A mitochondrial unfolded protein response-independent role of DVE-1 in longevity regulation

Abstract

The special AT-rich sequence-binding (SATB) protein DVE-1 is widely recognized for its pivotal involvement in orchestrating the retrograde mitochondrial unfolded protein response (mitoUPR) in C. elegans. In our study of downstream factors contributing to lifespan extension in sensory ciliary mutants, we find that DVE-1 is crucial for this longevity effect independent of its canonical mitoUPR function. Additionally, DVE-1 also influences lifespan under conditions of dietary restriction and germline loss, again distinct from its role in mitoUPR. Mechanistically, while mitochondrial stress typically prompts nuclear accumulation of DVE-1 to initiate the transcriptional mitoUPR program, these long-lived mutants reduce DVE-1 nuclear accumulation, likely by enhancing its cytosolic translocation. This observation suggests a cytosolic role for DVE-1 in lifespan extension. Overall, our study implies that, in contrast to the more narrowly defined role of the mitoUPR-related transcription factor ATFS-1, DVE-1 may possess broader functions than previously recognized in modulating longevity and defending against stress.

Keywords: ATFS-1; C. elegans; CP: Cell biology; CP: Molecular biology; DVE-1; cilia; dietary restriction; germline signaling; longevity; mitoUPR.

Figure 1.. Different ciliary mutants in lifespan regulation

(A) An illustration of C. elegans sensory cilium showing multiple different ciliary components involved in ciliogenesis and maintenance. The impacts of these components (highlighted in red) on longevity are studied in subsequent experiments (B–H). (B) The daf-10(p821) mutant with defective intraflagellar transport particle A is significantly longer lived than wild-type (WT) worms. (C) The osm-1(p808) mutant with defective intraflagellar transport particle B is significantly longer lived than WT worms. (D) The dyf-1(mn335) mutant with defective motor activator is significantly longer lived than WT worms. (E) The osm-3(p802) mutant with defective kinesin motor is significantly longer lived than WT worms. (F) The xbx-1(ok279) mutant with defective dynein motor is significantly longer lived than WT worms. (G) The klp-11(tm324) mutant with defective kinesin-II motor has a lifespan similar to that of WT worms. (H) The osm-12(n1606) mutant with defective BBSome is significantly shorter lived than WT worms. The comprehensive statistical analysis summaries for all lifespan experiments can be found in Table S2. Of note, in the case of multiple ciliary mutants within the same batch of lifespan experiments, the comparison may involve the use of a common WT control. For example, (B), (C), (D), (F), and (G) are conducted with the same control, whereas (E) and (H) share another control experiment.

Figure 2.. dve-1 RNAi suppresses ciliary mutations-conferred longevity

(A) In addition to daf-16 RNAi, dve-1 RNAi strongly suppresses the greatly extended lifespan of daf-10(p821) mutant animals. (B) dve-1 RNAi strongly suppresses the isp-1 RNAi-induced mitoUPR activation. The zcls13[Phsp-6::gfp] transcriptional reporter line for mitoUPR is used in this experiment. Compared to the basal condition on the empty vector (EV) RNAi bacteria (top panels), a 1:1 mixture of isp-1 RNAi feeding bacteria with EV RNAi bacteria triggers robust expression of Phsp-6::GFP (middle panels). However, dve-1 RNAi strongly suppresses isp-1 RNAi-induced Phsp-6::GFP expression (bottom panels). Scale bar, 100 μm. (C) Quantification of Phsp-6::GFP fluorescence intensity, as depicted in (B), was performed. The sample sizes are 15 for EV(RNAi), 15 for EV(RNAi) + isp-1(RNAi), and 13 for isp-1(RNAi) + dve-1(RNAi). Data are presented as mean ± SEM. Statistical significance was determined using one-way ANOVA. ****p < 0.0001. (D–H) dve-1 RNAi significantly shortens the lifespans of WT (D) as well as long-lived osm-1(p808) (E), dyf-1(mn335) (F), osm-3(p802) (G), and xbx-1(ok279) (H) mutant worms. (I and J) dve-1 RNAi has no significant impacts on the lifespans of klp-11(tm324) (I) and osm-12(n1606) (J) mutants. Of note, (I), Figure 3H, and Figure 4H are derived from the same experiment and share a common empty vector (RNAi) control.

Figure 3.. The mitoUPR transcription factor ATFS-1 is not involved in ciliary modulation of longevity

(A) Illustration of signaling pathways required for mitoUPR activation in C. elegans. Among them, two parallel pathways are centered around two transcription factors/regulators, ATFS-1 and DVE-1, both of which are highlighted in blue. (B–I) atfs-1 RNAi has no significant impacts on the lifespans of WT (B) and daf-10(p821) (C), osm-1(p808) (D), dyf-1(mn335) (E), osm-3(p802) (F), xbx-1(ok279) (G), klp-11(tm324) (H), and osm-12(n1606) (I) mutant worms. Of note, (C) and Figure 4C share the same empty vector (RNAi) control experiment, whereas (I) and Figure 4I share another empty vector (RNAi) control experiment.

Figure 4.. Ciliary mutations-conferred longevity does not require mitoUPR

(A) No changes of the hsp-6 mRNA level are observed in the long-lived daf-10(p821), normal-lifespan klp-11(tm324) or short-lived osm-12(n1606) mutant backgrounds, suggesting that mitoUPR is not induced in these ciliary mutants. Each genetic background is analyzed using three biological replicates, and the data are presented as mean ± SEM. ns, not significant (t test). (B) LIN-65, MET-2, UBL-5, and CLPP-1 have all been reported to interact with DVE-1 to regulate mitoUPR in C. elegans. However, unlike the lifespan-shortening effect of dve-1 RNAi, disruption of lin-65, met-2, ubl-5, and clpp-1 by RNAi does not shorten the lifespan in WT worms. clpp-1 RNAi significantly extends lifespan, which is a commonly seen phenomenon, by mildly inhibiting the functions of mitochondrially localized proteins. (C–G) Unlike the robust suppression of dve-1 RNAi on the lifespans of long-lived ciliary mutants, disruption of lin-65, met-2, ubl-5, and clpp-1 by RNAi does not have a similarly strong impact on the lifespans of daf-10(p821) (C), osm-1(p808) (D), dyf-1(mn335) (E), osm-3(p802) (F), and xbx-1(ok279) (G) mutant worms. (H and I) clpp-1 RNAi significantly extends the lifespan of klp-11(tm324) (H) and osm-12(n1606) (I) mutant worms, whereas lin-65, met-2, and ubl-5 RNAi have no or modest effects on their lifespans.

Figure 5.. Cytosolic DVE-1 mediates ciliary modulation of longevity

(A and B) Potent induction of mitoUPR by spg-7 RNAi, which can be largely suppressed by either atfs-1 RNAi or dve-1 RNAi. Shown are the representative images (A) and quantification of Phsp-6::GFP fluorescence intensity (B). The sample sizes are 17 for EV(RNAi), 20 for spg-7(RNAi) + EV(RNAi), 21 for spg-7(RNAi) + atfs-1(RNAi), and 27 for spg-7(RNAi) + dve-1(RNAi). Data are presented as mean ± SEM. ****p < 0.0001 (one-way ANOVA). Scale bar, 100 μm. (C and D) spg-7 RNAi promotes nuclear accumulation of DVE-1, as monitored using the zcIs39[Pdve-1::dve-1::gfp] translational reporter line. (C) Representative images of the expression pattern of DVE-1::GFP in the posterior intestine. (D) Quantifications of the DVE-1::GFP fluorescence intensity in the nucleus versus cytosol treated with EV RNAi (sample size 68) or spg-7 RNAi (sample size 129). Data are presented as mean ± SEM. **p < 0.01 (t test). Scale bar, 100 μm. (E and F) Opposite to the impact of spg-7 RNAi, the daf-10(p821) mutation reduces nuclear accumulation of DVE-1. (E) Representative images of the expression pattern of DVE-1::GFP in the posterior intestine. (F) Quantifications of the DVE-1::GFP fluorescence intensity in the nucleus versus cytosol of WT (sample size 105) and daf-10(p821) mutant worms (sample size 112). Data are presented as mean ± SEM. **p < 0.01 (t test). Scale bar, 100 μm. (G) Representative images of a group of aligned worms demonstrate that nuclear accumulation of DVE-1::GFP is greatly reduced in the posterior intestine of daf-10(p821) mutants (lower panels) compared to WT (upper panels) worms. Scale bar, 100 μm. (H and I) Two transgenic lines are created by overexpressing Pdve-1::dve-1::yfp::SL2::mCherry and Pdve-1::dve-1(ΔNLS)::yfp::SL2::mCherry in the N2 WT background, respectively. These lines utilize the endogenous dve-1 promoter to drive the expression of DVE-1::YFP and DVE-1(ΔNLS)::YFP fusion proteins. The inclusion of the SL2::mCherry cassette enables bicistronic expression, allowing simultaneous expression of DVE-1::YFP/DVE-1(ΔNLS)::YFP and mCherry. The NLS-deleted DVE-1 variant translocates from the nucleus to cytosol. (H) Representative images of the expression pattern of DVE-1::YFP and NLS-deleted DVE-1(ΔNLS)::YFP in the posterior intestine. (I) Quantification of the DVE-1::YFP (sample size 40) and DVE-1(ΔNLS)::YFP (sample size 30) fluorescence intensity in the nucleus versus cytosol. Data are presented as mean ± SEM. ****p < 0.0001 (t test). Scale bar, 100 μm. (J) The overexpression of either DVE-1 or NLS-deleted DVE-1(ΔNLS) variant can significantly extend lifespan.

Figure 6.. DVE-1 acts in a mitoUPR-independent fashion downstream of dietary-restriction-modulated and germline-signaling-modulated lifespan

(A and B) dve-1 RNAi does not significantly suppress the extended lifespans of daf-2(e1370) (A) and isp-1(qm150) (B) mutant worms, indicating that DVE-1 is not involved in the IIS-regulated and mitochondrial-signaling-regulated longevity. Of note, (A) and (B) share the same empty vector (RNAi) and dve-1 (RNAi) control experiments. (C and D) nuo-6 RNAi-induced mitoUPR is strongly suppressed by atfs-1 RNAi, but not dve-1 RNAi. Shown are the representative images (C) and quantification of Phsp-6::GFP fluorescence intensity (D). The sample sizes are 14 for EV(RNAi), 18 for nuo-6(RNAi), 35 for nuo-6(RNAi) + EV(RNAi), 43 for nuo-6(RNAi) + dve-1(RNAi), and 25 for nuo-6(RNAi) + atfs-1(RNAi). Data are presented as mean ± SEM. ns, not significant; ****p < 0.0001 (one-way ANOVA). Scale bar, 100 μm. (E) While dve-1 RNAi does not affect the mitoUPR induced by nuo-6 RNAi, it significantly shortens the lifespan of nuo-6 RNAi-treated worms. This lifespan reduction is even more pronounced than that caused by atfs-1 RNAi. (F and G) The extended lifespans conferred by the dietary-restricted eat-2(ad465) (F) and germline-lacking glp-1(e2141) (G) mutants are greatly suppressed by dve-1 RNAi, supporting that DVE-1 is involved in dietary-restriction-modulated and germline-signaling-modulated lifespan. (H and I) The eat-2(ad465) (H) and glp-1(e2141) (I) mutants do not elevate the expression level of hsp-6 mRNA, suggesting that mitoUPR is not activated by dietary restriction or germline ablation. Each genetic background is analyzed using three biological replicates, and data are presented as mean ± SEM. ns, not significant; ***p < 0.001 (t test). (J) Neither eat-2 RNAi nor glp-1 RNAi further extends the lifespan of daf-10(p821) mutants, suggesting that these factors may share a common genetic mechanism in regulating longevity.

Figure 7.. Bioinformatic analyses of DVE-1 target genes in the WT background

(A) Compared to the empty-vector RNAi-treated control worms, dve-1 RNAi significantly elevates (>2-fold increase) the expression levels of 1,817 genes while it represses (>2-fold decrease) the expression levels of 1,360 genes. Thus, DVE-1 downregulates 1,817 genes and upregulates 1,360 genes under the non-stressed resting condition. In a similar comparison, ATFS-1 downregulates 86 genes and upregulates 70 genes. Among all these target genes, only nine each are shared targets of DVE-1 and ATFS-1 between upregulated and downregulated categories. (B and C) Gene enrichment analysis of DVE-1 target genes under the non-stressed resting condition. Among the upregulated genes, many are involved in stress response, GPCR signaling, and proteolysis (B). By contrast, major sperm proteins, cilia, and extracellular matrix components are over-represented in the DVE-1 downregulated genes (C). (D) Under the mitochondrial stress condition (atp-2 RNAi), 176 extra DVE-1-dependent genes are induced compared to the non-stressed resting condition. However, the majority of DVE-1 target genes are not induced by atp-2 RNAi. (E) A working model of DVE-1’s roles in longevity modulation and mitoUPR activation.