Atf-6 Regulates Lifespan through ER-Mitochondrial Calcium Homeostasis

Abstract

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 (UPR^ER) 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 UPR^ER in determining the quality of aging.

Keywords: InsP3R; UPR; aging; calreticulin; interorganelle communication; longevity.

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). (C) 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).

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 (C), 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).

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.

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).