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Atf-6 Regulates Lifespan through ER-Mitochondrial Calcium Homeostasis

aging longevity calreticulin interorganelle communication upr insp3r

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#1 Engadin

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Posted 29 September 2020 - 07:07 PM








O P E N   A C C E S S   S O U R C E :   Cell Reports










  •  Loss of the UPRER mediator ATF-6 in C. elegans extends lifespan
  •  ATF-6 loss reverses age-associated increases in calreticulin, an ER calcium sink
  •  ER calcium efflux via the InsP3R is required to extend lifespan
  •  ER calcium release enhances mitochondrial dynamics and bioenergetics
Individually, dysfunction of both the endoplasmic reticulum (ER) and mitochondria has been linked to aging, but how communication between these organelles might be targeted to promote longevity is unclear. Here, we provide evidence that, in Caenorhabditis elegans, inhibition of the conserved unfolded protein response (UPRER) mediator, activating transcription factor (atf)-6, increases lifespan by modulating calcium homeostasis and signaling to mitochondria. Atf-6 loss confers longevity via downregulation of the ER calcium buffer, calreticulin. ER calcium release via the inositol triphosphate receptor (IP3R/itr-1) is required for longevity, while IP3R/itr-1 gain of function is sufficient to extend lifespan. Highlighting coordination between organelles, the mitochondrial calcium import channel mcu-1 is also required for atf-6 longevity. IP3R inhibition leads to impaired mitochondrial bioenergetics and hyperfusion, which is sufficient to suppress long life in atf-6 mutants. This study reveals the importance of organellar calcium handling as a critical output for the UPRER in determining the quality of aging.
Graphical Abstract
Aging is associated with failures in the ability to maintain homeostasis at the molecular and subcellular levels, and understanding how to prevent these age-related changes is essential to developing therapies for a variety of age-onset diseases. The largest organelle system in cells, the endoplasmic reticulum (ER), serves as a hub of metabolism through a variety of critical roles in the cell. These roles include housing the secretory pathway and associated proteostasis machineries, providing storage and timely release of intracellular calcium, and acting as the primary site of the synthesis of triacylglycerols and membrane lipids (Bravo et al., 2013). Perturbations in any of these processes can trigger cell dysfunction and lead to disease, necessitating a robust homeostatic network to maintain the health and function of the ER. The unfolded protein response (UPR) serves as this network, and as its name suggests, historical insights into the UPR tend to center around its activation by unfolded proteins in the secretory pathway (Ron and Walter, 2007).
Three conserved branches mediate the UPR in metazoans, namely the inositol-requiring enzyme-1 (Ire1) branch, PKR-like ER kinase (PERK), and activating transcription factor 6 (atf-6). Previous studies of the UPR in mammalian models revealed that the loss of Ire1 or PERK results in lethality or severe metabolic pathology, respectively, in mice, demonstrating critical roles for these branches in both organelle and organismal homeostasis (Zhang et al., 2002, 2005). In contrast, the first Atf6α knockout models surprisingly did not result in overt phenotypes (Wu et al., 2007; Yamamoto et al., 2007). Upon chronic, non-physiological dosing of tunicamycin, an inhibitor of protein glycosylation and maturation in the ER, Atf6α−/− animals succumb to liver failure, revealing its importance in long-term adaptation to ER stress conditions (Wu et al., 2007; Yamamoto et al., 2007). Similar studies in Caenorhabditis elegans confirmed the hierarchical, relative importance of the three branches, revealing that ire-1/xbp-1 and PERK/pek-1 mutants exhibit the strongest phenotypes and are highly sensitive to tunicamycin. Unlike ire-1 and pek-1, atf-6 mutants present few baseline phenotypes and appear to exhibit normal, wild-type responses to tunicamycin (Bischof et al., 2008; Shen et al., 2005; Springer et al., 2005). These results suggest that (1) targeting atf-6 may be more tolerable than the alternative UPR branches and that (2) atf-6 may play a role in maintaining ER and cellular homeostasis beyond protein folding.
Despite the established and essential role of the UPR in maintaining cell homeostasis, fundamental questions regarding its roles in aging persist. First, while activation of the UPR is most commonly associated with cytoprotection, the outputs of the UPR are not universally beneficial. Chronic UPR activation can promote cell death and inflammation, ultimately contributing to pathology and, in some cases, limiting lifespan, through excessive and persistent activation later in life (Hotamisligil, 2010; Wang et al., 2015a). Depending on the context therefore, not only activation but also UPR inhibition may provide therapeutic value (e.g., Harnoss et al., 2019; Tufanli et al., 2017), suggesting that UPR inhibitors may be an underexplored strategy in combating age-associated diseases. Second, the importance of the proteostasis outputs of the ER and UPR are well established and critical in promoting healthy aging (Frakes and Dillin, 2017). As mentioned above, however, the ER performs myriad roles in parallel to proteostasis. While these functions are also influenced by the UPR, how they affect aging or longevity is not well understood.
Here, we focus on understanding the roles of the understudied atf-6 branch of the UPR in aging in C. elegans. atf-6 loss promotes longevity, and we find that the regulation of lifespan in this case does not occur through canonical proteostasis pathways but instead through the modulation of ER calcium homeostasis. Reduction in the expression of the ER calcium sink, calreticulin, mediates longevity in atf-6 mutants, and ER calcium efflux through the conserved inositol triphosphate receptor/itr-1 (IP3R/itr-1) channel is necessary and sufficient for lifespan extension in this pathway. Finally, we reveal mitochondrial reprogramming as a downstream consequence of modulating ER calcium release, highlighting interorganelle calcium signaling as a key factor in promoting healthier aging.
Results and Discussion
Atf-6 Mutants Are Not Sensitive to Proteotoxic Stress and Exhibit Extended Lifespan
To define novel roles for ATF-6 in healthy aging, we used a C. elegans deletion mutant, atf-6(ok551). We confirmed that atf-6 mutants do not exhibit reduced survival in the presence of tunicamycin, an inhibitor of protein glycosylation and maturation in the ER lumen (Figure 1A). Indicating that ATF-6 still functions to regulate some aspect of ER homeostasis in the nematode, the loss of atf-6 results in synthetic lethality when combined with loss of the ire-1/xbp-1 pathway in C. elegans (Shen et al., 2005). Furthermore, the ER residency and activation of mammalian Atf6 are accomplished through a transmembrane domain and lumenal Golgi trafficking signals, which are conserved in the nematode (Shen et al., 2005).
Figure 1. Atf-6 Is Dispensable in Proteotoxic Stress, and Mutants Exhibit Extended Lifespans
(A) Survival analysis of UPR mutant nematodes exposed chronically to 30 mg/mL tunicamycin starting on the first day of adulthood (n = 72 per condition).
(B) Lifespan analysis of atf-6 mutants (n = 100 per condition).
© Pharyngeal pumping rates as a measure of health span in 7-day old nematodes (means ± SDs of n = 31 combined over 2 independent repeats).
(D) Period of the defecation motor program, an ultradian behavioral rhythm, in aging nematodes (means ± SDs of n = 20–40 total periods measured from 4–5 animals on each day).
(E) Survival of worms exposed to high temperatures for 3 and 4 h (means ± SDs of 3 independent assays of 100 animals).
(F) Fecundity in atf-6 mutants over the first 3 days of adulthood (means ± SDs of n = 5–19 worms per day).





To determine how atf-6 affects the aging process, we asked whether atf-6 loss alters the C. elegans lifespan. Counterintuitively for loss of a homeostatic regulator, we found that the atf-6 deletion mutant was significantly long-lived (57% increase in median, p < 0.0001; Figure 1B), as had been suggested previously (Henis-Korenblit et al., 2010; Wang et al., 2015b). Confirming that this lifespan effect was due to the loss of function of atf-6, we created a second null allele of atf-6 through an early CRISPR-Cas9-generated frameshift, and this also extended lifespan (43%, p < 0.0001; Figure 1B). Furthermore, a subset of health-span markers were improved in aged atf-6 mutants, including pharyngeal pumping (Figure 1C) but not rhythmicity of the defecation cycle (Figure 1D), suggesting that normal atf-6 function somehow promotes age-onset dysfunction and deterioration. We investigated whether the loss of atf-6 protected animals from heat, a more generalized form of proteotoxic stress that affects all intracellular compartments, but we found no differences from wild-type animals (Figure 1E). Because the ability to withstand extrinsic stress from either tunicamycin (Shen et al., 2005; Wu et al., 2007) or heat treatment (Figures 1A and 1E) was unaffected in atf-6 mutants, we hypothesized that atf-6 may instead play a role in basal ER functions, such as the biosynthetic demands associated with growth and reproduction. When we examined atf-6 mutants for developmental or brood-size effects, however, we observed no developmental delay (data not shown) or defects in reproductive rate relative to wild-type animals (Figure 1F). These findings suggest that in contrast to IRE1 and PERK, ATF-6 is pro-aging under basal conditions in C. elegans, providing an opportunity to discover longevity mechanisms downstream of this conserved transcription factor.
Atf-6 Regulates ER Function and Lifespan through Its Conserved Target, Calreticulin
Given the lack of obvious phenotypes and putative mechanisms for atf-6 longevity, we performed unbiased transcriptomic analyses to understand the physiological roles of ATF-6 in C. elegans. In agreement with previous analysis of atf-6-dependent transcripts in early development (Shen et al., 2005), we found that relatively few genes were differentially expressed (DE) in day 1 atf-6(ok551) adults when compared with wild-type N2 animals: only 26 genes were downregulated (Figure 2A; Table S1), while 101 genes were upregulated (Table S2) using cutoff criterion α < 0.01. Confirming previous studies in C. elegans and our hypothesis that atf-6 may specialize in roles that are separable from canonical proteostasis functions, we found virtually no signature markers of ire-1/xbp-1 activation among these DE genes (Bischof et al., 2008; Shen et al., 2005; Springer et al., 2005) (Tables S1 and S2). This finding argues strongly against one possible model of atf-6 longevity in which compensatory activation of an alternative UPR branch is responsible for the lifespan extension. Because constitutive activation of the ire-1/xbp-1 branch is sufficient to extend lifespan in C. elegans (Taylor and Dillin, 2013), we attempted to further rule out this compensatory model by inhibiting the proximal sensors of ER stress, ire-1 and pek-1, via RNAi and performing lifespan analyses in atf-6 mutants (Figure S1). We found that neither ire-1 nor pek-1 is fully required for atf-6 mutant longevity, although it is technically difficult to completely rule out contributions from the alternative branches considering the synthetic lethality of ire-1/xbp-1 and atf-6 mutants during development (Shen et al., 2005).
Figure 2. Atf-6 Regulates ER Function and Lifespan through Its Conserved Target, Calreticulin
(A) Volcano plot of differentially expressed transcripts in atf-6(ok551) relative to N2.
(B) Relative transcript abundance of conserved UPR target genes in 1-day old atf-6 mutants (means ± SDs of RNA sequencing [RNA-seq] read counts normalized to wild-type, n = 4).
(C and D) qRT-PCR analysis of changes in UPR transcripts in wild-type worms between days 1 and 7 of adulthood ©, contrasted with changes in UPR transcripts in aging atf-6 mutants (D) (means ± 95% confidence intervals of 3 independent samples of ∼100 worms; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001).
(E) Lifespan analysis of atf-6(ok551) and crt-1(bz29) mutants (n = 100 animals per curve).




The relatively small pool of DE genes suggests that atf-6-dependent phenotypes arise from its regulation of single or small groups of transcriptional targets, as opposed to a large network effect. We therefore set out to identify the most promising candidate mediators. Among the most significantly affected transcripts, we found two genes previously implicated in ER homeostasis and stress responses: calreticulin/crt-1 (Park et al., 2001), which was downregulated in atf-6 mutants, while choline kinase b-2/ckb-2 (Caruso et al., 2008) was upregulated (Figure 2B). Because atf-6 mutants exhibit both extended lifespan and health-span benefits after 1 week of adulthood, we reasoned that candidate aging factors may show the greatest differences in expression at later time points. We aged worms to 7 days and harvested RNA from wild-type and atf-6(ok551) mutants. While ckb-2 levels appeared more dramatically altered than crt-1 in young mutants, its expression declined similarly during aging in both wild-type and atf-6 backgrounds. However, whereas crt-1 levels are significantly upregulated with age in wild-type animals (Figure 2C), these transcripts are even further reduced with age in atf-6 mutants (Figure 2D), leading us to focus on calreticulin/crt-1 as the most promising candidate for an atf-6-dependent aging factor. If atf-6 deletion promotes lifespan through its effects on crt-1, then, we hypothesized, the loss of crt-1 may be sufficient to reproduce the longevity phenotype. We used a null mutant of calreticulin, crt-1(bz29) (Xu et al., 2001), and, consistent with our hypothesis, crt-1(bz29) mutants are long-lived to a similar extent as atf-6(ok551) (38%, p < 0.001 versus wild-type; Figure 2E). These results highlight calreticulin as a mediator of atf-6 functions in the C. elegans lifespan and are consistent with its evolutionary conservation as a direct target of Atf6 transcription in mammals (Yoshida et al., 1998).
ER Calcium Flux Functions Downstream of atf-6 in Regulating Lifespan
Calreticulin functions with calnexin as a quality control checkpoint during the glycosylation of ER proteins, but it plays a more outsized role as a calcium-buffering protein, capable of binding and sequestering up to half of the total ER Ca2+ pool (Michalak et al., 2009). Mirroring the lack of a proteostasis defect in atf-6 mutants here, calnexin can effectively compensate for the loss of calreticulin to maintain protein quality control in mammalian cells (Molinari et al., 2004). However, manipulating calreticulin levels causes overt alterations in calcium handling that ultimately drive changes in cell physiology (Michalak et al., 2009). These prior studies and our data suggest a model whereby atf-6 maintains a primary role in the regulation of changes in ER calcium handling in parallel to ire-1/xbp-1 and pek-1 control of ER proteostasis. To test this concept, we placed synchronized L1 larval nematodes on bacterial lawns in the presence of thapsigargin, an inhibitor of ER calcium uptake, and measured their ability to grow and develop during chronic ER calcium stress. In contrast to what we observed with the protein-folding inhibitor tunicamycin (Bischof et al., 2008; Shen et al., 2005; Springer et al., 2005), growth of atf-6 mutants is highly sensitive to thapsigargin and is reduced to the lowest levels among proximal UPR sensors (Figure 3A).
Figure 3. ER Calcium Flux Functions Downstream of atf-6 in Regulating Lifespan
(A) Relative growth of L1 larval worms exposed to 5 mg/mL of the SERCA inhibitor thapsigargin for 48 h (means ± SDs of n = 88–164 worms combined over 2 independent trials).
(B and C) Representative traces of GCaMP3 imaging in the spermatheca of worms beginning with oocyte entry and ending with spermathecal contraction.
(D and E) Area under the curve (AUC) (D) and time-to-maximum (E) summary measurements of the GCaMP3 calcium traces recorded in the spermatheca of wild-type and atf-6 mutant worms (means ± SDs of 11–16 animals combined over 2 repeats).
(F) Lifespan analysis of atf-6 mutants in the temperature-sensitive IP3R/itr-1(sa73) background at the semi-permissive temperature of 20°C (n = 100 per condition).
(G) Lifespan analysis of the atf-6 interaction with itr-1(sy290) gain-of-function mutation (n = 100 per condition). NS = p > 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by t test.




These data suggest that atf-6 may indeed play a role in maintaining ER calcium homeostasis, prompting us to determine whether there is evidence that in vivo calcium signaling is perturbed in these animals. One of the best-characterized calcium signaling paradigms in C. elegans is ovulation, in which oocytes entering the smooth muscle-like spermatheca trigger oscillatory ER calcium release via the IP3R, ultimately driving myosin-dependent contraction (Kovacevic et al., 2013). By expressing the genetically encoded calcium indicator GCaMP3 in the spermatheca, we observed the cytosolic calcium oscillations and contraction events in vivo in atf-6 mutants (Figures 3B and 3C). These experiments revealed a number of aberrant phenotypes in atf-6 mutants, including altered calcium oscillatory patterns, delayed calcium release and contraction, and greater total calcium release per contraction (Figures 3B–3E). Thus, atf-6 appears to be a regulator of calcium homeostasis in C. elegans.
Due to the differences observed in the spermathecal calcium signaling of atf-6 mutants and the central role for the IP3R in that process, we hypothesized that ER calcium release via the IP3R may also play a role in the atf-6-dependent lifespan effects. We crossed atf-6 deletion mutants with both gain-of-function (sy290) and temperature-sensitive reduction-of-function (sa73) alleles of the sole C. elegans IP3R ortholog, itr-1. At the sa73 semi-permissive temperature of 20°C, atf-6 longevity is fully suppressed in the itr-1(sa73) reduction-of-function mutants (p = 0.108; Figure 3F). Consistent with a model in which enhanced IP3R-dependent calcium release promotes longevity (Iwasa et al., 2010), the gain-of-function allele, itr-1(sy290), is sufficient to extend lifespan (p = 0.0005), but its effects are not additive when crossed to the atf-6 mutant (p = 0.292; Figure 3G). These results suggest a model in which reduced ER calcium retention in atf-6 mutants functions to extend lifespan.
ER-Mitochondrial Communication via Calcium Regulates Lifespan
We aimed next to better understand the mechanisms by which ER calcium release may be linked to longevity. Because excess cytosolic Ca2+ is more closely linked to pathology than to longevity (Arruda and Hotamisligil, 2015; Mattson and Arumugam, 2018), we hypothesized that another intracellular compartment may compensate for enhanced ER calcium release. Studies have shown that calreticulin not only regulates ER calcium storage and release through the IP3R (Michalak et al., 2009) but it also affects mitochondrial calcium levels (Arnaudeau et al., 2002). Specifically, enhanced ER Ca2+ storage promoted by calreticulin overexpression is counterbalanced by a loss of Ca2+ from mitochondrial pools (Arnaudeau et al., 2002), suggesting an inverse relationship between calcium storage in these two compartments. Furthermore, mild ER stress can result in organelle reorganization to increase communication between ER and mitochondria (Arruda et al., 2014; Bravo et al., 2011). To determine how altered ER calcium handling via atf-6 and itr-1 can affect mitochondrial behavior, we performed in vivo imaging of mitochondrial networks in the nematode intestine. While young adult atf-6 mutants exhibited mild if any alterations in gross mitochondrial morphology, itr-1(sa73) mutants promoted dramatic reorganization of the networks, which is consistent with enhanced mitochondrial fusion (Figures 4A and 4B). Because itr-1(sa73) both suppresses atf-6-mediated longevity and causes mitochondrial networks to become hyperfused, we next asked whether this change in mitochondrial morphology was sufficient to block lifespan extension in this context. We fed atf-6(ok551) mutants Escherichia coli-generating double-stranded RNA (dsRNA) to inhibit the conserved fission factor, dynamin-related protein 1 (drp-1), and this genetically imposed hyperfusion was sufficient to block the effects of atf-6 on the lifespan (p = 0.1212; Figure 4C). Conversely, enhancing mitochondrial fragmentation by feeding nematodes dsRNA to inhibit the ortholog of mammalian mitofusins, fzo-1, has no inhibitory effect on the longevity of atf-6 mutants (p < 0.0001 versus fzo-1(RNAi) alone; Figure 4D). Thus, reducing IP3R function causes mitochondrial hyperfusion, which is sufficient to suppress lifespan extension in atf-6 mutants.
Figure 4. ER-Mitochondrial Communication via Calcium Regulates Lifespan
(A) Representative fluorescence z stack projections of intestinal mitochondrial networks via TOMM-20(1–49)::EGFP in day 1 adult animals. Scale bars: 10 μm.
(B) Quantification of mitochondrial morphology (n = 14–27 worms combined over 2 trials; p < 0.0001 comparing control versus itr-1(sa73) and p = 0.001 comparing control versus itr-1(sy290)).
(C and D) Lifespan analysis of worms fed dsRNA for the mitochondrial fission (drp-1, C) and fusion (fzo-1, D) machineries in control and atf-6 mutant animals (n = 100 per condition).
(E) Quantification of western blots for P-AMPK normalized to wild-type animals (means ± SDs of n = 3 independent experiments using lysate from ∼600 young adult worms; ∗p = 0.024 by ANOVA).
(F) Quantification of the ratio of GCaMP7:mKate2 fluorescence as an indicator of mitochondrial calcium content (means ± SDs of n = 96 and 45 combined from 3 independent repeats; p < 0.0001 by t test).
(G) Lifespan analysis of atf-6 mutants harboring a deletion in mcu-1 to ablate acute mitochondrial calcium uptake (n = 100 per condition).


Edited by Engadin, 29 September 2020 - 07:08 PM.

#2 Mind

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Posted 30 September 2020 - 05:11 PM

Thinking about calcium metabolism in the body mad me think about the over-mineralization theory of aging once again: https://www.longecit...ut-memory-loss/



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Also tagged with one or more of these keywords: aging, longevity, calreticulin, interorganelle communication, upr, insp3r

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