Stress survival and longevity of Caenorhabditis elegans lacking NCS-1

Abstract

Although dysfunctional Ca²⁺ signaling can trigger biochemical reactions that lead to cell death, the role of calcium-binding proteins (CBPs) in this process is still a topic of debate. Neuronal calcium sensor 1 (NCS-1) is a CBP that is highly conserved and has been shown to increase cell survival against various types of injuries. As such, we hypothesized that NCS-1 could also be a stress-responsive protein with potential effects on survival and longevity. To explore this possibility, we conducted experiments to examine how Caenorhabditis elegans ncs-1 mutant nematodes fared under three different stress conditions: hyperosmotic, thermal, and chemical oxidant challenges. Our results showed that while the lack of NCS-1 had no effect on survival responses to hyperosmotic and thermal stresses, ncs-1 worms demonstrated remarkable resistance to the oxidant paraquat in a dose-dependent manner. Based on these findings, we conclude that C. elegans may employ adaptive mechanisms in the absence of NCS-1 to survive specific oxidative stress stimuli.

Background

Abnormalities in the intracellular calcium (Ca²⁺) signaling alter cellular homeostasis leading to death in biological organisms.^1,2 Although the role of Ca²⁺ as a death trigger is well-described in processes such as abnormal Ca²⁺ distribution³ and excessive Ca²⁺ influx into cells,⁴ the relevant issue to be addressed is the contribution of the calcium-binding proteins (CBPs) in the cytotoxic effects in response to Ca²⁺ imbalances.

CBPs bind selectively to Ca²⁺ and mediate several cellular functions by acting as Ca²⁺-modulated sensors.⁵ Neuronal calcium sensor 1 (NCS-1) is a highly conserved CBP that has been shown to regulate the evoked neurotransmission⁶ and synaptic plasticity.⁷ In the nematode, Caenorhabditis elegans, proper Ca²⁺ signaling via NCS-1 appears to be essential for associative learning, memory, regulation of the warming-evoked thermoreceptor currents, and locomotion.^8–10

However, evidence also suggests a putative role for NCS-1 on cell survival mechanisms both in rodent and in vitro models. For example, NCS-1 overexpression in mouse brains caused axonal sprouting, regeneration, and neuroprotection after corticospinal tract denervation.¹¹ Moreover, NCS-1 overexpression in cultured neurons turned the cells more tolerant to death induced by oxidative stress.¹² Nevertheless, it remains unclear whether an imbalance of the Ca²⁺ homeostasis due to NCS-1 loss-of-function compromises the whole-organism survival under exposure to stress agents. This knowledge will be useful not only for a better understanding of the NCS-1 function in survival mechanisms but also to increment information on the apparent involvement of NCS-1 in the pathophysiology of neuropsychiatric disorders.^13–15 For example, we showed recently that the NCS-1 knockout (KO) mice display phenotypic abnormalities that resemble symptoms of neuropsychiatric patients.¹⁵ Moreover, we also showed that adeno-associated virus-mediated Ncs-1 overexpression in the frontal cortex produced anxiolytic-like and pro-social behaviors in mice.¹⁶ Possibly, the absence of NCS-1 may decrease the capability of cells to recover under unfavorable conditions therefore contributing to the remarkable deficits in neuronal plasticity seen in patients.¹⁷

In this study, we investigated the ability of the nematode C. elegans lacking NCS-1 (ncs-1) to deal with three different stressor stimuli: hyperosmotic, thermal, and chemical oxidant stresses. We carried out these experiments in C. elegans because this animal model has been demonstrated to yield information on the cellular processes that influence both survival and lifespan.¹⁸

Methods

Strains and growth conditions

C. elegans strains were grown at 20 °C on nematode growth medium (NGM) agar plates seeded with a lawn of Escherichia coli strain OP50 as a food source.¹⁹ Drugs and vehicles were combined with the non-solid NGM at the indicated concentrations. After solidified, plates were seeded with bacteria and incubated overnight to allow bacterial growth under 20–22 °C. The phenotypical changes due to NCS-1 loss-of-function were investigated in C. elegans strain XA406 (ncs-1 (qa401) X), which are viable, with normal developmental timing, and total lack of NCS-1 expression according to Gomez et al.⁸ The wild-type strain was C. elegans N2 Bristol. All the worms were obtained from the Caenorhabditis Genetics Center. To synchronize larvae, we isolated embryos by hypochlorite treatment as previously shown (www.wormbook.org).

Hyperosmotic stress

Hyperosmotic stress was carried out essentially as described by Gal et al.²⁰ and Lamitina et al.²¹ with slight modifications. Briefly, ncs-1 or N2 worms at the L4 larval stage were cultured on NGM plates containing four concentrations of sucrose (305, 400, 484, and 652 mM) for 24 h at 20 °C (approximately 30 nematodes for each petri dish). We assessed viability in M9 buffer (42 mM Na₂HPO₄, 22 mM KH₂PO₄, 86 mM NaCl and 1 mM MgSO₄.7H₂O) at 22 °C. We scored the worms as dead if they displayed no spontaneous movement, pharyngeal pumping, or response when prodded.

Heat stress

Thermotolerance studies were conducted according to Lithgow et al.²² with modifications. We exposed ncs-1 and N2 worms at the L1 or L4 larvae stage to 35 °C ± 0.8 °C from 1 h to 8 h on small pre-warmed (35 °C) NGM plates (20–30 nematodes for each petri dish). At the indicated times, culture plates were returned to 20 °C, and the worms were scored for survival as above described. Counts were performed in three replicates of worms and plates were discarded after each scoring.

Lifespan

Synchronized N2 and ncs-1 worms at the L1 larval stage were chosen for analysis. Day 0 was defined when approximately 20 larvae were cultured on each NGM petri dish containing 0.4 mM paraquat (1,1 ′ -dimethyl-4,4 ′ -bipyridinium) dichloride or drug vehicle (PBS) and a lawn of E. coli strain OP50 onto the center of the plate. Worms were checked for viability (as described previously) every 1–2 days from day 1 until death. We transferred the animals to fresh dishes every 2 days until the cessation of progeny production as indicated elsewhere.²³ Dead worms with internally hatched progeny, extruded gonad, or desiccation caused by crawling off the agar were excluded from the analyses.

Effect of paraquat on survival and larval development

Paraquat (methyl viologen; Sigma) treatment was performed in N2 and ncs-1 at the L1 larval stage to evaluate the impact of oxidative stress on survival and larval development. About 15 larvae from both strains were grown on NGM plates seeded with E. coli OP50 containing paraquat at 0.4, 0.8, 1.0, 1.2, or 2.4 mM concentrations or drug vehicle (PBS). Development and survival were monitored daily in worms kept under 20 °C for 10 days. Larval development was assessed by microscopic examination of stage-specific anatomical characteristics such as body size, vulva and gonad development, and egg production. Animals were classified as viable adults (those that reached adulthood followed by egg-laying), development delayed (worms unable to reach maturity even after 10 days of observation), or dead.

Data collection and statistical analyses

All experiments were replicated at least three times, and the survival scores were calculated as an average from three plates in each independent experiment. We pooled the scores from each condition to calculate summary statistics. The total sample size (n), as well as the statistical tests and post-hoc analyses used for comparisons between ncs-1 and N2 worms are reported in each figure/table legend. All data sets were parametric according to the D’Agostino and Person omnibus normality test. All graphics and statistics were carried out using GraphPad Prism® (GraphPad Software Corporation).

Results

Hyperosmotic stress

To compare survival rates between ncs-1 and N2 strains under hyperosmotic stress, we transferred the synchronized nematodes at the L4 stage from NGF (containing 21 mM NaCl) to agar plates with increasing concentrations of sucrose (305 mM to 652 mM). Analyses 24 h after transfer found that all the worms survived to sucrose at 305 mM (Table 1). In contrast, N2 worms exposed to 400 and 484 mM of sucrose displayed a significant decrease in viability, which was not statistically different from the ncs-1-treated worms. On 400 mM sucrose agar, 87.2% ± 5.2 (n = 235) of N2 and 88.1% ± 4.7 (n = 237) of ncs-1 worms survived after 24 h (Table 1). This survival fraction was quite similar to that found in 484 mM sucrose (85.4% ± 3.8 [n = 230] and 84.8% ± 5.1 [n = 228] for N2 and ncs-1, respectively). We did not observe any worm alive in the 652 mM sucrose agar plates. Altogether, these findings indicate that the hyperosmotic stress with sucrose affects C. elegans viability independently of NCS-1.

Table 1.

Effect of hypertonic stress on survival of C. Elegans following 24 h of exposure to growth agar containing increasing concentrations of sucrose.

	Sucrose concentration (mM)
	305	400	484	652
N2	100 ± 0	87.2 ± 5.2	85.4 ± 3.8	0 ± 0
ncs-1	100 ± 0	88.1 ± 4.7	84.8 ± 5.1	0 ± 0

Values are mean (%) ± S.E.M. Comparison among groups was perfomerd by two-way ANOVA.

Heat shock

We then investigated whether the lack of NCS-1 affects the intrinsic thermotolerance of the L1 and L4 larvae of C. elegans. We assessed survival rates by scoring three replicate populations every hour throughout the period of 8 h of thermal stress at 35 °C. In agreement with several studies,^20,22,24 all N2 worms (L1 and L4) subjected to a 35 °C heat shock remained viable until 3 h (Fig. 1). However, we noted that survival of L1 and L4 N2 worms decreases starting from 4 h of exposure. After 8 h, the longest exposure, 77.2% and 75.1% of L1 and L4 N2 worms survived respectively (Fig. 1). Overall, both ncs-1 and N2 strains exhibited identical survival at all the evaluated intervals during heat-shock, suggesting that NCS-1 does not seem to influence the heat-shock viability of C. elegans.

Fig. 1.

Effect of the lack of NCS-1 on the intrinsic thermotolerance of the L1 (A) and L4 (B) larvae of C. Elegans. Survival rates (%) were scored every hour throughout the period of 8 h of thermal stress at 35 °C. Heat treatment impaired survival of the N2 and ncs-1 worms at the same levels. Bars represent standard deviations calculated from three repeats of each experiment. n = 270. Two-way ANOVA.

Lifespan

To investigate the possibility that the lack of NCS-1 influences lifespan in C. elegans, hermaphrodite worms at the L1 stage were cultured at 20 °C on the NGF plates with abundant E. coli OP50 (day 0) and were analyzed every 1–2 days from day 0 until death. We did not observe any difference in the mean lifespan of ncs-1 (18.1 days) in comparison to N2 (18.4 days) worms (Fig. 2). We also investigated if exposure to the reactive oxidant paraquat would exert different effects on the longevity of these worms. As expected, exposure to paraquat resulted in significant decreases in the mean lifespan in both ncs-1 and N2 worms from 18 days to 7–8 days (Fig. 2). Again, differences in the lifespan of ncs-1 (8.1 days) and N2 (7.4 days) worms were not statistically significant under this oxidative stress condition. Likewise, statistical analyses did not show differences in the maximum lifespan between the strain in both non-treated and treated conditions (Fig. 2A and B). Similar results were found when the NGF plates contained 5-fluorodeoxyuridine (FUdR) to prevent egg-laying by the worms.

Fig. 2.

Lifespan of ncs-1 in comparison to N2 worms. C. Elegans at the L1 larval stage were cultured at 20 °C on the NGF plates with abundant E. Coli OP50 (day 0) and were analyzed every 1–2 days from day 0 until death. (A) N2 and ncs-1 represent WT and NCS-1 mutant worms respectively, cultured with no drug. We did not observe any difference of the lifespan of ncs-1 in comparison to N2 worms. N2 + PQ and ncs-1 + PQ are WT and ncs-1 worms respectively, exposed to paraquat (PQ) at 0.4 mM. Although the paraquat treatment shortened the mean and maximum lifespan significantly, there was no difference in the lifespan between these strains. This graph represents the results of a single trial; combined data for three or more trials are presented in the table at the bottom. (B) Lifespan analysis of ncs-1 and N2 worms untreated or treated with paraquat. Mean and maximum (max) lifespan were compared between N2 versus ncs-1 or N2 + PQ versus ncs-1 + PQ using the Student’s t-test since the standard deviations of the both groups were similar. Maximum adult lifespan is the mean lifespan of 10% of the population that had the longest lifespans. N represents the number of worms analyzed, with number of independent experiments (replicates) in parentheses.

Resistance to paraquat

We also conducted a dose–response experiment with paraquat to investigate the effect of oxidative stress on the proportion of larvae at L1 that succeed in completing development in the absence of NCS-1. All the worms cultured in a medium containing 0.4 mM paraquat arrived at the adult stage without any apparent phenotypic abnormalities (Fig. 3A). In contrast, paraquat concentrations ranging from 0.8 to 2.4 mM ablated the full development and resulted in a range of developmental arrest and death rates (Fig. 3B and C). According to our analyses, the only difference found between ncs-1 and N2 worms under these conditions was in the 0.8 mM paraquat treatment, in which the mortality of ncs-1 (23%) was significantly lower than N2 worms (80%). This result was probably due to the greater susceptibility of ncs-1 worms to developmental arrest at this paraquat concentration. Compared to N2 worms (20%), the fraction of the ncs-1 worms treated with 0.8 mM of paraquat with delayed development was dramatically increased (77%). Therefore, ncs-1 worms appear somewhat more resistant to paraquat than N2 worms only at the 0.8 mM concentration. These data suggest that the ablation of NCS-1 in C. elegans induces prolonged interruption of development and influences adaptation to paraquat exposure in a dose-specific manner.

Fig. 3.

Effect of paraquat on the survival and larval development of nematodes C. Elegans lacking NCS-1. About 15 larvae from N2 and ncs-1 strains at the L1 stage were grown on NGM plates seeded with E. Coli OP50 containing paraquat at 0.4, 0.8, 1.0, 1.2 or 2.4 mM or drug vehicle (PBS). Development and survival was monitored daily in worms kept under 20 °C for 10 days. Animals were classified as (A) viable adults, (B) development delayed (worms unable to reach maturity even after 10 days of observation), or (C) dead. Paraquat concentrations ranging from 0.8 to 2.4 mM ablated the full development, resulting in increased scores of developmental arrest and death. At 0.8 mM paraquat, mortality of ncs-1 (23%) was significantly lower than N2 worms 80% (^**P < 0.01; two-way ANOVA following Bonferroni’s post-hoc test). Bars represent mean and standard error of the mean (SEM) calculated from three repeats of each experiment. n = 270 for each condition.

Discussion

Dysbalanced Ca²⁺ signaling is known to trigger biochemical reactions that are associated with cell death^1,25 though the involvement of the calcium-binding proteins such as NCS-1 in this process is still under scrutiny. Although NCS-1 has been recognized to increase cell survival against a range of injuries, no study up-to-date has addressed the impact of the NCS-1 loss-of-function on whole organism survival and longevity. Here, we presented results consistent with this hypothesis by evaluating how nematodes C. elegans ncs-1 tolerate three different stress conditions: hyperosmotic, thermal, and chemical oxidant stresses. We found that NCS-1 does not appear to be essential for survival response to hyperosmotic or thermal stresses (Table 1 and Fig. 1) since survival rates of ncs-1 and N2 worms were similar. Moreover, these worms displayed similar lifespans even under paraquat treatment (Fig. 2). However, ncs-1 worms have increased resistance to paraquat in a dose–response context. In contrast to other paraquat concentrations (0.4; 1.0; 1.2 and 1.4 mM), 0.8 mM paraquat treatment caused a significantly lower mortality of ncs-1 (23%) in comparison to N2 worms (80%) (Fig. 3).

Although our results do not contradict evidence supporting a putative role of NCS-1 in neuron survival,^11,12 they do suggest that loss of NCS-1 has a relatively weak effect on the survival of C. elegans. Given that C. elegans NCS-1 is expressed predominantly in sensory neurons⁸ and the physiological changes induced by stress affect the whole worm body, one possible explanation for our findings is that the deficiency of NCS-1 is not sufficient to drastically affect the whole organism. Current efforts in our laboratory are focused on investigating the survival of NCS-1-expressing neurons, rather than the whole-organism survival. Since ncs-1 worms are reported to display significant defects in behavioral tasks associated with these sensory neurons,^8–10 we do not rule out that NCS-1-expressing neurons are less resilient to injury in the absence of NCS-1.

The increased survival of ncs-1 worms treated with 0.8 mM paraquat contrasts with data showing that overexpression of NCS-1 increases the ability of neurons to withstand the effects of stress.^11,12 Possible explanations for this apparent discrepancy include evidence showing that the nematode C. elegans displays broad hormetic abilities, in which low exposure to otherwise harmful agents slightly lengthens lifespan and increases the antioxidant defense.^22,26,27 Since the genetic background influences on hormetic abilities,²⁷ we do not exclude that the lack of NCS-1 induces an initial intrinsic stress response that facilitates the adaptation of the ncs-1 worms to a subsequent environmental stressor. This possibility is reinforced by our results showing that low mortality of ncs-1 treated with 0.8 mM paraquat was followed by an increased fraction of worms with delayed development (77% in comparison with 20% of N2 worms). Therefore, it appears that loss of NCS-1 extends the propensity of C. elegans at early larval stages for developmental arrest, which in turn would ensure survival under unfavorable conditions such as paraquat exposure.

Evidence obtained on C. elegans revealed in the last years several genes that extend lifespan and enhance resistance under stress conditions.^18,28 Therefore, this nematode is considered a useful model for investigating the link between CBP genes and stress. Such as NCS-1, calreticulin (CRT) is a CBP found to be overexpressed under chemical stress in both mammals and C. elegans.^29,30 Surprisingly, C. elegans overexpressing calreticulin are resistant to thermal stress though they exhibit a reduced lifespan.³⁰ This observation suggests that stress resistance and lifespan extension are dissociated in C. elegans mutants of CBPs, in agreement with our results. Additionally, CBPs appear to exert different effects on longevity since the lifespan of C. elegans crt-1 null mutants is reduced,³⁰ in contrast to ncs-1 worms.

Conclusions

We hypothesized in this study that the lack of NCS-1 might decrease the tolerance of C. elegans to stress, which would affect some organism responses such as survival and longevity. However, we found that ncs-1 worms are not significantly more susceptible to stress. Conversely, these worms display a remarkable resistance to paraquat, suggesting that adaptive or compensatory mechanisms under the absence of NCS-1 may keep C. elegans alive under oxidative conditions. These observations open a new window for investigating the cellular mechanisms by which calcium-binding proteins, such as NCS-1, modulate survival under oxidative stress.

Author contributions

LAVM, SHDBP and PG performed the experiments and analyzed data. LAVM and APBO wrote the paper. LAVM, DFRV and MARS jointly conceived and supervised the study. All authors gave their final approval for publication.

Funding

This study was supported by Fundação de Amparo à Pesquisa do Estado de Minas Gerais – FAPEMIG (grant APQ-00075-09), Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (grant 573646/2008–2) and Fundação Coordenação de Aperfeiçoamento de Pessoal de Nível Superior – CAPES (Finance Code 001). MARS is a CNPq research fellow.

 

Conflict of interest statement: All authors declare that they have no conflicts of interest.

Data availability

Data used in this study are available upon request.

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