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      Fighting against Skin Aging : The Way from Bench to Bedside

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          Abstract

          As the most voluminous organ of the body that is exposed to the outer environment, the skin suffers from both intrinsic and extrinsic aging factors. Skin aging is characterized by features such as wrinkling, loss of elasticity, laxity, and rough-textured appearance. This aging process is accompanied with phenotypic changes in cutaneous cells as well as structural and functional changes in extracellular matrix components such as collagens and elastin. In this review, we summarize these changes in skin aging, research advances of the molecular mechanisms leading to these changes, and the treatment strategies aimed at preventing or reversing skin aging.

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          Inflamm-aging. An evolutionary perspective on immunosenescence.

          In this paper we extend the "network theory of aging," and we argue that a global reduction in the capacity to cope with a variety of stressors and a concomitant progressive increase in proinflammatory status are major characteristics of the aging process. This phenomenon, which we will refer to as "inflamm-aging," is provoked by a continuous antigenic load and stress. On the basis of evolutionary studies, we also argue that the immune and the stress responses are equivalent and that antigens are nothing other than particular types of stressors. We also propose to return macrophage to its rightful place as central actor not only in the inflammatory response and immunity, but also in the stress response. The rate of reaching the threshold of proinflammatory status over which diseases/disabilities ensue and the individual capacity to cope with and adapt to stressors are assumed to be complex traits with a genetic component. Finally, we argue that the persistence of inflammatory stimuli over time represents the biologic background (first hit) favoring the susceptibility to age-related diseases/disabilities. A second hit (absence of robust gene variants and/or presence of frail gene variants) is likely necessary to develop overt organ-specific age-related diseases having an inflammatory pathogenesis, such as atherosclerosis, Alzheimer's disease, osteoporosis, and diabetes. Following this perspective, several paradoxes of healthy centenarians (increase of plasma levels of inflammatory cytokines, acute phase proteins, and coagulation factors) are illustrated and explained. In conclusion, the beneficial effects of inflammation devoted to the neutralization of dangerous/harmful agents early in life and in adulthood become detrimental late in life in a period largely not foreseen by evolution, according to the antagonistic pleiotropy theory of aging.
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            Pathophysiology of premature skin aging induced by ultraviolet light.

            Long-term exposure to ultraviolet irradiation from sunlight causes premature skin aging (photoaging), characterized in part by wrinkles, altered pigmentation, and loss of skin tone. Photoaged skin displays prominent alterations in the collagenous extracellular matrix of connective tissue. We investigated the role of matrix-degrading metalloproteinases, a family of proteolytic enzymes, as mediators of collagen damage in photoaging. We studied 59 whites (33 men and 26 women, ranging in age from 21 to 58 years) with light-to-moderate skin pigmentation, none of whom had current or prior skin disease. Only some of the participants were included in each of the studies. We irradiated their buttock skin with fluorescent ultraviolet lights under standard conditions and obtained skin samples from irradiated and nonirradiated areas by keratome or punch biopsy. In some studies, tretinoin and its vehicle were applied to skin under occlusion 48 hours before ultraviolet irradiation. The expression of matrix metalloproteinases was determined by in situ hybridization, immunohistology, and in situ zymography. Irradiation-induced degradation of skin collagen was measured by radioimmunoassay of soluble cross-linked telopeptides. The protein level of tissue inhibitor of matrix metalloproteinases type 1 was determined by Western blot analysis. A single exposure to ultraviolet irradiation increased the expression of three matrix metalloproteinases -- collagenase, a 92-kd gelatinase, and stromelysin -- in skin connective tissue and outer skin layers, as compared with nonirradiated skin. The degradation of endogenous type I collagen fibrils was increased by 58 percent in irradiated skin, as compared with nonirradiated skin. Collagenase and gelatinase activity remained maximally elevated (4.4 and 2.3 times, respectively) for seven days with four exposures to ultraviolet irradiation, delivered at two-day intervals, as compared with base-line levels. Pretreatment of skin with tretinoin (all-trans-retinoic acid) inhibited the induction of matrix metalloproteinase proteins and activity (by 70 to 80 percent) in both connective tissue and outer layers of irradiated skin. Ultraviolet irradiation also induced tissue inhibitor of matrix metalloproteinases-1, which regulates the enzyme. Induction of the inhibitor was not affected by tretinoin. Multiple exposures to ultraviolet irradiation lead to sustained elevations of matrix metalloproteinases that degrade skin collagen and may contribute to photoaging. Treatment with topical tretinoin inhibits irradiation-induced matrix metalloproteinases but not their endogenous inhibitor.
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              A Mitochondrial Superoxide Signal Triggers Increased Longevity in Caenorhabditis elegans

              Introduction Mitochondrial function has been linked to the aging process in a number of ways [1]. In particular, mitochondria are crucial in energy metabolism and as such have been implicated in the aging process by one of the very first theories of aging [2], the rate-of-living theory of aging [3], which suggested that the rate of aging is proportional to the rate of energy metabolism (reviewed in [4]). Mitochondrial function in animals is also known to decline with age [5],[6], which, together with the finding that mitochondria are an important source of toxic reactive oxygen species (ROS), has led to the oxidative stress (or free radical) theory of aging [7],[8]. Two types of mutations that affect mitochondrial function have been found to affect the rate of aging in C. elegans, mutations that shorten lifespan, such as mev-1 [9] and gas-1 [10], and mutations that lengthen lifespan, such as clk-1 [11], isp-1 [12], lrs-2 [13], and nuo-6 [14]. lrs-2 encodes a mitochondrial leucyl-tRNA-synthetase, and its effect on the function of mitochondrial electron transport is likely relatively indirect, via partial impairment of mitochondrial translation. However, clk-1 encodes an enzyme necessary for the biosynthesis of ubiquinone, a lipid antioxidant and an electron transporter of the respiratory chain [15], and mev-1, gas-1, isp-1, and nuo-6 all encode subunits of mitochondrial respiratory complexes. On the strength of the oxidative stress theory of aging it has been suggested, and supported by a number of observations (reviewed in [16],[17]), that the mev-1 and gas-1 mutations reduce lifespan by increasing mitochondrial oxidative stress, and clk-1, isp-1, and nuo-6 increase lifespan by reducing it. In addition to genomic mutations that affect mitochondrial proteins, it has been found that knockdown by RNA interference of C. elegans genes that encode subunits of mitochondrial complexes, including isp-1 and nuo-6, also prolongs lifespan [13],[18],[19]. Although the effect of RNAi on ETC subunits, which is conserved in Drosophila [20], was initially believed to be similar to that of the mutations [21],[22],[23], it was recently found that it is in fact distinct and separable [14]. A recent study analyzed patterns of gene expression in isp-1 mutants together with those in clk-1 and cyc-1(RNAi) [23] and suggested that the overlap between these patterns could define the biochemical processes that underlie the effect of all interventions that impact mitochondria. However, our recent findings that isp-1(qm150) and isp-1(RNAi) trigger fully separable mechanisms suggests that the overlapping gene expression changes identified by Cristina et al. [23] might not be sufficient to prolong lifespan. Rather some of the gene expression changes that are specific to each type of intervention are necessary for their effect on lifespan and can act additively. isp-1 mutants show a trend toward low levels of oxidative damage to proteins, increased expression of the cytoplasmic Cu/Zn superoxide dismutase (SOD-1) and of the mitochondrial Mn superoxide dismutase (SOD-2) [24], and increased resistance to acute treatment with the prooxidant paraquat [14]. However, although knocking down the genes encoding the major superoxide dismutase by RNAi results in normal or elevated levels of oxidative damage, it had no effect on the lifespan of the mutants [24], suggesting that the reduced oxidative damage found in isp-1 mutants is not responsible for their longevity. Furthermore, the notion that mitochondrial oxidative stress could be the cause of aging has recently been challenged by a number of studies in C. elegans [24],[25],[26],[27],[28], in Drosophila [29], and in mice (reviewed in [30]). ROS are not just toxic metabolites that lead to oxidative stress but are also signaling molecules that are believed to be involved in a mitochondria-to-nucleus signaling pathway that could impact aging [1],[31],[32],[33]. Interfering with mitochondrial function has the potential to alter the rate and/or the pattern of production of ROS by mitochondria, including in counter-intuitive ways. For example, reducing oxygen concentration increases ROS production by mitochondrial complex III in vertebrate cells [34],[35], and the knockout of sod-2 in C. elegans can lead to normal [25] or increased lifespan in spite of increased oxidative damage [26]. Here we examined ROS production by mitochondrial mutants and found that isp-1 and nuo-6 mutants have increased generation of the superoxide anion but not increased levels of other ROS and that this increase is necessary and sufficient for longevity, suggesting that superoxide triggers mechanisms that slow down aging, presumably at the level of gene expression. Results isp-1, nuo-6, and daf-2 Mutant Mitochondria Display Elevated Generation of Superoxide But Not of Overall ROS To measure changes in mitochondrial ROS generation that could affect signaling, it is not adequate to measure the level of ROS damage, as a change in ROS damage levels can be brought about by changes in detoxification of ROS, in protein turnover, or in damage repair. However, it is notoriously difficult to directly visualize or measure ROS generation and ROS levels in intact organisms including in living worms. To overcome this difficulty we have adapted a technique originally developed for vertebrates that uses flow cytometry to sort isolated intact mitochondria and measure ROS levels with indicator dyes (Figure S1) [37]. Mitochondria were extracted from worms by standard techniques and loaded with either one of two fluorescent indicator dyes, H2DCFDA, a dye that is sensitive to a variety of ROS but rather insensitive to superoxide [38],[39], and MitoSox, a dye that is exclusively sensitive to superoxide [40]. The prooxidant paraquat (PQ) induces mitochondrial superoxide generation [41], and the antioxidant N-acetyl-cysteine (NAC) has an antioxidant effect on all types of ROS [42],[43]. As expected, when purified mitochondria were treated with PQ, the fluorescence of both H2DCFDA and MitoSox increased, and the fluorescence of both decreased when treated with NAC (Figures 1A, 1B, and S1B). One limitation of this technique is the need for a rather large amount of mitochondria. For example, a sufficient amount of worms is not readily obtained from worms treated by RNAi, and we have therefore focused on long-lived mutants only. 10.1371/journal.pbio.1000556.g001 Figure 1 Reactive oxygen species (ROS) in isolated mitochondria of long-lived mutants and in response to paraquat (PQ) and N-acetyl-cysteine (NAC) treatment. Global ROS levels were measured by quantifying the fluorescence of the reporter dye H2DCFDA, and superoxide with the dye MitoSox, in FACS-sorted mitochondria. Values are normalized to the value of the untreated sample or the wild type. PQ and NAC, respectively, increase and decrease the levels of both global ROS (A) and superoxide (B). Mitochondria isolated from isp-1(qm150) and nuo-6(qm200) mutants show slightly decreased global ROS generation (C) but significantly increased superoxide generation (D). Mitochondria from clk-1 mutants, but not from daf-2(e1370), eat-2(ad1116), and sod-2(ok1030) mutants, show significantly increased global ROS levels (E). Mitochondria from daf-2 mutants, but not from clk-1, eat-2, and sod-2 mutants, show increased superoxide levels (F). * p 10–15 mM) NAC can be deleterious even on the wild type, but at the concentration used (10 mM) NAC had no effect on the apparent health of the mutants, whose overall aspect after treatment was indistinguishable from that of the untreated worms (Figure S2A). We have also quantified several phenotypes, including defecation, swimming, brood size, and post-embryonic development, after NAC treatment of the wild type and of nuo-6, which is the mutant that is most sensitive to NAC (10 mM NAC completely abolishes its increased longevity). Treatment with 1 mM vitamin C also significantly shortened the lifespan of both isp-1 and nuo-6 mutants without affecting the wild type (Table S1). Most effects of NAC were quite small (Figure S2B–E), except on the post-embryonic development of the wild type (Figure S2C). Furthermore, for defecation, brood size, and post-embryonic development, the effect of NAC on the mutant produced a change in the same direction as on the wild type but of a lesser extent. Only for swimming is the effect greater on the mutant. But the effect consists of swimming faster after NAC treatment and thus bringing the mutant phenotype closer to the wild-type. We conclude that there is little evidence of an indirect deleterious effect of NAC. 10.1371/journal.pbio.1000556.g002 Figure 2 Lifespans of wild-type animals and mutants treated with 10 mM NAC. The treatment has no effect on wild type (A) but dramatically suppresses the lifespan extension of the respiratory chain subunit mutants nuo-6 (B) and isp-1 (C). In contrast, it has no lifespan-shortening effects on the long-lived clk-1 mutants (D). NAC treatment has only a moderate effect on the very long-lived daf-2 mutants (E) but also completely abolished the extended longevity of sod-2 mutants (F). See Tables 1 and S1 for details of genotype, sample size, and statistical analysis. 10.1371/journal.pbio.1000556.t001 Table 1 Longevity after paraquat and NAC treatment. Control PQ NAC Mean ± SD Sample Size Maximum Lifespan Mean ± SD Sample Size Maximum Lifespan % Change p Value Mean ± SD Sample Size Maximum Lifespan % Change p Value Wild Type (N2) 18.4±3.5 29 29.0±5.1 43 +58% 20.2±3.2 30 +10% (n = 400) (n = 200) p 0.2 mM) is severely deleterious. We thus first tested the ability of PQ to increase ROS damage in the animals at a very low concentration (0.1 mM). We found that this treatment indeed measurably increased the level of oxidative damage to proteins at the young adult stage as assessed by determination of protein carbonylation (Figure 3A) and increased the expression of both the main cytoplasmic (SOD-1) and the main mitochondrial (SOD-2) superoxide dismutases (Figure 3B and 3C). We then tested whether PQ could increase the lifespan of the wild type at three different concentrations (0.05, 0.1, and 0.2 mM) and found that at all three concentrations both the mean and maximum lifespan were increased, with a maximal effect at 0.1 mM (Figures 3D and 4A, and Tables 1 and S1). The effect of 0.2 mM was less pronounced than that of 0.1 mM and similar to that of 0.05 mM, likely because at 0.2 mM a toxic effect starts to balance the pro-longevity effect. The effect does not depend on the exact chemical structure of paraquat, as benzyl-viologen, a compound with similar activity as PQ but structurally different, also increases lifespan (Table S1). A small effect of the prooxidant juglone under different conditions has also been documented previously [56]. The effect did not depend on an effect of PQ on the E. coli (OP50) food source, as the effect was also observed with heat-killed cells (Table S1). Finally, the effect was not confined to development or adulthood as PQ prolongs lifespan whether provided only during adult lifespan or only during development (Table S1). 10.1371/journal.pbio.1000556.g003 Figure 3 Treatment with 0.1 mM of paraquat (PQ) increases protein oxidative damage, superoxide dismutase expression, and lifespan. (A) Young wild type adults treated with 0.1 mM PQ have higher protein oxidative damage compared to untreated control. (B) Young wild type adults treated with 0.1 mM PQ since hatching express significantly more SOD-1 protein than untreated animals. (C) Young wild type adults treated with 0.1 mM paraquat since hatching express significantly more SOD-2 protein compared to untreated wild type worms. (D) Treatment with 0.05, 0.1, or 0.2 mM PQ increases mean and maximum lifespan significantly (see also Tables 1 and S1). * 15, and it was 5 for muscles and the germline. In muscles and in the hypodermis, the difference between the ETC mutants and the wild type was significant (p<0.001), while the difference between PQ treatment and untreated wild type worms was not. Thus, the nuo-6 and isp-1 mutations, but not treatment with 0.1 mM PQ, affect mitochondrial abundance. PQ Is Able to Considerably Lengthen the Lifespan of a Variety of Mutants That Define Genetic Pathways of Aging daf-2 mutants have elevated superoxide levels, and they are sensitive to NAC (lifespan shortening by 15%). However, the level of superoxide in daf-2 appears not to be sufficient for a maximal effect as these mutants remain somewhat sensitive to PQ (lifespan lengthening by 9%). To further study how superoxide plays a role in the lifespan of daf-2 we studied genes that function downstream of daf-2. At least three genes are known to be required for the full lifespan extension of daf-2, that is, daf-16, aak-2, and hsf-1 [62],[63],[64]. If one of these genes were necessary for an activity that mediates the small effect of PQ on daf-2 mutants, PQ should not be able to prolong the lifespan of mutants of such a gene. In fact, however, we found that PQ prolonged the lifespan of all three mutants tested (Table 1). The lifespan increase upon PQ treatment of daf-16 (35% increase) and aak-2 (29% increase) is not as large as upon treatment of the wild type (58% increase). This suggests that part but not all of the lifespan increase determined by superoxide requires daf-16 and aak-2. These findings are consistent with the observations that the lifespan extension provided by nuo-6 and daf-2(e1370) are only partially additive (Table S1), similarly to what was found previously for isp-1 and daf-2 [12], and that elimination of daf-16 partially shortens the lifespan of isp-1 [12]. We also tested the sensitivity to PQ of mutants that are diagnostic of a variety of pathways of aging. In particular mutants of genes that, based on their known functions in C. elegans or that of their homologues in other systems, might encode the targets of superoxide signaling or be otherwise necessary for implementing superoxide signaling. The c-Jun N-terminal kinase 1 (JNK-1) is involved in stress responses in vertebrate cells and is a positive regulator of DAF-16 that acts in parallel to the effect of daf-2 on daf-16 [65]. We treated jnk-1(gk7) mutants with PQ and obtained a particularly large lifespan increase (Table 1). Although it is not clear what activities lie upstream of jnk-1 in C. elegans nor whether it has other targets than daf-16, its activity does not appear necessary for the effect of superoxide. The transcription factor SKN-1 defends against oxidative stress by mobilizing the conserved phase II detoxification response and can delay aging independently of DAF-16 [66]. Although PQ induces oxidative stress and induces enzymes that protect from oxidative stress (Figure 3), it was still able to prolong the lifespan of skn-1(zn67) mutants (Table 1), indicating that skn-1 does not act downstream of superoxide. wwp-1 encodes a conserved E3 ubiquitin ligase that is necessary for lifespan extension by dietary restriction [67]. Treatment of wwp-1(ok1102) with PQ prolonged lifespan of these mutants, which is consistent with our finding that PQ can considerably extend the lifespan of eat-2 mutants (Figure 4E). This confirmed that the lifespan increase produced by the superoxide increase in mitochondrial mutants is distinct from the mechanisms that support the lifespan effects of dietary restriction [14]. hif-1 encodes a worm homologue of the vertebrate hypoxia inducible factor 1α (HIF-1α), a transcription factor involved in a number of protective mechanisms. In C. elegans hif-1 is necessary for a lifespan pathway that involves proteolysis and that is distinct from insulin signaling [68] and has also been involved in the dietary restriction pathway [69]. In vertebrates HIF-1α is positively regulated by mitochondrial ROS [34],[35], which would make it an interesting candidate to mediate the effects of superoxide. However, PQ was fully capable of increasing the lifespan of the hif-1 mutants (Table 1). Several of the genes whose mutants remain sensitive to PQ, including daf-16, have been involved in stress responses, including oxidative stress, yet they do not seem necessary for the effect of PQ. Similarly we have shown previously that although the expression of SOD-1 and SOD-2 are elevated in isp-1(qm150) mutants, the elevation is not necessary for the extended lifespan of these mutants [24]. nuo-6(qm200) mutants also show elevated SOD-1 and SOD-2 expression [14], but this too is unnecessary for the longevity of the mutants, as RNAi against sod-1 an sod-2, which we have shown to be efficient in reducing enzyme levels [24], does not shorten the lifespan of nuo-6 mutants (Figure S5). We conclude that the mitochondrial mutants protect from an aspect of the aging process that has not yet been studied through mutants that affect stress. In addition, our observations suggest that the lifespan effect we observed is not hormetic, as neither superoxide-detoxifying enzymes, nor the regulatory factors that are involved in protection from oxidative stress, are crucially implicated. Discussion We have shown previously that mutations in isp-1 and nuo-6 prolong lifespan by a common mechanism [14]. Using direct measurement of ROS and superoxide we find here that this mechanism involves an increase in mitochondrial superoxide generation that is necessary and sufficient for the longevity of these mutants. As ROS, including superoxide [70],[71],[72], are known to be intracellular messengers, the increased superoxide might trigger a signal transduction pathway that ultimately results in changes in nuclear gene expression [23]. Superoxide is highly reactive and could trigger such a signal by modifying proteins in the mitochondria or in the nearby cytosol after having escaped from the mitochondria through an appropriate channel [73],[74]. Although no superoxide sensor has yet been identified, a similar type of mechanism, in which a highly reactive, quickly diffusing, molecule modifies a signal transduction protein, has been evidenced for nitric oxide (NO), which covalently and permanently modifies guanylyl cyclases. Similarly, hydrogen peroxide (H2O2), the product of superoxide dismutation, can inactivate phosphatases involved in signal transduction. Future work will aim at using forward and reverse genetic screens in C. elegans to uncover the molecular machinery that reacts to the superoxide signal, as well as the transcription factors that are needed to regulate nuclear gene expression in response to the pathway's activation. In addition, the pattern of gene expression that results in increased lifespan in these mutants could be defined very specifically by identifying changes in the gene expression patterns that are common to isp-1, nuo-6, and PQ treatment and that are suppressed by treatment with NAC. A number of studies in C. elegans have explored hormesis by treating animals with sub-lethal but clearly deleterious treatments for a short period of time and observing subsequent prolongation of lifespan [75]. These hormetic effects are different from what we have observed and describe here, as both the genetic mutations and the very low level PQ are present throughout life and as only a part of the effect we observe might require the insulin signaling pathway. Furthermore, although in nuo-6 and isp-1 mutants the expression levels of the superoxide dismutases SOD-1 and SOD-2 are elevated, likely in response to the elevated superoxide generation, and as one expects in the hormetic response, these elevations are not necessary for the lifespan prolongation of nuo-6 (Figure S5) or isp-1 [24]. CLK-1 is a mitochondrial protein that is required for ubiquinone biosynthesis and its absence affects mitochondrial function [50], although it could potentially affect many other processes as ubiquinone is found in all membranes. Furthermore, ubiquinone is both a prooxidant as co-factor in the respiratory chain and an anti-oxidant. Interestingly, the mechanism of lifespan prolongation induced by clk-1 appears to be entirely distinct from, but particularly synergistic with, that induced by elevated superoxide. Indeed, clk-1 mutants do not show elevated superoxide generation and are not affected by NAC. Furthermore, although double mutant combinations of clk-1 with nuo-6 and isp-1 are not viable (unpublished data) the lifespans of clk-1 mutants treated with PQ (Figure 4D), or of sod-2;clk-1 mutants [26], or of clk-1;daf-2 mutants [76] are much greater than expected from simple additivity of the effects of individual mutations or treatments. Studies in yeast [77] and in worms [78] have suggested that an increase in ROS from mitochondria might also be important in triggering the lifespan extension produced by glucose restriction. However, our results here with an eat-2 mutation, one of the ways in which global dietary restriction can be produced in worms, as well as with a wwp-1 and hif-1, which may function downstream of dietary restriction, did not reveal an involvement of superoxide signaling, providing further evidence for a distinction between the mechanisms of glucose restriction and dietary restriction. It remains possible, however, that DR could lead to superoxide or ROS production when it is induced by other methods than the use of an eat-2 mutant, as it is well documented that different types of DR induce different molecular mechanisms [79]. One question that our current experiments do not address is whether the mitochondrial dysfunction in the mutants, or the effect of PQ, is necessary in every tissue in order to increase longevity. There are indications for both the insulin signaling pathway mutants [80],[81] and dietary restriction [67],[82] that the entire effect might be mediated by action in particular cells that influence the physiology of the whole organism. Similarly, the presence or absence of the germline is sufficient for a dramatic effect on lifespan [83]. For mitochondrial dysfunction the question could be addressed in the future by mosaic analysis and by purifying and analyzing mitochondria from specific tissues using our flow cytometry technique to purify mitochondria expressing GFP in a tissue-specific manner. The oxidative stress theory of aging has been one of the most acknowledged theories of aging for the simple reason of the strikingly good correlation between the levels of oxidative stress and the aged phenotype [8]. A number of recent results in worms and in mice, however, have suggested that oxidative stress cannot be the cause of aging [24],[25],[26],[30]. Our findings suggest a conceptual framework for why oxidative stress and the aged phenotype are so tightly correlated [31]. In this model mitochondria, like the rest of the cell, sustain a variety of age-dependent insults (not only and not even principally from oxidative stress) that trigger an increase in superoxide, which acts as a signal that induces general protective and repair mechanisms. However, aging in most animals is clearly irreversible, indicating that the protective mechanisms, which must have evolved to control damage in young organisms, are unable to fully prevent the accumulation of age-related damage. Thus, as superoxide is a reactive molecule as well as a signal, and as age-dependent damage cannot be fully reversed, it is possible that at high ages the chronically elevated superoxide will participate in creating some of the damage itself. This could explain the strong tendency for aged animals to have high oxidative stress and high oxidative damage, although it does not imply that ROS cause aging or even that they are a major source of age-dependent damage. In this model, the nuo-6 and isp-1 mutations lead to increased longevity because they turn on the stress signal prematurely and thus slow down the entire process. Materials and Methods Lifespan Scoring Eggs were placed on plates at 20°C and left for 1 h to hatch. Larvae that had hatched during that period were placed onto fresh plates and monitored once daily until death. The animals were transferred once daily while producing eggs to keep them separate from their progeny. Animals were scored as dead when they no longer responded with movement to light prodding on the head and tail. Missing worms and worms that have died because of internal hatching (bagging) were replaced from a backup group. Survival was scored every day. Drug Treatment Drugs were added into NGM media from a high concentration stock solution (500 mM for NAC, 1 M for PQ, and 500 mM for vitamin C) before pouring of the plates. Plates were made fresh each week. Gravid adult worms were transferred from normal NGM plates to drug plates and left to lay eggs for 3 h. With each transfer of worms a substantial amount of bacteria was also transferred onto the new plates. The progeny was then scored for different phenotypes. Staining and Confocal Imaging All dyes except MitoSox were diluted in DMSO at high concentration (all at 5 mM except H2DCFDA, which is at 10 mM) and frozen at −20°C as a stock. MitoSox was prepared fresh at 5 mM for each use. Before staining stocks were diluted in M9 buffer at a 1∶1000 dilution. Young adult worms were transferred into staining solution and stained for 20 min. Worms were mounted on a thick layer of half-dried agar pad on microscopic glass slides and then subjected to confocal microscopy (Zeiss LSM 510 Meta). Pictures were taken by Zeiss LSM Imaging software and analyzed by Volocity V4.0 software. Oxygen Consumption Five young adult worms (1st day of adulthood) were placed into 0.25 µl M9 buffer in a 0.5 µl sealed chamber at 22°C. A fiber optical oxygen sensor (AL300 FOXY probe from Ocean Optics) was inserted into this chamber and oxygen partial pressure was monitored for 15 to 30 min. Oxygen consumption measured in this way was normalized to body volume. For this worms were photographed at each measurement day under a binocular microscope and their cross-section was calculated with ImageJ software. Worm volume was determined by the formula: volume (nl)  = 1.849 • 10–7 (nl/µm3) • area 1.5 (µm3) [84]. Expression Levels of Superoxide Dismutases (SODs) After RNAi treatment, 100 young adult worms of each genotype were picked, lysed in 2× loading buffer, and subjected to electrophoresis in 12% SDS–polyacrylamide gels (SDS–PAGE), and then blotted onto nitrocellulose membrane (Bio-Rad). After applying primary antibody (1∶1000, rabbit polyclonal antibody against worm SOD-1 or SOD-2) and secondary antibody (1∶10,000 mouse anti-rabbit IgG, Invitrogen), the membranes were incubated with the ECL plus detection reagent (Amersham Biosciences) and scanned using a Typhoon trio plus scanner. Band densities were analyzed by ImageQuant TL V2003.03. Fluorescence Activated Cell Sorting For fluorescence activated cell sorting [37], adult worms grown on large NGM plates were collected and washed 3 times with M9 buffer. Worms were then suspended in 5× isolation buffer (200 mM mannitol; 120 mM sucrose; 10 mM Tris; 1 mM EGTA; pH 7.4) and set on ice. Worms were broken up with a 5 ml glass-glass homogenizer and centrifuged at 600 g for 10 min, the supernatant was collected and re-centrifuged at 7,800 g for 10 min, and the pellet was washed once with isolation buffer and then suspended in isolation buffer and kept on ice. Different dyes were added from stocks into the analysis buffer (250 mM sucrose; 20 mM MOPS; 100 uM KPi; 0.5 mM MgCl2; 1 uM CsA pH 7.0) at a 1∶1000 dilution before staining. 100 µl of mitochondria was added to 900 µl of analysis buffer with dye and substrate and incubated for 1 h at room temperature. Mitochondria were recollected by 7,800 g centrifugation and then suspended in 500 µl analysis buffer. A FACSCalibur instrument equipped with a 488 nm Argon laser and a 635 nm red diode laser (Becton Dickinson) was used. Data from the experiments were analyzed using the CellQuest software (Becton Dickinson). To exclude debris, samples were gated based on light-scattering properties in the SSC (side scatter) and FSC (forward scatter) modes, and 20,000 events per sample were collected, using the “low” setting for sample flow rate (Figure S1). ATP Content 200 age-synchronized young adult worms were collected in M9 buffer and washed three times. Worm pellets were treated with three freeze/thaw cycles and boiled for 15 min to release ATP and destroy ATPase activity, and then spun at 4°C and 11,000 g for 10 min. ATP contents were measured with a kit (Invitrogen, Carlsbad, California, USA; Cat: A22066). The ATP content value was then normalized to the soluble protein level of the same preparation, measured with the protein assay from Bio-Rad. Dyes Used for Staining and FACS Mitotracker green (Invitrogen M7514) stock concentration 5 mM; H2DCFDA (Invitrogen D399) stock concentration 10 mM; Mitotracker red (Invitrogen M7512) stock concentration 5 mM. Supporting Information Figure S1 Selection of isolated mitochondria and ROS-sensitive dye analysis. Mitochondria were prepared as described in Materials and Methods. (A) A FACSCalibur flow cytometry cell sorter from Becton Dickinson equipped with a 488 nm Argon laser and a 635 nm red diode laser was used. Data from the experiments were analyzed using the CellQuest software (Becton Dickinson). To exclude debris, samples were gated based on light-scattering properties in the SSC (side scatter) and FSC (forward scatter) modes, and 20,000 events per sample within the region (gate) delimitated by a square in (A) were collected, using the “low” setting for sample flow rate. 99% of the particles in that region successfully stained with the mitochondria-specific dye Mitotracker Green. (B) Isolated mitochondria were incubated with analysis buffer containing substrate (see Materials and Methods) and MitoSox (1 µM) at room temperature for 1 h and then sorted and the fluorescence of mitochondria in the gate measured. The purple area represents un-stained control. Paraquat (red line) was able to increase ROS generation over the untreated control (green line), while NAC (blue line) decreased the superoxide signal. Note that the x-axis shows a log scale. (C) Isolated mitochondria (see A) from wild-type worms were stained with both H2DCF-DA (the signal plotted on FL1-H; 530±15 nm channel) and Mitotracker Red (the signal plotted on FL3-H; ≥670 nm channel). When particles were stained by both dyes (upper-right region), the signals were strongly correlated. Furthermore, 89.6%±2.4% (n = 4) of the particles stained by H2DCF-DA were also stained by Mitotracker Red. (0.11 MB PDF) Click here for additional data file. Figure S2 Absence of deleterious effects of N-acetyl-cysteine. (A) NAC (N-acetyl-cysteine) had no effect on the apparent health of isp-1 or nuo-6 mutants. Mutant animals were treated or not with 10 mM NAC throughout their lives and all pictures in the panel were taken on day 23 of their lifespan, when less than 25% of untreated mutants but more than 75% of NAC-treated mutants had already died. NAC-treated isp-1 and nuo-6 mutants did not show any visible ill effects from the treatment. All worms are shown at the same magnification; scale bar is 0.5 mm. Phenotypes possibly resulting from NAC treatment of nuo-6(qm200) mutants were also quantified. We chose to examine nuo-6 mutants because their longevity was the most sensitive to NAC (completely suppressed at 10 mM). Adult worms were allowed to lay eggs on NAC plates and phenotypes of the resulting F1 progeny were scored. (B) NAC significantly decreased defecation cycle length of the wild type (p = 0.0104), while it has no significant effect on that of nuo-6(qm200) mutant (n = 15). (C) NAC significantly increased post-embryonic development length of both the wild type and nuo-6(qm200) mutants (n = 100). (D) NAC has no significant effect on brood size of both the wild type and nuo-6(qm200) mutants (n = 50). (E) NAC has no significant effect on the swimming rate (frequency of thrashing) of the wild type, but it significantly increased that of nuo-6(qm200) mutants (p = 0.0024) (n = 15). (0.06 MB PDF) Click here for additional data file. Figure S3 Co-localization of Mitotracker Red and GFP signals in C. elegans mitochondria. We used Mitotracker Red to stain worms carrying the transgene qmIs16[Pclk-1::clk-1::gfp], which expresses the mitochondrial protein CLK-1 fused to GFP [50]. Staining was as described in Materials and Methods. Worms were mounted on agar pads on slides and subjected to confocal microscopical analysis. (A) Mitotracker Red expression in hypodermal tissue. (B) The same region as in (A) expressing the mitochondrial GFP fusion. (C) The merged images of (A) and (B). The Mitotracker Red and GFP signals are co-localized. (0.10 MB PDF) Click here for additional data file. Figure S4 Effect of paraquat (PQ) and N-acetylcysteine (NAC) on energy metabolism. Untreated wild type controls and animals treated with 0.1 mM PQ or 10 mM NAC since hatching were collected at the first day of adulthood for both experiments. (A) Animals in groups of 5 (n≥3) were transferred in 0.25 µl M9 buffer into a 0.5 µl chamber where oxygen concentration was measured with a fiber optic oxygen sensor (AL300 FOXY probe from Ocean Optics) for 15–30 min. The body volume of animals was calculated from pictures of the same worms and used for normalization. PQ had a small but significant consumption-increasing effect only on nuo-6 mutants. NAC increased oxygen consumption in all three genotypes, with the largest effect on isp-1 mutants. (B) The ATP content from 200 worms was normalized to the amount of soluble protein from the same sample (n≥6). Both PQ and NAC treatment had no effect on ATP content with the exception of PQ-treated nuo-6mutants, in which the treatment suppressed the high ATP content that is observed in the untreated animals. For all statistic analyses we used the Student's t test.* p<0.05, ** p<0.01 and *** p<0.001. (0.06 MB PDF) Click here for additional data file. Figure S5 SOD-1 and SOD-2 are not necessary for the longevity of nuo-6(qm200) . Knocking down sod-1 (red) or sod-2 (blue) does not shorten the long lifespan of nuo-6(qm200) mutant. In fact silencing these two genes slightly increases the lifespan of nuo-6 mutants. Mean lifespan of control (empty vector) is 33 d (green), mean lifespan after sod-1 RNAi treatment is 35 d, and mean lifespan after sod-2 RNAi treatment is 36.5 d; p<0.05 for both RNAi experiments compared to control, analyzed by curve comparison using the log-rank test. (0.02 MB PDF) Click here for additional data file. Table S1 Individual aging experiments and statistics. (0.14 MB PDF) Click here for additional data file.
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                Author and article information

                Journal
                Cell Transplant
                Cell Transplant
                CLL
                spcll
                Cell Transplantation
                SAGE Publications (Sage CA: Los Angeles, CA )
                0963-6897
                1555-3892
                25 April 2018
                May 2018
                : 27
                : 5 , Special Issue: American Society for Neural Therapy and Repair (ASNTR) Part 2
                : 729-738
                Affiliations
                [1 ]Department of Histology and Embryology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, China
                [2 ]Central laboratory of Molecular and Cellular Biology, School of Basic Medical Sciences, Anhui Medical University, Hefei, Anhui, China
                [3 ]State Key Lab of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, China
                Author notes
                [*]Enkui Duan, State Key Lab of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, 1 Beichen West Road, Chaoyang, Beijing 100101, China. Email: duane@ 123456ioz.ac.cn
                Article
                10.1177_0963689717725755
                10.1177/0963689717725755
                6047276
                29692196
                95367b4a-ceb5-4e2a-b8b9-6a37c01d8e18
                © The Author(s) 2018

                This article is distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 License ( http://www.creativecommons.org/licenses/by-nc/4.0/) which permits non-commercial use, reproduction and distribution of the work without further permission provided the original work is attributed as specified on the SAGE and Open Access pages ( https://us.sagepub.com/en-us/nam/open-access-at-sage).

                History
                : 1 November 2016
                : 5 July 2017
                : 14 July 2017
                Categories
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                skin aging,intrinsic aging,extrinsic aging,extracellular matrix,treatment strategy

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