Collectively, our findings point toward a mechanism involving mitochondrial dynamics to respond to trigger(s) of muscle degeneration via apoptosis in Caenorhabditis elegans.Mitochondria are double-membrane subcellular organelles with highly conserved metabolic functions including ATP production. Mitochondria shapes change continually through the combined actions of fission and fusion events rendering mitochondrial network very dynamic. Mitochondria are largely implicated in pathologies and mitochondrial dynamics is often disrupted upon muscle degeneration in various models. Currently, the exact roles of mitochondria in the molecular mechanisms that lead to muscle degeneration remain poorly understood. Here we report a role for DRP-1 in regulating apoptosis induced by dystrophin-dependent muscle degeneration. We found that:  Mitochondria are characterized by an important polymorphism of shapes due to permanent fusion and fission events that vary within a considered tissue2. Dynamin family GTPases regulates mitochondrial dynamics. In mammals, Mitofusin-1 and Mitofusin-2 (MFN1 and MFN2)3 allow for mitochondria outer membrane fusion whereas inner membrane fusion implicates Optic atrophy 1 (OPA1)4. Fission machinery of the outer membrane is composed of Dynamin-related protein1 (DRP1) and Dynamin-2 (DYN2)6. In Caenorhabiditis elegans (C. elegans), DRP-1 is also required for mitochondrial fission7 whereas EAT-3  and FZO-1  control inner and outer mitochondrial membrane fusion, respectively8. Mitochondrial fusion is essential for normal mitochondrial activity notably by diluting damaged mitochondrial DNA, lipids or proteins10. In mammals, mitochondrial fission is key for mitophagy to eliminate damaged mitochondria12. Also, fragmented mitochondria often appear in the early steps of cell death associated with permeabilization of the mitochondrial outer membrane, which induces the release of inter membrane space-stored proapoptotic factors, such as cytochrome c, Apoptosis-Inducing Factor (AIF) or Endonuclease G (EndoG)16. In C. elegans, fragmentation of mitochondria during apoptosis has been reported but the exact roles of mitochondria in cell death remain largely unknown17. Whether mitochondrial fusion is directly implicated in apoptosis is still under debate. In mammals, hyperfusion allows for survival under numerous stresses including nutrient deprivation21. Furthermore, the fusion proteins MFN1 and MFN2 seem to directly regulate apoptosis suggesting that longer mitochondria are more supportive for cell survival than fragmented mitochondria23. Interestingly, molecular links exist between mitochondrial fusion and apoptosis26.Mitochondria are double-membrane organelles that are crucial for many cellular processes such as aerobic ATP generation, calcium homeostasis, lipid biosynthesis or cell death28 and in the maintenance of the morphology and the function of the Endoplasmic Reticulum (ER)30. In C. elegans, DRP-1 can contribute to apoptotic processes independently from mitochondrial fission31. Furthermore, both DRP-1 and OPA1 allow for mitochondrial cristae remodeling separately from mitochondrial dynamics34. The alternative functions of OPA1 also include lipolysis regulation35. The pro-fusion protein MFN2 is an essential component of the contact sites between mitochondria and ER and contributes to mitochondrial Ca2+ uptake and ER morphology maintenance36. Additionally, levels of MFN2 are regulated by ER stress and can affect the ER-Unfolded Protein Response (ER-UPR) and in turn, cell death38. Furthermore, axonal mitochondrial transport requires MFN2 but not mitochondrial fusion39.Importantly, the main actors of mitochondrial fission and fusion have alternative functions separate from mitochondrial dynamics. It is well established that DRP-1 participates in peroximal divisiondys-1(cx18);hlh-1(cc561ts) C. elegans mutant, which exhibits progressive dystrophin-dependent muscle degeneration reflected by muscle weakness and dramatic loss of muscle cells40. DYS-1 is the homolog of dystrophin in mammals and mutations in this gene cause Duchenne Muscular Dystrophy in Human41. HLH-1 is a homolog of Myogenic Differentiation 1 (MyoD), a muscle transcription factor42. Despite the phylogenetic distance between mammals and C. elegans, the nematode presents striated body wall muscle similar to vertebrate skeletal muscle in terms of function and structural components; however, the overall architecture is less complex44. Mitochondrial fragmentation increases in muscle cells during dystrophin-dependent muscle degeneration in various animal models including nematode45, zebrafish45, mouse, which exhibits abnormalities of mitochondrial morphology and density as well as an up-regulation of DRP-1 levels47, dog48 and in biopsies from Duchenne Muscular Dystrophy patients49. Fragmentation of mitochondria is also observed during muscle aging in nematode50, fly51 and mouse52.Strength and function of muscle tissues are highly dependent on the energy produced by mitochondria. Compromised muscle tissues, due to deterioration and loss of muscle cells, and leading to muscle weakness, are hallmarks of muscle degeneration. The primary causes of muscle degeneration are various. For instance, it can be due to numerous genetic mutations causing muscular dystrophies. Muscle degeneration can also be caused by aging processes, and is called sarcopenia. To study muscle degeneration, we used the dys-1(cx18);hlh-1(cc51ts) mutant worms. Conversely, increased mitochondrial fusion can enhance the muscle degeneration phenotype of dystrophic nematodes. Interestingly, we showed that DRP-1 functions mainly in the apoptosis pathway downstream of the caspase 3 to impact muscle degeneration and that others apoptotic executors are important for muscle health. Finally, apoptosis induced by DRP-1 seems also to be implicated in aging-dependent muscle loss. Collectively, our results point toward a novel mechanism by which, DRP-1-mediated apoptosis provokes muscle degeneration.We hypothesized that genetic manipulations of the main actors of mitochondrial dynamics can improve muscle health. We found that decreasing mitochondrial fission or increasing mitochondrial fusion can partially rescue mitochondrial dynamics, reduce muscle degeneration and improve the mobility of C. elegans, fission is mediated by DRP-17, inner membrane fusion by EAT-3 (OPA1 homolog)53 and outer membrane fusion by FZO-1 (MFN1 homolog)8  was calculated for each of the tested conditions to evaluate mitochondrial dynamics. A value of 1 for the index of mitochondria circularity means that mitochondria are perfectly circular and a value of 0 means that mitochondria are perfectly flat. In wild-type C. elegans muscle cells, tubular and circular mitochondria were in equal proportions ;hlh-1(cc561ts) mutant worms, which present progressive muscle degeneration, exhibited mitochondrial dynamics perturbations with more fragmented mitochondria in muscle cells than wild-type worms ;hlh-1(cc561ts) mutant vs 0,52 +/− 0,009 in wild-type worms) ;hlh-1(cc561ts) mutant worms presented shorter and smaller mitochondria than that of wild-type worms 8 Fig. . As C. ells Fig. 55. As prons Fig.  as refleons Fig. 54. dys-1rms Fig.  as reflems) Fig. . By comprms Fig. . Furtherrms Fig. .Figure 1drp-1(tm1108) null mutation31 in the dys-1(cx18);hlh-1(cc561ts) mutant worms. Mitochondria of drp-1(tm1108) mutant worms formed bubbles-like shape  mutant worms exhibited increased circularity and size but were shorter compared to that of wild-type worms ;hlh-1(cc561ts);drp-1(tm1108) mutant worms appeared less circular, longer and with the occasional presence of interconnecting mitochondrial strings when compared to those of dys-1(cx18);hlh-1(cc561ts) mutant worms ;hlh-1(cc561ts) mutant worms was significantly decreased in absence of DRP-1 ;hlh-1(cc561ts) mutant vs 0,65 +/− 0,014 in dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms) ;hlh-1(cc561ts) mutant worms accompanied by a decreased mitochondrial circularity index when compared to that of dys-1(cx18);hlh-1(cc561ts) mutant worms ;hlh-1(cc561ts);Ex[eat-3] mutant and 0,69 +/− 0,013 in dys-1(cx18);hlh-1(cc561ts);Ex[fzo-1] mutant vs 0,75 +/− 0,014 in dys-1(cx18);hlh-1(cc561ts) mutant worms)  Fig. . SimilarNAi Fig. 45. To enion Fig.  while fzogy Fig. . Accordirms Fig. . These sms) Fig. . Overalldys-1(cx18);hlh-1(cc561ts) mutant worms. To that extent, we performed TUNEL  assays along with actin staining on wild-type and dys-1(cx18);hlh-1(cc561ts) mutant worms. TUNEL stains DNA fragments resulting from apoptosis-induced DNA fragmentation58. In wild-type worms, the actin network in muscle cells was nicely and regularly organized in filaments along the diamond-shaped cell with the nuclei, centered along the anterior-posterior axis, showing no staining by TUNEL. By contrast, in dys-1(cx18);hlh-1(cc561ts) mutant worms, muscle cells often presented abnormal phenotypes with actin network perturbations (i.e actin filaments form wavelets and/or aggregates) and with fragments of nuclei displayed over the entire cell and stained by TUNEL ;hlh-1(cc561ts) mutant worms in absence of the pro-fission protein DRP-1 or when eat-3 and fzo-1 were overexpressed (2.07 +/− 0.19 TUNEL positive cells in dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant vs 2,4 +/− 0,20 in dys-1(cx18);hlh-1(cc561ts);Ex[eat-3] mutant vs 2,3 +/− 0,21 in dys-1(cx18);hlh-1(cc561ts);Ex[fzo-1] mutant vs 5,28 +/− 0,26 in dys-1(cx18);hlh-1(cc561ts) mutant worms)  Fig. . These do-1 Fig. . Hence, dys-1(cx18);hlh-1(cc561ts) mutant worms, we further quantified muscle degeneration and estimated muscle activity. In C. elegans, the number of body-wall muscle cells is fixed from one individual to the other (95 muscle cells). By contrast to mammals, C. elegans muscle cells are mononuclear and do not fuse. Moreover, as C. elegans lacks satellite cells and therefore, regenerative capabilities, each muscle cell can be individually followed in vivo throughout its whole life60, and muscle degeneration can be quantified by actin staining allowing to visualize and to count abnormal or absent muscle cells. Wild-type and dys-1(cx18) single mutant worms did not present abnormal muscle cells; nor did drp-1(tm1108) mutant worms or eat-3 and fzo-1 overexpressing worms  mutant worms exhibited a weak number of abnormal muscle cells40. By contrast, dys-1(cx18);hlh-1(cc561ts) mutant worms exhibited a substantial number of abnormal muscle cells ;hlh-1(cc561ts) mutant worms ;hlh-1(cc561ts) mutant vs 3,3 +/− 0,20 in dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms) ;hlh-1(cc561ts) mutant worms ;hlh-1(cc561ts);Ex[eat-3] and in dys-1(cx18);hlh-1(cc561ts);Ex[fzo-1] mutant respectively vs 7,1 +/− 0,15 in dys-1(cx18);hlh-1(cc561ts) mutant worms) . However, dys-1(cx18);hlh-1(cc561ts) mutant worms fed with either fzo-1 or eat-3 RNAi exhibited enhanced levels of muscle degeneration ;hlh-1(cc561ts) mutant worms fed with eat-3 RNAi: 11,7 +/− 0,59 or fzo-1 RNAi: 10,0 +/− 0,70 vs fed with the empty vector L4440: 6,6 +/− 0,44)  mutant worms was slightly decreased compared to that of wild-type worms whereas eat-3 and fzo-1 overexpression or eat-3 and fzo-1 RNAi did not affect worm thrashing abilities ;hlh-1(cc561ts) mutant worms were unable to bend quickly and this poor muscle fitness was partially rescued by lowered DRP-1 levels ;hlh-1(cc561ts) mutant worms ;hlh-1(cc561ts) mutant worms  Fig.  similarlNAi Fig. 45. Both ms) Fig. . These d44) Fig. . Collecties Fig. . As expeels Fig. . Moreoverms Fig. . These do-1 Fig. . By contrms Fig. . Togethedys-1(cx18);hlh-1(cc561ts) mutant or wild-type worms carrying the ccIs4251 transgene. In wild-type worms, tubular and circular mitochondria were in equal proportions and organized along the actin network, which is arranged in filaments along the cell ;hlh-1(cc561ts) mutant worms, different stages of actin network perturbations were observed, likely reflecting various degrees of muscle degeneration. The actin staining can reveal an apparent totally preserved actin network or the apparition of wavelets or a more or less noticeable aggregation of actin. Muscle cells with mutated dystrophin and unperturbed actin network ;hlh-1(cc561ts) mutant worms  a wild-type drp-1 transgene called drp-1(+) and (ii) a transgene of drp-1 with the CED-3 cleavage site mutated called drp-1(D118A) ;hlh-1(cc561ts) and dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms expressing, or not, drp-1(+) or drp-1(D118A) transgenes. As shown above, dys-1(cx18);hlh-1(cc561ts) mutant worms presented fragmented mitochondria ;hlh-1(cc561ts);drp-1(tm1108) mutant worms expressing DRP-1(+) or DRP-1(D118A) showed important mitochondrial fragmentation, loss of the mitochondria bubbles caused by the drp-1(tm1108) mutation7 and an increased index of mitochondrial circularity compared to that of dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms ;hlh-1(cc561ts);drp-1(tm1108) mutant worms and that both drp-1(+) and drp-1(D118A) transgene are able to induce mitochondrial fission. Our data confirmed that cleavage of DRP-1 by CED-3 is at least in part dispensable for its function in regulating mitochondrial fission.The links between mitochondrial dynamics and cell death have been extensively studied8A) Fig. . The preria Figs  and 3B wria Figs  and 3C,Frms Fig. . These rdrp-1(+) or drp-1(D118A) transgenes on muscle degeneration and fitness of dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms. As shown above, dys-1(cx18);hlh-1(cc561ts) mutant worms exhibited an important number of abnormal muscle cells and weak thrashing abilities  transgene into dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms provoked an increased number of abnormal muscle cells and a drastic thrashing decrease ;hlh-1(cc561ts);drp-1(tm1108);Ex[drp-1(+)] mutant vs 8,59 +/− 0,35 abnormal muscle cells and 7,4 +/− 0,90 bends/30 seconds in dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms) ;hlh-1(cc561ts) mutant worms. We hypothesized that if drp-1(D118A) construct is unable to increase hallmarks of muscle degeneration in the dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms that would indicate that the role of DRP-1 in cell death is required for dystrophin-dependent muscle degeneration. We found that introducing drp-1(D118A) construct into dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms affected neither the number of abnormal muscle cells nor thrashing ;hlh-1(cc561ts) mutant worms. This strongly suggests that caspase cleavage of DRP-1 is required for dystrophin-dependent muscle degeneration in C. elegans.We next tested the effects of expressing ies Figs  and 3G,Hies Figs  and 3G,Hing Fig. . The mutC. elegans, we revealed the spontaneous apparition of more than two abnormal muscle cells by muscle quadrant at days 17 and 19 of adulthood  mutant worms showed impaired mobility early in adulthood life, which could be explained by energy production impairment64. Importantly, in absence of DRP-1, the apparition of abnormal muscle cells, over aging, was a very rare event  mutant worms expressing either the wild-type drp-1 transgene (drp-1(+)) or the drp-1 transgene with the CED-3 cleavage site mutated (drp-1(D118A)) supposed to be inefficient in apoptosis  mutant worms with drp-1(+) transgene exhibited apparition of abnormal muscle cells to the same extent as wild-type worms, demonstrating a role for DRP-1 in age-related muscle degeneration. At day 17 of adulthood, drp-1(tm1108) mutant worms expressing the drp-1(D118A) transgene presented very few abnormal muscle cells  mutant worms expressing the drp-1(D118A) transgene exhibited nearly the same level of muscle degeneration than wild-type worms ;hlh-1(cc561ts) mutant worms. We thus down-regulated CED-3, required for the execution of apoptosis via its activation by CED-4  and for DRP-1 to act in apoptosis  and CPS-6  which, upon cell death signal, are released from mitochondria and can interact together to translocate to the nucleus where they participate to DNA degradation67. CPS-6 can also interact with CRN-1, CRN-4, CRN-5 and CYP-13 to form the degradeosome68. Second, CRN-2 likely to encode an homolog of the TatD nuclease  and predicted to be mitochondrial68. CRN-2 acts in the same pathway as CRN-368. Third, NUC-1 and CRN-6  that seem involved in the degradation of DNA debris from apoptotic cells in late stage of apoptosis69. Concerning corpse engulfment, important for the phagocytic process70, we have tested the effects of knocking-down CED-1, which is involved in cell-corpse recognition with CED-6 and CED-771 and PSR-1, which participates in the migration of engulfing cell73.Having established that the functions of DRP-1 in apoptosis play a key role in dystrophin-dependent muscle degeneration, we asked whether other executors of apoptosis could be important. For this, we performed RNAi experiments against several executors of cell death and tested the effects of each RNAi constructs on mitochondrial morphology and the number of abnormal cells in sis Fig. 65,66. Wedys-1(cx18);hlh-1(cc561ts) mutant worms fed with bacteria expressing either the empty vector L4440 or RNAi constructs against ced-3 or each of the above described cell death executors tested. ced-3 RNAi treatment dramatically decreased the number of abnormal muscle cells compared to that observed in dys-1(cx18);hlh-1(cc561ts) mutant worms fed with the empty vector L4440 ;hlh-1(cc561ts) fed with ced-3 RNAi vs 9,63 +/− 0,24 abnormal muscle cells for dys-1(cx18);hlh-1(cc561ts) fed with L4440) ;hlh-1(cc561ts) mutant worms (dys-1(cx18);hlh-1(cc561ts) mutant worms fed with: cps-6 RNAi: 5,01 +/− 0,32 abnormal muscle cells; wah-1 RNAi: 5,00 +/− 0,37 abnormal muscle cells; crn-2 RNAi: 4,81+/0,19 abnormal muscle cells; nuc-1 RNAi: 6,25 +/− 0,25 abnormal muscle cells; crn-6 RNAi: 6,53 +/− 0,26 abnormal muscle cells; ced-1 RNAi: 5,50 +/− 0,29 abnormal muscle cells; psr-1 RNAi: 5,31 +/− 0,24 abnormal muscle cells vs dys-1(cx18);hlh-1(cc561ts) mutant worms fed with the empty vector L4440: 9,63 +/− 0,24 abnormal muscle cells)  Fig.  confirmils) Fig. . Our datdys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms. The effects of ced-3 RNAi and of the drp-1(tm1108) mutation on muscle cells of dys-1(cx18);hlh-1(cc561ts) were cumulative indicating that CED-3 probably has an additional role in dystrophin-dependent muscle degeneration, which is DRP-1-independent. Moreover, knock-down of ced-1 and psr-1, involved in corpse engulfment, could also further decrease the muscle degeneration of dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms (dys-1(cx18);hlh-1(cc561ts) mutant worms fed with: ced-1 RNAi: 2,77 +/− 0,24 abnormal muscle cells; psr-1 RNAi: 3,13 +/− 0,22 abnormal muscle cells vs dys-1(cx18);hlh-1(cc561ts) mutant worms fed with the empty vector L4440: 4,86 +/− 0,26 abnormal muscle cells)  could further decrease muscle degeneration of dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) mutant worms fed with the empty vector L4440 (dys-1(cx18);hlh-1(cc561ts) mutant worms fed with: cps-6 RNAi: 2,33 +/− 0,29 abnormal muscle cells; wah-1 RNAi: 3,68 +/− 0,33 abnormal muscle cells; crn-2 RNAi: 3,88 +/− 0,29 abnormal muscle cells vs dys-1(cx18);hlh-1(cc561ts) mutant worms fed with L4440: 4,86 +/− 0,26 abnormal muscle cells) ;hlh-1(cc561ts);drp-1(tm1108) mutant worms although nuc-1 and crn-6 RNAi decreased muscle degeneration in dys-1(cx18);hlh-1(cc561ts) mutant worms  mutant worms  DRP-1 may act in parallel of the DNA degradation pathway in dystrophin-dependent muscle cell death, (ii) DRP-1 may act upstream of the DNA degradation pathways.Then, we wondered whether DRP-1 was required for CED-3 and others cell death executors to impact dystrophin-dependent muscle degeneration. For that, we tested the effects of the different RNAi presented above on muscle degeneration of ls) Fig. . Similarls) Fig. . By contrms Fig. . None ofrms Fig. . Based ocrn-2 RNAi) had an effect on mitochondrial morphologies in wild-type worms carrying the ccIs4251 transgene ;hlh-1(cc561ts) mutant background, all the tested RNAi, with exception of psr-1 RNAi ;hlh-1(cc561ts) mutant worms fed with: wah-1 RNAi: 0,60 +/− 0,013; cps-6 RNAi: 0,63 +/− 0,020; crn-2 RNAi: 0,67 +/− 0,017; nuc-1 RNAi: 0,64 +/− 0,022; crn-6 RNAi: 0,67 +/− 0,019; ced-1 RNAi: 0,68 +/− 0,024 vs dys-1(cx18);hlh-1(cc561ts) mutant worms fed with the empty vector L4440: 0,76 +/− 0,009) ;hlh-1(cc561ts) mutant worms.We then tested the effect of cell death executors on mitochondrial dynamics. None of the RNAi tested  Fig. . Thus, d79 and sarcopenia83 in various species including Human. However, the exact molecular mechanisms leading to muscle apoptosis upon dystrophin deficiency or aging remain unclear. Here, we revealed a DRP-1-dependent apoptotic mechanism that participates in dystrophin-dependent and age-dependent muscle degeneration. DRP-1 is the principal and universal actor of mitochondrial fission. In mammals, mitochondrial fragmentation induced by DRP-1 can trigger apoptosis84. In C. elegans, a previous study suggested a marginal role for DRP-1 in apoptosis during development using the mutation drp-1(tm1108)31, whereas another study showed that the dominantly interfering mutation drp-1(K40A) caused mitochondria to form large blebs and induced excessive apoptosis in embryos56. Our study reveals that DRP-1 can play a key role in apoptosis that takes place during cellular stresses provoked either by the loss of dystrophin function or by aging. Our data also demonstrate that EAT-3 and FZO-1 overexpression in dystrophin deficient worms can reduce both mitochondrial fragmentation and muscle degeneration. A recent study showed that in mice, muscle-specific deletion of OPA1 (EAT-3 homolog in C. elegans) induces a precocious aging phenotype with muscle loss and weakness85 and a reduced muscle mass has been also reported in mice ablated for both Mfn1 (FZO-1 homolog in C. elegans) and Mfn285. These observations are nicely corroborating our findings and the fact that levels of mitochondrial fusion greatly impact muscle degeneration. Our data also suggest that manipulating mitochondrial fusion is sufficient to decrease apoptosis in dystrophic worms. In C. elegans, the direct links between mitochondrial dynamics and developmental apoptosis are highly controversial26; our study suggests that, upon cellular stress such as dystrophin deficiency, the major actors of mitochondrial dynamics in worms, namely DRP-1, EAT-3 and FZO-1, can directly interplay with the apoptotic pathways.Apoptotic muscle fibers have been frequently associated with Duchenne Muscular Dystrophyvia apoptosis rather than through mitochondrial fission to induce muscle cell death. In mammals, DRP-1-induced outer membrane fragmentation allows for release of mitochondrial pro-apoptotic factors such as cytochrome c, EndoG (CPS-6 in worms) or AIF (WAH-1 in worms)86. In C. elegans muscle cells, cytochrome c could be released from mitochondria to the cytosol during muscle degeneration74 but its role in activating APAF-1 (CED-4 homolog in worms) to form the apoptosome is unlikely to be conserved88. Strong evidence suggests that both WAH-1  and CSP-6  can be released from the mitochondria to the nucleus to degrade DNA upon apoptotic stimuli in C. elegans89. Our data emphasize a function for WAH-1/CSP-6 in dystrophin-dependent muscle degeneration potentially downstream of DRP-1. One possibility is that DRP-1, once activated by CED-3 induces mitochondrial outer membrane permeabilization that will, in turn, allows for WAH-1/CSP-6 release from the mitochondria and translocation into the nucleus. However, we cannot exclude that DRP-1 acts in a parallel pathway of WAH-1/CSP-6. Our data strongly suggest that DRP-1 does not participate in corpse engulfment during apoptosis and is likely to act upstream of NUC-1 and CRN-6. Collectively, our data converge toward a mechanism that underlies DRP-1-dependent mitochondrial pathways activated downstream of CED-3 to impact dystrophin-dependent muscle degeneration. The cleavage of DRP-1 by caspase CED-3 seems to be restricted to C. elegans31 suggesting that it could be an ancestral role of CED-3 that was lost over evolution. However, we also found that diminution of CED-3 expression can decrease muscle degeneration of a dystrophin C. elegans mutant in a DRP-1-independent manner. Our data are consistent with the fact that maintenance of C. elegans muscle integrity during aging depends on CED-390. Interestingly, skeletal muscles of aged rats exhibit a high level of activated caspase-391 and muscles of Duchenne Muscular Dystrophy patients present an increase of both caspase-3 expression92 and activity93. Collectively, the role of the caspase-3 in muscle degeneration appears to be likely conserved among species.We also showed that DRP-1 cleavage by the pro-caspase CED-3 is required for dystrophin-dependent and age-dependent muscle degeneration. Hence, our study demonstrates that DRP-1 sits downstream of CED-3 and strongly suggests that DRP-1 acts mostly 94. Control of intracellular concentration of Ca2+ ([Ca2+]i) homeostasis is critical for the maintenance of muscle contraction and relaxation. Elevated [Ca2+]i increases production of reactive oxygen species (ROS)95, activates calpain96, impairs autophagy97, increases mitochondrial Ca2+ accumulation96 and induces cell death99. Intriguingly, a drastic increase in the [Ca2+]i in myofibers and myotubes deficient in dystrophin was observed in several studies101. In mdx mice, abnormal elevation of [Ca2+]i is due to SERCA (sarco(endo)plasmic reticulum Ca2+ ATPase) inhibition suggesting an importance of the ER-dependent regulation of Ca2+ in Duchenne Muscular Dystrophy103. Moreover, muscle degeneration of the dys-1(cx18);hlh-1(cc561ts) mutants worms is likely to be a calcium-dependent105. Calcium is also a well-known activator of DRP-1. For instance, ER-calcium release induces uptake of calcium by mitochondria and DRP-1 activation allowing for calcium-dependent mitochondrial fission106. Calcineurin, a calcium-dependent phosphatase, dephosphorylates DRP1 and promotes mitochondrial fragmentation and cell vulnerability to apoptosis108. In neurons of C. elegans, phosphorylation of DRP-1 by Ca/calmodulin-dependent kinase II (CaMKII) inhibits DRP-1 activity109. One possibility is that the loss of dystrophin function could induce changes in intracellular calcium that would activate DRP-1 and cell death.Having established that DRP-1 acts mostly in apoptosis to impact muscle degeneration upon dystrophin deficiency, it would be interesting to investigate whether others molecular mechanisms than cleavage by caspase-3 can regulate DRP-1 activityIn conclusion, our findings point to mitochondrial dynamics as an early signaling hub that controls cell death in dystrophin-dependent muscle degeneration. A better understanding of the role of DRP-1 in muscle degeneration could allow in the long term to identify new targets for treatments of muscular dystrophies or to improve muscle degeneration in the elderly.Caenorhabditis Genetic Center with the exception of the drp-1(tm1108) strain provided by the National Bioresource Project for the Nematode to Sylvia Lee’s lab . N2 was used as the wild-type (WT) strain. The dys-1(cx18);hlh-1(cc561ts)40, dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) and the dys-1(cx18);hlh-1(cc561ts);ccIs4251 strains were constructed using standard genetic methods. The drp-1(tm1108) strain, which has no detectable DRP-1 protein expression, is likely to represent a strong loss-of-function or null mutant31. The integrated ccIs4251 transgene contains Pmyo-3::Ngfp-lacZ  and Pmyo-3::Mtgfp 55.Most of the strains used were obtained from the Escherichia coli strain OP50 or with HT115 bacteria for RNAi experiments.All strains were maintained at 15 °C on standard NGM (Nematode Growth Medium) agar plates seeded with All constructs were verified by sequencing and restriction fragment length. Sequences of the primers used for PCR are available upon request.drp-1 coding region was amplified from genomic DNA by PCR. A second 3.6 Kbp genomic fragment containing the 650 bp sequence upstream, the 2.5 Kbp coding region, and the 490 bp sequence downstream of the drp-1 coding region was amplified from the first 4.3 Kbp fragment. Gibson Assembly® Master Mix and KpnI enzyme was used to introduce this second fragment in pBSC vector.A first 4.3 Kbp genomic fragment containing the 950 bp sequence upstream and the 2.5 Kbp coding region, and the 1.1 Kbp sequence downstream of the drp-1 coding region was amplified from genomic DNA by PCR. Two fragments of 1050 pb and 2640 pb with overlapping end in drp-1 containing a mutation (A > G) on the CED-3 cleavage site of DRP-1 were amplified by PCR from the first 4.3 Kbp fragment. A 3.6 Kpb fragment containing the 650 bp sequence upstream, the 2.5 Kbp coding region with a mutation on the CED-3 cleavage site of DRP-1, and the 490 bp sequence downstream of the drp-1 coding region was amplified from the two fragments described above by Fusion-PCR. Gibson Assembly® Master Mix and KpnI enzyme was used to introduce this second fragment in pBSC vector.A first 4.3 Kbp genomic fragment containing the 950 bp sequence upstream, the 2.5 Kbp coding region, and the 1.1 Kbp sequence downstream of the eat-3 coding region were amplified from genomic DNA by PCR. Secondly, two fragments of 2.3 Kpb and 2.8 Kpb with overlapping end in eat-3 were amplified by PCR from the first two fragments described above. A 5.1 Kpb fragment containing the 182 bp sequence upstream, the 4.7 Kbp coding region, and the 216 bp sequence downstream of the eat-3 coding region was amplified from the two fragments described above by Fusion-PCR.First, two genomic fragments of 2.9 Kbp and 3.3 Kpb containing the 701 bp sequence upstream, the 4.7 Kbp coding region, and the 474 bp sequence downstream of the fzo-1 coding region was amplified from genomic DNA by PCR. A second 3.6 Kbp genomic fragment containing the 113 bp sequence upstream, the 2.8 Kbp coding region, and the 702 bp sequence downstream of the fzo-1 coding region was amplified from the first fragment described above.A first 3.8 Kbp genomic fragment containing the 163 bp sequence upstream, the 2.8 Kbp coding region, and the 762 bp sequence downstream of the drp-1(+) or drp-1(D118A) or eat-3(+) or fzo-1(+)); 2,5 ng/μl of the co-injection marker pCFJ90 (Pmyo-2::MCherry::unc-54utr) and 116,5 ng/μl Bluescript plasmid filler DNA were microinjected into the gonads of adult wild-type, dys-1(cx18);hlh-1(cc561ts);drp-1(tm1108) or dys-1(cx18);hlh-1(cc561ts) mutant worms using standard methods110. F1 progeny were selected on the basis of MCherry fluorescence. Individual F2 animals were isolated to establish independent lines.For generation of transgenic animals: 10 ng/μl of transgene (C. elegans RNAi collection (Ahringher laboratory-Gene Service Inc) were cultured overnight in LB medium supplemented with 100 mg/ml ampicillin and 12.5 mg/ml tetracycline (LBAT) at 37 C. The day after, 0.01 volume of this pre-culture was added in 1 volume of LBAT and incubated at 37 C. At 0.6–0.8 DO, bacteria were concentrated 5 times and then 300 μL of the bacteria culture was seeded on NGM plates containing ampicillin (100 ug/mL) and tetracycline . Plates were allowed to dry at room temperature for at least 3 days. Then, plates were stored at 4 C during 2 months utmost. The day before the experiment, 4 mM IPTG was added to induce bacteria overnight at room temperature. Gravid worms were allowed to lay eggs during 6 hours on NGM plates seeded with RNAi bacteria carrying specific dsRNA-expressing bacteria and progeny grew on RNAi plates until the day of experiment. Unless indicated differently, experiments were performed at the age of 3 days after the L4 larval stage (L4 + 3 days). As RNAi efficiency control, dpy-13 RNAi was performed in parallel and resulted in nearly 100% dumpy. As RNAi-negative control, plates seeded with HT115 carrying the empty vector RNAi L4440 were used.RNAi bacteria clones from the commercial 111. Synchronized adult  animals were fixed in 1 ml of PBS supplemented with 20 μl of 37% formaldehyde, extracted with acetone at −20 °C, and incubated in 3U of phalloidinAlexa Fluor® 488 (Molecular Probes) for 2 h. Imager Z1 microscope was used for microscopic observations. Only the two most visible quadrants of each animal were counted for the quantification of muscle degeneration.Phalloidin staining was carried out as described previouslyFor aging experiments, 1.3 μg/mL 5-FU  was added when the worms reached the L4 stage.Pmyo-2::mCherry::unc-54utr) were transferred. Synchronized adult animals , were moved onto a fresh 2% agarose 12 wells-plates containing M9 bufferpre-equilibrated at a temperature of 15 °C. Thrashing frequency was measured during the swimming sessions in the liquid. Videos of animal movements were acquired using a Macro Zoom microscope. The videos were recorded for 30 seconds at 30 frames per second. The thrashing frequency of the worms, measured in body bends per 30 seconds, was quantified using the open-source wrMTrck plugin for Fiji software112 (http://www.phage.dk/plugins/wrmtrck.html). One body bend was defined as a change indirection of bending at the midbody. For aging experiments, 1.3 μg/mL 5-FU  was added when the worms reached the L4 stage.L4 worms were transferred to a plate during 3 days. In the case of transgenic lines, only the MCherry-fluorescent L4 worms  was used in order to minimize the noise and to sharpen the contrast between mitochondria and the background. Nucleus of each image was removed by hand (with the unaided human eye). « Analyze particles » command was used to obtain values for circularity  for each mitochondrion in an image, ignoring mitochondria <5 square-pixels or on the edge of the image. Mitochondrial circularity at 0 refers to a straight line whereas mitochondrial circularity at 1 refers to a perfect circle.Gravid adults with integrated Fiji, we have compared the circularity indexes of 96 confocal images measured by visual counting and the circularity indexes measured by the macro processed. No statistical differences were found between the two methods.In order to determine the accuracy and consistency of the circularity measurements obtained with In Situ Cell Death Detection Kit, Fluorescein Roche) for 1.5 hours at 37 C. After incubation, worms were washed 3 times in PBST for 10 min. Then, phalloïdin staining with phalloïdin Alexa Fluor® 633 (Molecular Probes) was performed like previously described above. Worms were then mounted in Vectorshield mounting medium with DAPI (Vector Laboratories) and visualized using either a Zeiss Axiophot microscope or Zeiss LSM 710 confocal microscope. Only the two most visible quadrants of each animal were counted for the quantification of TUNEL positives muscle cells.Synchronized L4 + 3 day-old worms were fixed in 1 ml of PBS supplemented with 20 μl of 37% formaldehyde, extracted with acetone at −20 °C, and incubated in 50 μL of TUNEL solution (One-way ANOVA was used to test statistical differences between independent groups within the same experiment. Statistical significance was tested with Tukey and Dunnett post tests. Two-tailed unpaired Student’s t-test was used to examine direct differences between two independent groups.No data sets were generated or analyzed during the current study.Supplementary information


Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation.Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC’s stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H The corneal epithelium, an indispensable prerequisite for visual acuity, is postnatally maintained and regenerated by a pool of adult stem cells, termed limbal stem cells (LSC) –4. Solaret al. demonstrated that PAX6 in corneal epithelial cells responded to oxidative stress by nuclear-to-cytoplasmic translocation [Paired-box protein 6 (PAX6) is a master transcription factor guiding corneal morphogenesis and homeostasis by regulating cell cycle and fate of tissue progenitor cells ,15. Recelocation . HoweverBacillus polymyxa, Sigma-Aldrich, St. Louis, MO) at 4°C and subjected to trypsin digestion for 10 mins at 37°C. Cells were seeded at densities ranging from 100 to 400 cells/cm2 and cultured up to 12 days. Colonies were expanded in serum- and feeder cell-free conditions in Keratinocyte-Serum Free Medium  supplemented with 5 ng/mL human recombinant epidermal growth factor and 50 μg/mL bovine pituitary extract (both Gibco).This research has been approved by the Ethical Committee of the Medical University of Vienna . Human corneal tissues procured in MUW cornea bank not suitable for corneal transplantation were used in the current study. Limbal epithelial sheets from corneoscleral rims were surgically isolated after overnight incubation in dispase II . Sense and antisense sequences were as follows: ATG7 Knockdown (KD) #1 sense 5’-CGCUUAACAUUGGAGUUCAtt-3’ antisense 5’-UGAACUCCAAUGUUAAGCGag-3’, KD#2 sense 5’-GGAACACUGUAUAACACCAtt-3’ antisense 5’-UGGUGUUAUACAGUGUUCCaa-3’; PAX6, KD#1 sense 5’-GGCAAUCGGUGGUAGUAAAtt-3’ antisense 5’-UUUACUACCACCGAUUGCCct-3’, KD#2 sense 5’-CCAACUCCAUCAGUUCCAAtt-3’ antisense 5’-UUGGAACUGAUGGAGUUGGta-3’.Controls were transfected with two scrambled siRNAs (SCR) with no complementarity to any known human gene product . For a 60-mm petri dish, 12.5 pmol of each siRNA and 6.25 μl lipofectamine  were used to transfect colonies on clonal day 8–10 at 70% confluence. Transfection mixture was removed after overnight incubation and cells were cultured for additional three days in KSFM prior to UVA irradiation and further experiments. Transfection efficiency was assessed by quantitative PCR, western blot and immunofluorescene analysis.siRNA oligonucleotide duplexes targeting human PAX6 gene under control of the cytomegalovirus (CMV) promoter (Ad-CMV-PAX6) was used to ectopically express PAX6 in LSC colonies. To study whether the observed PAX6 gene functions were ocular tissue-specific, experiments of ectopic PAX7 gene expression in LSC colonies were performed in parallel. Adenoviral cassettes carrying viral backbones and an empty CMV promoter (Ad-CMV-null) served as controls. Colonies were infected on clonal day 10 with an MOI of 50 overnight. Cells were cultured for additional two days in adenovirus-free KSFM for protein expression before experiments were performed.Pre-packaged human adenovirus  expressing the human 2 was used throughout all experiments to study PAX6’s regulation on cell cycle progression in UVA-stressed LSCs  was used to generate radiation filtered for the emission of UVA light in the spectral range of 340–440 nm. During irradiation, cells were transferred to Dulbecco’s Phosphate Buffered Saline . A non-lethal dose of UVA titrated to the irradiance of 20 J/cmsed LSCs .2O2 for 6 hours served as positive controls for oxidative stress. ROS levels were visualized using CM-H2DCFDA  according to the manufacturer’s instructions. Live cell imaging was performed by laser scanning confocal microscopy 30 minutes after exposure to UVA. For quantification, mean fluorescence intensity of LSC colonies was determined.Cells were treated with an antioxidant mixture  for 16 hours prior to UVA irradiation. Cells treated with 20 μM HStreptomyces hygroscopicus, Sigma-Aldrich) for 6 hours served as positive controls. Autophagic cells were defined as cells displaying more than five perinuclear puncta [Streptomyces griseus, Sigma-Aldrich) with or without UVA irradiation was assessed at different time points up to 24 hours [Autophagy activity was determined by autophagosome formation and autophagic flux. Autophagosomes were visualized 6 hours after UVA exposure using a cationic amphiphilic tracer dye to stain autophagosomal vesicles . Cells treated with 10 μM rapamycin  according to the manufacturer’s protocol.GAPDH values of at least three biological repeats.Total RNA was isolated using RNeasy Plus Mini Kit  and reverse transcribed using iScript cDNA Synthesis Kit . qPCR was performed using TaqMan Gene Expression Assays and Gene Expression Master Mix . Amplification reactions were carried out according to a standardized qPCR program of the ABI Prism 7500 Fast Real-Time PCR System . Data were analyzed using the “delta-delta method” as suggested by Applied Biosystems and Pfaffl . ResultsFor immunofluorescent staining, cells were fixed in 4% (v/v) formaldehyde and subsequently immersed in DPBS containing 0.1% (v/v) Triton X-100 for permeabilization. Samples were blocked using serum-free Protein Block . Antibody diluent (Dako) was used to dilute primary and secondary antibodies. Mouse and rabbit IgG served as isotype controls . 4,6-diamidino-2-phenylindole dilactate (DAPI) and Hoechst 33342 (both Molecular Probes) were used as nuclear counterstains for fixed and viable cells, respectively. Images were acquired using an inverted laser scanning confocal microscope  equipped with a 405-nm laser diode and an argon ion laser capable of providing an illumination of 488 nm. A helium-neon laser was used to generate an excitation wavelength of 594 nm. Fluorescent signals were detected by a 32-channel spectral detection unit . Imaging was routinely performed with a 20x air objective (numeric aperture 0.8) and a 63x oil lens (numeric aperture 1.4) was used to detect autophagosomal vesicles using ZEN software . Image processing software ImageJ and Fiji  were used for image analysis ,25.Proteins of LSC colonies were isolated with a cell extraction buffer  supplemented with protease inhibitor cocktail . Forty μg total protein provided with loading buffer  and 100 mM dithiothreitol were electrophoretically separated on polyacrylamide gels . For LC3B-I/II immunoblotting, a precast 4–20% gradient gel  was used. Proteins were transferred to a nitrocellulose membrane . Five % (w/v) nonfat dried milk powder and 2% (w/v) bovine serum albumin  in tris buffered saline containing 0.05% (v/v) Tween20 was used for membrane blocking and as antibody diluent. Peroxidase-conjugated secondary antibodies were visualized using Novex ECL (Invitrogen). Chemiluminescence was detected using ChemiDoc XRS system with Image Lab software .Rabbit polyclonal antibody (Rb pAb) against ATG7 , rabbit monoclonal antibody (Rb mAb) against GAPDH , Rb pAb against LC3B , mouse monoclonal antibody (Ms mAb) against p21 , Ms mAb against PCNA , Rb pAb against PAX6 , AlexaFluor 488-conjugated Donkey Anti-Rb IgG (H+L) (1:1000), AlexaFluor 594-conjugated Donkey Anti-Ms IgG (H+L) , Peroxidase-conjugated IgG Fraction Monoclonal Ms Anti-Rb IgG, Light Chain Specific .t test, one-way ANOVA, Sidak’s, Dunnett’s and Tukey’s multiple comparisons . Data are shown as means ± s.e.m. with p < .05 considered statistically significant.Data represent average of at least 3 independent experiments. For IF data analysis, at least 10 colonies per condition were imaged, unless specified otherwise. Statistical analysis was performed using non-paired Student’s in vitro, colonies during exponential growth phase  were transfected with two siRNAs targeting human homolog of autophagy-related gene 7 (ATG7) while controls received scrambled siRNAs not complementary to any human gene sequence (SCR). The efficiency of siRNA-mediated ATG7KD was measured at both mRNA and protein levels. Quantitative PCR demonstrated a 65.1 ± 1.6% reduction of ATG7 mRNA expression in ATG7KD compared to SCR control (p < .05) (p < .01), while no inductive effect of rapamycin was observed in ATG7KD LSCs   . Westernp < .05) . To vali, p>.05) . Taken t2 UVA by CYTO-ID assay. UVA radiation induced autophagy in SCR LSCs evidenced by an increase in autophagic cells from 35.7 ± 4.9% to 59.4 ± 3.3% compared to non-irradiated controls (p < .01) (p>.05). Data indicate that UVA increases autophagosomes in an ATG7-dependent mechanism.To test whether autophagy mediates LSC’s ultraviolet stress response, formation of autophagosomal structures was assessed 6 hours after exposure to 20 J/cmp < .01) . In contp < .05 compared to drug vehicle-treated group), indicative of a low homeostatic autophagic flux in LSC colonies , suggesting that UVA activates autophagy in LSCs by increasing autophagic flux.To determine whether the observed autophagosome increase in irradiated SCR colonies results from UVA-impaired autophagosomal clearance or UVA-stimulated autophagic flux, we then employed BafA1, an autophagolysosomal inhibitor, to measure autophagic flux at homeostatic state and under UVA stress. In non-irradiated LSCs, LC3B-II was found elevated after 24 hours of BafA1 treatment  (p < .05) as well as irradiated, autophagy-competent LSCs . Conversely, treating ATG7KD LSCs with antioxidant mixture 16 hours prior to irradiation largely alleviated the accumulation of intracellular ROS in ATG7KD LSCs . These data suggest a crucial role of autophagy in regulating redox balance in LSC’s stress response to UVA.Next, we sought to determine whether autophagy contributes to restore redox balance after UVA stress. To this end, intracellular ROS were evaluated by CM-H, p>.05) . In cont2O2 and subcellular expression of PAX6 was examined by immunofluorescence. Notably, LSC colonies displayed high levels of nuclear PAX6 expression on clonal day 12 (70.9 ± 1.5%) , validating our in vitro ROS-responsive PAX6 model. Since we found that autophagy balances UVA-induced oxidative stress, we next asked whether PAX6 was differentially regulated in autophagy-competent and—deficient LSCs confronted with UVA-elicited ROS. To this end, the subcellular localization of PAX6 was studied in UVA-stressed SCR and ATG7KD LSCs by immunofluorescence. Interestingly, the percentage of LSCs expressing nuclear PAX6+ was significantly decreased in SCR LSC colonies after UVA exposure (8.0 ± 1.6%) compared to non-irradiated SCR LSCs  . Of note, the reduction of nuclear PAX6+ cells in irradiated SCR LSCs correlated with the increase of cells with cytoplasmic PAX6 expression. In addition, antioxidant pre-treatment abolished the UVA-triggered PAX6 nuclear depletion observed in SCR LSCs evidenced by 26.6 ± 1.3% of nuclear PAX6+ cells in irradiated SCR LSCs (p < .01 compared to 8.0 ± 1.6% in irradiated LSCs). Collectively, data presented here suggest: (1) Nuclear-to-cytoplasmic protein transport, rather than transcriptional repression or acceleration of cytoplasmic PAX6 protein degradation, is the dominant mechanism by which UVA induces nuclear reduction of PAX6. (2) The UVA-induced PAX6 nucleocytoplasmic transportation is both ROS- and autophagy-dependent.To determine whether our model is suitable to study PAX6 response to oxidative stress, colonies were challenged with 20 μM H ± 1.5%) . H2O2-inp < .01) . In cont+ PAX6+ cells (p < .01) . The number of PCNA+ PAX6+ cells remained high in ATG7KD LSCs following UVA irradiation . Interestingly, cells co-expressing PCNA and nuclear PAX6 after UVA exposure were significantly lower in SCR cells compared to ATG7KD LSCs under UVA stress .Next, we aimed to determine whether nucleocytoplasmic translocation of PAX6 plays a role in cell cycle response during UVA stress. Therefore, we performed dual immunofluorescent staining against PAX6 and PCNA, a proliferative marker. Accompanying nuclear export of PAX6, UVA irradiation led to a significant reduction of PCNA expression in autophagy-competent LSC colonies from 52.8 ± 6.5% to 42.3 ± 4.0% PCNAp < .01) , indicat+ PCNA+ cells was significantly lower in UVA-irradiated, Ad-CMV-null vector-infected LSCs (4.7 ± 1.1%) compared to Ad-CMV-null vector-infected controls  (p>.05). Furthermore, the percentages of cycling PCNA+ PAX6+ cells in irradiated and non-irradiated Ad-CMV-null vector-infected ATG7KD LSCs were found similar to that in non-irradiated Ad-CMV-null vector-infected SCR LSCs . Lastly, adenoviral forced PAX6 expression in ATG7-KD LSCs did not correct the aberrant cell cycle progression under UVA stress, as the percentage of PAX6+ PCNA+ cells did not differ between non-irradiated Ad-CMV-null vector-infected ATG7KD, UVA-irradiated Ad-CMV-null vector-infected ATG7KD and UVA-irradiated Ad-CMV-PAX6 vector-infected ATG7KD .To determine whether reduced PCNA levels following UVA exposure results from nuclear depletion of PAX6, we restored the nuclear expression of PAX6 by adenoviral overexpression . Comparep < .05)  & S3 FigPAX7, a PAX family member normally absent from ocular tissue. qPCR analysis was used to determine PAX7 expression in Ad-CMV-PAX7-infected and control LSC colonies 72 hours after viral transduction. PAX6 mRNA was found highly expressed in Ad-CMV-null controls and not affected by adenoviral forced PAX7 overexpression . In contrast, PAX7 was undetectable in Ad-CMV-null controls, while PAX7 expression was found significantly increased in colonies transfected with Ad-CMV-PAX7 , confirming PAX7 overexpression. To determine the effect of ectopic PAX7 on cell cycle regulation, PCNA expression was assessed by immunofluorescence in Ad-CMV-PAX7 LSCs. As expected, the number of PCNA-expressing cells was found significantly reduced by irradiation in scrambled KD and control vector-transduced LSCs  . Taken together, these results suggest a PAX6-specific mediation on UVA-induced PCNA downregulation.To determine whether the UVA-induced PCNA downregulation in LSCs is a gene function unique to ocular tissue-resident PAX6, we ectopically overexpressed p < .01) . In contp < .01) , PCNA exp < .01) , PAX6KD LSCs displayed equally low levels of PCNA+ proliferative cells as irradiated SCR LSCs . Importantly, concomitant knockdown of both ATG7 and PAX6 restored the UVA-induced cycle arrest response in irradiated LSCs, as the percentage of PCNA+ cells in irradiated ATG7/PAX6 double KD LSCs was reduced to a level of only 16.2 ± 4.9%, significantly lower than those observed in non-irradiated SCR LSC  and irradiated ATG7KD LSC . Taken together, our data suggest PAX6 functions as a unique molecular intermediate downstream to ATG7-mediated autophagy to regulate the cell cycle kinetics during LSC’s ultraviolet stress response.To verify autophagy-dependent PAX6’s regulatory effects on LSC’s clonal proliferation, we knocked down PAX6 by siRNA  . While tp < .01) (p < .05). In contrast, UVA radiation failed to induce p21 expression in autophagy-deficient LSCs . Moreover, p21 expression was not induced by UVA irradiation in PAX6-overexpressing ATG7KD colonies (6.5 ± 0.9% p21+ cells) compared to irradiated Ad-CMV-null controls .To further dissect the mechanism by which autophagy induces cell cycle arrest in response to UVA, we investigated nuclear expression of p21, a negative regulator for cell cycle progression. Following UVA irradiation, autophagy-competent LSCs upregulated p21 expression from 0.6 ± 0.3% to 43.5 ± 11.6% compared to non-irradiated controls (p < .01)  & S5 Fig+ cells in the UVA group versus 3.1 ± 0.5% in non-irradiated controls, p < .01), p21 induction was not observed in UVA-irradiated ATG7KD (30.1 ± 3.8%)  . NotablyIn current study, we identified autophagy as an UV stress sensor in LSCs. Dual functional roles for autophagy in cellular stress response were further suggested by alteration of redox status and cell cycle progression in UVA-irradiated LSC. Activation of autophagy in LSC contributes to restoration of intracellular redox balance and adaptive cell cycle response. We propose a novel PAX6-p21 molecular mechanism underpinning the autophagy-mediated stress response .Autophagosome accumulation has been reported in the UV stress response of epidermal keratinocytes , whetheret al. show that autophagy exerts a cytoprotective effect by selective degradation of UV-oxidized macromolecules [Zhao olecules . Here, wi.e. mediation of cell cycle response. In our experiment, PAX6 and PCNA were found mostly co-localized in the nuclear compartment of proliferative LSCs in unstressed conditions Click here for additional data file.S2 Figp < .01 of 3 independent experiments.(A) Western blot of PAX6 expression in Ad-CMV-null and Ad-CMV-PAX6 infected cells. (B) Densitometry of PAX6 expression normalized to GAPDH levels. **(TIF)Click here for additional data file.S3 Fig+PCNA-, PAX6+PCNA+, PAX6-PCNA+, and PAX6-PCNA- cells. *p < .05 compared to SCR, Ad-CMV-null controls, #p < .05 compared to SCR, Ad-CMV-null, UVA-irradiated counterparts.(A) Stacked bar diagram and (B) statistics of PAX6(TIF)Click here for additional data file.S4 Figp < .001.(A) PAX6 immunofluorescence in SCR and PAX6KD LSC colonies. Scale bar, 100 μm. (B) Mean fluorescence intensity of PAX6 in SCR and PAX6KD LSCs. ***(TIF)Click here for additional data file.S5 Fig+p21-, PAX6+p21+, PAX6-p21+, and PAX6-p21- cells. *p < .05 compared to SCR, Ad-CMV-null controls, #p < .05 compared to SCR, Ad-CMV-null, UVA-irradiated counterparts.(A) Stacked bar diagram and (B) statistics of percentages of PAX6(TIF)Click here for additional data file.


Next-generation sequencing is making it critical to robustly and rapidly handle genomic ranges within standard pipelines. Standard use-cases include annotating sequence ranges with gene or other genomic annotation, merging multiple experiments together and subsequently quantifying and visualizing the overlap. The most widely-used tools for these tasks work at the command-line (e.g. BEDTools) and the small number of available R packages are either slow or have distinct semantics and features from command-line interfaces.To provide a robust R-based interface to standard command-line tools for genomic coordinate manipulation, we created bedr. This open-source R package can use either BEDTools or BEDOPS as a back-end and performs data-manipulation extremely quickly, creating R data structures that can be readily interfaced with existing computational pipelines. It includes data-visualization capabilities and a number of data-access functions that interface with standard databases like UCSC and COSMIC.bedr package provides an open source solution to enable genomic interval data manipulation and restructuring in R programming language which is commonly used in bioinformatics, and therefore would be useful to bioinformaticians and genomic researchers.The online version of this article (doi:10.1186/s13029-016-0059-5) contains supplementary material, which is available to authorized users. With the advent of high-throughput sequencing technologies, data scientists are facing immense challenges in large-scale sequence analysis and in integrating genomic annotations. For instance, comparing new experiments with previously published datasets, translating genomic coordinates between different assemblies of an organism as well as finding cross-species orthologues are some of the common use-cases in basic science experiments. To assist these tasks genomic features are routinely represented and shared using Browser Extensible Display , Distride facto standard for statistical analysis and visualization in computational biology ]x = region,engine = "unix",method = "natural")bedr.sort.regionbedr.sort.regionbedr.sort.regionbedr.sort.region"chr1:10-100" "chr1:101-200""chr1:200-210" "chr1:211-212""chr2:10-50" "chr2:40-60""chr10:50-100" "chr20:1-5"The above code will generate the following outputs of sorted regions:# lexicographical sort (R)"chr1:10-100" "chr1:101-200""chr1:200-210" "chr1:211-212""chr10:50-100" "chr2:10-50""chr2:40-60" "chr20:1-5"# lexicographical sort (bedtools)"chr1:10-100" "chr1:101-200""chr1:200-210" "chr1:211-212""chr10:50-100" "chr2:10-50""chr2:40-60" "chr20:1-5"# lexicographical sort (bedops)"chr1:10-100" "chr1:101-200""chr1:200-210" "chr1:211-212""chr10:50-100" "chr2:10-50""chr2:40-60" "chr20:1-5"As shown above, various types of sorting results are presented in a similar R data structures regardless of which sorting engine is used  and their respective output style. Also, BEDTools and BEDOPS do not support natural sorting, and if method = “natural” is requested with these two engines, bedr automatically defaults to using engine = “unix” of “R” to perform sorting. Note, sorting of large number of regions through R will be slow and may also result in high memory overhead.Much of the command-line interaction with BEDTools and BEDOPS is performed through temporary files followed by efficient piping/parsing of the output straight into R data structures. This ensures that memory intensive sorting tasks (or any other genomic operations discussed below) are managed by the optimized engines such as (BEDTools or BEDOPS), and therefore memory operations in R are limited to subsequent parsing of output.bedr.merge.region(x = region)In addition to sort operations, bedr also supports identification of overlapping regions which can be collapsed to avoid downstream analytical challenges such as many:many join results Fig. , e.g.Fig"chr1:10-100" "chr1:101-210""chr1:211-212" "chr10:50-100""chr2:10-60" "chr20:1-5"The above code will generate the following output of merged regions:bedr.snm.region(x = region)Sort and merge can be combined into one step given they are generally run as a tandem preprocessing step:"chr1:10-100" "chr1:101-210""chr1:211-212" "chr10:50-100""chr2:10-60" "chr20:1-5"The above code will generate the following vector output of sorted and merged regions:x = regions[[1]])regions.a <- bedr.merge.region(x = regions[[2]])regions.b <- bedr.merge.region(x = regions[[4]])regions.c <- bedr.merge.regionbedr.join.region. Regions in regions.a with no overlap are encoded as: . and -1a = regions.a,b = regions.b,c = regions.c)x = listbedr.join.multiple.region passed to the function. For instance, sub-region chr1:1-10 (column: index) overlaps with 2 region objects (b and c). This presence is shown as comma separated list in ‘names’ column as well as a truth table in the subsequent columns. The number of columns representing the truth table will match the number of region objects passed to the function bedr.join.multiple.region.x = regions.a,y = regions.b)bedr.subtract.regionin.regionregions.d <- ccosmic.vcf.example <- system.filetabixhead:which identifies regions overlapping with COSMIC coding mutations file resulting in the following vector as well as data.frame data structure, it is easily transformable to other third-party sequence and region objects. For instance, bedr provides a utility adaptor to convert regions into BED data.frame as shown below:x = regions.a)regions.a.bed <- convert2bed(Given that bedr can process regions data as R’s GRanges [library("GenomicRanges")df = regions.a.bed)makeGRangesFromDataFrame,input = listbedr.plot.region can be generated automatically . The oveThe above code will generate a base pair level overlap of sequence objects regions.a and regions.b, and show the results as a Venn diagram highlighting lengths of exclusive and overlapping regions as shown below:Further, bedr output is ideally suited for alternative complex set visualization tools such as UpSetR  and GvizBED and GRanges data.frame/objects makes it an easy-to-fit component in existing genomic pipelines. bedr is freely available as an open-source package through CRAN and lends itself for customized extensions needed for in-house sequencing-analysis pipelines as well as future bioinformatics protocols.We created bedr; an R package to support genomic operations using the BEDTools  and BEDOProject name: bedrProject home page:http://cran.r-project.org/web/packages/bedrOperating system(s): OSX, Linux/UnixProgramming language: ROther requirements: BEDTools, BEDOPSLicense: e.g. GNU GPL-2Any restrictions to use by non-academics: None


We thus propose that the sequential administration of DAC-TNPs and 5-FU could be significant in the development of novel targeted therapies.Hypermethylation of the transcription factor AP-2 epsilon (TFAP2E) gene affects 5-fluorouridine (5-FU) resistance in gastric cancer (GC) patients. The epigenetic inhibitor 5-Aza-2′-deoxycytidine (DAC), which reverses DNA methylation by targeting DNA methyltransferases (DNMTs), has potential to sensitize GC to 5-FU. Nevertheless, DNA demethylation only DAC transiently occurs since DAC is unstable in aqueous solutions, which limits its potential. Here we developed intelligent nanoparticles (NPs) comprising gelatinase with polyethylene glycol (PEG) and poly-ε-caprolactone) (PCL) to specifically deliver DAC (DAC-TNPs) to tumors. DAC-carrying PEG-PCL NPs (DAC-NPs) lacking gelatinase features served as controls. 72 hours after administration of DAC-TNPs or DAC-NPs, 5-FU was sequentially applied to GC cells and human GC xenografts in nude mice. Both  Gastric cancer (GC) ranks 4th in incidence among malignancies, but constitutes the 3rd deadliest cancer worldwide . Most GCin vivo [Abnormal gene methylation, an epigenetic modification, regulates the expression of several genes, changing patient sensitivity to chemotherapeutics. For instance, hypermethylation of the transcription factor AP-2 epsilon (TFAP2E) affects the therapeutic efficacy of 5-FU based chemotherapy in colorectal cancer  and GC . The hypin vivo .The properties of nanoparticles (NPs) could help deliver drugs to target sites through enhanced permeability and retention (EPR) . Thus, NIn our previous study, we demonstrated that 5-FU and DAC encapsulated with gelatinase-stimuli NPs effectively suppress MKN45 cell proliferation . HoweverDAC-NP and DAC-TNP preparation was performed with the double-emulsion solvent evaporation method; Control nanoparticles had no drug included. The average nanoparticle size ranged from 176.3 to 196.3 nm as measured by TEM, which could facilitate DAC delivery by the EPR effect . This fiThe concentration of free DAC in PBS quickly decreased within 24 h. However DAC in NPs and TNPs degraded at a reduced rate compared with free DAC (72 h) Figure , indicatMatrix metalloproteinase 2 (MMP)2 and MMP9 protein levels in MKN45 and MKN28 cells were determined by immunohistochemistry (IHC). Expression was reflected by brown cytoplasmic signals Figure . We confGC cells were treated with Rhodamine B-loaded NPs and TNPs for 2 h. Cellular internalization was investigated by detecting red fluorescence in the cytoplasm. Red fluorescence was found in the cytoplasm of both MKN45 and MKN28 cells, while nuclear signals were low Figure , indicatP > 0.05, Figures P < 0.05, Figure P > 0.05, Figure Figure P < 0.01); the differences among the three groups were not significant during the first 7 days of treatment (P > 0.05). DAC-NPs 72 h+5-FU and DAC-TNPs 72 h+5-FU began to show greater antitumor effects in comparison with other test articles in the first 7 days, with anticancer advantages becoming increasingly pronounced with time (P < 0.05). Interestingly, DAC-TNPs 72 h+5-FU completely halted tumor growth, with overtly superior outcome compared with DAC-NPs 72 h+5-FU (P < 0.05). Tumors treated with DAC-TNPs 72 h+5-FU were the most reduced in size (P < 0.05) , concomitant to DKK4 downregulation (P < 0.01). Additionally, TFAP2E levels after treatment with DAC-TNPs were significantly increased compared with those of the DAC-NPs group (P < 0.05), with lower levels of DKK4 accordingly (P < 0.05). Nevertheless, TFAP2E and DKK4 amounts remained unchanged upon treatment with free DAC at 10 μmol/L  and TNPs-DAC (10 μmol/L DAC eq) resulted in TFAP2E upregulation in the MKN45 cell line  because minutes . In the o Figure .in vivo study are more pronounced than the in vitro ones. However, our in vivo study was based on xenograft models, and orthotopic transplantation is needed for further investigation. More experiments are needed in additional cancers in order to further evaluate this sequential treatment strategy. The proposed chemotherapy enhancement of gelatinase-stimuli DAC-TNPs in vitro and in vivo provides a rationale for the clinical translation of this technology.In conclusion, our strategy of applying 5-FU followed by 72-hour DAC-TNPs treatment efficiently enhanced 5-FU efficacy via demethylation of TFAPE. Therefore, sequential treatment with DAC-TNPs and 5-FU is promising for GC treatment. Since simultaneous treatment with DAC-TNPs and 5-FU showed no effects (data not shown), epigenetic alterations are likely critical for DAC-induced sensitization to chemotherapy drugs. GC cells were first treated with DAC-TNPs for cells to undergo at least 2 divisions before 5-FU administration . The str® F68 (40 mg/ml) before storage. Control TNPs and NPs were obtained in a similar fashion without DAC supplementation.PEG-PCL di-block copolymers and gelatinase-stimuli NPs were obtained by applying previous methods . Drug-loNPs were characterized by their size, polydispersity, zeta potential, and morphology. Hydrodynamic size and polydispersity were measured by dynamic light scattering (DLS) . Zeta potential was determined using Zetaplus . The samples were kept at 37°C in phosphate buffered saline (PBS), and their size was monitored for 16 days for stability. The morphology of the NPs was determined by transmission electron microscopy . One drop of properly diluted NP suspension was placed on a copper grid covered with nitrocellulose membrane and air-dried at room temperature. The sample was negatively stained with phosphotungstic sodium solution (1% w/v) before observation.The drug loading and encapsulating process were carried out at 4°C since the decomposition rate of DAC in solution at 5°C is minimal (< 1% for 24 h) . A predeThe following steps were carried out in DAC decomposition experiments. Lyophilized TNPs-DAC and NPs-DAC (50 mg) in 2 mL PBS were dialyzed  at 37°C for 72 hours. Samples were collected at various times to assess residual DAC in both DAC-NPs and DAC-TNPs.Previous study showed that MKN45 cells are hypermethylated and MKN28 cells hypomethylated . These tin vitro cellular uptake studies. Gelatinase levels in GC cells were measured by immunohistochemistry; results were confirmed by zymography as previously described [4/well) were seeded in 6-well plates and incubated for 24 h, before exposure to Rhodamine B loaded NPs for 2 h. After fixation with 4% paraformaldehyde (PFA) for 30 minutes at room temperature, the cells were assessed for fluorescent signals by fluorescence microscopy .Biocompatibility of NPs was assessed in escribed . For celWe utilized the MTT assay to assess the inhibitory effects of NPs as previously described . Cells w6 MKN45 cells in 0.1 ml RPMI 1640 medium were subcutaneously injected into right posterior flanks of BALB/c nude mice . Tumor volumes (mm3) were derived as W × L2/2 . When 80% of tumors were at least 100 mm3, the animals were randomly assigned to treatment cohorts (n = 7), treated intravenously with saline control, NPs, TNPs, 5-Fu (20 mg/kg), DAC (15 mg/kg) 72 h + 5-Fu (20 mg/kg), DAC-NPs  72 h + 5-FU (20 mg/kg). DAC-TNPs (15 mg/kg DAC eq) 72 h +5-FU (20 mg/kg). The tumors were measured every 3 days for 21 days after treatment. Data were expressed as relative tumor volumes . Then, qRT-PCR was carried out with SYBR Green and primers specific for TFAP2E , DKK4 . β-actin was used for normalization . The data were analyzed by the comparative ΔΔCt method.RT-PCR was performed to assess TFAP2E and DKK4 gene expression levels . Total RU test. A P value below 0.05 indicated statistical significance.Data are mean ± SD, and were assessed by Mann-Whitney


Background: More than 75 species/species-level phylotypes of oral treponeme bacteria inhabit the oral cavity. However, their respective genomic compositions and clinical distributions remain poorly understood.Objectives: To compare distributions of phylogroup 1 and 2 oral treponemes in subjects with various periodontal health conditions, via sequence analysis of a highly-conserved treponeme ‘housekeeping’ gene.Methods: Subgingival plaque samples were collected from Chinese subjects with chronic periodontitis (n=5), aggressive periodontitis (n=4), gingivitis (n=5), and healthy controls (n=4). Samples were analyzed by a PCR/plasmid clone sequencing-based approach, using primer sets targeting the pyrH gene. Data was analyzed using various computational/bioinformatic approaches.Results: 1,227 quality-filtered pyrH gene sequences were obtained (mean 66.2±9.6 sequences per subject), which were assigned to 33 ‘pyrH genotypes’ (97% identity cut-off). 538 pyrH sequences (17 pyrH genotypes) corresponded to phylogroup 1 treponemes . 689 pyrH sequences (16 pyrH genotypes) corresponded to phylogroup 2 taxa. Correlations between pyrH genotype distributions and disease status were complex. One pyrH genotype, which was phylogenetically-related to T. denticola GM-1/MS25 strains, was highly prevalent: being detected in 17/18 subjects.Conclusions: Both healthy and periodontally-diseased subjects harbor multiple genetic lineages corresponding to the same treponeme species/phylotype within their subgingival niches.


Smad proteins have been implicated in metabolic processes, but little is known about how they regulate metabolism. Because Smad 2, 3, 4, and 7 have previously been shown to interact with murine protein serine–threonine kinase 38 (MPK38), an AMP‐activated protein kinase (AMPK)-related kinase that has been implicated in obesity-associated metabolic defects, we investigated whether Smad proteins regulate metabolic processes via MPK38. Smads2/3/4 increased, but Smad7 decreased, MPK38-mediated apoptosis signal-regulating kinase-1 (ASK1)/transforming growth factor-β (TGF-β)/p53 signaling. However, MPK38-mediated phosphorylation-defective Smad mutants  had no such effect. In addition, Smads2/3/4 increased, but Smad7 decreased, the stability of MPK38. Consistent with this, Smads2/3/4 attenuated complex formation between MPK38 and its negative regulator thioredoxin (Trx), whereas Smad7 increased this complex formation. However, an opposite effect was observed on complex formation between MPK38 and its positive regulator zinc-finger-like protein 9 (ZPR9). When Smads were overexpressed in high-fat diet (HFD)-fed obese mice using an adenoviral delivery system, Smads2/3/4 improved, but Smad7 worsened, obesity-associated metabolic parameters and inflammation in a MPK38 phosphorylation-dependent manner. These findings suggest that Smad proteins have class-specific impacts on obesity-associated metabolism by differentially regulating MPK38 activity in diet-induced obese mice. The TGF-β signaling pathway regulates a broad range of cellular processes, which include cell proliferation, differentiation, apoptosis, migration, extracellular matrix remodeling, immune functions, and tumor metastasis. This occurs through the combined use of TGF-β signaling pathway components, such as Smads and Smad-interacting transcription factors, cross-talk with other intracellular signaling pathways, and the ability of TGF-β receptors to activate other signaling modules6. Many studies have shown that Smads are phosphorylated by multiple intracellular kinases, including mitogen-activated protein kinases, Ca2+/calmodulin-dependent kinase II, cyclin-dependent kinase (CDK), protein kinase C, G protein-coupled receptor kinase 2, extracellular signal-regulated kinase, apoptosis signal-regulating kinase-1 (ASK1), and murine protein serine–threonine kinase 38 (MPK38)/maternal embryonic leucine zipper kinase (MELK)8, suggesting that the TGF-β pathway is closely integrated with other intracellular signaling pathways to achieve tightly regulated TGF-β responses. However, most of these studies have focused on the regulatory role of Smad phosphorylation in the TGF-β signaling pathway. Additional studies are required to investigate the effect of Smad proteins on the activity of these interacting kinases in order to decipher the molecular interplay between TGF-β and other intracellular signaling pathways.The identification of a growing list of intracellular kinases that phosphorylate Smad proteins suggests that the transforming growth factor-β (TGF-β)/Smad signaling pathway cross-talks with a variety of other intracellular signaling pathways13, although its exact physiological functions still remain to be determined. MPK38 and its interacting partner Smad3 have recently been shown to serve as components of a multi-protein complex linking ASK1 and TGF-β signaling pathways, which are involved in glucose and lipid metabolism in mice, and to contribute to the activation of ASK1 signaling via a direct interaction with ASK111. TGF-β1 was previously reported to positively regulate the 3-phosphoinositide-dependent protein kinase-1 (PDK1)/AKT1 pathway14, although PDK1 was shown to inhibit TGF-β signaling through direct interactions with Smads15. These findings suggest potential roles of Smads in the regulation of key kinases involved in intracellular signaling pathways that are integrated with TGF-β signaling.MPK38/MELK, an AMP‐activated protein kinase (AMPK)-related kinase, has been shown to mediate various cellular functions, including proliferation, spliceosome assembly, gene expression, carcinogenesis, apoptosis, and metabolism18. Smad3 deficiency in mice resulted in improved glucose tolerance and insulin sensitivity, accompanied by reduced white adipose tissue (WAT) mass and browning. The associated increase in mitochondrial biogenesis resulted in the dissipation of the excess energy stored in WAT by thermogenesis17. Higher TGF-β1 in humans has been shown to positively correlate with greater adiposity and a poor metabolic profile, and to negatively correlate with fitness17. Several recent studies have demonstrated that TGF-β signaling regulates insulin gene transcription in pancreatic β cells19. Moreover, the Smad3 gene was identified in a genome-wide association study for type 2 diabetes risk20. These findings implicate Smad3 as a potential target for the treatment of obesity and its associated disorders. Conversely, targeted disruption of Smad2 in mouse pancreatic β cells caused islet cell hyperplasia and impaired insulin secretion by attenuating ATP-sensitive K+ channel activity21. However, inhibition of Smad4 in pancreatic β cells conferred minor but significant improvements in blood glucose and glucose tolerance in high-fat diet (HFD)-induced obese mice22. Nevertheless, the molecular mechanisms involved in the regulation of metabolic homeostasis by TGF-β signaling remain poorly understood.Recent discoveries have shed some light on the important role that TGF-β signaling plays in adipose physiology and metabolismIn this study, we show that there are direct physical and functional interactions between MPK38 and Smads . Smads2/3/4 stimulate MPK38-dependent ASK1/TGF-β/p53 signaling pathways, whereas Smad7 inhibits these signaling pathways through differential regulation of MPK38 activity. Furthermore, overexpression of Smads2/3/4 improves, whereas Smad7 overexpression worsens, obesity-associated metabolic parameters by differentially regulating MPK38 activity in HFD-induced obese mice.7, we reasoned that Smad proteins would affect MPK38 activity through direct interaction and phosphorylation. To assess this possibility, we performed immunoblot analysis using CRISPR/Cas9-mediated Smad knock-in (KI) HEK293 cells , which are defective in MPK38-mediated phosphorylation7, to determine the endogenous kinase activity of MPK38 in the presence or absence of ASK1/TGF-β/p53 signals, including H2O2, TGF-β1, and 5-fluorouracil (5FU). The endogenous kinase activity of MPK38 was markedly lower in the S245A, S204A, and S343A KI cells when compared with wild-type control cells, whereas T96A KI cells had higher MPK38 kinase activity  on MPK38 protein stability. HEK293 cells were transfected with expression vectors encoding wild-type and MPK38-mediated phosphorylation-defective Smad mutants, and MPK38 protein levels were quantified using immunoblot analysis. As shown in Fig. 24. HEK293 cells were transfected with expression vectors for wild-type and MPK38-mediated phosphorylation-defective Smad mutants. Smads2/3/4 markedly decreased MPK38-Mdm2 complex formation in a MPK38 phosphorylation-dependent manner, whereas Smad7 increased complex formation -expressing adenovirus (Ad-GFP)  Fig. . The mRNFP) Fig. .Fig. 5SpFasting blood glucose was lower in HFD-fed mice infected with Ad-Smads2/3/4 than in uninfected HFD-fed mice Fig.  and Ad-S27. Therefore, we also examined the expression of proinflammatory genes in blood samples. Ad-Smads2/3/4 infection considerably decreased serum proinflammatory proteins when compared with uninfected HFD-fed mice, whereas Ad-Smad7 infection increased the serum levels of proinflammatory proteins , sterol CoA desaturase 1, and sterol regulatory element-binding transcription factor 1c, consistent with lower circulating free fatty acid levels, whereas Ad-Smad7 infection had the opposite effect in both tissues Fig. . Consistins Fig. . HoweverAd-Smads2/3/4 infections also increased the mRNA expression of adipose lipolytic genes, including hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and beta-3 adrenergic receptor (ADRB3), in a MPK38 phosphorylation-dependent manner, whereas Ad-Smad7 infection decreased the expression of these genes  by which Smad proteins differentially regulate MPK38 activity, we also found that they modify the stability and ubiquitination of MPK38. However, such effects were not detected in MPK38-mediated phosphorylation-defective Smad mutants, indicating that Smad phosphorylation by MPK38 is required for these regulatory effects  was from Sigma-Aldrich. All other chemicals used was described23. Cell culture and isolation of hepatocytes and adipocytes were also described23.Antibodies and plasmids used for experiments have been described previously23. Briefly, single-guide (sg) RNAs were designed to target the genomic areas adjacent to the Smad mutation sites -α, IL-1β, IL-6, and monocyte chemoattractant protein 1 (MCP1) were determined using analysis kits from Peprotech (ADI-900-047), RayBio (ELM-IL1beta-001), Invitrogen (KMC0061), and R&D Systems (MJE00), respectively. Other blood metabolic parameters were measured as described previously8.Plasma glucose levels for GTTs/ITTs were measured using blood obtained from the tail vein at 0, 10, 20, 30, 45, 60, 90, and 120 min post-injection according to the Mouse Metabolic Phenotyping Center recommendations for data presentation33. Total ketone levels were measured using a colorimetric assay from Wako Chemicals8. Fatty acid oxidation rate was determined using [1-14C] palmitic acid, as described previously8.Serum insulin and glucose levels, liver triglyceride content, and 2-deoxy-glucose uptake in epididymal WAT and soleus muscles were measured as described previously34. To prepare adenoviruses expressing wild-type and MPK38 phosphorylation-defective Smad mutants, FLAG-tagged wild-type and mutant Smad plasmids were used as templates for PCR with the primers shown in Supplementary Table 9 plaque-forming units) were directly injected into the tail vein or epididymal fat pads of 12- to 14-week-old HFD-fed mice.Four-week-old male C57BL/6 mice purchased from Orient  were fed a HFD  for 8–10 weeks under a 12:12-h light:dark cycle. Male 12- to 14-week-old C57BL/6 mice fed with a standard rodent diet were used as controls. All procedures were performed in a specific pathogen-free facility at Chungbuk National University, in accordance with the approved animal protocols and the guidelines established by the Ethics Review Committee of Chungbuk National University for Animal Experiments (CBNUA-966-16-02). Recombinant adenoviruses were generated as previously describedData are expressed as mean ± standard error and are representative of at least three independent experiments. Statistical significance was determined by one-way or two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparison test, using GraphPad Prism software (GraphPad Software).Supplemental Information


Arabidopsis. In this study, we showed that SGS3 from Nicotiana benthamiana (NbSGS3) is required for sense-RNA induced post-transcriptional gene silencing (S-PTGS) and initiating sense-RNA-triggered systemic silencing. Further, the deficiency of NbSGS3 inhibited geminivirus-induced endogenous gene silencing (GIEGS) and promoted geminivirus infection. During TRV-mediated NbSGS3 or N. benthamiana RDR6 (NbRDR6) silencing process, we found that their expression can be effectively fine-tuned. Plants with the knock-down of both NbSGS3 and NbRDR6 almost totally blocked GIEGS, and were more susceptible to geminivirus infection. These data suggest that NbSGS3 cooperates with NbRDR6 against GIEGS and geminivirus infection in N. benthamiana, which provides valuable information for breeding geminivirus-resistant plants.RNA silencing has an important role in defending against virus infection in plants. Plants with the deficiency of RNA silencing components often show enhanced susceptibility to viral infections. RNA-dependent RNA polymerase (RDRs) mediated-antiviral defense has a pivotal role in resistance to many plant viruses. In RDR6-mediated defense against viral infection, a plant-specific RNA binding protein, Suppressor of Gene Silencing 3 (SGS3), was also found to fight against some viruses in  Arabidopsis  labeled GFP probes using the Prime-a-Gene® Labeling System . Hybridization signals were detected with a Typhoon 9200 imager . For siRNA blotting and RT-qPCR analysis, the procedures have been described previously [Total RNA was isolated from virus-infected plants and different plant organs using Trizol reagent . For Northern analyses of GFP mRNA, total RNA was separated by electrophoresis on a 1.5% formaldehyde gel, transferred to a Hybond-Neviously .N. benthamiana plants) or from the newly-emerged upper leaves of 16c N. benthamiana plants infected by TRV-VIGS as described previously [Total protein was extracted from infiltrated leaf patches (wild type or 16c eviously . ImmunobN. benthamiana 16c leaves was performed. Agrobacterium tumefaciens cultures harboring a binary vector capable of expressing a sense GFP mRNA (35S:GFP) and a hairpin RNAi construct containing NbSGS3 (dsSGS3) or NbRDR6 (dsRDR6) sequences under control of the 35S promoter of cauliflower mosaic virus (CaMV), were co-infiltrated into leaves of 16c plants. Agroinfiltration of 16c leaves with 35S:GFP and an empty vector (Vec) induced GFP RNA silencing, and led to reduced GFP fluorescence under UV light at 5 days post infiltration (dpi)  and empty vector were co-infiltrated into wild type (Wt), dsSGS3 and dsRDR6 transgenic N. benthamiana leaves. Leaves co-infiltrated with 35S:GFP, 35S:FP and p19 served as positive control. The IR-PTGS suppression assay was designed in a similar manner to that of S-PTGS, except that 35S:dsFP (dsRNA of FP) substituted for 35S:FP as the silencing trigger. As shown in N. benthamiana was silenced by expression of either the sense RNA trigger (35:FP) or the dsRNA inducer (35S:dsFP). Similar to the experiments shown in NbSGS3 or NbRDR6 (due to expression of dsSGS3 or dsRDR6) suppressed the GFP silencing triggered by 35S:FP, leading to bright green fluorescence in co-infiltrated areas of leaves -PTGS were investigated. For S-PTGS, f leaves C. The prf leaves D,E. GFP-P siRNAs D and secP siRNAs E during P siRNAs E. These Agrobacterium cultures harboring 35S:GFP were infiltrated into N. benthamiana line 16c and observed at 6 dpi for efficient induction of RNA silencing. Plants were then further agroinfiltrated with recombinant tobacco rattle virus (TRV) vectors carrying partial fragments of NbSGS3 or NbRDR6. Knockdown of NbSGS3 or NbRDR6 was verified by RT-qPCR, which showed an approximate 80% reduction in the respective mRNA levels as compared to TRV-GUS-treated plants at 7 dpi  as the helper virus in association with a geminivirus alphasatellite derivative (2mDNA1) [NbSu in plants inoculated with 10A + 2mDNA1-NbSu, displaying a yellow leaf color phenotype as a result of the inhibition of chlorophyll II biosynthesis. Typical phenotypes associated with Su-silencing developed in mock or TRV-GUS-treated plants, but not in NbSGS3 or NbRDR6-silenced plants at 30 days post inoculation (dpi) (NbSu mRNA in mock or TRV-GUS-treated plants silenced for NbSGS3 or NbRDR6 were measured using RT-qPCR. The data showed that expression of NbSu mRNA in mock or TRV-GUS-treated plants was less than 23% of levels detected in non-silenced control plants (infected by 10A + 2mDNA1)  . This leon (dpi) A. To fur 2mDNA1) B. Howevel plants B. It is d plants C. This iN. benthamiana plants which either overexpressed (35S:SGS3) or silenced NbSGS3 (35S:dsSGS3) were generated by Agrobacterium-mediated leaf disc transformation. Although transcription levels of NbSGS3 varied in individual plants, the variation did not cause any detectable phenotypic changes  or NbRDR6 (dsRDR6) as compared to Wt plants. In contrast, less than 50% viral DNA accumulations of Wt plants were detected in transgenic plants overexpressing NbSGS3 (35S:NbSGS3). Based on the observation that overexpression of NbSGS3 alleviated geminivirus symptoms and deficiency of NbSGS3 promoted viral infection, we concluded that NbSGS3 plays an important role in host defense against geminivirus infection.To determine whether NbSGS3 has a general role in defense against geminivirus infection, transgenic  changes . TYLCCNVt plants D–F. Soutt plants G–I and rt plants J–L showeNbSGS3 and NbRDR6 mRNA levels in silenced plants showed that when NbSGS3 expression was knocked down, expression of NbRDR6 was unexpectedly enhanced. Similarly, expression of NbSGS3 was higher when NbRDR6 was down-regulated (RDR6 (dsRDR6), and subsequently inoculated with 10A + 2mDNA1-NbSu. Relatively efficient silencing of NbSGS3 and NbRDR6 was observed  induced much more severe symptoms and accumulated higher levels of viral DNA than that in mock, NbSGS3 or NbRDR6-silenced plants  or TVCV [Glycine max SGS3 (GmSGS3)-silenced soybean than non-silenced plants [SGS3 [Arabidopsis or wild type CMV oilseed rape was negatively related to the expression of SGS3 [Plants with the knock-out of f NbSGS3  were mor or TVCV ,18,19. B or TVCV . Therefod plants , suggestd plants . The potts [SGS3 ,43, sugg of SGS3 ,18,19, sTaken together, the diversity of SGS3-mediated resistance reflects the complexity and diversity of RNA silencing pathways, and these pathways may be more complex and connected when dealing with viruses with VSRs. However, SGS3-mediated S-PTGS and resistance to geminiviruses are evidence-based, which will provide valuable strategy for breeding geminivirus-resistant plants.