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. Author manuscript; available in PMC: 2016 Feb 29.
Published in final edited form as: Cell. 2013 Jun 20;153(7):1435–1447. doi: 10.1016/j.cell.2013.05.042

XBP-1 Is a Cell-Nonautonomous Regulator of Stress Resistance and Longevity

Rebecca C Taylor 1, Andrew Dillin 1,*
PMCID: PMC4771415  NIHMSID: NIHMS554127  PMID: 23791175

SUMMARY

The ability to ensure proteostasis is critical for maintaining proper cell function and organismal viability but is mitigated by aging. We analyzed the role of the endoplasmic reticulum unfolded protein response (UPRER) in aging of C. elegans and found that age-onset loss of ER proteostasis could be reversed by expression of a constitutively active form of XBP-1, XBP-1s. Neuronally derived XBP-1s was sufficient to rescue stress resistance, increase longevity, and activate the UPRER in distal, nonneuronal cell types through a cell-nonautonomous mechanism. Loss of UPRER signaling components in distal cells blocked cell-nonautonomous signaling from the nervous system, thereby blocking increased longevity of the entire animal. Reduction of small clear vesicle (SCV) release blocked nonautonomous signaling downstream of xbp-1s, suggesting that the release of neurotransmitters is required for this intertissue signaling event. Our findings point toward a secreted ER stress signal (SERSS) that promotes ER stress resistance and longevity.

INTRODUCTION

Cells are faced throughout their lifetime with a variety of stresses that lead to protein misfolding and aggregation (Balch et al., 2008). The disruption of protein homeostasis (proteostasis) mechanisms that guard the proteome against these stresses can be catastrophic to cellular viability. To protect against proteotoxicity, cells have evolved compartment-specific stress responses that allow for the targeted induction of chaperone and degradation machineries within select organelles or sub-cellular locations. These stress responses include the heat shock response (HSR), the endoplasmic reticulum unfolded protein response (UPRER), and the mitochondrial unfolded protein response (UPRmt) (Akerfelt et al., 2010; Ron and Walter, 2007; Haynes and Ron, 2010). These collective responses give the cell the capacity to manipulate its chaperone networks and protein degradation pathways in response to subcellular stress.

The UPRER responds to the presence of misfolded proteins in the ER, an important site for folding and modification of many proteins, including those destined for secretion and membrane localization (Ron and Walter, 2007). The UPRER is comprised of three canonical branches. Each of these branches transmits information on ER proteostasis to the cytosol through a trans-membrane regulator—PERK, IRE1, or ATF6—that activates a distinct downstream signal transduction pathway, culminating in a program that reduces ER load and increases the capacity of the organelle to deal with protein misfolding (Ron and Walter, 2007). Activation of the ancestral IRE1 pathway leads to the regulated splicing of a transcription factor, XBP1, by the IRE1 endoribonuclease activity (Sidrauski and Walter, 1997). Thus activated, spliced XBP1, XBP1s, is translated and regulates a range of transcriptional targets required for ER proteostasis (Shen et al., 2005; Acosta-Alvear et al., 2007).

The maintenance of proteostasis both within the ER and throughout the cell becomes increasingly difficult with age, as damaged, misfolded, and aggregated proteins progressively accrue. The accumulation of damaged proteins is exacerbated by what appears to be an organism-wide loss in the ability to mount cellular stress responses. As such, a decline in the capacity of the cell to protect its proteome has been highly correlated with age-onset disease. During aging, ER chaperones are down-regulated, and activation of the UPRER upon ER stress may be abrogated with age (Ben-Zvi et al., 2009). Reduction of the IRE-1/XBP-1 UPR branch also shortens the long lifespan caused by reduced insulin/IGF1 signaling (IIS) in C. elegans (Henis-Korenblit et al., 2010). However, it is unknown whether ectopic induction of the UPRER is sufficient to prevent age-onset decline of proteostasis and delay age-related pathologies.

We hypothesized that the controlled induction of the UPRER might reverse the loss of ER proteostasis in aging. We find that age-onset loss in UPRER function can be rescued by the expression of constitutively active xbp-1s. The expression of xbp-1s in the neurons initiates a cell-nonautonomous response in which the UPRER is activated in non-neuronal tissue, protecting against ER stress and extending lifespan in the whole organism. Moreover, cell-nonautonomous UPRER activation requires neurotransmitter release from small clear vesicles (SCVs). We therefore conclude that constitutive activation of XBP-1 in neurons can rescue stress resistance and extend lifespan through a cell-nonautonomous signaling mechanism.

RESULTS

The Ability to Activate the UPRER Declines with Age

Previous studies indicate that the ability to activate protective stress responses declines early in adulthood (Ben-Zvi et al., 2009; Dillin et al., 2002a, 2002b; Durieux et al., 2011). We hypothesized that the maintenance of the ER proteome would also decline with age in C. elegans. We used an hsp-4p::GFP strain as a reporter for UPRER activation (Calfon et al., 2002). Transcription of hsp-4, the worm ortholog of the mammalian ER-localized Hsp70 chaperone BiP, is upregulated upon ER stress in a manner dependent upon the IRE-1 kinase/endoribonuclease and the XBP-1 transcription factor. We treated hsp-4p::GFP worms with tunicamycin, an inhibitor of N-linked glycosylation that induces ER stress, at a range of ages from day 1 to day 10 of adulthood. We found that activation of the hsp-4p::GFP reporter was dependent upon xbp-1 and ire-1 (Figure S1A available online and data not shown), consistent with Calfon et al. (2002). In a wild-type background, reporter activation was robust at days 1–2 of adulthood but reduced sharply by days 3–4 and was essentially nonexistent by days 7–10 of adulthood (Figures 1A and 1B).

Figure 1. The Ability to Activate the UPRER Declines with Age.

Figure 1

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(A) hsp-4p::GFP UPRER reporter worms were treated at days 1, 4, 7, and 10 of adulthood with 25 ng/μl tunicamycin in M9 buffer, or buffer only, for 4 hr, and GFP fluorescence assessed.

(B) hsp-4p::GFP UPRER reporter worms were treated as above at days 1, 2, 3, 4, 7, and 10 of adulthood, and GFP expression measured by fluorimetry. Fold inductions relative to untreated animals are shown, with error bars indicating mean + standard error of the mean (SEM) (n = 3). A Student's t test was used to assess statistical significance of induction at each age: *p < 0.05, n/s = not significant.

(C) N2 animals were treated as above at day 1 and day 5 of adulthood. Transcript levels of UPR regulators and targets in tunicamycin-treated (red) and untreated (black) animals were measured by quantitative RT-PCR, with error bars indicating mean ± standard deviation (SD). A Student's t test was used to assess significance: ***p < 0.005, n/s = not significant.

(D) Schematic describing the measurement of age-dependent ER stress resistance. Animals are transferred to plates containing tunicamycin at either day 1 or day 7 of adulthood.

(E) N2 (black) and xbp-1(zc12) (red) animals were transferred at day 1 and day 7 of adulthood to plates containing 50 μg/ml tunicamycin, and survival monitored (N2 day 1, median lifespan 7 days; xbp-1(zc12) day 1, median lifespan 5 days; p < 0.0001. N2 day 7, median lifespan 4 days; xbp-1(zc12) day 7, median lifespan 4 days; p = 0.9211. See Table S1).

See also Figure S1 and Table S2.

A similar loss in UPRER activation was evident upon examination of transcript levels (Figure 1C). Levels of total xbp-1 transcript increased 2-fold following tunicamycin treatment at day 1 of adulthood but were virtually unchanged upon tunicamycin treatment at day 5. Levels of spliced xbp-1 transcript were increased around 20-fold by tunicamycin treatment at day 1, but very little increase in spliced transcript was observed by day 5. Transcriptional upregulation of UPRER target genes hsp-4, crt-1, T14G8.3, and Y41C4A.11 was also greatly reduced by day 5. Interestingly, whereas ire-1 transcript levels were increased roughly 2-fold by ER stress at day 1, by day 5, basal transcript levels were increased around 2-fold relative to day 1 basal levels, and no further increase was seen upon ER stress. xbp-1 splicing and UPRER-induced transcriptional changes therefore seem to be abrogated with age, within early adulthood.

Tunicamycin treatment inhibits the formation of N-acetylglucosamine lipid intermediates, preventing the glycosylation of newly synthesized glycoproteins, and therefore requires active protein translation to induce ER stress (Heifetz et al., 1979). C. elegans are robustly translationally active through day 3 of adulthood (Hansen et al., 2007), whereas the ability of tunicamycin to activate the UPRER is substantially reduced by day 3 (Figure 1B), suggesting that this diminished activation is not caused by a loss of translation. As an independent measure of ER stress that is not dependent upon active protein translation, we treated animals with thapsigargin, which is an inhibitor of the sarco-endoplasmic reticulum Ca2+-transport ATPase (SERCA) and induces ER stress by depletion of ER Ca2+ stores (Lytton et al., 1991; Kijima et al., 1991). Treatment with thapsigargin at day 1 of adulthood induced the UPRER, although to a lesser degree than treatment with tunicamycin, as previously shown (Figures S1B and S1C) (Caruso et al., 2008). However, UPRER activation by thapsigargin was abolished by day 4 of adulthood with similar kinetics to the loss of UPRER activation upon tunicamycin treatment.

In addition to tunicamycin and thapsigargin, heat stress and exposure to pathogenic bacteria also induce the UPRER (Galbadage and Hartman, 2008; Richardson et al., 2010). We treated hsp-4p::GFP animals with exposure to pathogenic Pseudomonas aeruginosa strain PA14 or with a 34° C heat shock at day 1 and day 7 of adulthood. As seen with tunicamycin treatment, we observed upregulation of GFP fluorescence at day 1 but not at day 7, suggesting that the response of the UPRER to these stresses also declines with age (Figures S1D and S1E). Taken together, loss of UPRER activation with age is not simply a consequence of reduced rates of translation but rather reflects the ability of the animal to mount a response to ER stress.

Protection against UPRER-Inducing Stress Declines with Age

Because induction of the UPRER declined with age, we hypothesized that physiological resistance to ER stress would also decline. We challenged wild-type N2 and xbp-1(zc12) mutant animals, which lack an intact UPRER, to the ER stressor tunicamycin at day 1 and day 7 of adulthood and monitored their survival (Figure 1D). We found that when animals were transferred to tunicamycin at day 1, N2 worms survived substantially longer than their xbp-1(zc12) mutant counterparts (Figure 1E). However, whentransferred at day 7, the survival of N2 animals was no longer than that of xbp-1(zc12) mutant animals, consistent with an age-onset loss of ER stress resistance. No difference in longevity between wild-type and xbp-1 mutants was seen upon treatment with paraquat, an agent that increases oxidative stress and does not activate the UPRER, demonstrating that xbp-1 mutant animals are specifically susceptible to ER stress (Figures S2A and S2B).

We then asked whether the youthful susceptibility to ER stress that we observe in xbp-1 animals affected longevity under standard culture conditions and found that the lifespan of xbp-1 mutants was consistently shorter than that of N2 animals (Figure S2C). Importantly, pharyngeal pumping rates (indicative of food intake) were similar between these genotypes, and animals were still actively feeding at day 8 of adulthood (Figure S2D). These results suggest that the presence of functional XBP-1 offers a protection against ER stress in young animals that is no longer present in older animals, potentially due to a decline in XBP-1 activation and target regulation during early adulthood.

Expression of xbp-1s Constitutively Activates the UPRER Late in Life

Because xbp-1 splicing and expression of UPRER targets are lost with age, we hypothesized that the expression of a constitutively active, spliced form of xbp-1 might be sufficient to induce UPRER activation past the age at which stress-induced splicing of endogenous xbp-1 ceases. Activation of C. elegans xbp-1 is achieved by IRE-1-mediated removal of a 23 nucleotide intron, allowing the translation of an active transcription factor (Calfon et al., 2002). We amplified xbp-1 from C. elegans complementary DNA (cDNA) and removed this intronic sequence to create a cDNA sequence identical to that created by IRE-1-mediated splicing, xbp-1s (Figure 2A). We then expressed this gene in C. elegans under the ubiquitously expressed sur-5p promoter (Gu et al., 1998). Expression of sur-5p::xbp-1s in an hsp-4p::GFP background led to robust GFP expression throughout the animal at all ages, even those at which stress-induced splicing of xbp-1 and target upregulation are no longer observed in wild-type animals (Figures 1C, 2B, and 2C). Transcript levels of UPRER targets were also elevated in sur-5p::xbp-1s animals (Figure 2D).

Figure 2. Expression of xbp-1s under the sur-5p Promoter Constitutively Activates the UPRER and Prevents a Decline in ER Stress Resistance.

Figure 2

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(A) Schematic of the exon/intron boundaries of the 23 bp intron removed from the xbp-1 sequence to activate the transcription factor.

(B) Fluorescent micrographs of hsp-4p::GFP UPRER reporter worms expressing an integrated sur-5p::xbp-1s transgene at days 1, 4, 7, and 10 of adulthood.

(C) A COPAS Biosort was used to measure length (TOF) and GFP fluorescence of hsp-4p::GFP (−) and hsp-4p::GFP; sur-5p::xbp-1s (sur-5p::xbp-1s) animals at day 1 of adulthood. Mean GFP fluorescence/ TOF and standard deviation of each genotype were calculated and normalized to control hsp-4p::GFP animals.

(D) Transcript levels of UPR targets in sur-5p::xbp-1s animals at day 1 of adulthood were measured by quantitative RT-PCR. Results are shown relative to transcript levels in N2 worms, with error bars indicating mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, **p < 0.01, n/s = not significant.

(E) N2 (black) and sur-5p::xbp-1s (red) animals were transferred at day 1 and day 7 of adulthood to plates containing 50 mg/ml tunicamycin, and survival monitored (sur-5p::xbp-1s day 1, median lifespan 7 days; N2 day 1, median lifespan 7 days; p = 0.0078. sur-5p::xbp-1s day 7, median lifespan 7 days; N2 day 7, median lifespan 3 days; p < 0.0001. See Table S1).

(F) Survival of sur-5p::xbp-1s (red) and N2 (black) worms on E. coli OP50 at 20° C (sur-5p::xbp-1s: median lifespan 17 days; N2: black; median lifespan 19 days; p = 0.0925. See Table S3).

See also Figure S2 and Table S2.

If the constitutive expression of xbp-1s circumvents an age-associated loss of XBP-1 activation, we would predict that this would lead to increased resistance to ER stress at older ages. sur-5p::xbp-1s worms had resistance to ER stress similar to that of wild-type when transferred to plates containing tunicamycin at day 1 of adulthood (Figure 2E). However, when transferred to stress conditions at day 7 of adulthood, sur-5p::xbp-1s animals lived substantially longer. This difference was not caused by a reduced uptake of tunicamycin in the food, as pharyngeal pumping rates were similar in both geno-types (Figure S2E). This effect was also specific, as resistance to paraquat was unaffected by sur-5p::xbp-1s expression (Figure S2F). However, and surprisingly, longevity of sur-5p::xbp-1s animals in the absence of ER stress was not increased; these animals exhibited a lifespan slightly shorter than that of N2 (Figure 2F).

Tissue-Specific Expression of xbp-1s Extends Lifespan

Ubiquitous expression of xbp-1s did not extend lifespan, despite increasing ER stress resistance. This suggested that xbp-1s expression might have positive effects on some tissues, while having negative effects on others. To test this hypothesis, we asked how lifespan was affected if we expressed xbp-1s under tissue-specific promoters (Figure 3A). Neuronal xbp-1s transgenic animals had a surprising and significant extension of longevity. Expression in the intestine also resulted in a modest increase in longevity, whereas expression in the muscle cells shortened lifespan (Figure 3B). We observed in these animals no defect in muscle cell structure or function that could directly explain their reduced longevity (Figures S3A and S3B).

Figure 3. Neuronal and Intestinal Expression of xbp-1s Increases Lifespan, and Neuronal xbp-1s Can Induce Cell-Nonautonomous Activation of the UPRER.

Figure 3

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(A) Schematic of a tissue-specific xbp-1s expression system driving xbp-1s expression in neurons, intestine, and body-wall muscle cells.

(B) Survival of N2 (black) and transgenic strains on E. coli OP50 at 20° C: (i) gly-19p::xbp-1s (line 1 [red], median lifespan 22 days; p = 0.0013; line 2 [orange], median lifespan 24 days; p < 0.0001), (ii) rab-3p::xbp-1s (line 1 [red], median lifespan 22 days; p < 0.0001; line 2 [orange], median lifespan 22 days; p < 0.0001), and (iii) unc-54p::xbp-1s (line 1 [red], median lifespan 16 days; p = 0.0003; line 2 [orange], median lifespan 14 days; p < 0.0001). See Table S3.

(C) Fluorescent micrographs of hsp-4p::GFP UPRER reporter worms expressing (i) gly-19p::xbp-1s, (ii) rab-3p::xbp-1s, and (iii) unc-54p::xbp-1s transgenes at day 1 of adulthood (i and ii) and L2/L3 (iii).

(D) Fluorescent micrograph of hsp-4p::GFP UPRER reporter worms expressing rgef-1p::xbp-1s.

See also Figure S3.

Neuronal xbp-1s Activates the UPRER Cell Nonautonomously

We then used the hsp-4p::GFP UPRER reporter to examine in which tissues activation of the UPRER occurred in each of these tissue-specific xbp-1s expression strains. As expected, gly-19p::xbp-1s animals had increased GFP fluorescence exclusively in their intestine, and unc-54p::xbp-1s animals had GFP expression exclusively in their body-wall muscle cells (Figure 3C). We found that muscle-specific UPRER activation was strongest in larvae, as is typical for promoters driving muscle expression in C. elegans (Honda and Epstein, 1990). Animals showing the highest degree of GFP fluorescence at larval stages tended to arrest before development to adulthood (data not shown). Surprisingly, worms expressing xbp-1s under the pan-neuronal rab-3p promoter had GFP expression not only in neuronal cell bodies and processes but also in the intestine, particularly the posterior intestine and the anterior two intestinal cells (Figure 3C). We confirmed the intestinal induction of hsp-4p::GFP using an additional neuronal-specific promoter, rgef-1p (Figure 3D) (Brignull et al., 2006). In contrast, animals expressing RFP under the same promoter (rab-3p:: tdTomato) showed RFP fluorescence only in the neurons (Nonet et al., 1997) (Figure S4A).

Intrigued by the ability of neuronal xbp-1s to drive cell-nonautonomous UPRER activation, we derived integrated rab-3p::xbp-1s transgenic lines. Neuronal xbp-1s transgenic animals had a 32% increase in longevity, one of the longest caused by transgene expression in the worm (Henderson and Johnson, 2001; Hsu et al., 2003; Carrano et al., 2009; Bishop and Guarente, 2007; Viswanathan and Guarente, 2011). We then crossed these into an hsp-4p::GFP background. Intestinal as well as neuronal UPRER activation was evident from larval stages throughout adulthood (Figures 4A–4C). Transcript levels of UPRER targets were also increased in rab-3p::xbp-1s animals (Figure 4D). The extended longevity of rab-3p::xbp-1s worms (Figure 4E) could not be explained by toxicity of xbp-1s and functional ablation of specific neurons previously shown to affect lifespan (Apfeld and Kenyon, 1999; Alcedo and Kenyon, 2004), as both the dye-filling capacity of the sensory neurons and chemotaxis behavior in these worms were normal (Figures S4B and S4C).

Figure 4. Expression of xbp-1s in Neurons Activates the UPRER in the Intestine and Prevents a Decline in Stress Resistance.

Figure 4

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(A) Fluorescent micrographs of hsp-4p::GFP UPRER reporter worms expressing an integrated rab-3p::xbp-1s transgene at days 1, 4, 7, and 10 of adulthood.

(B) Tail region of hsp-4p::GFP UPRER reporter worms expressing an integrated rab-3p::xbp-1s transgene showing neurons at 633 magnification.

(C) Length (TOF) and GFP fluorescence of hsp-4p::GFP (−) and hsp-4p::GFP; rab-3p::xbp-1s (rab-3p::xbp-1s) animals were measured using a COPAS Biosort at day 1 of adulthood. Mean GFP fluorescence/TOF and standard deviation of each genotype were calculated and normalized to control hsp-4p::GFP animals.

(D) Transcript levels of UPR targets in rab-3p::xbp-1s animals were measured at day 1 of adulthood by quantitative RT-PCR. Results are shown relative to transcript levels in N2 worms, with error bars indicating mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, *p < 0.05, n/s = not significant.

(E) Survival of rab-3p::xbp-1s (red) and N2 (black) worms on E. coli OP50 at 20° C (rab-3p::xbp-1s: median lifespan 25 days; N2: median lifespan 19 days; p < 0.0001. See Table S3).

(F) rab-3p::xbp-1s (red) and N2 (black) animals were transferred at day 1 and day 7 of adulthood to plates containing 50 mg/ml tunicamycin, and survival monitored (rab-3p::xbp-1s day 1, median lifespan 9 days; N2 day 1, median lifespan 7 days; p = 0.0078; rab-3p::xbp-1s day 7, median lifespan 7 days; N2 day 7, median lifespan 4 days; p < 0.0001. See Table S1).

See also Figure S4 and Table S2.

Neuronal xbp-1s Rescues ER Stress Resistance

We asked whether expression of xbp-1s in the nervous system was sufficient to rescue ER stress resistance in older animals. We found that rab-3p::xbp-1s expression increased resistance to tunicamycin to a small but significant degree at day 1 of adulthood and to a much greater extent at day 7 (Figure 4F). Again, pharyngeal pumping was comparable at both ages, and paraquat resistance was unaffected (Figures S4D and S4E). Expression of xbp-1s in neurons is therefore sufficient to increase resistance to ER stress in older animals and to increase lifespan substantially.

Expression of xbp-1s Specifically Activates the UPRER

We then asked whether cell-nonautonomous stress-response activation by xbp-1s was specific to the UPRER. We found that neither the hsp-6p::GFP UPRmt nor the hsp-16.2p::GFP HSR reporter, nor transcriptional targets of either response, were activated by the neuronal expression of xbp-1s (Figures 5A–5E). In addition, RNA interference (RNAi) against ubl-5, essential for longevity in mitochondrial mutant animals in which the UPRmt is activated (Durieux et al., 2011), did not abolish lifespan extension in rab-3p::xbp-1s expressers (Figure S5A). Therefore the expression of xbp-1s in neurons specifically activates the UPRER distally and promotes lifespan extension by a mechanism distinct from the activation of the UPRmt or HSR.

Figure 5. Neuronal xbp-1s Specifically Activates the UPRER Cell Nonautonomously.

Figure 5

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(A) Fluorescent micrograph of hsp-4p::GFP UPRER and hsp-16.2p::GFP HSR reporter worms with and without the rab-3p::xbp-1s transgene at day 1 of adulthood. Fluorescence in the pharyngeal area of rab-3p::xbp-1s animals is due to the myo-2p::tdTomato injection marker. As controls, separate populations of hsp-16.2p::GFP and hsp-16.2p::GFP; rab-3p::xbp-1s worms were incubated at 34° C for 6 hr.

(B) Fluorescent micrograph of hsp-4p::GFP UPRER and hsp-6p::GFP UPRmt reporter worms, with and without the rab-3p::xbp-1s transgene at day 1 of adulthood. Fluorescence in the pharyngeal area of rab-3p::xbp-1s animals is due to the myo-2p::tdTomato injection marker. As controls, separate populations of hsp-6p::GFP and hsp-6p::GFP; rab-3p::xbp-1s animals were treated with cco-1 RNAi from hatch.

(C) A COPAS Biosort was used to measure length (TOF) and GFP fluorescence of hsp-4p::GFP, hsp-6p::GFP, and hsp-16.2p::GFP animals, with and without the rab-3p::xbp-1s transgene, at day 1 of adulthood. Mean GFP fluorescence/TOF and standard deviation of each genotype were calculated, with rab-3p::xbp-1s animals normalized to reporter controls.

(D) Transcript levels of HSR targets in rab-3p::xbp-1s and sur-5p::xbp-1s animals were measured by quantitative RT-PCR. Results are shown relative to transcript levels in N2 worms, with error bars indicating mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, **p < 0.01, n/s = not significant.

(E) Transcript levels of UPRmt targets in rab-3p::xbp-1s and sur-5p::xbp-1s animals were measured by quantitative RT-PCR. Results are shown relative to transcript levels in N2 worms, with error bars indicating mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, **p < 0.01, n/s = not significant.

See also Figure S5.

We also analyzed the role of other regulators of lifespan in rab-3p::xbp-1s animals. RNAi against pha-4, the FOXA transcription factor required for longevity in C. elegans models of dietary restriction (Panowski et al., 2007), did not shorten the lifespan of rab-3::xbp-1s animals (Figure S5B). In contrast, RNAi against daf-16, the FOXO homolog required for the long lifespan of mutants with reduced insulin signaling (Kenyon et al., 1993), reduced longevity in this model (Figure S5C). However, rab-3p::xbp-1s expression further extended the long lifespan of animals with reduced levels of daf-2, the sole insulin receptor in C. elegans (Kimura et al., 1997) (Figure S5D). These results suggest that rab-3::xbp-1s extends longevity by a mechanism distinct from previously described lifespan-extension pathways but partially requiring daf-16/FOXO.

Cell-Nonautonomous Activation of the UPRER Is Dependent upon Endogenous xbp-1 and ire-1

The activation of hsp-4p::GFP expression upon ER stress is dependent on the presence of functional IRE-1 and XBP-1 (Calfon et al., 2002) (Figure S1A). We tested whether the cell-nonautonomous activation of the hsp-4p promoter observed in the intestine of rab-3p::xbp-1s animals was also dependent upon an intact UPRER. We first expressed rab-3p::xbp-1s in hsp-4p::GFP animals that had an ire-1(v33) mutant background, with an 878 bp deletion extending from 199 bp upstream of the ATG start codon to bp 679 of the ire-1 gene. We found that the ire-1(v33) mutation prevented UPRER activation in the intestine of rab-3p::xbp-1s animals, whereas neuronal UPRER activation was still significantly upregulated, suggesting that endogenous IRE-1 signaling is required for cell-nonautonomous UPRER activation in distal cells (Figures 6A, 6B, S6A, and S6C). As an additional measure for the requirement of an intact UPRER in distal cells, we fed worms xbp-1 RNAi. Under these conditions, the UPRER in neurons was significantly activated by neuronally expressed xbp-1s, whereas distal UPRER induction by rab-3p::xbp-1s was entirely abolished, suggesting that endogenous xbp-1 is required for perception of this signal (Figures 6C, 6D, S6B, and S6D). Taken together, loss of either ire-1 or xbp-1 blocks induction of the UPRER in distal cells but not cell-autono mous UPRER induction in the nervous system by neuronal expression of xbp-1s.

Figure 6. Cell-Nonautonomous Activation of the UPRER Requires ire-1 and xbp-1.

Figure 6

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(A) Fluorescent micrograph of hsp-4p::GFP and hsp-4p::GFP; ire-1(v33) worms expressing rab-3p::xbp-1s at day 1 of adulthood.

(B) Using a COPAS Biosort, fluorescence in the nerve ring and intestine were approximated by measuring GFP fluorescence in equidistant sections through the proximal 25% (nerve ring) and distal 75% (intestine) of hsp-4p::GFP (−) and hsp-4p::GFP; rab-3p::xbp-1s or hsp-4p::GFP; rab-3p::xbp-1s; ire-1(v33) (+) worms. Total fluorescence in each region was divided by number of sections to give average fluorescence per section, which was then averaged among animals and normalized to control hsp-4p::GFP worms. Error bars indicate mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, n/s = not significant. See Figure S6C.

(C) Fluorescent micrograph of hsp-4p::GFP; rab-3p::xbp-1s worms grown on empty vector and xbp-1 RNAi.

(D) Fluorescence in the nerve ring and intestine were approximated as above in hsp-4p::GFP animals on control empty vector RNAi (−) and hsp-4p::GFP; rab-3p::xbp-1s animals on control or xbp-1 RNAi (+). Error bars indicate mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, n/s = not significant. See Figure S6D.

(E) Survival of rab-3p::xbp-1s and N2 worms grown on empty vector control or xbp-1 RNAi (rab-3p::xbp-1s on xbp-1 [red], median lifespan 18 days; rab-3p::xbp-1s on empty vector [orange], median lifespan 22 days; p < 0.0001. See Table S3).

See also Figure S6.

Cell-Nonautonomous Activation of the UPRER Is Required for Extended Longevity

As animals expressing xbp-1s in their nervous systems are exceptionally long-lived, we next asked whether the communication of UPRER activation between the nervous system and distal cells was required for increased longevity of these animals. We grew rab-3p::xbp-1s animals upon bacteria producing xbp-1 RNAi to abolish non-neuronal UPRER activation and asked whether this cell-nonautonomous activation was required for the extended longevity of rab-3p::xbp-1s worms. rab-3p::xbp-1s animals cultured on xbp-1 RNAi bacteria had a wild-type lifespan, whereas rab-3p::xbp-1s animals on control RNAi were substantially long-lived (Figure 6E). The cell-nonautonomous activation of the UPRER is therefore required for lifespan extension caused by neuronal UPRER activation. However, as xbp-1 RNAi also knocked down xbp-1 in tissues other than the intestine, the specific tissues contributing to this lifespan suppression remain unknown.

Cell-Nonautonomous Activation of the UPRER Requires unc-13

We hypothesized that the ability of neuronal xbp-1s to activate the UPRER in a distal non-neuronal tissue suggested the existence of a neuroendocrine signaling pathway through which neurons are able to influence the UPRER status of distant cells. To test this, we crossed rab-3p::xbp-1s; hsp-4p::GFP animals with strains deficient in the release of neuronal signaling molecules. Specifically, we utilized the unc-13(e450) mutant strain, which is defective in the release of SCVs, and the unc-31(e928) mutant strain, which has a defect in the Ca2+-mediated release of dense core vesicles (DCVs) (Figure 7A) (Madison et al., 2005; Speese et al., 2007). When crossed with unc-13(e450), cell-nonautonomous UPRER activation in the intestine of rab-3p::xbp-1s; hsp-4p::GFP animals was reduced, but autonomous activation of the UPRER in the nervous system remained intact (Figures 7B and S6E). unc-13(e450) animals could activate the UPRER in the intestine upon expression of gly-19::xbp-1s, indicating that the ability to activate the UPRER cell autonomously in an unc-13(e450) mutant background was not lost (Figures S6F and S6G). In contrast to a loss of unc-13, when the rab-3::xbp-1s strain was crossed with unc-31(e928), we found very little, if any, effect on distal UPRER activation or activation in the nervous system (Figures 7B and S6E). Taken together, the release of SCVs from xbp-1s-expressing neurons by an unc-13-mediated mechanism is required for cell-nonautonomous activation of the UPRER in distal tissues. The unc-13 dependence suggests that the signaling molecule mediating cell-nonautonomous activation of the UPRER may be released from SCVs and is therefore most likely a small-molecule neurotransmitter (Figure 7A).

Figure 7. Cell-Nonautonomous Activation of the UPRER Requires unc-13.

Figure 7

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(A) Schematic of the roles of UNC-13 and UNC-31 in neurosecretory vesicle release. UNC-13 is required for the release of SCVs containing small-molecule neurotransmitters. UNC-31 is involved in the release of DCVs primarily containing neuropeptides.

(B) (i) Fluorescent micrograph of hsp-4p::GFP, hsp-4p::GFP; rab-3p::xbp-1s, hsp-4p::GFP; rab-3p::xbp-1s; unc-13(e450), and hsp-4p::GFP; rab-3p::xbp-1s; unc-31(e928) worms at day 1 of adulthood.

(ii) Using a COPAS Biosort, fluorescence in the nerve ring and intestine of hsp-4p::GFP; rab-3p::xbp-1s animals in a wild-type (WT), unc-13(e450), or unc-31(e928) background was approximated by measuring GFP fluorescence in equidistant sections through the proximal 25% (nerve ring) and distal 75% (intestine) of worms. Total fluorescence in each region was divided by number of sections to give average fluorescence per section, which was then averaged among animals and normalized to hsp-4p::GFP controls. Error bars indicate mean ± SD. A Student's t test was used to assess significance: ***p < 0.005, n/s = not significant. See Figure S6E.

(C) Survival of (left panel) rab-3p::xbp-1s and N2 and (right panel) rab-3::xbp-1s; unc-13(e450) and unc-13(e450) animals was assessed at 15° C, following a single treatment with FUDR at t = 0 (left panel: rab-3p::xbp-1s [red], median lifespan 28 days; N2 [black], median lifespan 24 days; p < 0.0001; right panel: rab-3::xbp-1s; unc-13(e450) [red], median lifespan 34 days; unc-13(e450) [black], median lifespan 43 days; p < 0.0001. See Table S3).

(D) (Left panel) rab-3p::xbp-1s and N2 and (right panel) rab-3::xbp-1s; unc-13(e450) and unc-13(e450) animals were transferred at day 1 and day 7 of adulthood to plates containing 50 mg/ml tunicamycin, and survival monitored (left panel: rab-3p::xbp-1s; unc-13(e450) day 1 [red], median lifespan 9 days; unc-13(e450) day 1 [black], median lifespan 8 days; p = 0.1295; right panel: rab-3p::xbp-1s; unc-13(e450) day 7 [red], median lifespan 6 days; unc-13(e450) day 7 [black], median lifespan 5 days; p = 0.0908. See Table S1).

(E) Model for the communication of UPRER activation between neurons and distal cells. ER stress in neurons induces splicing of xbp-1 and activation of target gene transcription that leads to UNC-13-mediated release of a secreted ER stress signal (SERSS) from SCVs. This signal induces the UPRER in distal intestinal cells through activation of endogenous IRE-1 and XBP-1, leading to target gene transcription and increased stress resistance and longevity.

See also Figure S7.

unc-13 Is Required for Lifespan Extension and Increased Stress Resistance by Neuronal xbp-1s Expression

Because unc-13-mediated vesicle release was required for cell-nonautonomous UPRER activation, we asked whether loss of unc-13 could suppress the long lifespan and stress resistance of rab-3p::xbp-1s animals. We found that rab-3p::xbp-1s expression increased neither lifespan nor ER stress resistance in a unc-13(e450) mutant background, strongly suggesting that communication between neurons and intestine through release of SCVs is essential for increased lifespan and stress resistance downstream of neuronal xbp-1s expression (Figures 7C and 7D). However, it should be noted that unc-13(e450) itself increases lifespan, complicating the interpretation of longevity data, and that as the muta tion was not specific to neurons, loss of unc-13 activity in other tissues may have contributed to this loss of longevity and stress resistance.

Intrigued to identify the molecular mediator originating from SCVs, we treated hsp-4p::GFP and hsp-4p::GFP; rab-3p::xbp-1s; unc-13(e450) animals with a range of small-molecule neurotransmitters and biogenic amines known to be secreted in an unc-13-mediated manner. Neurotransmitters and amines included (at concentrations previously demonstrated to be physiologically effective) acetylcholine, levamisole, glutamate, GABA, octopamine, tyramine, dopamine, and serotonin (Lewis et al., 1980; McIntire et al., 1993; Dent et al., 1997; Wragg et al., 2007; Schafer and Kenyon, 1995). However, none of these molecules consistently induced activation of the UPRER, suggesting that they are unlikely to act as mediators of the cell-nonautonomous UPRER response (Figures S7A and S7B). The identity of this signaling molecule remains to be defined.

We therefore propose a model whereby the detection of ER stress and activation of the IRE-1/XBP-1 UPRER-signaling module in neurons leads to the release of one or more as-yet-uncharacterized small-molecule neurotransmitters from SCVs, leading to activation of the IRE-1/XBP-1 branch of the UPRER in the intestine, with beneficial effects on ER stress resistance and longevity (Figure 7F).

DISCUSSION

Cellular processes face an inevitable decline with age, exacerbated by a lack of evolutionary imperative to maintain proteostasis beyond early adulthood. Because many of the diseases associated with ER dysfunction are age onset and because prior evidence has suggested that the UPRER becomes attenuated in older organisms, we hypothesized that a restoration of the UPRER might have a protective effect on the viability of older animals.

Consistent with this hypothesis, we report that activation of xbp-1 is lost in an age-dependent manner, leading to a decrease in ER stress resistance. It is not clear why xbp-1 splicing is lost with age given that both ire-1 and xbp-1 transcripts are present in older animals; possible explanations include a failure of the xbp-1 transcript to localize to the ER or a reduced ability to activate IRE-1 at the protein level (Aragón et al., 2009; Korennykh et al., 2009). Furthermore, and significantly, the reintroduction of a single element that declines with age in this system, xbp-1s, is sufficient to restore ER stress resistance in aged populations. Neuronal xbp-1s expression activates the UPRER in distal cells nonautonomously, in a manner dependent upon the presence of endogenous ire-1 and xbp-1 in the receiving cells. Intriguingly, we only find nonautonomous UPRER activation between neurons and the intestine, and only in one direction, with expression of xbp-1s in the intestine having no apparent effect on UPRER activation in neurons. It is interesting to note that in C. elegans, the neurons and intestine are the tissues most prone to UPRER activation when animals are faced with ER stress (Shim et al., 2004), and the intestine in particular is extremely sensitive to perturbations in ER proteostasis (Richardson et al., 2010, 2011). This communication of ER stress from the nervous system is required for extension of longevity by neuronal xbp-1s.

At this time, we do not yet understand the fundamental mechanisms by which this signal is generated or perceived. We do, however, find a dependency of nonautonomous signaling on unc-13, suggesting that one or more small molecules released from SCVs are required for the signal. UNC-13 function is also essential for increases in longevity and stress resistance by neuronal UPRER activation. The fact that either knockdown of UPRER signaling in distal cells or the abolishment of neuronal secretion through mutations in unc-13—very different interventions with different effects on physiology and longevity—can eliminate lifespan extension by neuronal xbp-1s expression suggests that cell-nonautonomous UPRER activation plays a pivotal role in the long lifespan and stress resistance of these animals.

What, then, is the mechanism by which this increase in health-span is achieved? It is possible that neuronal xbp-1s expression leads to the release of a signal that directly rejuvenates all tissues of the animal, activating the UPRER in the intestine and potentially other responses in other tissues. Indeed, as longevity induced by intestinal xbp-1s expression does not extend lifespan to the extent of neuronal xbp-1s expression, the possibility remains that effects on other tissues contribute to this longevity. This hypothesis would be supported by evidence suggesting that xbp-1s delays phenotypes of age-related decline, such as tissue decay or necrosis, similarly in all tissues. Alternatively, however, our evidence strongly suggests a specific, pivotal role for the intestine in the lifespan extension seen in rab-3p::xbp-1s animals, implying that this longevity is due to increased protection from stress specifically in the intestine following the activation of the UPRER in neurons. This is supported by the striking activation of the UPRER in the intestine following xbp-1s expression in neurons, the increase in lifespan when xbp-1s is specifically expressed in the intestine, and the abrogation of lifespan extension in rab-3p::xbp-1s animals when intestinal UPRER activation is blocked by loss of unc-13 or xbp-1.

Our data therefore suggest a central role for the intestine in the maintenance of youthfulness, and indeed, this tissue has been previously implicated as a site in which insulin-signaling perturbations and stress-response activation extend longevity (Libina et al., 2003; Durieux et al., 2011). xbp-1 and ire-1 are required for the extension of lifespan by reduced IIS, and it has been proposed that XBP-1 and DAF-16 might function together to promote longevity (Henis-Korenblit et al., 2010). We have found that lifespan extension by rab-3p::xbp-1s is reduced, but not eliminated, upon knockdown of daf-16. It is therefore possible that these two transcription factors work together in the cell-nonautonomous promotion of longevity. Coincidentally, daf-16, like xbp-1s, is required in the nervous system and intestinal cells to modulate longevity (Wolkow et al., 2000; Libina et al., 2003).

Our findings address a long-standing question—how the tissues of an organism age in a coordinate and seemingly orchestrated manner. It increasingly seems that cell-nonautonomous activation of stress responses, mediated by neurons, may be a common mechanism to coordinate stress resistance and aging in different tissues across an organism.

EXPERIMENTAL PROCEDURES

Strains

SJ4005 (zcIs4[hsp-4p::GFP]), SJ17 (xbp-1(zc12);zcIs4[hsp-4p::GFP]), SJ4100 (zcIs13[hsp-6p::GFP]), CL2070 (dvIs[hsp-16.2::GFP]), RE666 (ire-1(v33)), and N2 were obtained from the Caenorhabditis Genetics Center. Strain CB928 (unc-31(e928)) was a gift from the Jin lab (UCSD). Strain CB450 (unc-13(e450)) was a gift from the Chalasani lab (The Salk Institute). Strain AGD972 (xbp-1(zc12)) was derived by further out-crossing strain SJ17 four times to N2. To make strains expressing spliced xbp-1, xbp-1 cDNA was amplified from N2 worms by PCR. A 23 nucleotide intron sequence was removed using the QuikChange site-directed mutagenesis kit (QIAGEN). The xbp-1s cDNA was inserted into sequence-verified vectors containing the sur-5, gly-19, rab-3, unc-54, and rgef-1 promoters and the unc-54 3′ untranslated region (UTR) sequence. DNA was injected into N2 worms at 25 ng/ml, with the exception of gly-19p::xbp-1s, which was injected at 75 ng/ml, in the presence of myo2p::tdTomato at 15 ng/ml. sur-5p::xbp-1s and rab-3p::xbp-1s extrachromosomal arrays were integrated and backcrossed to N2 seven and eight times, respectively. All transgenic lines were also crossed with strain SJ4005 (zcIs4[hsp-4p::GFP]). The integrated rab-3p::xbp-1s line AGD927 was crossed with strains SJ4100 (zcIs13[hsp-6p::GFP]), CL2070 (dvIs [hsp-16.2::GFP]), RE666 (ire-1(v33)), CB928 (unc-31(e928)), and CB450 (unc-13(e450)).

Induction of ER Stress

Eggs were isolated by bleaching and allowed to develop on E. coli OP50. At the indicated ages, animals were incubated for 4 hr at 20° C in a 25 ng/μl solution of tunicamycin in M9 buffer prepared from a 1 mg/ml stock solution in DMSO. Control animals were incubated in an equivalent dilution of DMSO in M9.

Analysis of Fluorescence

For fluorescence microscopy, worms were anesthetized with 50 mM sodium azide at specified ages, and a Leica S6E dissecting microscope and a Zeiss Axio Observer Z1 used. Fluorimetry assays were performed on a TECAN infinite M1000 fluorescence plate reader. A COPAS Biosort (Union Biometrica) was also used to measure fluorescence of individual worms for quantification. Detailed methodologies for fluorescence protocols can be found in the Extended Experimental Procedures.

qPCR

Total RNA was harvested from populations of 500 animals using Qiazol reagent (QIAGEN). RNA was purified on an RNeasy mini column (QIAGEN), and cDNA synthesized using the QuantiTect reverse transcription kit (QIAGEN). SybrGreen quantitative RT-PCR experiments were performed as described in the manual using an ABI Prism7900HT (Applied Biosystems), and data analyzed using the comparative 2ΔΔCt method. Experiments were repeated at least three times.

ER Stress Survival Assays

Synchronized eggs were harvested by bleaching, and worms were grown on E. coli OP50 without tunicamycin until day 1 or day 7 of adulthood. Tunicamycin was added to NG plates at a final concentration of 50 μg/ml and a final DMSO concentration of 0.2%, and plates seeded with E. coli OP50 from an overnight culture for 24 hr before animals were transferred. Upon transfer to tunicamycin-containing plates, viability was scored daily. All assays were performed with 60–100 animals at 20° C at least three times.

Lifespan Analyses

Lifespan analyses were performed at 20° C, unless otherwise indicated, with the pre-fertile period of adulthood as t = 0 and were repeated at least three times. Eighty to one hundred animals were used per condition and scored every day or every second day. Lifespans were performed on E. coli OP50, unless otherwise indicated, and animals were treated with 100 mg/ml FUDR at t = 0 and again at day 5 of adulthood. All lifespans were also repeated without FUDR. Prism 5 and JMP 8 software were used for statistical analysis. In all cases, p values were calculated using the log-rank (Mantel-Cox) method.

Supplementary Material

Document S1. Tables S1-S3
01

ACKNOWLEDGMENTS

We thank E. Kapernick, Z. Liu, K. Berendzen, and Drs. S. Panowski, C. Riera, and V. Ramachandran for technical assistance. We also thank Dr. T. Lamitina, E. Morton, and R.S. Taylor for help with MATLAB analysis. We are grateful to Drs. S. Wolff and V. Ramachandran for insightful comments on the manuscript, to Dr. J. Durieux for help with the graphical abstract, and to Drs. Y. Jin and S. Chalasani and the Caenorhabditis Genetics Center for strains. R.C.T. is an Ellison Medical Foundation/AFAR Postdoctoral Fellow. A.D. is cofounder of Proteostasis Therapeutics, Inc. and declares no financial interest related to this work. This work was supported by NIH R01 AG042679.

Footnotes

SUPPLEMENTAL INFORMATION

Supplemental Information includes Extended Experimental Procedures, seven figures, and three tables and can be found with this article online at http://dx.doi.org/10.1016/j.cell.2013.05.042.

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Supplementary Materials

Document S1. Tables S1-S3
NIHMS554127-supplement.pdf (15.9KB, pdf)
01
NIHMS554127-supplement-01.pdf (1.7MB, pdf)

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