370 allele is a temperature-sensitive, partial loss-of-function (LOF) allele of daf-2. At 20°C, smn-1(ok355);daf-2(e1370) double mutants exhibited a median survival time of 10 d, living significantly longer than smn-1(ok355), which have a median survival time of 7 d (p < 0.0001). Recently, a second described SMA worm model (smn-1(rt248); smn-1(rt248);daf-2(e1370) mutant animals and observed that they exhibited a significant prolongation survival . Taken together, our results suggest that loss of daf-2 or daf-2 inactivation can provide a survival benefit in worm models of SMA.To verify the survival benefit of 10.1523/ENEURO.0289-18.2018.f1-1Extended Data Figure 1-1smn-1(ok355) mutant animals. A, Lifespan analysis of wild-type N2 worms fed either EV  or corresponding RNAi clones, including age-1 RNAi , daf-2 RNAi , daf-15 RNAi , C27B7.7 RNAi , cdc25 RNAi , ril-1 RNAi , sca-1 RNAi , yrs-1 RNAi , or rha-1 RNAi ; p < 0.005 was considered significant after Bonferroni correction. B, Lifespan analysis of smn-1(ok355) mutant animals fed either EV  or longevity-promoting RNAi clones validated in Figure 1A. Only daf-2 RNAi feeding significantly prolongs lifespan in smn-1(ok355) mutant animals ; p < 0.006 was considered significant after Bonferroni correction. C, Table for statistical analysis of lifespan data in RNAi screen and reference of selected RNAi candidates. After conducting a literature search, nine candidate RNAi clones, which can extend lifespan in C. elegans, were selected for RNAi screen experiment. Download Figure 1-1, TIF file.An approach of RNAi screen identified Daf-2 as a disease modifier to promote lifespan in smn-1(ok355) mutant animals may die prematurely when maternally derived SMN mRNA and protein levels fall below a specific threshold although this threshold varies as a function of developmental stage and in a cell type-specific manner. The striking beneficial effect of loss of daf-2 on smn-1(ok355) mutant animals’ survival could arise from two possibilities. First, loss of daf-2 might slow the degradation of smn-1 mRNA or protein and thereby maintaining them above the critical threshold. Second, loss of daf-2 might render animals resistant to reduced SMN protein levels.A critical function of SMN protein is the assembly of small nuclear RNPs (snRNPs), which are required for pre-mRNA splicing machinery . This susmn-1 RNA level in wild-type N2, smn-1(ok355), and smn-1(ok355);daf-2(e1370) mutant animals at various developmental time points  and smn-1(ok355);daf-2(e1370) strains, across multiple developmental time points. This result suggests that loss of daf-2 activity prolongs lifespan in smn-1(ok355);daf-2(e1370) strain despite the lack of smn-1 mRNA. The absence of good tools for monitoring SMN protein levels leaves a caveat that loss of daf-2 might slow SMN protein degradation. In mammalian cells, the half-life of full length SMN protein is ∼4.5 h (daf-2 renders smn-1(ok355) mutant animals insensitive to the absence of SMN protein.To address these possibilities, we conducted real-time quantitative PCR to monitor e points C. In wils ∼4.5 h . It seemC. elegans are capable to provide survival benefit in smn-1 null animals. Only reduction of daf-2 signaling pathway has such unique ability to extend the life of animals lacking SMN-1. The beneficial effects of reducing daf-2 appear to be independent of changes in SMN mRNA and probably protein.Taken together, our results reveal that not every longevity promoting pathways identified in daf-2 signaling can double the lifespan ;daf-2(e1370) is also daf-16/FOXO dependent. Both smn-1 and daf-16 reside on Chromosome I (about one centimorgan apart), and it is difficult to generate animals that contain mutations of both genes in this case. To examine whether DAF-16 is required and sufficient for lifespan extension in smn-1 null worms, we used both RNAi and overexpression approaches. smn-1(ok355) mutant animals fed with daf-16 RNAi showed 12.5% median lifespan decrease in comparison with EV-fed animals . Feeding daf-16 RNAi to smn-1(ok355);daf-2(e1370) and smn-1(rt248);daf-2(e1370) mutant animals suppressed the longevity-promoting effect by daf-2(e1370)  on smn-1(ok355) mutant animals’ longevity  strain in which a 6 kB of daf-16 promoter drives the expression of DAF-16-GFP fusion proteins (smn-1(ok355);daf-16::gfp(TJ356) mutant animals comparison with smn-1(ok355) mutant animals . Our results suggest that daf-16 is required for daf-2-dependent lifespan extension of smn-1(ok355) mutant animals.In wild-type worm, it has been shown that a reduction of lifespan  and suchignaling . Thus, w2(e1370) B,C. The ene sets . Thus, w daf-16f , was lesongevity C. This iproteins . There idaf-2 LOF we explored our main interest – the impaired locomotion of the smn-1(ok355) mutant animals. (smn-1(ok355) mutant animals display impaired locomotor activity, there is neither neuronal death nor gross anatomic abnormalities in motor circuit wiring  mutant animals have been shown to exhibit a progressive decline in a single measure - thrashing rate after the second larva (L2) stage  mutant animals. Choosing the best comparison groups for the late stage is not straightforward; late smn-1(ok355) mutant animals cannot be compared with 5 days-old N2 animals because they have arrested at an earlier larval stage. We selected control groups based on the most biomechanically relevant developmental parameter, the body length. The typical body lengths for the two experimental groups are 0.39 ± 0.01 mm (L2) and 0.63 ± 0.08 mm  and dynamic  properties of tion M9; . Previou2) stage . With thmm late; .smn-1(ok355) mutant animals move slowly and beat less frequently than N2 and daf-2(e1370) mutant animals  mutant animals demonstrate a qualitative improvement in swimming gait, leading to a pattern that more closely resemble N2 animals. Next, we present kymographs of the worm’s body curvature over one second of forward swimming (smn-1(ok355) mutant animals have a region along their body near the tail that exhibits no curvature. This region stretches from approximately the middle of the worm to just before the tail ;daf-2(e1370) genotype).Simple observation of the swimming gait of individuals from each genotype over one second demonstrates that  animals A. In the10.1523/ENEURO.0289-18.2018.f3-1Extended Data Figure 3-1smn-1(ok355), daf-2(e1370), and smn-1(ok355);daf-2(e1370) mutant animals. Statistical analysis of normalized body curvature in N2, Statistical analysis for normalized body curvature data shown in Figure 3C. Multiple t tests followed by Holm–Sidak method correction for significant; * reach to statistical significance. Download Figure 3-1, TIF file.daf-2(e1370) mutant animals, we observed the mean normalized curvature decreases from head to tail. The daf-2 group shows a significant lower curvature at its tail region compared to the N2 group. The curvature pattern in the smn-1(ok355) mutant animals are very distinct from N2 and daf-2. It decreases rapidly after the worm’s mid-point and then sharply increase right near the tail (p < 0.00001 by multiple t tests and Holm–Sidak method correction). This pattern corresponds to the region of low curvature observed in smn-1(ok355);daf-2(e1370) mutant animals displayed a normalized mean curvature between those of N2 and daf-2(e1370) mutant animals. It is clear that the defects in body curvature in the midsection and tail incurred by loss of smn-1 are rescued in the daf-2 mutant background.To interrogate this failure of curvature issue more closely, we recall that the body curvature of the nematode is dependent on its local orientation and its parameterized body shape. By quantifying the body curvature of all the nematodes for each genotype, we systematically probe this aspect of worm locomotion with high sensitivity. The curvature of a worm appears to have nodes when we take an average in time over each population . For theGenetics (daf-2(e1370), smn-1(ok355), and smn-1(ok355);daf-2(e1370) mutant animals . With developmental progression , wild-type N2 and daf-2(e1370) animals significantly increase their swimming speed by 63% and 65%, respectively. In contrast, at the late stage smn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals trend toward a decrease in swimming speed, there was no statistically significant difference between swimming speed at the early and late time points. This suggests that the loss of smn-1 impairs the normal, age-dependent increase in locomotion rate.Next, we undertook a quantitative analysis of worm swimming using in-house biomechanical profiling algorithms first published in Genetics , and exaGenetics . The bod.16 mm/s B, and thp > 0.05, Kruskal–Wallis test), both N2 and daf-2(e1370) animals did not show significant increase in frequency (smn-1(ok355) mutant animals at the L2 stage is significantly higher than N2-L2 animals  and decreases by 32% at the late stage . We also found that the beating frequency of smn-1(ok355);daf-2(e1370) mutant animals at the L2 stage is similar to the age-matched smn-1(ok355) (2.6 vs 2.5 Hz). Interestingly, unlike the smn-1(ok355) group, smn-1(ok355);daf-2(e1370) mutant animals are able to maintain beating frequency at the late stage in a fashion similar to wild-type N2. A pathologic decrement in neuromuscular activity over time in the smn-1(ok355) mutant animals could account for the observed decrease in beat frequency. Such pathologic decrement does not occur in the smn-1(ok355);daf-2(e1370) mutant animals.Next, we investigated beating frequency in these animals. Although N2 animals increases modestly over time  mutant animals, this does not occur in the smn-1(ok355);daf-2(e1370) mutant animals . While swimming frequency and speed are important factors in describing locomotion, there are of other determinants that contribute to propulsive force and mechanical power. We used the hydrodynamic model resistive force theory  animals exhibited an increase in propulsive force of 39% and 83%, respectively, when they progress from L2 to late stage  mutant animals, the propulsive force remains the same during this developmental period, suggesting that motor output is impaired. In contrast, smn-1(ok355);daf-2(e1370) mutant animals show an increase in propulsive force by 24%  over this period. Thus, the normal increase in motor output as worms mature fails to occur in smn-1 mutant animals. Reduced daf-2 signaling can rescue this defect.We expect that as a worm develops, the propulsive force of the organism will increase. Indeed, we found that both N2 and daf-2(e1370) animals significantly increase their mechanical power  as they develop (smn-1(ok355) mutant animals produced the same mechanical power at the L2 and the late stage; in addition, the mechanical power of the smn-1(ok355) group is significantly lower than N2, late stage . Reduction of daf-2 signaling (smn-1(ok355);daf-2(e1370)) significantly improves mechanical power when compared to smn-1(ok355) mutant animals. Mechanical power for locomotion of smn-1(ok355);daf-2(e1370) mutant animals is statistically indistinguishable from that of the late stage wild-type group  mutant animals. While these modifications do not directly increase the swimming speed of the organism, they improved the beating patterns, muscle output, and the energy expended during locomotion.In summary, the kinematic and dynamic biomechanical properties  show that a reduction of daf-2(e1370) background, (3) presynaptic or postsynaptic reporter in the smn-1 mutant background, and (4) presynaptic or postsynaptic reporter in the smn-1(ok355);daf-2(e1370) background.To assess the contribution of abnormalities in the NMJ structure to the locomotor defects defined above, we examined the morphology of inhibitory synapses by using a presynaptic (Punc-25/GAD-synaptobrevin::GFP fusion protein) and postsynaptic [Punc-49/GABA receptor subunit (UNC-49B)::GFP fusion protein] marker . To quansmn-1(ok355);daf-2(e1370) mutant animals was 33% stronger than smn-1 mutant animals  mutant animals had 30% fewer puncta  and smn-1(ok355);daf-2(e1370) mutant animals were significantly (∼100%) wider than controls (N2 or daf-2(e1370) mutant animals, p < 0.01 for N2 vs smn-1(ok355) and p < 0.001 for daf-2(e1370) vs smn-1(ok355);daf-2(e1370), one-way ANOVA followed by post hoc Tukey HSD for multiple comparisons; smn-1(ok355) than in N2, p < 0.001; 39% less dense in smn-1(ok355);daf-2(e1370) than in daf-2(e1370), p < 0.005, one-way ANOVA with Tukey test; smn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals are ∼300% greater than in N2 or daf-2(e1370) (p < 0.01 for N2 vs smn-1(ok355) and p < 0.001 for daf-2(e1370) vs smn-1(ok355);daf-2(e1370), one-way ANOVA with Tukey test; daf-2(e1370) animals were compared or when smn-1(ok355) versus smn-1(ok355);daf-2(e1370) mutant animals were compared. Thus, over development, presynaptic GABAergic terminals of smn-1(ok355) mutant animals appear to be larger and have undergone structural reorganization. A reduction in daf-2 signaling appears to have no impact on this phenotype.At the later developmental point, a qualitative comparison of presynaptic puncta revealed substantial differences in puncta distribution between the four groups (daf-2(e1370) animals have ∼2-fold wider and 2.7-fold brighter puncta on body wall muscles compared to N2 animals , and this is associated with a reduction in puncta number  mutant animals (smn-1(ok355);daf-2(e1370) and smn-1(ok355) mutant animals (mn-1(ok355);daf-2(e1370) mutant animals have ∼300% brighter puncta than in the smn-1(ok355) mutant animals  mutant animal leads to a large increase in GABA receptor content at/or near synapses  mutant animals appear to have a normal GABAergic NMJs. Over time there are two major changes to these synapses: (1) presynaptic elements decrease in number but are restructured, and (2) the number of postsynaptic elements increase without an effect on width or GABAergic receptor content. The main effect of daf-2 reduction is to decrease postsynaptic element number to the N2 level and to increase the synaptic content of GABA receptors. It is difficult to predict the functional consequence of these diametrically opposing effects of the daf-2 mutant animals on postsynaptic GABA receptors.Together, these observations reveal that young To investigate the functional consequence of morphologic abnormality of GABAergic NMJs observed above, we used pharmacological tools: pyridostigmine bromide , an acetsmn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals exhibited a levamisole-resistance phenotype (smn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals are ∼66% and 100%, respectively, longer than the time in the wild-type N2 animals . There is no significant difference between smn-1(ok355) and smn-1(ok355);daf-2(e1370). Thus, smn-1(ok355) mutant animals are resistant to direct cholinergic agonists. When we assayed animal’s sensitivity to pyridostigmine bromide, however, we found no significant differences between wild-type N2, smn-1(ok355), and smn-1(ok355);daf-2(e1370) at L2 stage (smn-1(ok355) genotype have a shift in the balance between excitatory and inhibitory neurotransmission toward decreased excitability. Either a reduction in cholinergic neurotransmission or an enhancement in GABAergic neurotransmission (or a combination of both) would be compatible with these observationsAt the L2 stage, both henotype A. The meL2 stage B. These smn-1(ok355) mutant animals (smn-1(ok355) is 50% shorter than the time in the N2 . Loss of daf-2 signaling rescues this phenotype; the levamisole sensitivity of smn-1(ok355);daf-2(e1370) mutant animals is the same as N2 animals. For pyridostigmine test in late stage of animals, both smn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals show hypersensitivity phenotype (smn-1(ok355) and smn-1(ok355);daf-2(e1370) mutant animals are 3.5-fold and 2-fold shorter time course to reach the median paralyzed time when compared to wild-type N2 . The smn-1(ok355);daf-2(e1370) mutant animal are slightly less hypersensitive to pyridostigmine than the smn-1(ok355) mutant animals (smn-1(ok355) mutant animals have now shifted the balance between excitatory and inhibitory neurotransmission toward increased excitability . Either an enhancement in cholinergic neurotransmission or a reduction in GABAergic neurotransmission (or a combination of both) would be compatible with these observations. That the HIC phenotype is suppressed in the daf-2(e1370) background suggests that equipoise in the excitation/inhibition balance has been normalized.Interestingly, at the late stage, we observed a sensitivity switch to levamisole from resistance to hypersensitivity in  animals C. The mehenotype D. smn-1 mutant animals is predominantly due to reduced number of presynaptic elements, and (2) benefit of reduced daf-2 in the smn-1(ok355) mutant animals is due to increased density of GABA receptors at synapses.To the extent that changes in GABAergic transmission are contributing to these observations, we interpret our morphologic observations to indicate that: (1) reduced GABAergic transmission in smn-1(ok355) mutant animals by loss of daf-2 is mainly due to increased GABAergic transmission, a functional enhancement of the GABAergic system in the smn-1(ok355) mutant animals should phenocopy loss of daf-2 function. To test this idea, we identified two strains of worms that are likely to exhibit enhanced GABAergic neurotransmission without affecting the baseline swimming behaviors: RM2710 (snf-11(ok156)) and FY297 (oxls22(UNC-49::GFP)). The snf-11(ok156) mutant animals lack the sodium-dependent GABA transporter (which clears synaptic GABA). These animals are aldicarb- resistant supporting the view that they have enhancement of GABAergic transmission (oxls22(UNC-49::GFP) animals probably have enhanced GABAergic transmission, although to our knowledge, this has never been formally tested.If the locomotor benefit conferred on smission . The ovesmn-1(ok355), oxls22(UNC-49::GFP), and smn-1(ok355);oxls22(UNC-49::GFP). The mean normalized curvature (from head to tail) in N2 and oxls22(UNC-49::GFP) are similar, except the head region (0.07–0.13% of body length from the head) of oxls22(UNC-49::GFP) group, which shows a significantly lower curvature than N2 (t tests). Consistent with the data observed previously (smn-1(ok355) mutant animals display a significantly small curvature at the tail region (0.68–0.87% of body length from the head) compared with N2 (p < 0.00001 by multiple t tests and Holm–Sidak method correction). Interestingly, the normalized mean curvature of smn-1(ok355);oxls22(UNC-49::GFP) animals shows no significant difference to those of N2 and UNC-49::GFP animals ;unc-49::GFP mutant animals.Statistical analysis of normalized body curvature in  Statistical analysis for normalized body curvature data of smn-1(ok355);unc-49::GFP mutant animals shown in Figure 8A. Multiple t tests followed by Holm–Sidak method correction for significant; * reach to statistical significance. Download Figure 8-1, TIF file.10.1523/ENEURO.0289-18.2018.f8-2Extended Data Figure 8-2smn-1(ok355);snf-11(ok156) mutant animals.Statistical analysis of normalized body curvature in  Statistical analysis for normalized body curvature data of smn-1(ok355);snf-11(ok156) mutant animals shown in Figure 8B. Multiple t tests followed by Holm–Sidak method correction for significant; * reach to statistical significance. Download Figure 8-2, TIF file.smn-1(ok355), snf-11(ok156), and smn-1(ok355);snf-11(ok156). Both N2 and snf-11(ok156) animals show very similar curvature pattern (smn-1(ok355) mutant animals display a distinct curvature shape. The normalized mean curvature in smn-1(ok355) animals decreases rapidly after the worm’s mid-point and then sharply increases right near the tail. The smn-1(ok355);snf-11(ok156) mutant animals display a curvature pattern similar to smn-1(ok355) mutant animals but distinct from the N2 and snf-11(ok156) mutant animals, especially from the mid-point to tail region (p < 0.00001 by multiple t tests and Holm–Sidak method correction). This result indicates that loss of the GABA uptake transporter ) does not improve the curvature phenotype of the smn-1(ok355) mutant animals  mutant animals display a progressive increase in swimming speed between L2 and late stage, the smn-1(ok355) mutant animals display a significant decrease in swimming speed. This decrement in swimming speed was prevented in the smn-1(ok355);oxls22(UNC-49::GFP) and smn-1(ok355);snf-11(ok156) mutant animals (oxls22(UNC-49::GFP) or snf-11(ok156), while beating frequency declines over this period in smn-1(ok355), smn-1(ok355);oxls22(UNC-49::GFP), and smn-1(ok355);snf-11(ok156) mutant animals (smn-1(ok355) animals have an age-dependent decline in swimming speed and beat frequency. Enhancing GABAergic neurotransmission in these animals selectively abrogates the swimming speed defect.Furthermore, we undertook a quantitative analysis of their kinematic and biomechanical properties . Between animals B. There  animals C. In sumsnf-11(ok156) mutant animals show an increase over the time  and snf-11(ok156) animals display a progressively significant increase between L2 and late stage, while the smn-1(ok355) mutant animals do not display this age-dependent change. In contrast, the smn-1(ok355);oxls22(UNC-49::GFP) and smn-1(ok355);snf-11(ok156) mutant animals do display a significant age-dependent increase in mechanical power (smn-1(ok355) mutant animals, it improves their mechanical power but not the propulsive force.Next, we studied the propulsive force and mechanical power of these animals. With regard to propulsive force, only N2 and al power E. In sumdaf-2, insulin/IGF signaling (IIS) prolongs life and ameliorates aspects of motor dysfunction in C. elegans models of SMA. The effects of reduced daf-2 signal depend, at least in part, on the daf-16, a Forkhead box transcription factor. The daf-2/daf-16 signaling pathway is conserved in metazoans, and is implicated in the control of human longevity and health-span  mutations into the transgenic worms expressing G85R  is, to a large degree, morphologically intact  this is associated with enhanced GABAergic neurotransmission, and (3) simply enhancing GABAergic transmission also improves locomotion. We propose that diminished GABAergic neurotransmission in the worm nervous system impairs the function of circuitry required for neuromuscular system operation in the smn-1(ok355) mutant animals. Consideration of the fly and worm data together supports the view that neurochemically defined populations of premotor interneurons are dysfunctional in the reduced SMN condition, and the sum of this interneuron dysfunction manifests as impaired motor behavior. It is noteworthy that the mouse (and likely human) spinal cord contains GABAergic and cholinergic subtypes of interneurons  reduction in rneurons . We suggC. elegans GABAergic neurotransmission is a fascinating topic. Classic work identifies 26 GABAergic neurons in the worm based on the criteria: (1) they contain GABA, (2) express the GABA biosynthetic enzyme glutamic acid decarboxylase , and (3) express the GABA vesicular transporter  for loading GABA into synaptic vesicles (snf-11), have been dubbed “GABA clearance neurons” and a second class of neurons, containing GABA, GABA uptake transporter (snf-11) and GABA vesicular transporter (unc-47), have been dubbed “GABA recycling neurons.” Based on their anatomy and position, both subtypes are poised to release GABA in addition to classical GABAergic neurons. Since 30–40% of all synapses in the worm have GABA receptors, GABA may have both widespread and precise influence on circuit behavior (vesicles . Recent behavior .smn-1(ok355) mutant animals led to improved locomotion function, strong evidence that improvement in mechanical power of the SMA mutant animals is a result of increased GABAergic tone. There are 7 known GABA receptor subtypes in the worm  mutant animals may have a quantitatively and qualitatively unique GABA receptor phenotype. As mentioned above, the snf-11 locus encodes the sole GABA uptake transporter in the worm and in the snf-11(ok156) mutant animals there is likely to be enhanced GABAergic neurotransmission owing to decreased GABA clearance. Considering the existence of GABA clearance and GABA recycling neurons, the snf-11(ok156) mutant animals may have a more widespread and perhaps less specific effect on interneuronal GABAergic communication. Although both mutant animals enhance the mechanical power of the smn-1(ok355) mutant animals, their different modes of action provide an explanation for why there is a restoration of body curvature when swimming in the smn-1(ok355);UNC-49::GFP, but not smn-1(ok355);snf-11(ok156), mutant animals.We find that two different ways of enhancing GABAergic transmission in the the worm  and the  variant . Part ofsmn-1(ok355) mutant animals. Studies of other neuronal populations come to complementary conclusions. For example, smn-1(ok355). This raises the possibility that pharmacological manipulation of these systems could be of benefit to individuals with SMAOur anatomic , 6, funcsmn-1(ok355) mutant animals to be resistant to aldicarb and levamisole (smn-1(ok355) mutant animals switch from levamisole-resistant phenotype at L2 stage to both levamisole- and pyridostigmine-hypersensitive phenotype at the late stage. The origin of the disparity between these observations probably lies in differences in developmental stages, definition, and timing of paralyses, the Hart lab performed the functional tests at the L4 stage, while we performed the functional tests at the L2 and late  stage.We note that the Hart lab reported vamisole , while wDo observations in mouse models of SMA provide support for the view that anatomic/functional abnormalities in segmental spinal cord circuitry contribute to motor deficits? Study of the intrinsic properties of motor neurons and the monosynaptic reflex arc between proprioceptive afferents and motor neurons has revealed that SMAΔ7 motor neurons are hyperexcitable . Despite