Further Extension of Lifespan by Unc-43/CaMKII and Egl-8/PLCβ Mutations in Germline-Deficient Caenorhabditis elegans

Reduction of insulin/insulin-like growth factor 1 (IGF1) signaling (IIS) promotes longevity across species. In the nematode Caenorhabditis elegans, ablation of germline stem cells (GSCs) and activity changes of the conserved signaling mediators unc-43/CaMKII (calcium/calmodulin-dependent kinase type II) and egl-8/PLCβ (phospholipase Cβ) also increase lifespan. Like IIS, these pathways depend on the conserved transcription factor daf-16/FOXO for lifespan extension, but how they functionally interact is unknown. Here, we show that altered unc-43/egl-8 activity further increases the lifespan of long-lived GSC-deficient worms, but not of worms that are long-lived due to a strong reduction-of-function mutation in the insulin/IGF1-like receptor daf-2. Additionally, we provide evidence for unc-43 and, to a lesser extent, egl-8 modulating the expression of certain collagen genes, which were reported to be dispensable for longevity of these particular daf-2 mutant worms, but not for other forms of longevity. Together, these results provide new insights into the conditions and potential mechanisms by which CaMKII- and PLCβ-signals modulate C. elegans lifespan.


Introduction
Studies in invertebrate model organisms, such as the roundworm Caenorhabditis elegans and the fruit fly Drosophila melanogaster, provided key insights into the biology of lifespan regulation that apparently are also applicable to human aging [1]. A prototypic example is provided by reduced insulin/insulin-like growth factor 1 (IGF1) signaling (IIS). IIS was first recognized in C. elegans to cause a dramatic,~two-fold increase in lifespan and, subsequently, was confirmed to promote longevity in mammals [1]. In C. elegans, lifespan extension by reduced IIS, e.g., by reduction-of-function mutations in the daf-2 gene (the common ortholog of mammalian insulin and IGF1 receptors), is dependent on daf-16 (the ortholog of mammalian FOXO transcription factors) [1]. Of note, genetic variants in FOXO3 have been repeatedly and robustly associated with lifespan in humans [2].
Beyond daf-2 mutation, additional mechanisms for lifespan extension have been described in C. elegans, including two pathways that can even further extend the extraordinary long lifespan of daf-2 mutant worms: the absence of germline stem cells (GSCs) [3,4], and a gain-of-function mutation in the calcium/calmodulin-dependent kinase type II (CaMKII), unc-43 [5]. This further extension of daf-2 lifespan raises the possibility that the germlineand the unc-43 signaling pathways are at least in part mechanistically different from the IIS/daf-2 pathway. Interestingly, both the germline-and the unc-43 pathway, just as the daf-2 pathway, depend on daf-16 for lifespan extension [3,5]. Whether other factors that promote longevity in GSC-and in daf-2 deficient animals, such as hlh-30 (TFEB), hsf-1 (HSF1), and skn-1 (NRF2) [6][7][8][9], are also shared with the unc-43 pathway has not yet been investigated.
Several lines of evidence indicate that activation and function of the FOXO-transcription factor DAF-16 differ between GSC(−) and daf-2(−) C. elegans. For example, only GSC(−) worms require the conserved adaptor protein KRI-1 and the translation elongation factor TCER-1 for lifespan extension [10,11]. Moreover, whilst DAF-16 acts in the intestine to ensure GSC(−) longevity, it acts in the intestine and neurons to ensure daf-2(−) longevity [12]. DAF-16/FOXO function is strongly regulated by multiple protein kinases [13], including, but not limited to, the CaMKII-ortholog UNC-43. UNC-43 promotes DAF-16 nuclear localization by direct phosphorylation at a site different from the sites at which DAF-16 is phosphorylated and inhibited by the kinase AKT downstream of IIS [5]. CaMKII, in turn, is activated upon a sudden increase in intracellular calcium ion (Ca 2+ ) levels by binding to Ca 2+ -calmodulin complexes [14]. Many additional mediators of Ca 2+ -signaling are conserved across metazoans and present in C. elegans, including multiple types of sarcoplasmic/endoplasmic reticulum or plasma membrane channels, G-protein coupled receptors (GPCRs), phospholipase C (PLC) enzymes, and Ca 2+ -pumps [15]. Among these, the G-protein α-subunit egl-30, the γ-aminobutyric acid type B (GABA B ) receptor gbb-1, and the PLCβ-ortholog egl-8, through their function in neurons, also contribute to intestinal daf-16 activation and longevity [16][17][18].
Given that both, germline-ablation and unc-43 gain-of-function mutation, activate DAF-16 and further extend daf-2(−) lifespan, we hypothesized that the two pathways are largely overlapping. We tested this hypothesis in the present study and provide evidence for a novel longevity-promoting mechanism triggered by unc-43(gf) and also, albeit more weakly, by unc-43 and egl-8 loss, which may explain their different effects on various lifespan-extending pathways in C. elegans.

C. elegans Strains and Culture
Strains used in this study are listed in Supplementary Table S1. Worms were cultured following standard protocols on NG agar plates seeded with E. coli OP50 [19]. Worms carrying the glp-1(e2144ts) mutation served as a genetic model for germline ablation [4,9]. To eliminate germ cells, glp-1(ts) strains (referred to as GSC(−) in Results/Discussion), and corresponding glp-1(+) (i.e., GSC(+)) control strains were incubated at 25 • C for the first 24 h of postembryonic development and subsequently shifted to 20 • C for the remainder of the experiment. daf-2(e1370) worms and corresponding daf-2(+) control worms were continuously cultured at 20 • C. For experiments, all strains were routinely cultured on plates containing 20 µM 5-Fluoro-2 -deoxyuridine (FUDR; Sigma-Aldrich/Merck, Munich, Germany) from L4 onward to avoid progeny development and internal hatching in fertile strains carrying the unc-43(gf) mutation (allele n498).

Lifespan Analysis
Worms were synchronized by hypochlorite treatment, cultured under the appropriate temperature regimen (cf. above), and scored for survival every other day starting on days 8-10 of adulthood. From the late L4 stage onward, animals were maintained on 6 cm plates at a density of 40 worms/plate and every 10 days, they were transferred to fresh OP50seeded FUDR-containing NG agar plates to prevent progeny development and desiccation, respectively. Worms were considered dead if they did not respond to gentle touching with a worm pick. Animals that showed a protruding vulva or had ruptured, died from internal progeny hatching (bagging), or escaped from the plate were censored.

Stress Resistance Assays
Worms were synchronized by hypochlorite treatment, cultured under the appropriate temperature regimen (cf. above), and transferred to assay plates on day 2 of adulthood (20-30 worms per 3 cm plate for heat stress experiments, 50-60 worms per 3 cm plate for oxidative stress experiments). Survival was scored every 1-2 h. Oxidative stress assay plates contained 15.4 mM tert-butyl hydroperoxide (TBHP) and were prepared 12 h before starting the experiment.

Growing Worms for RNA-Extraction
To obtain synchronized populations, gravid adults were treated with hypochlorite, and eggs were allowed to hatch in M9 overnight.~700 L1 larvae per strain were plated on 10 cm NG agar plates seeded with concentrated E. coli OP50 (for RNA-seq: 1600 worms/2 plates) and cultured at the required temperatures (cf. above). At the L4-stage, 500 worms per strain were manually transferred to two E. coli OP50-seeded 6 cm NG agar plates supplemented with 20 µM FUDR to inhibit germ cell proliferation and progeny production (for RNA-seq: 1500 worms to two 10 cm plates). At day 1 of adulthood, worms were harvested by washing them off their plates with M9 buffer. After additional washing with M9 and RNAse-free water, worms were suspended in 1 mL Trizol, snap-frozen in liquid nitrogen, and stored at −80 • C until RNA extraction.

RNA-Extraction
RNA was extracted using Trizol and cleaned up with the Monarch Total RNA Miniprep Kit or RNA Cleanup Kit (New England Biolabs, Ipswich, MA, USA) according to the manufacturer's instructions. Equal amounts of RNA extracted from three biological replicates were pooled for RNA-sequencing to mitigate batch effects.

Overlap of DEG-Lists
The statistical significance of overlaps between gene lists was calculated as the hypergeometric probability of detecting at least as many common genes as observed in the two lists using the phyper function in R. Representation factors were calculated as the number of overlapping genes divided by the expected number of overlapping genes in the two lists (http://nemates.org/MA/progs/overlap_stats.html; accessed on 31 August 2020). For all calculations, the number of genes in the genome was set to 10,602, i.e., the number of genes in Wormbase WS276 that passed CPM-filtering during NOIseq-analysis. DEG-lists from published studies were, if necessary, converted to WBGene-IDs using WormMine (http://intermine.wormbase.org/tools/wormmine/begin.do; accessed on 10 August 2020) and adjusted to genes in WS276 using custom R-scripts and manual curation.

Dauer Analysis
Synchronized (hypochlorite treatment) L1 larvae were plated at a density of~50 larvae/6 cm plate (2 plates per strain), incubated at 20 • C or 25 • C, and inspected for the presence of dauers every 12 h. At the 60 h (20 • C) or 48 h (25 • C) timepoint, when wildtype worms reached adulthood, worms were morphologically classified as "dauer" or "non-dauer". Daf-2(−) strains, which all formed 100% dauers when incubated at 25 • C, were allowed to recover from dauer by incubation at 15 • C for 120 h before classification. Experiments in daf-2(+) strains were either conducted in parallel with experiments in daf-2(−) strains or included daf-2(−) control plates to provide a reference for the typical dauer morphology.

qPCR
A total of 1 µg total RNA was reverse transcribed using LunaScript RT SuperMix (New England Biolabs, Ipswich, MA, USA). qPCR-reactions were performed in duplicates or triplicates in a 20 µL reaction volume on an CFX Connect Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) with iTaq Universal SYBR Green Supermix (Bio-Rad Laboratories). The thermal cycling protocol comprised one activation step at 95 • C for 3 min, followed by 40 cycles of denaturation at 95 • C for 10 s and combined annealing/extension at 60 • C for 30 s. Melting curve analysis was performed from 65 • C to 95 • C with 0.5 • C increments at 5 s per step. Data were analyzed by the ∆∆Ct method, and target gene expression levels were normalized to the geometric means of cdc-42, tba-1, and Y45F10D.4 expression [32,33]. Primer sequences are listed in Supplementary Table S2.

Statistical Analysis
Statistical analysis was performed using Prism 5 or 9 (GraphPad Software, San Diego, CA, USA). Details on the particular tests used are specified in the figure legends. To test the hypothesis that germline ablation extends C. elegans lifespan at least in part by activating unc-43/CaMKII, we generated a set of relevant double mutants and measured their lifespans. Thereby, we took advantage of a widely used genetic model for germline stem cell deficiency, glp-1(e2144ts) (hereafter referred to as GSC(−); [4]) and combined this allele with the previously studied [5] unc-43 alleles n498 and n498n1186 (referred to as unc-43(gf) and unc-43(−), respectively). All experiments were conducted in the presence of FUDR (cf. Materials and Methods), to avoid internal progeny hatching in fertile unc-43(gf) worms and to facilitate comparison with similar previous studies that also took advantage of FUDR to prevent progeny development in fertile strains [5,8,17,18,34]. In GSC(+), i.e., otherwise wildtype worms, unc-43(gf) robustly extended lifespan (36-87%; Figure 1A, Supplementary Table S3), consistent with a previous report [5]. However, in contrast to this previous report, unc-43(−) also consistently produced a small to moderate lifespan increase in otherwise wildtype worms (6-21%; Figure 1A, Supplementary Table S3). Similarly, in GSC(−) worms, unc-43(gf) substantially extended lifespan (67-92%), while unc-43(−) had a more modest effect (2/3 experiments; 22-24%; Figure 1B, Supplementary Table S3). Collectively, these data cannot easily be reconciled with our original hypothesis and instead indicate that unc-43(gf) and unc-43(−) both trigger longevity-promoting mechanisms. Moreover, these mechanisms apparently are not, or are not maximally, triggered by germline ablation.

Discussion
In this study, we report lifespan-regulatory functions of the conserved signaling mediators unc-43 and egl-8 in long-lived GSC-deficient C. elegans. Moreover, our experiments confirm previously published positive roles of unc-43 hyperactivation and egl-8 loss for

Discussion
In this study, we report lifespan-regulatory functions of the conserved signaling mediators unc-43 and egl-8 in long-lived GSC-deficient C. elegans. Moreover, our experiments confirm previously published positive roles of unc-43 hyperactivation and egl-8 loss for longevity of otherwise wildtype animals and further support the concept that unc-43 and egl-8 modulate the activity of the conserved lifespan-regulatory key transcription factor DAF-16 [5,[16][17][18]. In addition, gene expression analyses indicate, to at least some extent, the induction of specific non-dauer longevity-associated collagens in response to unc-43 and egl-8 mutations. Currently available data do not support a prominent role for daf-16 in promoting expression of these collagens, raising the interesting possibility that they consti-tute a second, largely independent mechanism through which unc-43 and egl-8 regulate C. elegans lifespan (Figure 6). longevity of otherwise wildtype animals and further support the concept that unc-43 and egl-8 modulate the activity of the conserved lifespan-regulatory key transcription factor DAF-16 [5,[16][17][18]. In addition, gene expression analyses indicate, to at least some extent, the induction of specific non-dauer longevity-associated collagens in response to unc-43 and egl-8 mutations. Currently available data do not support a prominent role for daf-16 in promoting expression of these collagens, raising the interesting possibility that they constitute a second, largely independent mechanism through which unc-43 and egl-8 regulate C. elegans lifespan ( Figure 6). A key finding of our study consists in the observation that altered unc-43 and egl-8 activities further extend the lifespan of wildtype and GSC-deficient but not of Daf-2(−) (i.e. daf-2(e1370) at 20 °C) worms. This result may be explained by at least two, not mutually exclusive, models. First, unc-43(gf), unc-43(−) and egl-8(−) all may trigger processes that are already maximally induced by Daf-2(−), but not by GSC(−) and wildtype worms. This model is supported by the fact that all three mutations induce similar gene expression changes than Daf-2(−). Alternatively, processes triggered by unc-43(gf), unc-43(−) and egl-8(−) may not be critical to daf-2(e1370) longevity. Consistent with this possibility, unc-43(gf) and, to a lesser extent, unc-43(−) and egl-8(−), induce the expression of certain collagen genes, which are required for longevity under conditions that do not induce dauer-like traits in adults. These conditions include germline deficiency but not the daf-2(e1370) allele under our culture conditions of 20 °C [34]. Thus, it is tempting to speculate that these collagens constitute a second mechanism besides the regulation of daf-16, by which in particular unc-43(gf), but also unc-43(−) and egl-8(−) modulate lifespan. Of note, overexpression of a single key collagen, such as col-120, also examined by us by qPCR, is sufficient for lifespan extension [34]. Conversely, in daf-2(e1370), mild induction of collagen expression, which is still observed, at least in response to unc-43(gf), may not be functionally relevant. Clearly, it will be extremely interesting to directly examine the role of non-dauer longevity-associated collagens in unc-43(gf), unc-43(−) and egl-8(−) dependent C. elegans lifespan regulation and, eventually, to define the mechanisms that link unc-43/egl-8 to Thereby, these specific collagens may act in parallel with other longevity-promoting factors, e.g., daf-16. Daf-16, but not these specific collagens, is also required for the dauer-predisposing daf-2(e1370) mutation to extend lifespan. Note that daf-16 dependency of lifespan extension has been shown for GSC(−), unc-43(gf) and egl-8(−), but not yet for unc-43(−). See the main text for details and references.
A key finding of our study consists in the observation that altered unc-43 and egl-8 activities further extend the lifespan of wildtype and GSC-deficient but not of daf-2(−) (i.e., daf-2(e1370) at 20 • C) worms. This result may be explained by at least two, not mutually exclusive, models. First, unc-43(gf), unc-43(−) and egl-8(−) all may trigger processes that are already maximally induced by daf-2(−), but not by GSC(−) and wildtype worms. This model is supported by the fact that all three mutations induce similar gene expression changes than daf-2(−). Alternatively, processes triggered by unc-43(gf), unc-43(−) and egl-8(−) may not be critical to daf-2(e1370) longevity. Consistent with this possibility, unc-43(gf) and, to a lesser extent, unc-43(−) and egl-8(−), induce the expression of certain collagen genes, which are required for longevity under conditions that do not induce dauer-like traits in adults. These conditions include germline deficiency but not the daf-2(e1370) allele under our culture conditions of 20 • C [34]. Thus, it is tempting to speculate that these collagens constitute a second mechanism besides the regulation of daf-16, by which in particular unc-43(gf), but also unc-43(−) and egl-8(−) modulate lifespan. Of note, overexpression of a single key collagen, such as col-120, also examined by us by qPCR, is sufficient for lifespan extension [34]. Conversely, in daf-2(e1370), mild induction of collagen expression, which is still observed, at least in response to unc-43(gf), may not be functionally relevant. Clearly, it will be extremely interesting to directly examine the role of non-dauer longevity-associated collagens in unc-43(gf), unc-43(−) and egl-8(−) dependent C. elegans lifespan regulation and, eventually, to define the mechanisms that link unc-43/egl-8 to expression of these collagens. Although we have not yet experimentally confirmed or excluded the possibility that the known mediator of unc-43(gf)/egl-8(−) longevity, daf-16, regulates non-dauer longevityassociated collagen expression in the specific context of unc-43/egl-8 mutation, even though it does not do so in other contexts, an alternative candidate is emerging from the literature: skn-1 [34]. In support of this hypothesis, we already showed that unc-43(gf), unc-43(−) and egl-8(−) modulate the expression of similar gene sets, including similar collagens, than skn-1.
Yet, our study and a previous report [5] on the longevity effects of unc-43(−) and unc-43(gf) differ from each other in several details despite the use of the same alleles. In the case of unc-43(−), these discrepancies may be explained by different experimental conditions, i.e., different temperatures, and by the use of FUDR, both of which have been shown to affect lifespan in certain genetic backgrounds [43,44]. Specifically, we conducted the respective assays at 20 • C rather than 25 • C [5] and analyzed the unc-43(−) and unc-43(gf) alleles within the same experiments, which required the addition of FUDR not just to unc-43(gf) and corresponding control [5], but also to unc-43(−) strains. Importantly, FUDR does not affect wildtype lifespan nor daf-2(−)'s and GSC(−)'s basic ability to promote longevity [8,34,43].
In agreement with published studies [16][17][18], we found that loss of egl-8 further extends wildtype lifespan. However, in contrast to previous work [16], we did not observe decreased oxidative stress resistance upon egl-8 loss. Again, this difference may be explained by different experimental conditions, such as different egl-8(−) alleles (md1971 vs. n488), different assay temperatures (20 • C vs. 25 • C) and treatment with different chemicals to impose oxidative stress (TBHP vs. arsenite). Indeed, TBHP and arsenite have already been shown to trigger different transcriptional responses [49]. In a current model, egl-8 modulates lifespan cell non-autonomously through its function in neurons: egl-8 loss reduces neuronal secretion of insulin-like peptides, which in turn increases daf-16 activity in the intestine; in addition, evidence suggests that this pathway is further mediated by the EGL-8-generated second messenger diacylglycerol (DAG) and the DAG-dependent kinase dkf-2 [16][17][18]50]. This model predicts that worms with compromised insulin/IGF1-like receptor function in the intestine will not display increased longevity upon an egl-8(−) mutation, while GSC(−) worms may display further intestinal daf-16 activation and lifespan extension. Results from our lifespan analyses are in line with this prediction. Conceptually, egl-8 loss also decreases the levels of another second messenger, inositol-1, 4, 5-trisphosphate and subsequently, intracellular Ca 2+ levels, and CaMKII/UNC-43 activity [15]. Yet, additional studies are necessary to determine how egl-8(−) and unc-43(−) interact to modulate daf-16 activity and C. elegans lifespan.
In summary, our study identified new genetic backgrounds in which unc-43 and egl-8 can/cannot modulate lifespan in C. elegans and suggests collagen expression as a second lifespan-regulatory mechanism functioning downstream of unc-43 and egl-8, in addition to the known regulator daf-16.