Lysosomal Signaling Promotes Longevity by Adjusting Mitochondrial Activity

Abstract

Lysosomes and mitochondria are both crucial cellular organelles for metabolic homeostasis and organism health. However, mechanisms linking their metabolic activities to promote organism longevity remain poorly understood. We discovered that the induction of specific lysosomal signaling mediated by a LIPL-4 lysosomal acid lipase and its lipid chaperone LBP-8 increases mitochondrial ß-oxidation to reduce lipid storage and promote longevity in Caenorhabditis elegans. We further discovered that increased mitochondrial ß-oxidation reduces mitochondrial electron transport chain complex II activity, contributing to the induction of reactive oxygen species in mitochondria (mtROS) and the longevity effect conferred by LIPL-4-LBP-8 signaling. Moreover, by activating the JUN-1 transcription factor downstream of mtROS, the LIPL-4-LBP-8 signaling pathway induces antioxidant targets and oxidative stress tolerance. Together, these results reveal regulatory mechanisms by which lysosomal signaling triggers adjustments in mitochondrial activity and suggest the significance of these metabolic adjustments for improving metabolic fitness, redox homeostasis, and longevity.

Keywords: aging; inter-organelle coordination; longevity; lysosomal signaling; metabolism; mitochondrial signaling; redox homeostasis.

Figure 1:. Lysosomal signaling induces mitochondrial ß-oxidation and fat mobilization.

A, B) Mitochondrial ß-oxidation dependent oxygen consumption is increased in C. elegans transgenic strains overexpressing lipl-4 (lipl-4 Tg), compared to wild type (WT) animals. The oxygen consumption rate (OCR) is measured with a Seahorse XF24 Analyzer at baseline (A), and after the addition of the mitochondrial ß-oxidation inhibitor etomoxir, the percent decrease in OCR following etomoxir addition is shown (B). N=200 for each genotype in 10 technical replicates. Error bars represent mean ± standard error of the mean (s.e.m.) *P<0.05, n.s. P>0.05, Student’s t-test. C) lipl-4 Tg and C. elegans transgenic strains overexpressing lbp-8 (lbp-8 Tg) have reduced fat content levels compared to WT. Fat storage is visualized by Stimulated Raman Scattering (SRS) microscopy, and the average signal intensity in anterior intestinal cells is quantified (Ramachandran et al., 2015). Scale bar, 50μm. N=20 for each genotype in 3 biological replicates. Error bars represent mean ± s.e.m. **P<0.01, Student’s t-test. D) Reduction of fat content levels in lipl-4 Tg and lbp-8 Tg is suppressed by RNAi knockdown of acs-2, the acyl-coA synthetase. N=20 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. ***P<0.001, **P<0.01, *P<0.05, two-way ANOVA with Holm-Sidak correction. E) Reduced fat content in lipl-4 Tg and lbp-8 Tg is suppressed by RNAi knockdown of the acyl-coA dehydrogenase acdh-1. N=20 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. ***P<0.001, *P<0.05, two-way ANOVA with Holm-Sidak correction. See also Figure S1.

Figure 2:. Lysosomal signaling promotes longevity through inducing mitochondrial ß-oxidation.

A, B) Both lipl-4 Tg and lbp-8 Tg exhibit extended lifespan compared to WT (P<0.001, log-rank test). C, D) RNAi knockdown of acs-2 partially and fully suppresses the longevity of lipl-4 Tg (C) and lbp-8 Tg (D), respectively. (P<0.001, RNAi vs. ctrl for Tg animals, log-rank test). WT lifespan is unaffected by acs-2 RNAi knockdown (P<0.05, log-rank test). N=60–100 for each genotype/condition. E, F) RNAi knockdown of acdh-1 fully suppresses the longevity of both lipl-4 Tg (E) and lbp-8 Tg (F). (P<0.001, RNAi vs. ctrl for Tg animals, log-rank test). WT lifespan is unaffected by acdh-1 RNAi knockdown (P<0.05, log-rank test). N=60–100 for each genotype/condition. G) Transgenic animals carrying acs-2 overexpression specifically in the intestine, the fat storage tissue of C. elegans (acs-2 Tg) live longer compared to WT and non-transgenic siblings (P<0.001, log-rank test). N=60–100 for each genotype/condition H) The fat content level is decreased in acs-2 Tg compared to WT and non-transgenic siblings. N=20 for each genotype in 3 biological replicates. Error bars represent mean ± s.e.m. ***P<0.001, one-way ANOVA, Holm-Sidak correction. See also Table S1 and Table S2 for lifespan data.

Figure 3:. Lysosomal pro-longevity signaling selectively alters mitochondrial ETC activities.

A) Neither lipl-4 Tg nor lbp-8 Tg shows changes in mitochondrial DNA content, measured by the levels of mitochondrial encoded genes nduo-1 and ctb-1. N=1000 for each genotype in 3 biological replicates. Error bars represent mean ± s.e.m. n.s. P>0.05, one-way ANOVA, Holm-Sidak correction. B) lipl-4 Tg and lbp-8 Tg do not transcriptionally activate hsp-6 or hsp-60, two mitochondrial chaperones induced by UPR^mt. N=1000 for each genotype in 3 biological replicates. Error bars represent mean ± s.e.m. n.s. P>0.05, one-way ANOVA, Holm-Sidak correction. C) Enzymatic activities of mitochondrial ETC complexes I (NADH:ubiquinone oxidoreductase), II (succinate dehydrogenase), III (ubiquinol:cytochrome c oxidoreductase) and IV (cytochrome c oxidase), as well as citrate synthase, are tested in lipl-4 Tg and lbp-8 Tg and normalized to WT levels. Only the activity of complex II (succinate dehydrogenase) is decreased in both lipl-4 Tg and lbp-8 Tg. N=20000 for each genotype in 10 technical replicates. Error bars represent mean ± s.e.m. ***P<0.001, n.s. P>0.05, two-way ANOVA, Holm-Sidak correction. D-E) Reduced activity of ETC complex II (succinate dehydrogenase) in lipl-4 Tg or lbp-8 Tg is suppressed upon reduction of mitochondrial ß-oxidation by acs-2 (D) and acdh-1 (E) RNAi knockdown. N=1000 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. ***P<0.001, one-way ANOVA, Holm-Sidak correction. F) The enzymatic activity of complex II (succinate dehydrogenase) is decreased in acs-2 Tg compared to WT. N=1000 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. ***P<0.001, unpaired t-test, two tailed method.

Figure 4:. Lysosomal signaling interacts with mitochondrial ETC to promote longevity.

Lifespans of lipl-4 Tg (A, C, E, G and I) or lbp-8 Tg (B, D, F, H and J) are examined upon the inactivation of ETC complex I (nuo-4 RNAi; C and D), complex II (mev-1 RNAi; E and F), complex III (isp-1 RNAi; G and H) or complex IV (cco-1 RNAi; I and J). lbp-8 Tg does not enhance the lifespan extension conferred by the inactivation of either complex I, III or IV (P>0.05, log-rank test). lipl-4 Tg further enhances the lifespan extension conferred by the inactivation of complex I or IV (P<0.001, log-rank test), but not III (P>0.05, log-rank test). Neither lipl-4 Tg nor lbp-8 Tg can extend lifespan with the inactivation of complex II (P>0.05, log-rank test). N=60–100 for each genotype/condition. See also Table S1 for lifespan data. See also Figure S2.

Figure 5:. Lysosomal pro-longevity signaling acts through JUN-1 transcription factor.

Lifespans of lipl-4 Tg (A, C, E, G, I, K and M) and lbp-8 Tg (B, D, F, H, J, L and N) are measured in the background of taf-4 (C and D), hif-1 (E and F), aha-1 (G and H), ceh-18 (I and J), nhr-27 (K and L) or jun-1 (M and N) inactivation by RNAi knockdown. Only jun-1 (c-Jun homolog) inactivation fully suppresses the longevity of both lipl-4 Tg and lbp-8 Tg (P<0.05 Tg vs. WT with RNAi, log-rank test). N=60–100 for each genotype/condition See also Table S1 for lifespan data.

Figure 6:. Lysosomal pro-longevity signaling regulates redox homeostasis.

A, B) Intestine are dissected from WT, lipl-4 Tg and lbp-8 Tg adult worms and stained with mitoSOX™ Red. Mitochondrial superoxide levels in lipl-4 Tg and lbp-8 Tg are increased compared to WT, which is suppressed by acs-2 RNAi knockdown. Representative images and the quantification of fluorescence intensity are shown in (A) and (B), respectively. N=20 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. **P<0.01, ***P<0.001, one-way ANOVA with Holm-Sidak correction. Scale bar, 70.6 μm. C, D) lipl-4 Tg and lbp-8 Tg show transcriptional induction of oxidative stress responsive genes gst-4 (C) and sod-4 (D). The induction of these antioxidant genes is suppressed by RNAi inactivation of the jun-1 transcription factor or the mitochondrial ß-oxidation gene acs-2. N=1000 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. n.s. P>0.05, *P<0.05, **P<0.01, one-way ANOVA, Holm-Sidak correction. E) The Nrf2 homolog skn-1 is transcriptionally induced in lipl-4 Tg and lbp-8 Tg, which is suppressed by RNAi inactivation of jun-1. N=1000 for each genotype/condition in 3 biological replicates. Error bars represent mean ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, one-way ANOVA, Holm-Sidak method. F) lipl-4 Tg and lbp-8 Tg have increased resistance to oxidative stress induced by paraquat compared to WT. N=60–70 for each genotype in 3 biological replicates. Error bars represent mean ± s.e.m. n.s. P>0.05, *P<0.05, two-way ANOVA, Holm-Sidak method. G, H) Inactivation of skn-1 by RNAi fully suppresses the lifespan extension of lipl-4 Tg (G) and lbp-8 Tg (H). (P<0.05 WT vs. Tg, log-rank test). N=60–100 for each genotype/condition. See also Table S1 for lifespan data.

Figure 7:

Schematic representation of the molecular mechanism by which lysosomal lipid signaling regulates mitochondrial activity and retrograde signaling to improve longevity, metabolic and redox homeostasis.