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. 2017 Aug 1;31(15):1561-1572.
doi: 10.1101/gad.301648.117. Epub 2017 Sep 7.

Neuronal Inhibition of the Autophagy Nucleation Complex Extends Life Span in Post-Reproductive C. elegans

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Free PMC article

Neuronal Inhibition of the Autophagy Nucleation Complex Extends Life Span in Post-Reproductive C. elegans

Thomas Wilhelm et al. Genes Dev. .
Free PMC article

Abstract

Autophagy is a ubiquitous catabolic process that causes cellular bulk degradation of cytoplasmic components and is generally associated with positive effects on health and longevity. Inactivation of autophagy has been linked with detrimental effects on cells and organisms. The antagonistic pleiotropy theory postulates that some fitness-promoting genes during youth are harmful during aging. On this basis, we examined genes mediating post-reproductive longevity using an RNAi screen. From this screen, we identified 30 novel regulators of post-reproductive longevity, including pha-4 Through downstream analysis of pha-4, we identified that the inactivation of genes governing the early stages of autophagy up until the stage of vesicle nucleation, such as bec-1, strongly extend both life span and health span. Furthermore, our data demonstrate that the improvements in health and longevity are mediated through the neurons, resulting in reduced neurodegeneration and sarcopenia. We propose that autophagy switches from advantageous to harmful in the context of an age-associated dysfunction.

Keywords: BEC-1; C. elegans; aging; antagonistic pleiotropy; autophagy; neurodegeneration.

Figures

Figure 1.
Figure 1.
Screening for AP genes uncovers novel regulators of post-reproductive longevity. (A) Screening for AP genes. The force of natural selection (white line) declines over age. AP genes have positive fitness effects (blue region) early in life and negative effects (red region) late in life. Inhibiting genes post-reproductively can identify novel regulators of longevity. (Blue circles) Conventional RNAi screens’ initiation points; (red circle) initiation point of our RNAi screen. (B) Potential longevity genes identified from the AP RNAi screen. The percentage of rrf-3(pk1426) worms alive at day 30 is shown. Controls used were empty vector (EV), nontargeting GFP (black), novel longevity genes (gray), known longevity genes (khaki), and our top five candidate genes (blue). The red line indicates the threshold for consideration as a longevity candidate. Data represent an average of four replicates ± the SEM (Supplemental Table 2). (C) Day 9 RNAi against our top candidate genes—eri-1, dot-1.1, cyk-3, ego-1, and pha-4—extended mean treated life span (MTL). All life spans were rrf-3(pk1426). Days represent days after first egg lay. Life span statistics are in Supplemental Table 4.
Figure 2.
Figure 2.
pha-4 and bec-1 extend life span in an AP manner and improve post-reproductive health span. (A) Day 9 RNAi against bec-1 extends MTL by twice that of pha-4. For knockdown validation, see Supplemental Figure S2, F and G. (B) Inactivation of pha-4 at day 9 in combination with bec-1 reduces MTL extension compared with bec-1 knockdown alone. Quantitative PCR (qPCR) validation is in Supplemental Table S12. (C) Inhibition of pha-4 and bec-1 at different times across life shows differing effects, depending on the RNAi initiation time point. Depicted is the relative percentage change in MTL ± SEM compared with control. qPCR validation is in Supplemental Table S12. (D) Day 9 RNAi-treated worms were scored at day 20 for pharynx degradation on a three-point scale. n = 90. White arrows indicate damage of the corpus, isthmus, and terminal bulb. Bar, 30 µm. Magnification, 63×. (****) P < 0.0001. (E) Day 9 RNAi-treated worms were scored at day 20 for phalloidin-stained muscle damage on a five-point scale. n = 120. White arrows indicat sites of muscle damage. Bar, 30 µm. Magnification, 63×. (****) P < 0.0001. (F) Day 20 quantification of body movement in rrf-3(pk1426) worms, as measured by body bend counts. Data are depicted as a box plot with minimum to maximum values. RNAi against bec-1 was initiated at day 9. n = 50. (****) P < 0.0001. (G) Day 20 quantification of pharynx pumping rate in rrf-3(pk1426) worms. Data are depicted as a box plot with minimum to maximum values. RNAi against bec-1 was initiated at day 9. n = 50. (****) P < 0.0001. All life spans were rrf-3(pk1426). Days represent days after first egg lay. Life span statistics are in Supplemental Tables S4 and S5.
Figure 3.
Figure 3.
Inactivation of bec-1 extends life span through autophagy and is independent of classical longevity pathways. (A) bec-1 RNAi treatment shows no reduction in effect on MTL when combined with simultaneous ced-4 RNAi treatment at day 9 in rrf-3(pk1426) worms. qPCR validation is in Supplemental Table S12. (B) bec-1, vps-34, and epg-8 extend the MTL of rrf-3(pk1426) worms equally when inactivated at day 9. (C) Day 9 RNAi against bec-1 extends the MTL of glp-1(e2141) worms. (D) Day 9 RNAi against bec-1 extends the MTL of rrf-3(pk1426);daf-2(e1370) worms. (E) Combined inactivation of bec-1 and let-363(TOR) at day 9 in wild-type (N2) worms further extends their MTL compared with let-363 inactivation alone. qPCR validation is in Supplemental Table S12. Days represent days after first egg lay. Life span statistics are in Supplemental Tables 6 and 7.
Figure 4.
Figure 4.
Autophagy is dysfunctional in aged worms, and inhibition of its initiation increases life span. (A) Day 9 RNAi against genes involved in autophagosome regulation (I), induction (II), nucleation (III), expansion/maturation (IV), lysosomal fusion (V), and degradation (VI). Relative percentage change in MTL ± SEM compared with control is shown. Statistics are in Supplemental Table 3. Gene knockdown validation is shown in Supplemental Figure S4E. (B) Quantification of GFP::LGG-1 foci over time in hypodermal cells of rrf-3(pk1426);lgg-1p::GFP::lgg-1. Foci were quantified in young and old worms from images at 100× magnification. n = 50. Red lines show median and interquartile range. (****) P < 0.0001. (C) Representative Western blot of free GFP across life balanced to total GFP::LGG-1. The cleaved GFP bands correspond to the GFP::LGG-1 degradation products in the autolysosome. (D) Day 15 Quantification of GFP::LGG-1-positive foci in hypodermal cells of rrf-3(pk1426);lgg-1p::GFP::lgg-1 worms following day 9 RNAi knockdown. Foci were quantified from images with 100× magnification. n = 50. Red lines show median and interquartile range. (****) P < 0.0001. LGG-1 knockdown validation is shown in Supplemental Figure S4G. (E) Representative Western blot of free GFP (balanced to GFP::LGG-1) in young and old worms treated with chloroquine. The cleaved GFP bands correspond to the GFP::LGG-1 degradation products in the autolysosome. (F) Representative Western blot of day 9 RNAi against bec-1, vha-15, and lgg-1 and its effect on free GFP compared with GFP:LGG-1 measured at day 15. The cleaved GFP bands correspond to the GFP::LGG-1 degradation products in the autolysosome. (G) Day 9 inactivation of lysosomal degradation or acidification in rrf-3(pk1426) has no effect on MTL or the MTL increase mediated by bec-1 inhibition when used in combination. qPCR validation is shown in Supplemental Table S12. All Western blots used antibodies against GFP. Days represent days after first egg lay. Life span statistics are in Supplemental Tables S8 and S9.
Figure 5.
Figure 5.
Neuronal inhibition of bec-1 mediates health and life span effects. (A) Day 9 RNAi against members of the autophagic flux extended MTL in sid-1(pk3321);unc-119p::sid-1 worms. (B) Day 20 quantification of pharynx pumping rate in sid-1(pk3321);unc-119p::sid-1 worms. Data are depicted as a box plot with minimum to maximum values. RNAi against bec-1 was initiated at day 9. n = 50. (****) P < 0.0001. (C) Quantification of worm mobility measured by body bend counts in sid-1(pk3321);unc-119p::sid-1 worms at day 20. Data are depicted as a box plot with minimun to maximum values. RNAi against bec-1 was initiated at day 9. n = 50. (****) P < 0.0001. (D) Day 20 quantification of phalloidin-stained muscle damage following day 9 RNAi against bec-1 in sid-1(pk3321);unc-119p::sid-1 worms. The scoring system is the same as used in Figure 2D. n = 100. (****) P < 0.0001. (E) Day 20 quantification of longitudinal neuron morphology and integrity in unc-119p::GFP;rrf-3(pk1426) worms treated with RNAi against bec-1 at day 9. Neurons were scored on a three-point scale based on the amount of visible bubbling and neuron integrity. Neurons were quantified from images with 100× magnification. n = 100. Bar, 40 µm. (****) P < 0.0001. Days represent days after first egg lay. Life span statistics are in Supplemental Table 11.
Figure 6.
Figure 6.
Model depicting age-associated antagonistic effects of autophagy on longevity. (Left panel) Young worms have functional autophagy, and inhibition of vesicle nucleation in these young worms shortens life span. (Right panel) Old worms still have effective autophagy induction but exhibit an age-related dysfunction in autophagic degradation. This is accompanied by age-associated neurodegeneration. Inhibition of autophagic nucleation in old worms through an unknown mechanism leads to improved neuronal integrity and an accompanying decrease in sarcopenia. This improvement in health acts either causally or in concert with an increase in post-reproductive life span.

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