Nuclear Export Inhibition Enhances HLH-30/TFEB Activity, Autophagy, and Lifespan

Abstract

Transcriptional modulation of the process of autophagy involves the transcription factor HLH-30/TFEB. In order to systematically determine the regulatory network of HLH-30/TFEB, we performed a genome-wide RNAi screen in C. elegans and found that silencing the nuclear export protein XPO-1/XPO1 enhances autophagy by significantly enriching HLH-30 in the nucleus, which is accompanied by proteostatic benefits and improved longevity. Lifespan extension via xpo-1 silencing requires HLH-30 and autophagy, overlapping mechanistically with several established longevity models. Selective XPO1 inhibitors recapitulated the effect on autophagy and lifespan observed by silencing xpo-1 and protected ALS-afflicted flies from neurodegeneration. XPO1 inhibition in HeLa cells enhanced TFEB nuclear localization, autophagy, and lysosome biogenesis without affecting mTOR activity, revealing a conserved regulatory mechanism for HLH-30/TFEB. Altogether, our study demonstrates that altering the nuclear export of HLH-30/TFEB can regulate autophagy and establishes the rationale of targeting XPO1 to stimulate autophagy in order to prevent neurodegeneration.

Keywords: HLH-30; SINE; TFEB; XPO-1; XPO1; autophagy; longevity; lysosome; mTOR; nuclear export.

Figure 1. XPO-1 Modulates Nuclear Localization of HLH-30/TFEB and Autophagy

(A) Nematodes expressing HLH-30::GFP were fed control bacteria or bacteria expressing RNAi against xpo-1 during development or during adulthood for 48 hr (1003 magnification). (B) Expression levels of xpo-1, lgg-1, lgg-2, sqst-1, and hlh-30 were measured by qPCR in animals fed control bacteria or bacteria expressing RNAi against xpo-1 from day 1 to day 5 of adulthood. *p < 0.05; **p < 0.01; n = 4. Error bars represent ± SD, t test. (C and D) Autophagosome (AP) and autolysosome (AL) formation was measured in hypodermal seam cells (C) and pharynx (D) of wild-type and hlh-30(tm1978) animals expressing tandem autophagy reporter mCherry::GFP::LGG-1 and fed control bacteria or bacteria expressing RNAi against xpo-1 for 48 hr during early adulthood. *p < 0.05; **p < 0.01; N = 8. Error bars represent ± SD, t test. (E) Effect of xpo-1 silencing during 7 days of adulthood on heat resistance was assayed. p < 0.05, n = 200, Mantel-Cox log rank. See Table S1 for statistical analyses and repeats. (F–H) Accumulation of Aβ42 (F) and Q35::YFP punctae (Gand H) were measured in transgenic animals fed control bacteria or bacteria expressing RNAi against xpo-1 during 5 days of adulthood; images are shown in (G), and quantification is given in (H). *p < 0.05. Q35::YFP, n = 5; Aβ42, n = 100. Error bars represent ± SD, t test.

Figure 2. Silencing xpo-1 Extends Lifespan in C. elegans

(A–I) Lifespan analyses of (A) wild-type (WT), (B) hlh-30(tm1978), (C) daf-16(mu86), (D) atg-7(bp411), (E) atg-18(gk378), (F) daf-36(k114), (G) glp-1(e2144), (H) eat-2(ad1116), and (I) rsks-1(sv31) fed control bacteria or bacteria expressing RNAi against xpo-1 from day 1 of adulthood. n = 100, Mantel-Cox log rank. See also Table S1 for statistical analyses and repeats.

Figure 3. Pharmacological Inhibition of XPO-1/Embargoed Promotes Autophagy and Longevity

(A) Day 1 animals expressing HLH-30::GFP were fed E. coli OP50 bacteria with DMSO (0.1%) or KPT-330 (25, 50, or 100 µM). 100× magnification. (B) Lifespan analysis of wild-type animals fed bacteria with DMSO (0.1%) or KPT-330 (25, 50, or 100 µM). (C and D) Autophagosome (AP) and autolysosome (AL) were quantified in the pharynx (C) and in hypodermal seam cells (D) of animals expressing mCherry::GFP::LGG-1 and fed bacteria with DMSO (0.1%) or KPT-330 (50 or 100 µM) from day 1 to day 3 of adulthood. **p < 0.01; n = ~10. Error bars represent ± SD, t test. (E) Heat stress assay of animals fed bacteria with DMSO (0.1%) or KPT-330 (100 µM) from day 1 to day 5 of adulthood. p < 0.05; n~100. (F) Lifespan analysis of the ALS model in flies (dsod^H71Y) fed food with DMSO (0.1%) or KPT-330 (100 µM). p < 0.05; n > 300, Mantel-Cox log rank. See Table S2 for statistical analyses and repeats.

Figure 4. Nuclear Enrichment of TFEB and Autophagy Are Stimulated by XPO1 Inhibition

(A) TFEB-GFP-expressing HeLa cells were imaged after incubation for 6 hr in a medium containing vehicle or compounds (Torin 1, 5 µM; KPTs, 1 µM). Scale bars, 20 µm. (B) Percentage of cells with TFEB nuclear localization was quantified from four independent experiments. ***p < 0.001. Error bars represent ± SD, t test. (C and D) HeLa cells were grown in medium containing vehicle or compounds for 6 hr, lysosomes were visualized with Lysotracker Red (C), and signal intensities were quantified (D). **p < 0.01; ***p < 0.001. Scale bars, 50 µm. ^#Image taken at half the exposure for representational purposes. (E) HeLa cells were grown in a medium containing vehicle or compounds for 24 hr, and proteins were visualized by immunoblotting. (F) Levels of LC3 I and II were quantified by densitometry. *p < 0.05; **p < 0.01; ***p < 0.001. Error bars represent ± SD, one-way ANOVA. Images and blots are representative of three independent experiments.