Temporal requirements of SKN-1/NRF as a regulator of lifespan and proteostasis in Caenorhabditis elegans

Danielle Grushko; Hana Boocholez; Amir Levine; Ehud Cohen

doi:10.1371/journal.pone.0243522

. 2021 Jul 1;16(7):e0243522. doi: 10.1371/journal.pone.0243522

Temporal requirements of SKN-1/NRF as a regulator of lifespan and proteostasis in Caenorhabditis elegans

Danielle Grushko ^1,^#, Hana Boocholez ^1,^#, Amir Levine ¹, Ehud Cohen ^1,^*

Editor: Jamuna Subramaniam²

PMCID: PMC8248617 PMID: 34197476

Abstract

Lowering the activity of the Insulin/IGF-1 Signaling (IIS) cascade results in elevated stress resistance, enhanced protein homeostasis (proteostasis) and extended lifespan of worms, flies and mice. In the nematode Caenorhabditis elegans (C. elegans), the longevity phenotype that stems from IIS reduction is entirely dependent upon the activities of a subset of transcription factors including the Forkhead factor DAF-16/FOXO (DAF-16), Heat Shock Factor-1 (HSF-1), SKiNhead/Nrf (SKN-1) and ParaQuat Methylviologen responsive (PQM-1). While DAF-16 determines lifespan exclusively during early adulthood and governs proteostasis in early adulthood and midlife, HSF-1 executes these functions foremost during development. Despite the central roles of SKN-1 as a regulator of lifespan and proteostasis, the temporal requirements of this transcription factor were unknown. Here we employed conditional knockdown techniques and discovered that in C. elegans, SKN-1 is primarily important for longevity and proteostasis during late larval development through early adulthood. Our findings indicate that events that occur during late larval developmental through early adulthood affect lifespan and proteostasis and suggest that subsequent to HSF-1, SKN-1 sets the conditions, partially overlapping temporally with DAF-16, that enable IIS reduction to promote longevity and proteostasis. Our findings raise the intriguing possibility that HSF-1, SKN-1 and DAF-16 function in a coordinated and sequential manner to promote healthy aging.

Introduction

For decades, aging was thought to be an entirely stochastic, uncontrolled process driven by the accumulation of cellular damage [1,2]. This view has changed as it became evident that manipulating the activities of several genetic and metabolic pathways elevates stress resistance, enhances protein homeostasis (proteostasis) and extends lifespans of various organisms. Dietary restriction (DR) [3], reducing Insulin/IGF-1 signaling (IIS) [4], lowering the activity of the mitochondrial electron transport chain (ETC) [5] and of signaling that emanates from the reproductive system [6], were all found to slow the aging process. The IIS, probably the most prominent aging-regulating pathway, is a key regulator of development, stress resistance, metabolism and longevity of various organisms [4,7–9].

In the nematode Caenorhabditis elegans (C. elegans), upon binding of one of its ligands, the lone insulin/IGF-1 receptor DAF-2 activates a signaling cascade which regulates the activity of a nexus of transcription factors through a highly conserved set of molecular components. DAF-2’s downstream kinases mediate the phosphorylation of the transcription factors DAF-16 [10,11] and SKN-1 [12]. These phosphorylation events retain DAF-16 and SKN-1 in the cytosol, preventing them from regulating their target gene networks. Analogously, the IIS negatively regulates the activity of HSF-1 by preventing the phosphorylation of DDL-1, a protein that interacts with this transcription factor. Non-phosphorylated DDL-1 along with DDL-2 and HSB-1 form a complex of proteins that binds HSF-1 and retains it in the cytosol [13]. The IIS also governs the cellular localization of PQM-1, a transcription factor which responds to IIS reduction in opposition to DAF-16, and plays key roles in the IIS-controlled lifespan determining mechanism [14]. Thus, knocking down the activity of DAF-2 by either mutation or RNA interference (RNAi) hyper-activates HSF-1, DAF-16 and SKN-1, creating long-lived, youthful and stress-resistant worms. These longevity and stress resistance effects of daf-2 knockdown are dependent upon each of the aforementioned transcription factors [12,15,16]. Similarly to worms, reduced IGF-1 signaling was shown to extend the lifespan of mice [9], and mutations in components of the same pathway correlate with extreme longevity of humans [7,17], indicating that the aging-regulating roles of the IIS are conserved from worms to mammals.

The alteration of aging protects worms and mammals from toxic protein aggregation

Maintaining the integrity of the proteome is vital for organismal functionality and viability. However, as an organism ages, its ability to maintain proteostasis declines [18,19], enabling subsets of proteins to form potentially toxic aggregates that accrue within the cell [20]. In some cases, the accumulation of aggregated proteins underlies the development of a myriad of late-onset maladies including neurodegenerative disorders such as Alzheimer’s disease (AD) [21] and Huntington’s disease (HD) [22]. Aging is the major risk factor for the manifestation of neurodegeneration, a common feature in these late-onset diseases [23]. This raises the prospect that the alteration of aging could maintain proteostasis in the late stages of life thereby preventing, or at least delaying, the emergence of neurodegeneration. Indeed, IIS reduction [24–26], DR [27], ETC impairments [28] and germ cell ablation [29], were all found to promote proteostasis and protect model nematodes from toxic protein aggregation (proteotoxicity). These mechanistic links define proteostasis collapse as an inherent aspect of aging [30]. Importantly, all the aforementioned IIS regulated transcription factors; DAF-16, HSF-1 [24,25], SKN-1 [31] and PQM-1 [32] are involved in the regulation of proteostasis, raising the prospect that modulating the activities of these factors could extend healthspan through late stages of life. However, to maintain proteostasis and extend healthspan without affecting lifespan, it is critical to ascertain the temporal requirements of these factors as lifespan and proteostasis regulators.

The temporal requirements of DAF-16 and HSF-1

Reducing the IIS at different stages of life via daf-2 RNAi, identified that IIS reduction during reproductive adulthood (days 1–6) and no other stage of life, extends the lifespan of C. elegans [33]. Consistently, an increase in lifespan was observed in the fruit fly Drosophila melanogaster when dFOXO (the ortholog of DAF-16) was over-expressed during reproductive adulthood but not during any other stages of life [8]. Surprisingly, we discovered that HSF-1 is of foremost importance for the determination of lifespan during the L2 larval stage, but also has a marginal effect on lifespan during reproductive adulthood [34]. DAF-16 and HSF-1 also exhibit distinct temporal requirements for proteostasis maintenance. While DAF-16 is dispensable for proteotoxicity protection during development and plays its counter-proteotoxic protective roles exclusively during adulthood, HSF-1 executes these functions mainly during development [35]. These distinct temporal patterns raise questions about the functional relationship between these two transcription factors, SKN-1 and the IIS.

Despite the central roles of SKN-1 as a regulator of lifespan downstream of the IIS [12] and via the DR pathway [36], as well as its influence on proteostasis [31], the temporal requirements of SKN-1 for these functions were unknown. To address this, we used the nematode C. elegans and a conditional RNAi knockdown technique and found that SKN-1 governs lifespan and proteostasis primarily during late development and early adulthood.

Materials and methods

Worm and RNAi strains

N2 (wild type, Bristol), CB1370 (daf-2 (e1370)), CL2006 (unc-54p::human Aβ_3–42), CF512 (fer-15(b26)II; fem-1(hc17)IV) and DA1116 (eat-2 mutant) worms were obtained from the Caenorhabditis Genetics Center (CGC, Minneapolis, MN), which is funded by the National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). AGD1246 (rgef-1p::Aβ_1–42; RF4 rol-6) worms were a generous gift of Dr. Andrew Dillin (University of California at Berkeley). AM140 (rmIs141[unc-54p::Q35::YFP]) and AM1126 (rmIs383[Pf25b3.3::Q(35)::YFP]) worms were a generous gift of Dr. Richard Morimoto (Northwestern University). All worm strains were routinely grown at 15°C for maintenance. For experimentation, all worms were kept at 20°C throughout life except for CF512 animals which are heat-sensitive feminized and were therefore grown at 25°C throughout development to prevent progeny, and then maintained at 20°C throughout adulthood. To reduce gene expression, we used bacterial strains expressing dsRNA: empty vector (pAD12), skn-1 and dcr-1 dsRNA expressing bacteria from the M. Vidal RNAi library. Each RNAi bacteria colony was grown at 37°C in LB with 100μg/ml ampicillin and then seeded on NG-ampicillin plates and supplemented with 100mM Isopropyl β-D-1-thiogalactopyranoside (IPTG 1mM final concentration).

Expression analysis by quantitative real-time PCR (qPCR)

Synchronized eggs were placed on NGM plates seeded with the indicated bacteria. The worms were grown from hatching until day one of adulthood unless otherwise indicated. The worm samples were then harvested and washed with M9 buffer to remove bacteria from the samples. Each worm pellet was re-suspended in 1M DTT and RA1 (solution from the NucleoSpin® RNA kit (Macherey-Nagel, Duren Germany #740955.50)) and frozen at -80°C overnight. After thawing the samples on ice, zirconium oxide beads (Next Advance, ZrB05) were added to the samples and the samples were homogenized at 4°C using a Bullet Blender® (Next Advance). To separate RNA from protein and other materials, samples underwent centrifugation at room temperature in a tabletop centrifuge. The NucleoSpin RNA isolation Kit (Macherey Nagel, Duren Germany #740955.50) was used according to the manufacturer instructions to extract RNA. cDNA was generated by reverse transcription of the total RNA samples with iScriptRT Advanced cDNA Synthesis Kit for RT-PCR (Bio-Rad, Hercules, CA; #170–8891;). qPCR was performed in triplicates using the iTaq^TM Universal SYBR® Supermix (Bio-Rad; #172–5124) and quantified in a CFX96^TM Real-Time PCR Detection System (Bio-Rad). The levels were normalized to the levels of cdc-42, act-1 and/or pmp-3 cDNA.

Primer name	Forward sequence	Reverse sequence
*act-1*	*`GAG CAC GGT ATC GTC ACC AA`*	*`TGT GAT GCC AGA TCT TCT CCA T`*
*pmp-3*	*`GTT CCC GTG TTC ATC ACT CAT`*	*`ACA CCG TCG AGA AGC TGT AGA`*
*cdc-42*	*`CTG CTG GAC AGG AAG ATT ACG`*	*`CTC GGA CAT TCT CGA ATG AAG`*
*skn-1*	*`CGA GAT CGT TCA TAT TCA AGC`*	*`CAC ATA CTG GCC AGA TGG`*

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Lifespan assays

Lifespan assays were conducted as previously described [34]. Briefly, synchronized eggs were placed on master NG-ampicillin 9cm plates seeded with the indicated RNAi bacterial strain and supplemented with 100mM IPTG (~1mM final concentration). After synchronization by bleach solution (0.75N KOH, 1.8% hypochlorite), the worms were places on RNAi plates as indicated. L2 larvae were collected 28 hours after bleach and L4 larvae 48 hours. The developmental stages of larvae were validated by visualization by light microscopy. CF512 worms were grown at 25°C throughout development to avoid progeny, then transferred to 20°C for the duration of their life. daf-2 (e1370) mutant animals (strain CB1370) as well as N2 and DA1116 worms were developed and maintained at 20°C. At day 1 of adulthood, 120 animals per treatment were transferred onto 5cm NG-ampicillin plates (12 animals per plate). Worms that failed to move their heads when tapped twice with a platinum wire or when a hot pick was placed proximally to their body were scored as dead. Survival rates were recorded daily.

Proteotoxicity assays

To follow Aβ-mediated toxicity by the “paralysis assay” [25], synchronized CL2006 or AGD1246 worms were grown on NG plates containing 100μg/ml ampicillin, spotted with E. coli cultures that express dsRNA as indicated. On day one of adulthood, 120 worms were transferred onto 10 5mm NG-ampicillin plates (12 animals per plate). These 10 plates were randomly divided into 5 sets (2 plates, 24 worms per set) to prevent potential bias. Paralysis of these worms was scored daily by gently tapping their noses with a platinum wire or placing a hot pick proximally to their bodies. Worms that were capable of moving their noses but unable to move the trunk of their bodies were scored as "paralyzed" and removed from the plates. The assay was terminated at day 12 or 13 of adulthood in order to avoid scoring old animals as paralyzed. As a control, this assay was also performed using wild type N2 worms.

To follow the toxicity of polyQ35-YFP stretches by the “thrashing assay” [37], synchronized eggs of AM140 or AM1126 worms were placed on plates seeded with control bacteria (EV) or bacteria that express RNAi towards skn-1. At the indicated ages, one worm was placed in a 10μL drop of M9 buffer and the number of body bends per 30 seconds was scored. At each time point at least 20 animals were used. As a control, this assay was also performed using wild type N2 worms.

Statistical analyses

To quantitatively measure statistical significance for the paralysis assay and thrashing assay, two-way ANOVA followed by post hoc Holm-Šídák’s corrections for multiple comparisons were used since in this statistical method the assumption is that each comparison is independent of the others [31]. For qPCR experiments the statistical significance of differences was assessed using Student T-test using two-tailed distribution and two-sample equal variance. The analyses of the experiments were conducted using a minimum of three independent biological repeats of each experiment as indicated. Statistical information of lifespan experiments is presented in the supplemental tables. All the statistical analyses and plotting of the data were performed using GraphPad Prism 9 (GraphPad Software, Inc., La Jolla, USA).

Results

skn-1 is involved in lifespan determination in late development and early adulthood

We sought to determine when the knockdown of skn-1 regulates lifespan and proteostasis. To properly characterize the efficiency of conditional RNAi-mediated knockdown we took two measures. First, we tested the efficiency of skn-1 RNAi using quantitative real-time PCR (qPCR). To avoid possible effects of developing embryos on gene expression, we used feminized CF512 worms, a strain whose lifespan is similar to that of wild type animals [38]. The worms were grown from hatching on bacteria that harbor the empty RNAi vector (EV) or cultured on skn-1 RNAi bacteria, harvested at day 1 of adulthood and skn-1 expression levels were measured via qPCR analysis. S1A and S1B Fig shows that skn-1 RNAi treatment is highly efficient when applied from hatching, as it reduced the expression of the gene to less than 10% compared to untreated animals (grown on EV bacteria). This reduction was observed when three different normalizing genes, act-1, pmp-3 (A) and cdc-42 (B) were used.

Secondly, to estimate the lag time between transferring worms to skn-1 RNAi bacteria and a notable reduction in skn-1 expression, we examined the kinetics of skn-1 RNAi-mediated knockdown. CF512 worms were cultured on EV bacteria and on day 1 of adulthood were transferred onto plates seeded with skn-1 RNAi 3, 6, 9, or 12 hours prior to harvest. qPCR was used to compare the levels of skn-1 expression to those of untreated animals. As expected, treating worms with skn-1 RNAi from hatching resulted in an approximate 95% reduced expression. Exposure to RNAi for longer periods led to a gradual reduction that reached ~50% reduction after 12 hours (S1C Fig). An exposure of 24 hours resulted in a similar rate of ~55% reduction (S1D Fig). Therefore, exposing worms to skn-1 RNAi requires approximately 12 hours to reach an efficient reduction in skn-1 expression. Although the depletion of SKN-1 protein is not known, it is likely that 12 hours is sufficient to reduce the activity of this transcription factor. Thus. a lag of 12 hours in the knockdown of skn-1 expression was considered throughout the study.

Next, we asked when SKN-1 regulates the lifespan of wild type worms (strain N2). To address this, we knocked down the expression of skn-1 by RNAi from different stages during the worm’s lifecycle. Synchronized eggs of N2 animals were placed on plates seeded with EV bacteria, or with skn-1 RNAi expressing bacteria. At larval stages L2, L4 or at day 1, 5 or 9 of adulthood, groups of 120 worms were picked from EV plates and transferred onto skn-1 RNAi bacteria. Lifespans were followed by daily scoring of dead animals. While worms that were grown throughout life on EV bacteria had a mean lifespan of 18.12±0.51 days (±SEM), animals that were treated from hatching with skn-1 RNAi exhibited a significantly (p<0.001) shorter mean lifespan of 16.17±0.32 days (Fig 1A and 1B and S1 Table). This result is consistent with the previously reported shortening effect of skn-1 mutation on the lifespan of these worms [12]. Interestingly, the knockdown of skn-1 from the L2 or L4 larval stages resulted in similar lifespan shortening effects of 11.8% (mean lifespan 15.98±0.24 days, p<0.001) and 14.23% (mean lifespan 15.54±0.35 days, p<0.001), respectively (Fig 1A and S1 Table). These lifespans were very similar to the lifespan of animals grown on skn-1 RNAi throughout life (mean lifespan of 16.17±0.32 days, p<0.001). Considering the 12 hours delay from the application of skn-1 RNAi until an efficient reduction in the expression of skn-1 is achieved and the similar lifespans of worms that were grown from hatching on skn-1 RNAi and their counterparts that were treated from the L4 larval stage, we conclude that SKN-1 is dispensable as a regulator of lifespan during almost the entirety of larval development. In contrast, the knockdown of skn-1 from day 1 and 5 of adulthood resulted in a trend of lifespan shortening, however, this effect was not significant (Fig 1B and S1 Table, mean lifespan 17.83±0.31 days, p = 0.31 and mean lifespan 17.51±0.42 days, p = 0.18, respectively). To further test whether SKN-1 is needed for longevity assurance during adulthood we conducted an additional set of lifespan experiments using CF512 worms. Two groups of animals were grown on EV or skn-1 RNAi throughout life, or cultured on EV bacteria and transferred at day 1 of adulthood onto plates seeded with skn-1 RNAi. Our results show that the lifespans of these animals are somewhat longer than these of N2 worms, however, they also indicate that the knockdown of skn-1 from day 1 of adulthood significantly shortened the lifespan of CF512 worms (Fig 1C and S2A and S2B Table). These results show that skn-1 is primarily needed later than the L4 larval developmental stage through early adulthood to regulate lifespan.

Fig 1 — (**A-B**) The lifespan of wild type animals (WT, strain N2) treated from hatching with empty vector bacteria (EV, control), *skn-1* RNAi, or transferred from EV bacteria onto *skn-1* RNAi either during developmental stages L2 or L4 (A), or at day 1, 5, or 9 of adulthood (B) was measured. Worms treated with *skn-1* RNAi throughout life, or during developmental stages L2 or L4 showed significant reductions in lifespan (10.75%, 11.80%, and 14.23%, respectively, S1 Table). Worms treated with *skn-1* RNAi from day 1 or 5 of adulthood showed a trend of reduction in lifespan, though the observed lifespan shortening was not significant (S1 Table). Treating worms with *skn-1* RNAi from day 9 of adulthood did not affect lifespan (S1 Table). (C) CF512 animals were grown throughout life on EV or *skn-1* RNAi bacteria or let hatch on EV bacteria and transferred onto *skn-1* RNAi bacteria at day 1 of adulthood. Lifespans of all groups were followed daily. The knockdown of *skn-1* from day 1 of adulthood resulted in a significant shortening of lifespan compared to untreated animals (S2A and S2B Table). (**D-E**) The knockdown of *skn-1* throughout life or from day 1 of adulthood, resulted in lifespan shortening of *daf-2* (*e1370*) mutant worms (strain CB1370, D and S3A and S3B Table) and of *eat-2* (strain DA1116) mutant animals (D and S4A and S4B Table).

To further examine the timing requirements of SKN-1 for longevity assurance, we employed long-lived mutant worm strains. daf-2 (e1370) mutant worms (strain CB1370) carry a weak daf-2 allele and thus, exhibit exceptional longevity [4]. The animals were grown throughout life either on EV or on skn-1 RNAi bacteria. An identical group of worms was hatched on EV bacteria and transferred onto skn-1 RNAi bacteria at day 1 of adulthood. While the knockdown of skn-1 from hatching resulted in lifespan shortening of 31.6% compared to the lifespan of untreated worms (mean lifespan of 30.63±0.94 and 44.79±1.97 days, respectively), knocking down the expression of this gene exclusively during adulthood shortened lifespan by only 17.34% (Fig 1D and S3A Table, mean lifespan of 37.02±1.02). These results, which were verified with an additional biological experimental repeat (S3B Table), indicate that skn-1 is needed from day 1 of adulthood as a modulator of lifespan. Nevertheless, the observation that knocking down skn-1 from day 1 of adulthood did not shorten lifespan as efficiently as skn-1 RNAi treatment throughout life, suggests that this transcription factor is also needed during larval development to allow daf-2 mutant worms to exhibit their full longevity potential. We repeated this experiment using DA1116 worms which carry a mutation in the eat-2 gene, resulting in a pharyngeal defect that leads to constitutive dietary restriction, and thus, are long-lived [39]. We found that similarly to skn-1 RNAi-treated daf-2 (e1370) mutant worms (CB1370), the knockdown of skn-1 throughout life shortens the lifespan of DA1116 animals by 27.89% (mean lifespan of 14.93±0.35 days). The lifespan of their counterparts who were treated with skn-1 RNAi from day 1 of adulthood was shortened by 13.58% (mean lifespan of 17.89±0.60 days), relative to the control worms (Fig 1E and S4A Table, mean lifespan 20.70±0.90 days). These results were confirmed with an additional biological experimental repeat (S4B Table). Together, these results indicate that SKN-1 is at least partially required during developmental stages as a regulator of lifespan. However, SKN-1 is also needed during adulthood to promote the natural lifespan of wild type animals and confer the full longevity of long-lived mutant worms.

The temporal roles of skn-1 in proteostasis maintenance

We next investigated when during the worm’s lifecycle SKN-1 regulates proteostasis. To determine this, we utilized CL2006 worms which express the AD-causing amyloid beta (Aβ) peptide in their body wall muscles [40]. This expression results in a progressive paralysis within the worm population; a phenotype that can be tracked by the “paralysis assay” via a daily scoring of paralyzed animals [25]. First, we tested whether the knockdown of skn-1 throughout life enhances the paralysis phenotype of these animals and found that it does (Fig 2A). To confirm that the observed results stem from the counter-proteotoxic roles of skn-1 and that its knockdown does not induce paralysis independently, we tested whether the knockdown of skn-1 confers paralysis in wild type worms. We found that the knockdown of skn-1 in wild type worms did not enhance the rate of aging-associated paralysis up until day 12 of adulthood (S2A Fig). To test whether the paralysis phenotype is tissue specific, we performed an identical experiment using AGD1246 worms which express the Aβ peptide under the regulation of the rgef-1 pan-neuronal promoter [28] and found that, similarly to the observed phenotype in muscles, that the knockdown of skn-1 by RNAi results in an increased rate of paralysis of this worm population (S2B and S2C Fig).

Fig 2 — (A) The knockdown of *skn-1* significantly enhances the rate of paralysis of Aβ worms as observed in three independent repeats (p<0.001 see also S2C Fig). EV: n = 282, 78 censored; *skn-1* RNAi: n = 352, 8 censored. (B) Treating Aβ worms with *skn-1* RNAi from the L2 larval stage enhances the rate of paralysis as efficiently as lifelong treatment with *skn-1* RNAi. In contrast, knocking down *skn-1* RNAi from the L4 larval stage less pronouncedly, but significantly enhances the rate of paralysis. EV: n = 269, 91 censored; *skn-1* RNAi: n = 338, 22 censored; EV → *skn-1* RNAi (L2): n = 332, 28 censored; EV → *skn-1* RNAi (L4): n = 342, 18 censored. (C) Worms grown from hatching on EV and transferred at day 1 of adulthood onto *skn-1* RNAi show a marginal increase in paralysis compared to worms grown for life on EV. In contrast, worms transferred onto *skn-1* RNAi at day 5 or 9 of adulthood show no increased rate of paralysis compared to their counterparts grown on EV throughout life. EV: n = 282, 78 censored; *skn-1* RNAi: n = 342, 18 censored; EV → *skn-1* RNAi (Day 1): n = 326, 34 censored; EV → *skn-1* RNAi (Day 5): n = 296, 65 censored; EV → *skn-1* RNAi (Day 9): n = 287, 73 censored. (D) Treating worms with *dcr-1* RNAi throughout life, or growing worms on *skn-1* RNAi from hatching and transferring them to *dcr-1* RNAi during the L2 or L4 stages of development did not enhance the rate of paralysis within the population, apart from a very slight increase at day 12 of adulthood in the population transferred at the L4 stage. EV: n = 279, 81 censored; *skn-1* RNAi: n = 320, 40 censored; *dcr-1* RNAi: n = 300, 60 censored; *skn-1* RNAi → *dcr-1* RNAi (L2): n = 313, 47 censored; *skn-1* RNAi → *dcr-1* RNAi (L4): n = 292, 68 censored. (E) Aβ worms that were grown on *skn-1* RNAi from hatching then transferred onto *dcr-1* RNAi at either day 1, 5 or 9 of adulthood and animals that were treated with *skn-1* RNAi throughout life, exhibited similarly enhanced rates of paralysis compared to the EV treated population. EV: n = 292, 68 censored; *skn-1* RNAi: n = 333, 27 censored; *dcr-1* RNAi: n = 300, 60 censored; *skn-1* RNAi → *dcr-1* RNAi (Day 1): n = 308, 52 censored; *skn-1* RNAi → *dcr-1* RNAi (Day 5): n = 335, 25 censored; *skn-1* RNAi → *dcr-1* RNAi (Day 9): n = 332, 28 censored. (F) CL2006 worms were grown from hatching on untreated (EV) bacteria or grown on one of the following mixtures of RNAi bacteria: EV+*skn-1*, EV+*daf-2* or *skn-1*+*daf-2*. Rates of paralysis were recorded daily. Worms that were treated with a mixture of *skn-1* RNAi+*daf-2* RNAi bacteria exhibited higher rates paralysis than their counterparts that were grown on a mixture of EV+*daf-2* RNAi. EV: *n =* 340, 58 censored; EV+*skn-1* RNAi: *n =* 350, 10 censored; EV+*daf-2* RNAi: *n =* 325, 35 censored; *daf-2+skn-1* RNAi: *n =* 325, 15 censored.

To establish the temporal requirement of skn-1 as a modulator of proteostasis we treated CL2006 worms with skn-1 RNAi throughout the experiment or from the L2 or L4 larval stages. An identical group of CL2006 worms was grown throughout the experiment (until day 12 of adulthood) on EV bacteria (see illustration in S3 Fig). Three independent experiments indicated that worms which were treated with skn-1 RNAi throughout the experiment and their counterparts that were transferred onto skn-1 RNAi bacteria at the L2 developmental stage, were paralyzed at similar rates, significantly higher than that of untreated animals (EV). These results indicate that skn-1 has no role during early development (L2 stage and earlier) as a modulator of Aβ-mediated proteotoxicity. Worms that were transferred onto skn-1 RNAi from the L4 larval stage exhibited a higher rate of paralysis than the control group (EV). However, the rate of paralysis within this worm population was lower than that of nematodes that were treated from the L2 stage (Fig 2B). This shows that even when considering the 12 hours delay in the knockdown of skn-1 by RNAi (S1C Fig), SKN-1 activity in late developmental stages is needed for partial protection from Aβ.

To test whether skn-1 is required during adulthood as a modulator of proteostasis, we conducted a similar experiment in which CL2006 worms were grown on EV bacteria and then transferred onto skn-1 RNAi at either day 1, 5 or 9 of adulthood (S3 Fig). Rates of paralysis were scored daily. Three independent experiments showed that the knockdown of skn-1 at day 1 of adulthood enhances the rate of paralysis within the population (Fig 2C). This effect, however, was less prominent than that of knocking down skn-1 during development (Fig 2B). No significant enhancement in the paralysis phenotype, compared to untreated worms, was observed when worms were treated with skn-1 RNAi from day 5 or 9 of adulthood (Fig 2C).

These results propose that SKN-1 is foremost required as a proteostasis regulator during late larval development through early reproductive adulthood. To further test this conclusion, we conducted a reciprocal set of experiments using dcr-1 RNAi. DICER, encoded by dcr-1, is a nuclease that cleaves double stranded RNA to create small interfering RNA (siRNA) and thus, is crucial for the functionality of the RNAi machinery [41]. Accordingly, the knockdown of dcr-1 by RNAi inactivates the RNAi machinery and partially restores the expression of the knocked down gene [33]. We utilized this technique to conditionally knockdown skn-1 and followed the rates of paralysis of CL2006 worm populations that hatched on skn-1 RNAi bacteria and were then transferred onto plates seeded with dcr-1 RNAi at the L2 or L4 larval stages. Three experimental repeats indicated that the knockdown of dcr-1 had no effect on the rate of paralysis, as animals grown on control bacteria (EV) and their counterparts that were treated with dcr-1 RNAi throughout the experiment, had similar rates of paralysis (Fig 2D). As expected, the knockdown of skn-1 throughout the assay increased paralysis. However, knocking down skn-1 solely during early development, from hatching up until the L2 larval stage, did not increase the rate of paralysis. The knockdown of skn-1 from hatching up until the L4 larval stage had a small deleterious effect, as the rate of paralysis was significantly higher than that of untreated animals at days 11 and 12 of adulthood. These results suggest that SKN-1 is needed as a regulator of proteostasis from a late stage of larval development.

To further scrutinize the temporal requirements of skn-1 as a regulator of proteostasis, we tested how the knockdown of skn-1 affects the paralysis of Aβ worms during adulthood. Synchronized eggs were placed on plates seeded with skn-1 RNAi bacteria and transferred onto dcr-1 RNAi plates on either day 1, 5 or 9 of adulthood. Our results (Fig 2E) indicate that worms treated with skn-1 RNAi throughout development and transferred onto dcr-1 RNAi at either day 5 or 9 of adulthood, exhibited similar rates of paralysis to animals fed with skn-1 RNAi bacteria throughout life. Animals that were transferred onto dicer RNAi plates at day 1 of adulthood exhibited significantly elevated rates of paralysis within the population.

Together our results demonstrate that SKN-1 is required for protection against Aβ induced proteotoxicity from the L4 stage of larval development through the first day of adulthood. However, the restoration of skn-1 expression at day 5 or 9 of adulthood did not rescue the enhanced paralysis phenotype, indicating that this transcription factor is dispensable as a proteostasis regulator in late stages of adulthood. These temporal requirements support the notion that SKN-1 is required for proteostasis maintenance during late developmental stages and early adulthood.

To test whether the effects of skn-1 RNAi on the rates of paralysis stem from its roles as an IIS component, we asked whether a concomitant knockdown of daf-2 and of skn-1 prevent IIS reduction from providing its full protection from Aβ-mediated proteotoxicity. First, we utilized qPCR to test whether the dilution of skn-1 RNAi with EV bacteria (50:50) affects its effectiveness. We found that the level of skn-1 expression in worms grown on this mixture was approximately 20% compared to the levels seen in untreated worms and very similar to the level that we observed in worms that were fed solely with skn-1 RNAi bacteria (S4 Fig). Next, we cultured Aβ worms on a mixture of daf-2 RNAi and skn-1 RNAi bacteria, subjected them to the paralysis assay and found that the knockdown of skn-1 reduces the rate of the counter-proteotoxic protective effect that is conferred by daf-2 RNAi (Fig 2F). It is important to note that since SKN-1 is activated by additional aging-regulating pathways [36], additional experiments are needed to test whether the effect of skn-1 RNAi emanates from its role as a component of the IIS.

The knockdown of skn-1 exacerbates proteotoxicity of polyQ35-YFP

We next sought to test whether this temporal pattern of SKN-1 as a proteostasis modulator is also true for worms that are challenged by the aggregation of a proteotoxic protein other than Aβ. To address this, we utilized worms that express a chimeric, fluorescently-tagged polyglutamine protein of 35 repeats (polyQ35-YFP) in their body wall muscles (strain AM140). Abnormally long polyglutamine stretches in different proteins underlie the development of several human neurodegenerative maladies, including HD [22] and Machado Joseph Disease (MJD) [42]. AM140 animals accumulate aggregates and exhibit progressive motility impairment, [26], a phenotype that can be followed by the “thrashing assay” [37]. First, we tested whether the knockdown of skn-1 affects polyQ35-YFP toxicity by comparing the thrashing rates of AM140 worms that were treated from hatching with skn-1 RNAi to those of untreated animals (EV). We found that the knockdown of skn-1 results in a significantly reduced rate of motility on days 2, 4 and 6 of adulthood (Fig 3A), while knocking down skn-1 in wild type worms results in only a slight reduction in motility at day 6 (S5A Fig). Similar results to those observed in the AM140 worms were obtained when thrashing experiments were conducted using worms that express polyQ35-YFP under the rgef-1 pan-neuronal promoter (strain AM1126, S5B Fig), indicating that this phenotype is not tissue specific (In these worms, thrashing was measured at days 4 and 8 as they exhibit the proteotoxic phenotype later in life than AM140 animals). We next tested when SKN-1 protects worms from polyQ35-YFP by growing AM140 animals on EV bacteria and transferring them onto plates seeded with skn-1 RNAi bacteria at the L2 or L4 larval stages or at day 1 or 3 of adulthood. Thrashing rates were scored at days 3 and 6 of adulthood. Our results indicate that analogously to its roles in the mitigation of Aβ proteotoxicity, SKN-1 is foremost important as a regulator of proteostasis during late larval development through early stages of adulthood (Fig 3B and 3C).

Fig 3 — (A) The knockdown of *skn-1* by RNAi significantly decreased the thrashing rates of worms that express polyQ35-YFP in their muscles (strain AM140) at day 2, 4 and 6 of adulthood (reduction of 11.64%, 12.29% and 27.60%, respectively). 80 worms were scored per treatment per timepoint. (B) Growing AM140 worms on control bacteria from hatching followed by transferring them onto *skn-1* RNAi at the L2 or L4 developmental stage resulted in enhanced proteotoxicity as measured by the thrashing assay (Reduction of 10.45% and 11.29% at day 3, respectively, and a reduction of 33.64% and 23.6% at day 6, respectively). 60 worms were scored per treatment per time point. (C) The knockdown of *skn-1* from day 1 (reduction of 10.38% and 7.35% when measured at day 3 and day 6, respectively) resulted in a significant trend of reduced motility only when measured on day 3 of adulthood. The transfer of EV treated worms to *skn-1* RNAi on day 3 of adulthood, resulted in a non-significant trend of reduced motility when measured on day 6 of adulthood (reduction of 10%). 60 worms were scored per treatment per time point. (D) A schematic illustration of the temporal requirements for SKN-1 as a regulator of lifespan and proteostasis.

Discussion

Our temporal analysis indicates that skn-1 is predominantly required for lifespan determination and for protection from proteotoxicity, from late stages of larval development through early adulthood (Fig 3D). Because the IIS regulates lifespan [33] and proteostasis [35] during adulthood, it is likely that during the late stages of larval development through early adulthood, SKN-1 regulates the expression of gene networks that enable IIS reduction to promote these functions later in life. This conjecture is supported by the observation that a concurrent knockdown of daf-2 and of skn-1 reduces the rate of protection that emanates from IIS reduction (Fig 2F). Yet, it is also possible that SKN-1 provides protection from proteotoxicity by a different mechanism and thus, the paralysis rates that were observed in animals that were concurrently fed with skn-1 and daf-2 RNAi bacteria, is the sum of the protective effect of daf-2 RNAi and the deleterious outcome of knocking down skn-1. More experimental work is needed to better clarify this issue.

SKN-1 is required as a lifespan and proteostasis regulator in a time window which is subsequent to that of HSF-1, and partially overlapping and preceding the time window in which DAF-16 executes these functions [33–35]. Interestingly, SKN-1 is also, at least partially, needed during development for DR-promoted longevity (Fig 1D). In contrast, the transcription factor PHA-4, which is also crucial for DR-mediated longevity, is solely needed during adulthood to enable this phenotype [43].

These observations substantiate that different transcription factors are needed in a sequential manner during the nematode’s lifecycle and raise the question of how SKN-1 acts, and what genes it regulates during late stages of development through early adulthood to enable the promotion of longevity and proteostasis in later stages of life. One possibility suggests that the regulation of stress responses, such as oxidative stress [44], by SKN-1, reduces damage during early stages of life. Accordingly, the knockdown of skn-1 during these early stages, in which the organism may be more vulnerable to metabolic insults, results in a less efficient activation of stress response mechanisms, higher rates of damage accumulation, and accelerates the process of aging.

An alternative model suggests that during late larval development through early adulthood, SKN-1 regulates the expression of genes whose products are needed for IIS reduction and DR to promote lifespan and proteostasis in adulthood. This theme may be supported by the observation that skn-1 is highly expressed during the L2 larval stage (S6 Fig), a stage preceding the time window in which SKN-1 regulates lifespan and proteostasis, compared to the observed expression during adulthood of CF512 worms (S6 Fig). It is important to note that CF512 and wild type worms exhibit similar temporal requirements for skn-1 as a lifespan determinant (Fig 1C and S2A and S2B Table). Similar requirements were also observed in lifespan experiments in which long-lived worms were used (Fig 1). It would be interesting to compare the gene networks that are differentially regulated by SKN-1 in late larval development through early adulthood to those which are regulated in other stages of life. Such target genes might encode constitutive heat shock proteins and inducible protective proteins. It would also be interesting to test whether SKN-1 and HSF-1 co-regulate target genes during development and whether the products of these genes are needed for the IIS to promote longevity and/or proteostasis during adulthood.

Another key question is where SKN-1 executes its longevity and proteostasis-promoting functions. Together, the known roles of neurons in the regulation of proteostasis [45,46], the prominent expression of skn-1 in ASI neurons [36] and the differential regulation of DAF-16 and HSF-1 by a neuronal gene [38], suggest that the developmental functions of SKN-1 may be regulated at the organismal level by neurons. It would also be interesting to test whether the activity of signaling complexes that reside on caveolae, a membrane structures that we previously found to regulate Aβ-mediated proteotoxicity [47], is affected by the knockdown of skn-1. Further research is needed to test these possibilities.

An additional important aspect of the temporal analyses of IIS regulated transcription factors is the tight correlations between longevity and proteostasis. While DAF-16 regulates both lifespan and proteostasis during adulthood [33,35], and HSF-1 primarily during the L2 stage of larval development [34,35], SKN-1 govern these functions primarily from the late stages of larval development through early adulthood. This correlation supports the notion that the formation of an efficient proteostasis assurance mechanism is needed for IIS reduction, and perhaps also for DR, to slow the progression of aging and promote longevity [48].

The requirement of skn-1 during early adulthood as a regulator of lifespan and proteostasis overlaps with the reproductive adulthood time window in which daf-16 is needed to enable longevity of daf-2 mutant worms [33]. It is tempting to speculate that DAF-16 and SKN-1 may co-regulate the expression of certain genes during reproductive adulthood. Indeed, SKN-1 and DAF-16 were shown to co-regulate the mitophagy mediator dct-1 which promotes mitochondrial health [49]. Interestingly, two genes, dod-17 and dod-24 are regulated by SKN-1 and DAF-16 in opposing manners. While the knockdown of daf-16 elevates the expression of these genes [50,51], the knockdown of skn-1 reduces their expression levels [52]. These observations highlight the complex relations between aging-regulating pathways and their downstream transcription factors. The theory that aging-regulating pathways are coordinated is also supported by the observation that the IIS and proteostasis-maintaining signaling that originate from the reproductive system are linked at the post-translational level [53]. In accordance, it was reported recently that signals that originate from the reproductive system and from the IIS integrate to promote longevity [54].

Supporting information

S1 Fig. Analysis of skn-1 RNAi efficiency by quantitative real-time PCR.

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Click here for additional data file.^{(128.7KB, pdf)}

S2 Fig. The effects of skn-1 RNAi on rates of paralysis of wild type animals and worms that express Aβ in their neurons.

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Click here for additional data file.^{(128.9KB, pdf)}

S3 Fig. An illustration of the temporal knockdown of skn-1 in Figs 1–3.

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Click here for additional data file.^{(94.4KB, pdf)}

S4 Fig. Diluting skn-1 RNAi expressing bacteria with EV bacteria has a minor effects on its efficiency as measured by quantitative real-time PCR.

(PDF)

Click here for additional data file.^{(99.4KB, pdf)}

S5 Fig. Thrashing assays show that the knockdown of skn-1 by RNAi mildly reduces thrashing of wild-type worms at day 6 of adulthood and more prominently affects the thrashing rates of animals that express polyQ35-YFP in their neurons.

(PDF)

Click here for additional data file.^{(131.2KB, pdf)}

S6 Fig. A quantitative real-time PCR experiment to compare the skn-1 expression levels in different stages of the nematode’s lifecycle.

(PDF)

Click here for additional data file.^{(96.6KB, pdf)}

S1 Table. Numerical data of lifespan experiments presented at Fig 1A and 1B.

(PDF)

Click here for additional data file.^{(39.4KB, pdf)}

S2 Table

A: Numerical data of a lifespan experiment presented at Fig 1C. B: Numerical data of a lifespan experiment of CF512 worms treated throughout life with EV or skn-1 RNAi or transferred from EV bacteria onto skn-1 RNAi at day 1 of adulthood.

(PDF)

Click here for additional data file.^{(31.1KB, pdf)}

S3 Table

A: Numerical data of a lifespan experiment presented at Fig 1D. B: Numerical data of a lifespan experiment of CB1370 worms treated throughout life with EV or skn-1 RNAi or transferred from EV bacteria onto skn-1 RNAi at day 1 of adulthood.

(PDF)

Click here for additional data file.^{(34.9KB, pdf)}

S4 Table

A: Numerical data of a lifespan experiment presented at Fig 1E. B: Numerical data of a lifespan experiment of DA1116 worms treated throughout life with EV or skn-1 RNAi or transferred from EV bacteria onto skn-1 RNAi at day 1 of adulthood.

(PDF)

Click here for additional data file.^{(29.2KB, pdf)}

Acknowledgments

We thank all members of the Cohen laboratory for insightful discussions throughout the project.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by the Israel Science Foundation (ISF#981/16) awarded to EC (isf.org.il). The Israeli Ministry of Science and Technology (MOST#80884) awarded to EC (https://www.gov.il/en/Departments/ministry_of_science_and_technology) Henri J. and Erna D. Leir Chair for Research in Neurodegenerative Diseases - awarded to EC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

1.Reichel W. The biology of aging. J Am Geriatr Soc. 1966;14(5):431–6. doi: 10.1111/j.1532-5415.1966.tb03069.x [DOI] [PubMed] [Google Scholar]
2.Medawar P. An unsolved problem of biology. HK Lewis and co. 1952. [Google Scholar]
3.Soultoukis GA, Partridge L. Dietary Protein, Metabolism, and Aging. Annual review of biochemistry. 2016;85:5–34. doi: 10.1146/annurev-biochem-060815-014422 . [DOI] [PubMed] [Google Scholar]
4.Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. A C. elegans mutant that lives twice as long as wild type. Nature. 1993;366(6454):461–4. doi: 10.1038/366461a0 . [DOI] [PubMed] [Google Scholar]
5.Wolff S, Dillin A. The trifecta of aging in Caenorhabditis elegans. Experimental gerontology. 2006;41(10):894–903. doi: 10.1016/j.exger.2006.06.054 . [DOI] [PubMed] [Google Scholar]
6.Hsin H, Kenyon C. Signals from the reproductive system regulate the lifespan of C. elegans. Nature. 1999;399(6734):362–6. Epub 1999/06/09. doi: 10.1038/20694 . [DOI] [PubMed] [Google Scholar]
7.Suh Y, Atzmon G, Cho MO, Hwang D, Liu B, Leahy DJ, et al. Functionally significant insulin-like growth factor I receptor mutations in centenarians. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(9):3438–42. doi: 10.1073/pnas.0705467105 . [DOI] [PMC free article] [PubMed] [Google Scholar]
8.Giannakou ME, Goss M, Jacobson J, Vinti G, Leevers SJ, Partridge L. Dynamics of the action of dFOXO on adult mortality in Drosophila. Aging Cell. 2007. doi: 10.1111/j.1474-9726.2007.00290.x . [DOI] [PubMed] [Google Scholar]
9.Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2003;421(6919):182–7. doi: 10.1038/nature01298 . [DOI] [PubMed] [Google Scholar]
10.Henderson ST, Johnson TE. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr Biol. 2001;11(24):1975–80. doi: 10.1016/s0960-9822(01)00594-2 . [DOI] [PubMed] [Google Scholar]
11.Paradis S, Ruvkun G. Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 1998;12(16):2488–98. doi: 10.1101/gad.12.16.2488 . [DOI] [PMC free article] [PubMed] [Google Scholar]
12.Tullet JM, Hertweck M, An JH, Baker J, Hwang JY, Liu S, et al. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell. 2008;132(6):1025–38. doi: 10.1016/j.cell.2008.01.030 . [DOI] [PMC free article] [PubMed] [Google Scholar]
13.Chiang WC, Ching TT, Lee HC, Mousigian C, Hsu AL. HSF-1 Regulators DDL-1/2 Link Insulin-like Signaling to Heat-Shock Responses and Modulation of Longevity. Cell. 2012;148(1–2):322–34. doi: S0092-8674(11)01572-8 [pii] doi: 10.1016/j.cell.2011.12.019 . [DOI] [PMC free article] [PubMed] [Google Scholar]
14.Tepper RG, Ashraf J, Kaletsky R, Kleemann G, Murphy CT, Bussemaker HJ. PQM-1 Complements DAF-16 as a Key Transcriptional Regulator of DAF-2-Mediated Development and Longevity. Cell. 2013;154(3):676–90. Epub 2013/08/06. doi: 10.1016/j.cell.2013.07.006 [pii]. . [DOI] [PMC free article] [PubMed] [Google Scholar]
15.Hsu AL, Murphy CT, Kenyon C. Regulation of aging and age-related disease by DAF-16 and heat-shock factor. Science (New York, NY. 2003;300(5622):1142–5. doi: 10.1126/science.1083701 . [DOI] [PubMed] [Google Scholar]
16.Lee RY, Hench J, Ruvkun G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol. 2001;11(24):1950–7. doi: 10.1016/s0960-9822(01)00595-4 . [DOI] [PubMed] [Google Scholar]
17.Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, et al. FOXO3A genotype is strongly associated with human longevity. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(37):13987–92. doi: 10.1073/pnas.0801030105 . [DOI] [PMC free article] [PubMed] [Google Scholar]
18.Hipp MS, Kasturi P, Hartl FU. The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol. 2019. doi: 10.1038/s41580-019-0101-y . [DOI] [PubMed] [Google Scholar]
19.Carvalhal Marques F, Volovik Y, Cohen E. The roles of cellular and organismal aging in the development of late-onset maladies. Annu Rev Pathol. 2015;10:1–23. doi: 10.1146/annurev-pathol-012414-040508 . [DOI] [PubMed] [Google Scholar]
20.David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C. Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans. PLoS biology. 2010;8(8). doi: 10.1371/journal.pbio.1000450 . [DOI] [PMC free article] [PubMed] [Google Scholar]
21.Long JM, Holtzman DM. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell. 2019;179(2):312–39. doi: 10.1016/j.cell.2019.09.001 ; PubMed Central PMCID: PMC6778042. [DOI] [PMC free article] [PubMed] [Google Scholar]
22.Jimenez-Sanchez M, Licitra F, Underwood BR, Rubinsztein DC. Huntington’s Disease: Mechanisms of Pathogenesis and Therapeutic Strategies. Cold Spring Harb Perspect Med. 2017;7(7). doi: 10.1101/cshperspect.a024240 . [DOI] [PMC free article] [PubMed] [Google Scholar]
23.Amaducci L, Tesco G. Aging as a major risk for degenerative diseases of the central nervous system. Curr Opin Neurol. 1994;7(4):283–6. doi: 10.1097/00019052-199408000-00001 . [DOI] [PubMed] [Google Scholar]
24.Teixeira-Castro A, Ailion M, Jalles A, Brignull HR, Vilaca JL, Dias N, et al. Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways. Human molecular genetics. 2011. doi: 10.1093/hmg/ddr203 . [DOI] [PMC free article] [PubMed] [Google Scholar]
25.Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against age-onset proteotoxicity. Science (New York, NY. 2006;313(5793):1604–10. doi: 10.1126/science.1124646 . [DOI] [PubMed] [Google Scholar]
26.Morley JF, Brignull HR, Weyers JJ, Morimoto RI. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(16):10417–22. Epub 2002/07/18. doi: 10.1073/pnas.152161099 [pii]. ; PubMed Central PMCID: PMC124929. [DOI] [PMC free article] [PubMed] [Google Scholar]
27.Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, et al. Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans. Aging Cell. 2008;7(3):394–404. doi: 10.1111/j.1474-9726.2008.00385.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
28.Berendzen KM, Durieux J, Shao LW, Tian Y, Kim HE, Wolff S, et al. Neuroendocrine Coordination of Mitochondrial Stress Signaling and Proteostasis. Cell. 2016;166(6):1553–63 e10. doi: 10.1016/j.cell.2016.08.042 . [DOI] [PMC free article] [PubMed] [Google Scholar]
29.Shemesh N, Shai N, Ben-Zvi A. Germline Stem Cell Arrest Inhibits the Collapse of Somatic Proteostasis Early in Caenorhabditis elegans Adulthood. Aging Cell. 2013. Epub 2013/06/06. doi: 10.1111/acel.12110 . [DOI] [PubMed] [Google Scholar]
30.Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194–217. doi: 10.1016/j.cell.2013.05.039 ; PubMed Central PMCID: PMC3836174. [DOI] [PMC free article] [PubMed] [Google Scholar]
31.Steinbaugh MJ, Narasimhan SD, Robida-Stubbs S, Moronetti Mazzeo LE, Dreyfuss JM, Hourihan JM, et al. Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence. Elife. 2015;4. doi: 10.7554/eLife.07836 ; PubMed Central PMCID: PMC4541496. [DOI] [PMC free article] [PubMed] [Google Scholar]
32.O’Brien D, Jones LM, Good S, Miles J, Vijayabaskar MS, Aston R, et al. A PQM-1-Mediated Response Triggers Transcellular Chaperone Signaling and Regulates Organismal Proteostasis. Cell Rep. 2018;23(13):3905–19. doi: 10.1016/j.celrep.2018.05.093 . [DOI] [PMC free article] [PubMed] [Google Scholar]
33.Dillin A, Crawford DK, Kenyon C. Timing requirements for insulin/IGF-1 signaling in C. elegans. Science (New York, NY. 2002;298(5594):830–4. doi: 10.1126/science.1074240 . [DOI] [PubMed] [Google Scholar]
34.Volovik Y, Maman M, Dubnikov T, Bejerano-Sagie M, Joyce D, Kapernick EA, et al. Temporal requirements of heat shock factor-1 for longevity assurance. Aging Cell. 2012;11(3):491–9. Epub 2012/03/01. doi: 10.1111/j.1474-9726.2012.00811.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
35.Cohen E, Du D, Joyce D, Kapernick EA, Volovik Y, Kelly JW, et al. Temporal requirements of insulin/IGF-1 signaling for proteotoxicity protection. Aging Cell. 2010;9(2):126–34. doi: 10.1111/j.1474-9726.2009.00541.x . [DOI] [PMC free article] [PubMed] [Google Scholar]
36.Bishop NA, Guarente L. Two neurons mediate diet-restriction-induced longevity in C. elegans. Nature. 2007;447(7144):545–9. doi: 10.1038/nature05904 . [DOI] [PubMed] [Google Scholar]
37.Volovik Y, Marques FC, Cohen E. The nematode Caenorhabditis elegans: a versatile model for the study of proteotoxicity and aging. Methods. 2014;68(3):458–64. doi: 10.1016/j.ymeth.2014.04.014 . [DOI] [PubMed] [Google Scholar]
38.Volovik Y, Moll L, Marques FC, Maman M, Bejerano-Sagie M, Cohen E. Differential regulation of the heat shock factor 1 and DAF-16 by neuronal nhl-1 in the nematode C. elegans. Cell Rep. 2014;9(6):2192–205. doi: 10.1016/j.celrep.2014.11.028 . [DOI] [PubMed] [Google Scholar]
39.Lakowski B, Hekimi S. The genetics of caloric restriction in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(22):13091–6. doi: 10.1073/pnas.95.22.13091 ; PubMed Central PMCID: PMC23719. [DOI] [PMC free article] [PubMed] [Google Scholar]
40.Link C. Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(20):9368–72. doi: 10.1073/pnas.92.20.9368 [DOI] [PMC free article] [PubMed] [Google Scholar]
41.Knight SW, Bass BL. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science (New York, NY. 2001;293(5538):2269–71. doi: 10.1126/science.1062039 ; PubMed Central PMCID: PMC1855227. [DOI] [PMC free article] [PubMed] [Google Scholar]
42.Da Silva JD, Teixeira-Castro A, Maciel P. From Pathogenesis to Novel Therapeutics for Spinocerebellar Ataxia Type 3: Evading Potholes on the Way to Translation. Neurotherapeutics. 2019. doi: 10.1007/s13311-019-00798-1 . [DOI] [PMC free article] [PubMed] [Google Scholar]
43.Panowski SH, Wolff S, Aguilaniu H, Durieux J, Dillin A. PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature. 2007;447(7144):550–5. doi: 10.1038/nature05837 . [DOI] [PubMed] [Google Scholar]
44.Blackwell TK, Steinbaugh MJ, Hourihan JM, Ewald CY, Isik M. SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic Biol Med. 2015;88(Pt B):290–301. doi: 10.1016/j.freeradbiomed.2015.06.008 ; PubMed Central PMCID: PMC4809198. [DOI] [PMC free article] [PubMed] [Google Scholar]
45.Maman M, Carvalhal Marques F, Volovik Y, Dubnikov T, Bejerano-Sagie M, Cohen E. A Neuronal GPCR is Critical for the Induction of the Heat Shock Response in the Nematode C. elegans. J Neurosci. 2013;33(14):6102–11. Epub 2013/04/05. doi: 10.1523/JNEUROSCI.4023-12.2013 [pii]. . [DOI] [PMC free article] [PubMed] [Google Scholar]
46.Prahlad V, Morimoto RI. Neuronal circuitry regulates the response of Caenorhabditis elegans to misfolded proteins. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(34):14204–9. Epub 2011/08/17. doi: 10.1073/pnas.1106557108 [pii]. ; PubMed Central PMCID: PMC3161566. [DOI] [PMC free article] [PubMed] [Google Scholar]
47.Roitenberg N, Bejerano-Sagie M, Boocholez H, Moll L, Marques FC, Golodetzki L, et al. Modulation of caveolae by insulin/IGF-1 signaling regulates aging of Caenorhabditis elegans. EMBO Rep. 2018;19(8). doi: 10.15252/embr.201745673 ; PubMed Central PMCID: PMC6073070. [DOI] [PMC free article] [PubMed] [Google Scholar]
48.Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science (New York, NY. 2008;319(5865):916–9. doi: 10.1126/science.1141448 . [DOI] [PubMed] [Google Scholar]
49.Palikaras K, Lionaki E, Tavernarakis N. Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature. 2015;521(7553):525–8. doi: 10.1038/nature14300 . [DOI] [PubMed] [Google Scholar]
50.Zhang P, Judy M, Lee SJ, Kenyon C. Direct and indirect gene regulation by a life-extending FOXO protein in C. elegans: roles for GATA factors and lipid gene regulators. Cell Metab. 2013;17(1):85–100. Epub 2013/01/15. doi: 10.1016/j.cmet.2012.12.013 [pii]. . [DOI] [PMC free article] [PubMed] [Google Scholar]
51.Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature. 2003;424(6946):277–83. doi: 10.1038/nature01789 . [DOI] [PubMed] [Google Scholar]
52.Oliveira RP, Porter Abate J, Dilks K, Landis J, Ashraf J, Murphy CT, et al. Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell. 2009;8(5):524–41. doi: 10.1111/j.1474-9726.2009.00501.x ; PubMed Central PMCID: PMC2776707. [DOI] [PMC free article] [PubMed] [Google Scholar]
53.Moll L, Roitenberg N, Bejerano-Sagie M, Boocholez H, Carvalhal Marques F, Volovik Y, et al. The insulin/IGF signaling cascade modulates SUMOylation to regulate aging and proteostasis in Caenorhabditis elegans. Elife. 2018;7. doi: 10.7554/eLife.38635 ; PubMed Central PMCID: PMC6277199. [DOI] [PMC free article] [PubMed] [Google Scholar]
54.Lan J, Rollins JA, Zang X, Wu D, Zou L, Wang Z, et al. Translational Regulation of Non-autonomous Mitochondrial Stress Response Promotes Longevity. Cell Rep. 2019;28(4):1050–62 e6. doi: 10.1016/j.celrep.2019.06.078 ; PubMed Central PMCID: PMC6684276. [DOI] [PMC free article] [PubMed] [Google Scholar]

PLoS One. doi: 10.1371/journal.pone.0243522.r001

Decision Letter 0

Jamuna Subramaniam

31 Dec 2020

PONE-D-20-36759

Temporal Requirements of SKN-1/NRF as a Regulator of Lifespan and Proteostasis in Caenorhabditis elegans

PLOS ONE

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Comments to the Author

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Reviewer #2: Partly

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: No

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5. Review Comments to the Author

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Reviewer #1: Several transcription factors are key regulators of longevity and proteostasis in C. elegans including SKN-1/Nrf, DAF-16/FOXO, and HSF-1. DAF-16 and HSF-1 have been shown to be required during certain stages of development and adulthood to regulate longevity, but a temporal analysis of SKN-1 has not previously been done. Grushko et al., use RNAi to temporally reduce SKN-1 activity to define the developmental or life stages in which it functions to regulate lifespan and proteostasis and discovered that SKN-1 is required in late larval development through young adult to regulate lifespan and proteostasis.

Major comments:

Although there is precedent for RNAi being used to determine the temporal requirements for daf-2, daf-16 and hsf-1, and the use of dcr-1 RNAi to temporally turn off the RNAi effect, there is always the inherent concern regarding the efficacy of RNAi to any specific gene. Expression levels and the stability of the protein may be determinants of how rapidly RNAi knockdown of a specific target occurs. The authors demonstrate that skn-1(RNAi) from hatching to day 1 adult (~48 hours) results in ~90% reduction in skn-1 mRNA. Thus, there may be up to a two-day delay from the time of initiation of RNAi to the reduction of skn-1 mRNA, and the timing of protein depletion may be longer. Ideally a time course of skn-1(RNAi) would have determined how quickly RNAi takes effect at the mRNA level, and how effective RNAi is at later life stages. Additionally, assaying SKN-1 protein levels should be considered, there are reported antibodies that could be used to test this. Barring these additional controls, the authors should at least address the limitations of their studies regarding the resolution of the timing requirements in the discussion.

Minor comments:

1) Last sentence of the introduction regarding that SKN-1 might regulate the expression of DAF-16 co-factors is intriguing, but is very speculative. I suggest removing or referencing expression data that would support this idea.

2) Strain nomenclature in the materials and methods and elsewhere is inconsistent. N2, not N2. Gene names and alleles in parentheses should be in italics. For example, eat-2(ad1116), please also provide the strain name (DA1116?). Indicate the genotypes of AGD1246, AM140 and AM1126 in the materials and methods.

3) In the methods for the proteotoxicity assays: “These 10 plates were randomly divided into 5 sets (2 plates, 24 worms per set).” It is unclear to me the purpose of this step. Are you randomizing with the controls to reduce bias, or are the sets being analyzed by different people? Please clarify.

4) In the discussion neurons are considered as a potential site of SKN-1 action. However, neurons are refractory toward RNAi. Would not your results suggest that SKN-1 functions anywhere but neurons? This should be considered in the discussion.

5) In the discussion, it is speculated that SKN-1 and DAF-16 might co-regulate the expression of certain genes during reproductive adulthood. Oliveira et al (2009) Aging Cell PMID: 19575768 performed gene expression analysis with SKN-1 under various conditions and there are several studies on DAF-16 target genes, summarized well by Murphy (2006) Exp Gerontol PMID: 16934425. Seems that some data mining might identify candidates.

6) Last sentence of the Discussion is difficult to understand. These ideas need to be broken into two parts and stated more clearly.

7) Figure 2, why show panel A when the same data is being shown in panel B?

8) Although mentioned in the materials and methods, it is helpful to know the number of animals scored in figure legends.

Reviewer #2: This study, by Grushko et al., deals with the role of skn-1 transcription factor in C. elegans. Using a series of RNAi knockdown experiments, authors have concluded that skn-1 acts during the late larval and early adult stages to regulate the lifespan and proteostasis. The approach is essentially the same as described earlier for the hsf-1 study, albeit less in depth (Volovik et al., Aging Cell, 2012).

The current manuscript proposes that lifespan and proteostasis regulation depend on sequential and partially overlapping functions of skn-1 and two other transcription factors. While it is an interesting model, the data does not fully support it. More experiments are needed to strengthen the story. Specific comments are listed below.

Major comments

1. More details need to be provided in Methods on how animals were staged to obtain specific larval stage populations.

2. Stages of worms for various treatments and analyses are not very consistent. For example,

a. lifespans in wild-type background were analyzed after skn-1 knockdowns at L2, L4, adult day 1, adult day 5 and adult day 9 stages. But it was not the same for daf-2 and eat-2 mutants, making it hard to compare results.

b. Why did authors chose adult day 1 and day 3 stages for temporal analysis of skn-1 RNAi in AM140 worms (Fig. 3C, 3D) but day 2 and day 6 for others shown in Fig. 3A and 3B? Likewise, Sup 4B, C present data on day 4 and day 8 animals.

3. Some of the conclusions are confusing. For example, the authors claim that skn-1 is primarily needed later than the L4 larval stage to regulate lifespan (page 8). What is the evidence that skn-1 is not required during early larval (L1, L2 and L3) stages?

4. Paralysis phenotype is much stronger when skn-1 was knocked down in CL2006 animals starting the L2 stage compared to the L4 stage. Doesn’t this suggest that the gene plays a role during early larval stages?

5. Why are graphs 2A and 2B plotted differently? This is also the case for a few other figures. No explanation has been provided.

6. Authors have assumed that increased paralysis caused by skn-1 knockdown in aggregation-prone strains is due to increased proteotoxicity but did not provide any evidence for it. Could it be possible that skn-1 RNAi increases paralysis without affecting proteotoxicity?

7. Authors have argued that skn-1 mediates IIS pathway function by regulating downstream genes, but they have not demonstrated that skn-1 interacts with IIS signaling in their paralysis assays. They need to perform more experiments before making broad conclusions.

8. Sentences are confusing in many cases. For example,

a. ‘skn-1 RNAi throughout the experiment…’ on page 10 (used several times). What exactly does that mean? What duration is that?

b. On the same page, ’skn-1 plays no roles during development…’ is hard to understand.

c. The term ‘rate of paralysis’ has been used often. The data is, however, presented as a percentage of animals. The two are not the same.

8. While the number of batches of animals analyzed in some cases have been stated, this is not always the case. No information is provided in the Methods section either.

9. The efficiency of skn-1 RNAi was demonstrated just once at the beginning of the experiment. However, since many different stages of knockdowns were performed, additional evidence should be provided for the effectiveness of knockdowns. Also, does dcr-1 RNAi restore skn-1 levels?

10. Statistical analyses do not appear to be adequate, especially in cases where multiple paralysis phenotypes are being compared with controls. Consider using one-way ANOVA with Bonferroni correction or Tukey’s post hoc or Dunnett’s post hoc tests.

Other comments

- It is recommended that Results are divided into sub-sections to make it easier to follow different sets of findings.

- Manuscript should be carefully edited to eliminate all grammatical and sentence structure errors.

- Authors should consider providing a schematic to help understand the timings of various RNAi treatments and stages of analyses.

- Supplementary figure 4A title says 'Motility of neuronal polyQ35-YFP expressing worms', which seems to be incorrect.

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PLoS One. 2021 Jul 1;16(7):e0243522. doi: 10.1371/journal.pone.0243522.r002

Author response to Decision Letter 0

20 May 2021

A point-by-point rebuttal:

Major comments:

We thank the referee for this important comment, conducted the experimental work to address it and modified the text accordingly. To test the relative knockdown efficiencies of skn-1 RNAi after exposure, we cultured CF512 worms on control bacteria (harboring the empty RNAi vector (EV)) and transferred groups of worms to plates that were seeded with skn-1 RNAi for 3, 6, 9, 12 or 24 hours prior to harvest. qPCR was used to assess the levels of skn-1 expression in all worm groups. We observed a gradual reduction in the levels of skn-1 mRNA as 12 hours of exposure reduced the expression of skn-1 by approximately 50% (revised supplemental figure S1C. N=4). Exposure of CF512 worms to skn-1 RNAi has led to a similar rate of reduction of ~55% (Fig. S1D of the revised manuscript).

We modified the text to describe these experiments and explain the limitations of RNAi-mediated conditional knockdown. Please see the beginning of the “results” section of the revised manuscript, pages 8 and top of page 9. In addition, we changed the discussion to claim that SKN-1 is needed at “late stages of development” instead of at L4. This better reflects the delay of ~12h in the reduction of skn-1 by RNAi (see for instance the first sentence of the “discussion” section at the bottom of page 14).

Additionally, assaying SKN-1 protein levels should be considered, there are reported antibodies that could be used to test this. Barring these additional controls, the authors should at least address the limitations of their studies regarding the resolution of the timing requirements in the discussion.

We appreciate this comment and made an effort to obtain SKN-1 antibodies, alas with no success. Unfortunately the sc-9244 SKN-1 antibody is discontinued and the company could not provide it.

Yet, we address the limitations of RNAi-mediated gene knockdown in the revised manuscript and mentioned the possible effects of protein stability (see page 9, first paragraph).

In addition, the change in phenotype observed under conditions of skn-1 knockdown indicates that whilst protein stability might mask the full effects of skn-1 reduction, the reduction in skn-1 expression is enough to elicit phenotypical differences.

Minor comments:

We agree, tuned down the sentence and added a reference (last sentence of the introduction, page 5 of the revised manuscript).

We have added the details regarding each strain as requested and corrected the nomenclature where it was needed throughout the manuscript.

We clarified this issue by explaining that it was done to prevent potential bias. Please see page 7, line of the revised manuscript.

We agree with the referee that RNAi works less efficiently in neurons compared to other cell types. Nevertheless, the knockdown of neuronal genes can be achieved by RNAi. For instance, we have previously shown that RNAi towards the neuronal GPCR, gtr-1 renders the treated worm sensitive to heat stress (Maman et al., Journal of Neuroscience 2013). Thus, since the knockdown of skn-1 by RNAi results in clear phenotypes, this technique is an adequate method to conduct the temporal analysis that is described here.

We thank the referee for this comment and expanded the discussion to further elaborate on this this speculation. In the revised manuscript we provide more examples of genes that are regulated by both transcription factors: DAF-16 and SKN-1 (dod-17 and dod-24). We also added the relevant references. Please see page 17 of the revised manuscript.

6) Last sentence of the Discussion is difficult to understand. These ideas need to be broken into two parts and stated more clearly.

We revised the sentence as suggested and believe that is much clearer now.

7) Figure 2, why show panel A when the same data is being shown in panel B?

We modified figure 2 and all depicted panels display the sum of at least three independent experiments.

8) Although mentioned in the materials and methods, it is helpful to know the number of animals scored in figure legends.

We added the number of worms in each figure legend as suggested.

Major comments

1. More details need to be provided in Methods on how animals were staged to obtain specific larval stage populations.

We expanded the explanation of our synchronization protocol. Please see page 7 of the revised manuscript under “Lifespan assays”.

2. Stages of worms for various treatments and analyses are not very consistent. For example,

The experiments that were conducted using e1370 or eat-2 animals were aimed to address the question of whether SKN-1 plays any role during development. Accordingly, we do not claim anything beyond the importance of SKN-1 for lifespan determination during development. We believe that our results sufficiently support these claims.

We agree with this critique and improved this point in the revised manuscript. First, figure 3 has been modified. In the new figure 3A we present the relative rates of thrashing of untreated (EV) and skn-1 RNAi-treated AM140 worms at days 2, 4 and 6 of adulthood. We chose to test this at three ages since we did not know when the proteotoxic effect of knocking down skn-1would be apparent. Once we determined that day 2 old nematodes exhibit enhanced paralysis upon the knockdown of skn-1, we focused on two ages: day 2 and 6 of adulthood.

In supplemental figure 5B of the revised manuscript (previously Fig S4, B and C), we used AM1126 worms that express polyQ35-YFP under the regulation of the rgef-1 neuronal promoter. The appearance of measurable phenotypes is different in distinct proteotoxicity strains. These differences emanate from various factors including the activity of the promoter and the site of integration of the exogenous gene. Thus, to observe a measurable phenotype we followed the worms at days 4 and 8.

We thank the referee for this comment and modified the text to better explain this issue (page 14, lines 6-7 from bottom).

Since the knockdown of skn-1 from the L4 larval stage and on (Fig. 1A, gray line) and knocking it down throughout life (red line) result in a nearly identical lifespan shortening effects, we conclude that an uninterrupted expression of skn-1 during early development (prior to the application of RNAi) has no effect of lifespan determination. We clarified this issue in the revised manuscript (please see the bottom of page 9 and top of page 10).

The knockdown of skn-1 from the L2 stage and on (Fig. 2B, light green) and throughout life (2B, red) exhibit nearly identical effects of increased rates of paralysis. Therefore, we conclude the activity of SKN-1 in early developmental stages, between hatching and L2, has no role in protecting from Aβ-mediated proteotoxicity. In contrast, the knockdown of skn-1 from the L4 stage results in a partial restoration of the phenotype. This means that the activity of SKN-1 prior to L4 (when it was knocked down) is important for the worm to resist the toxicity of Aβ. Accordingly, we stated that SKN-1 is not needed during early development (up to the L2 stage) but is needed in later stages of development, for protection from proteotoxicity (page 11, second paragraph).

Nevertheless, we further clarified this issue in page 11 of the revised manuscript.

5. Why are graphs 2A and 2B plotted differently? This is also the case for a few other figures. No explanation has been provided.

We agree and modified the results accordingly. In figure 2 of the revised manuscript all panels represent three or more independent repeats of the paralysis assay.

This a matter of definition. We refer to “proteotoxicity” as the damage that is elicited by toxic protein aggregation. Accordingly, reduction in motility is used here to measure proteotoxicity (please see Volovik et al., Methods 2014, for more details). We make no claim regarding the rate of protein aggregation as in this project we only used physiological assays.

However, to better address this issue we present a control experiment (Fig. S2A) showing that the knockdown of skn-1 in wild type (N2) worms has only a marginal effect on the rate of paralysis. This minor effect is exclusively seen in the last day of the experiment – day 12 of adulthood.

We agree with the referee and conducted an experiment to directly test whether the roles of SKN-1 as a regulator of proteostasis are associated with the IIS. To test this, we conducted a set of paralysis assays in which we treated CL2006 worms with a mixture of bacteria that express RNAi towards daf-2 and skn-1 (a dilution of skn-1 RNAi bacteria with EV bacteria efficiently knocks down the expression of skn-1 – Fig. S4). Our new results which are displayed as figure 2F of the revised manuscript, show that a concurrent knockdown of these two genes prevents daf-2 RNAi from providing its full protective effect from the paralysis phenotype. This observation supports the result of the involvement of SKN-1 in the protective mechanism that is acted upon the knockdown of daf-2. Alternatively, it may be possible that SKN-1 provides protection by a different mechanism. According to this notion, the paralysis rates that were observed in animals that were concurrently fed with skn-1 and daf-2 RNAi bacteria, is the sum of the protective effect of daf-2 RNAi and the deleterious outcome of knocking down skn-1.

We discussed this result at the discussion section of the revised manuscript (page 15, first paragraph).

8. Sentences are confusing in many cases. For example,

a. ‘skn-1 RNAi throughout the experiment…’ on page 10 (used several times). What exactly does that mean? What duration is that?

We thank the referee for this comment and clarified the sentence. The worms that were subjected to the paralysis assay were followed up until day 12 of adulthood and thus, it is not accurate to claim that they were treated throughout life.

b. On the same page, ’skn-1 plays no roles during development…’ is hard to understand.

The sentence was modified to better clarify it.

c. The term ‘rate of paralysis’ has been used often. The data is, however, presented as a percentage of animals. The two are not the same.

We thank the referee for this comment and clarified it throughout the manuscript. Now it appears as “the rate of paralysis within the worm population”.

8. While the number of batches of animals analyzed in some cases have been stated, this is not always the case. No information is provided in the Methods section either.

We apologize for not providing sufficient information and corrected this issue throughout the manuscript.

We agree with the referee and re-analyzed the data using adequate methods: two way ANOVA followed by post hoc Holm-Šídák's corrections for multiple comparisons were used since in this statistical method the assumption is that each comparison is independent of the others (please see for reference: Steinbaugh et al. 2015 Elife. 2015 Aug 24;4:e07836. doi: 10.7554/eLife.07836).

Other comments

- It is recommended that Results are divided into sub-sections to make it easier to follow different sets of findings.

We added sub-headings as suggested.

- Manuscript should be carefully edited to eliminate all grammatical and sentence structure errors.

We apologize for these errors and corrected them throughout the manuscript.

- Authors should consider providing a schematic to help understand the timings of various RNAi treatments and stages of analyses.

We added illustrations as suggested to figures 2 and 3. This illustration is displayed as supplemental figure 3A of the revised manuscript.

- Supplementary figure 4A title says 'Motility of neuronal polyQ35-YFP expressing worms', which seems to be incorrect.

We apologize for this mistake and corrected it in the revised version.

Attachment

Submitted filename: Grushko et al - Response to reviewers 20-5.docx

Click here for additional data file.^{(27.2KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0243522.r003

Decision Letter 1

Jamuna Subramaniam

14 Jun 2021

Temporal Requirements of SKN-1/NRF as a Regulator of Lifespan and Proteostasis in Caenorhabditis elegans

PONE-D-20-36759R1

Dear Dr. Cohen,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Jamuna Subramaniam, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

I am glad to say that the manuscript will be accepted provided the minor revision required by Reviewer one is addressed satisfactorily.

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

Reviewer #2: Yes

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6. Review Comments to the Author

Using RNAi to determine temporal gene requirements. My major concern with the first draft was the efficiency of skn-1 knockdown, how long this took so as to accurately assess the temporal requirements as mRNAs and their corresponding proteins can display varying degrees of stability. The authors make a good attempt to address this using qRT-PCR. They demonstrate that after animals fed skn-1 RNAi at hatching have a >90% depletion of skn-1 mRNA by day one of adulthood. Day one adult animals fed skn-1 RNAi for 12 or 24 hours results in ~50% reduction. Since many of their assays are over the course of adulthood it would have been nice to see if this level continues to go down, or if 50% reduction is the best achievable depletion by RNAi. It is unfortunate that they were not able to assess SKN-1 protein levels, as this makes it difficult to know the true temporal effects of skn-1 RNAi. It is proposed that there is a 12-hour lag time, but this could be much longer. It should be acknowledged as a caveat at the end of this section that the depletion of SKN-1 protein is not known. In the future, other methods for rapid depletion of proteins, like the AID system would be more effective than RNAi. Nevertheless, I find that the conclusions accurately reflect their results.

The last paragraph of the introduction continues to be too speculative. None of the experiments in this paper address the relationship between skn-1 and daf-16. The speculation that SKN-1 might regulate DAF-16 cofactors is over selling the paper. This should be left to the discussion. Something more general about the timing of SKN-1 requirements for lifespan and proteostasis relative to other TFs might be more appropriate.

Different lifespan requirements between N2 and CF512 strain.

Using N2 (wild-type) worms, the authors demonstrate that starting skn-1 RNAi feeding at the L4 larval stage resulted in a decrease in lifespan, but starting at day 1 did not significantly reduce lifespan. Without a particular rationale, these experiments were repeated using the CF512 strain. Here they find that skn-1 RNAi started at day 1 adult significantly suppressed lifespan. Why do you think there is a difference? The skn-1 efficacy experiments were also done in this strain and it is mentioned that it has similar lifespan as N2. But comparing the supplementary tables 1 and 2, it appears that in empty vector control, CF512 animals live 3.5-4 days longer than N2, which I suspect would be significantly different (CF512 is even more long lived than DA1116). In either case, the time difference between L4 and day 1 adult is not long, but the difference should be more prominently acknowledged.

Does skn-1 RNAi effects on paralysis stem from its role as an IIS component?

Please add a concluding sentence for the data in Figure 2F. My interpretation is that we don’t know from the experiment shown. Partial reduction of the daf-2 effect by loss of skn-1 could be consistent with skn-1 being one of the outputs of IIS signaling, but is also consistent with daf-2 and skn-1 functioning in independent, but opposite pathways. OK – this is discussed in the discussion, however a concluding sentence in the results would be good. I don’t think there is much added value in this experiment.

Continues to not follow C. elegans nomenclature

Although some changes were made, there is still many inconsistencies in the nomenclature. I did not intend for the eat-2 allele, ad1116, to be replaced throughout by the strain name DA1116 (but this is more in line with the rest of the paper), but that in general there seems to be confusion regarding what is an allele and what is a strain. In Figure 1, e1370 is referred to as a strain. The strain is CB1370, the allele is e1370. N2, not N2. Italics are not used consistently where needed. I don’t mean to be nit-picky, however I wouldn’t expect these types of nomenclature errors from an established C. elegans lab. It reflects a lack of attention to detail and suggests that the rest of the work might also be sloppy.

http://www.wormbook.org/chapters/www_nomenclature/caenornomenclature.html

Reviewer #2: Authors have addressed all of my concerns adequately. The revised manuscript is greatly improved. I support publication in PLOS ONE.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

PLoS One. doi: 10.1371/journal.pone.0243522.r004

Acceptance letter

Jamuna Subramaniam

22 Jun 2021

PONE-D-20-36759R1

Temporal Requirements of SKN-1/NRF as a Regulator of Lifespan and Proteostasis in Caenorhabditis elegans

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Analysis of skn-1 RNAi efficiency by quantitative real-time PCR.

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S2 Fig. The effects of skn-1 RNAi on rates of paralysis of wild type animals and worms that express Aβ in their neurons.

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S3 Fig. An illustration of the temporal knockdown of skn-1 in Figs 1–3.

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S4 Fig. Diluting skn-1 RNAi expressing bacteria with EV bacteria has a minor effects on its efficiency as measured by quantitative real-time PCR.

(PDF)

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S6 Fig. A quantitative real-time PCR experiment to compare the skn-1 expression levels in different stages of the nematode’s lifecycle.

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Click here for additional data file.^{(96.6KB, pdf)}

S1 Table. Numerical data of lifespan experiments presented at Fig 1A and 1B.

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S2 Table

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S3 Table

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S4 Table

(PDF)

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Attachment

Submitted filename: Grushko et al - Response to reviewers 20-5.docx

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

[pone.0243522.ref001] 1.Reichel W. The biology of aging. J Am Geriatr Soc. 1966;14(5):431–6. doi: 10.1111/j.1532-5415.1966.tb03069.x [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref002] 2.Medawar P. An unsolved problem of biology. HK Lewis and co. 1952. [Google Scholar]

[pone.0243522.ref003] 3.Soultoukis GA, Partridge L. Dietary Protein, Metabolism, and Aging. Annual review of biochemistry. 2016;85:5–34. doi: 10.1146/annurev-biochem-060815-014422 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref004] 4.Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R. A C. elegans mutant that lives twice as long as wild type. Nature. 1993;366(6454):461–4. doi: 10.1038/366461a0 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref005] 5.Wolff S, Dillin A. The trifecta of aging in Caenorhabditis elegans. Experimental gerontology. 2006;41(10):894–903. doi: 10.1016/j.exger.2006.06.054 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref006] 6.Hsin H, Kenyon C. Signals from the reproductive system regulate the lifespan of C. elegans. Nature. 1999;399(6734):362–6. Epub 1999/06/09. doi: 10.1038/20694 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref007] 7.Suh Y, Atzmon G, Cho MO, Hwang D, Liu B, Leahy DJ, et al. Functionally significant insulin-like growth factor I receptor mutations in centenarians. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(9):3438–42. doi: 10.1073/pnas.0705467105 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref008] 8.Giannakou ME, Goss M, Jacobson J, Vinti G, Leevers SJ, Partridge L. Dynamics of the action of dFOXO on adult mortality in Drosophila. Aging Cell. 2007. doi: 10.1111/j.1474-9726.2007.00290.x . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref009] 9.Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Even PC, et al. IGF-1 receptor regulates lifespan and resistance to oxidative stress in mice. Nature. 2003;421(6919):182–7. doi: 10.1038/nature01298 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref010] 10.Henderson ST, Johnson TE. daf-16 integrates developmental and environmental inputs to mediate aging in the nematode Caenorhabditis elegans. Curr Biol. 2001;11(24):1975–80. doi: 10.1016/s0960-9822(01)00594-2 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref011] 11.Paradis S, Ruvkun G. Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor. Genes Dev. 1998;12(16):2488–98. doi: 10.1101/gad.12.16.2488 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref012] 12.Tullet JM, Hertweck M, An JH, Baker J, Hwang JY, Liu S, et al. Direct inhibition of the longevity-promoting factor SKN-1 by insulin-like signaling in C. elegans. Cell. 2008;132(6):1025–38. doi: 10.1016/j.cell.2008.01.030 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref013] 13.Chiang WC, Ching TT, Lee HC, Mousigian C, Hsu AL. HSF-1 Regulators DDL-1/2 Link Insulin-like Signaling to Heat-Shock Responses and Modulation of Longevity. Cell. 2012;148(1–2):322–34. doi: S0092-8674(11)01572-8 [pii] doi: 10.1016/j.cell.2011.12.019 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref014] 14.Tepper RG, Ashraf J, Kaletsky R, Kleemann G, Murphy CT, Bussemaker HJ. PQM-1 Complements DAF-16 as a Key Transcriptional Regulator of DAF-2-Mediated Development and Longevity. Cell. 2013;154(3):676–90. Epub 2013/08/06. doi: 10.1016/j.cell.2013.07.006 [pii]. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref015] 15.Hsu AL, Murphy CT, Kenyon C. Regulation of aging and age-related disease by DAF-16 and heat-shock factor. Science (New York, NY. 2003;300(5622):1142–5. doi: 10.1126/science.1083701 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref016] 16.Lee RY, Hench J, Ruvkun G. Regulation of C. elegans DAF-16 and its human ortholog FKHRL1 by the daf-2 insulin-like signaling pathway. Curr Biol. 2001;11(24):1950–7. doi: 10.1016/s0960-9822(01)00595-4 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref017] 17.Willcox BJ, Donlon TA, He Q, Chen R, Grove JS, Yano K, et al. FOXO3A genotype is strongly associated with human longevity. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(37):13987–92. doi: 10.1073/pnas.0801030105 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref018] 18.Hipp MS, Kasturi P, Hartl FU. The proteostasis network and its decline in ageing. Nat Rev Mol Cell Biol. 2019. doi: 10.1038/s41580-019-0101-y . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref019] 19.Carvalhal Marques F, Volovik Y, Cohen E. The roles of cellular and organismal aging in the development of late-onset maladies. Annu Rev Pathol. 2015;10:1–23. doi: 10.1146/annurev-pathol-012414-040508 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref020] 20.David DC, Ollikainen N, Trinidad JC, Cary MP, Burlingame AL, Kenyon C. Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans. PLoS biology. 2010;8(8). doi: 10.1371/journal.pbio.1000450 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref021] 21.Long JM, Holtzman DM. Alzheimer Disease: An Update on Pathobiology and Treatment Strategies. Cell. 2019;179(2):312–39. doi: 10.1016/j.cell.2019.09.001 ; PubMed Central PMCID: PMC6778042. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref022] 22.Jimenez-Sanchez M, Licitra F, Underwood BR, Rubinsztein DC. Huntington’s Disease: Mechanisms of Pathogenesis and Therapeutic Strategies. Cold Spring Harb Perspect Med. 2017;7(7). doi: 10.1101/cshperspect.a024240 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref023] 23.Amaducci L, Tesco G. Aging as a major risk for degenerative diseases of the central nervous system. Curr Opin Neurol. 1994;7(4):283–6. doi: 10.1097/00019052-199408000-00001 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref024] 24.Teixeira-Castro A, Ailion M, Jalles A, Brignull HR, Vilaca JL, Dias N, et al. Neuron-specific proteotoxicity of mutant ataxin-3 in C. elegans: rescue by the DAF-16 and HSF-1 pathways. Human molecular genetics. 2011. doi: 10.1093/hmg/ddr203 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref025] 25.Cohen E, Bieschke J, Perciavalle RM, Kelly JW, Dillin A. Opposing activities protect against age-onset proteotoxicity. Science (New York, NY. 2006;313(5793):1604–10. doi: 10.1126/science.1124646 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref026] 26.Morley JF, Brignull HR, Weyers JJ, Morimoto RI. The threshold for polyglutamine-expansion protein aggregation and cellular toxicity is dynamic and influenced by aging in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 2002;99(16):10417–22. Epub 2002/07/18. doi: 10.1073/pnas.152161099 [pii]. ; PubMed Central PMCID: PMC124929. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref027] 27.Steinkraus KA, Smith ED, Davis C, Carr D, Pendergrass WR, Sutphin GL, et al. Dietary restriction suppresses proteotoxicity and enhances longevity by an hsf-1-dependent mechanism in Caenorhabditis elegans. Aging Cell. 2008;7(3):394–404. doi: 10.1111/j.1474-9726.2008.00385.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref028] 28.Berendzen KM, Durieux J, Shao LW, Tian Y, Kim HE, Wolff S, et al. Neuroendocrine Coordination of Mitochondrial Stress Signaling and Proteostasis. Cell. 2016;166(6):1553–63 e10. doi: 10.1016/j.cell.2016.08.042 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref029] 29.Shemesh N, Shai N, Ben-Zvi A. Germline Stem Cell Arrest Inhibits the Collapse of Somatic Proteostasis Early in Caenorhabditis elegans Adulthood. Aging Cell. 2013. Epub 2013/06/06. doi: 10.1111/acel.12110 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref030] 30.Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194–217. doi: 10.1016/j.cell.2013.05.039 ; PubMed Central PMCID: PMC3836174. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref031] 31.Steinbaugh MJ, Narasimhan SD, Robida-Stubbs S, Moronetti Mazzeo LE, Dreyfuss JM, Hourihan JM, et al. Lipid-mediated regulation of SKN-1/Nrf in response to germ cell absence. Elife. 2015;4. doi: 10.7554/eLife.07836 ; PubMed Central PMCID: PMC4541496. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref032] 32.O’Brien D, Jones LM, Good S, Miles J, Vijayabaskar MS, Aston R, et al. A PQM-1-Mediated Response Triggers Transcellular Chaperone Signaling and Regulates Organismal Proteostasis. Cell Rep. 2018;23(13):3905–19. doi: 10.1016/j.celrep.2018.05.093 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref033] 33.Dillin A, Crawford DK, Kenyon C. Timing requirements for insulin/IGF-1 signaling in C. elegans. Science (New York, NY. 2002;298(5594):830–4. doi: 10.1126/science.1074240 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref034] 34.Volovik Y, Maman M, Dubnikov T, Bejerano-Sagie M, Joyce D, Kapernick EA, et al. Temporal requirements of heat shock factor-1 for longevity assurance. Aging Cell. 2012;11(3):491–9. Epub 2012/03/01. doi: 10.1111/j.1474-9726.2012.00811.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref035] 35.Cohen E, Du D, Joyce D, Kapernick EA, Volovik Y, Kelly JW, et al. Temporal requirements of insulin/IGF-1 signaling for proteotoxicity protection. Aging Cell. 2010;9(2):126–34. doi: 10.1111/j.1474-9726.2009.00541.x . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref036] 36.Bishop NA, Guarente L. Two neurons mediate diet-restriction-induced longevity in C. elegans. Nature. 2007;447(7144):545–9. doi: 10.1038/nature05904 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref037] 37.Volovik Y, Marques FC, Cohen E. The nematode Caenorhabditis elegans: a versatile model for the study of proteotoxicity and aging. Methods. 2014;68(3):458–64. doi: 10.1016/j.ymeth.2014.04.014 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref038] 38.Volovik Y, Moll L, Marques FC, Maman M, Bejerano-Sagie M, Cohen E. Differential regulation of the heat shock factor 1 and DAF-16 by neuronal nhl-1 in the nematode C. elegans. Cell Rep. 2014;9(6):2192–205. doi: 10.1016/j.celrep.2014.11.028 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref039] 39.Lakowski B, Hekimi S. The genetics of caloric restriction in Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 1998;95(22):13091–6. doi: 10.1073/pnas.95.22.13091 ; PubMed Central PMCID: PMC23719. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref040] 40.Link C. Expression of human beta-amyloid peptide in transgenic Caenorhabditis elegans. Proceedings of the National Academy of Sciences of the United States of America. 1995;92(20):9368–72. doi: 10.1073/pnas.92.20.9368 [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref041] 41.Knight SW, Bass BL. A role for the RNase III enzyme DCR-1 in RNA interference and germ line development in Caenorhabditis elegans. Science (New York, NY. 2001;293(5538):2269–71. doi: 10.1126/science.1062039 ; PubMed Central PMCID: PMC1855227. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref042] 42.Da Silva JD, Teixeira-Castro A, Maciel P. From Pathogenesis to Novel Therapeutics for Spinocerebellar Ataxia Type 3: Evading Potholes on the Way to Translation. Neurotherapeutics. 2019. doi: 10.1007/s13311-019-00798-1 . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref043] 43.Panowski SH, Wolff S, Aguilaniu H, Durieux J, Dillin A. PHA-4/Foxa mediates diet-restriction-induced longevity of C. elegans. Nature. 2007;447(7144):550–5. doi: 10.1038/nature05837 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref044] 44.Blackwell TK, Steinbaugh MJ, Hourihan JM, Ewald CY, Isik M. SKN-1/Nrf, stress responses, and aging in Caenorhabditis elegans. Free Radic Biol Med. 2015;88(Pt B):290–301. doi: 10.1016/j.freeradbiomed.2015.06.008 ; PubMed Central PMCID: PMC4809198. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref045] 45.Maman M, Carvalhal Marques F, Volovik Y, Dubnikov T, Bejerano-Sagie M, Cohen E. A Neuronal GPCR is Critical for the Induction of the Heat Shock Response in the Nematode C. elegans. J Neurosci. 2013;33(14):6102–11. Epub 2013/04/05. doi: 10.1523/JNEUROSCI.4023-12.2013 [pii]. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref046] 46.Prahlad V, Morimoto RI. Neuronal circuitry regulates the response of Caenorhabditis elegans to misfolded proteins. Proceedings of the National Academy of Sciences of the United States of America. 2011;108(34):14204–9. Epub 2011/08/17. doi: 10.1073/pnas.1106557108 [pii]. ; PubMed Central PMCID: PMC3161566. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref047] 47.Roitenberg N, Bejerano-Sagie M, Boocholez H, Moll L, Marques FC, Golodetzki L, et al. Modulation of caveolae by insulin/IGF-1 signaling regulates aging of Caenorhabditis elegans. EMBO Rep. 2018;19(8). doi: 10.15252/embr.201745673 ; PubMed Central PMCID: PMC6073070. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref048] 48.Balch WE, Morimoto RI, Dillin A, Kelly JW. Adapting proteostasis for disease intervention. Science (New York, NY. 2008;319(5865):916–9. doi: 10.1126/science.1141448 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref049] 49.Palikaras K, Lionaki E, Tavernarakis N. Coordination of mitophagy and mitochondrial biogenesis during ageing in C. elegans. Nature. 2015;521(7553):525–8. doi: 10.1038/nature14300 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref050] 50.Zhang P, Judy M, Lee SJ, Kenyon C. Direct and indirect gene regulation by a life-extending FOXO protein in C. elegans: roles for GATA factors and lipid gene regulators. Cell Metab. 2013;17(1):85–100. Epub 2013/01/15. doi: 10.1016/j.cmet.2012.12.013 [pii]. . [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref051] 51.Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, et al. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature. 2003;424(6946):277–83. doi: 10.1038/nature01789 . [DOI] [PubMed] [Google Scholar]

[pone.0243522.ref052] 52.Oliveira RP, Porter Abate J, Dilks K, Landis J, Ashraf J, Murphy CT, et al. Condition-adapted stress and longevity gene regulation by Caenorhabditis elegans SKN-1/Nrf. Aging Cell. 2009;8(5):524–41. doi: 10.1111/j.1474-9726.2009.00501.x ; PubMed Central PMCID: PMC2776707. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref053] 53.Moll L, Roitenberg N, Bejerano-Sagie M, Boocholez H, Carvalhal Marques F, Volovik Y, et al. The insulin/IGF signaling cascade modulates SUMOylation to regulate aging and proteostasis in Caenorhabditis elegans. Elife. 2018;7. doi: 10.7554/eLife.38635 ; PubMed Central PMCID: PMC6277199. [DOI] [PMC free article] [PubMed] [Google Scholar]

[pone.0243522.ref054] 54.Lan J, Rollins JA, Zang X, Wu D, Zou L, Wang Z, et al. Translational Regulation of Non-autonomous Mitochondrial Stress Response Promotes Longevity. Cell Rep. 2019;28(4):1050–62 e6. doi: 10.1016/j.celrep.2019.06.078 ; PubMed Central PMCID: PMC6684276. [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Temporal requirements of SKN-1/NRF as a regulator of lifespan and proteostasis in Caenorhabditis elegans

Danielle Grushko

Hana Boocholez

Amir Levine

Ehud Cohen

Roles

Abstract

Introduction

The alteration of aging protects worms and mammals from toxic protein aggregation

The temporal requirements of DAF-16 and HSF-1

Materials and methods

Worm and RNAi strains

Expression analysis by quantitative real-time PCR (qPCR)

Lifespan assays

Proteotoxicity assays

Statistical analyses

Results

skn-1 is involved in lifespan determination in late development and early adulthood

Fig 1. skn-1 regulates lifespan primarily from late larval development through early adulthood.

The temporal roles of skn-1 in proteostasis maintenance

Fig 2. skn-1 is foremost important from late stages of larval development through day 1 of adulthood to protect against Aβ-induced proteotoxicity.

The knockdown of skn-1 exacerbates proteotoxicity of polyQ35-YFP

Fig 3. skn-1 expression is most critical from the late larval development through day 1 of adulthood to counter polyglutamine (polyQ) induced proteotoxicity.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

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Jamuna Subramaniam

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Author response to Decision Letter 0

Decision Letter 1

Jamuna Subramaniam

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Acceptance letter

Jamuna Subramaniam

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Associated Data

Supplementary Materials

Data Availability Statement

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