Neuronal inhibition of the autophagy nucleation complex extends life span in post-reproductive C. elegans

Next Section 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.

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Aging represents the functional deterioration of an organism, which compromises fitness and predisposes for prevalent diseases such as cancer and neurodegeneration. Many genes that modulate aging have been identified in Caenorhabditis elegans through mutagenesis or RNAi screens. These screens inactivate genes across the whole life span from early development (Klass 1983; Ni and Lee 2010). Genes identified through these studies often work through a small set of common pathways, including the target of rapamycin (TOR) pathway and the insulin-like signaling pathway (Ni and Lee 2010). However, genetic pathways specific to aging in older individuals remain largely undiscovered. The existence of alleles belonging to such pathways is predicted by the antagonistic pleiotropy (AP) theory of aging. This evolutionary-based theory states that strong natural selection early in life enriches alleles that mediate fitness while simultaneously accumulating their harmful effects after reproduction, when natural selection is ineffectual (Williams 1957). Thus, some genes that reduce fitness when inhibited in young worms should conversely extend life span when inhibited in older worms. Two screens in late L4 worms identified AP genes critical to development (Chen et al. 2007; Curran and Ruvkun 2007). However, the potential of these screens was limited with regard to detecting AP genes, as they initiated RNAi early in life at a point of maximal selection pressure (Fig. 1A).

View larger version: Download as PowerPoint Slide 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.

Macroautophagy (referred to here as autophagy) is a conserved process that encloses cytoplasmic components and organelles in double-membrane structures called autophagesomes, which then fuse with the lysosome, where the contents are degraded and recycled (Mizushima et al. 1998). As such, autophagy is essential for proteostasis, in which long-lived, unfolded, misfolded, or damaged proteins are removed from circulation. Autophagy is required for normal development and health and is crucial for the extended life span by reduced germline, insulin, or TOR signaling (Rubinsztein et al. 2011). Autophagy is predominantly cytoprotective; however, when autophagy becomes dysregulated, it is associated with adverse effects such as cell death or failure to clear aggregated proteins, resulting in neurodegeneration (Komatsu et al. 2006). Autophagy has in fact been called a double-edged sword in that it can be detrimental in certain disease contexts, either being causal or exacerbating their pathologies (Shintani and Klionsky 2004). However, the role of autophagy in post-reproductive life span has not been investigated.

Here, we introduce a novel post-reproductive screening approach for the identification of unknown longevity genes functioning according to the AP model of aging (Fig. 1A). In our screen, we identified 30 novel regulators of longevity, including the FOXA transcription factor pha-4. Surprisingly, we found that PHA-4 inactivation increases life span through its role in autophagy via BEC-1. Importantly, the inhibition of pha-4 or bec-1 modulates life span conversely over aging, fulfilling the criteria for AP. Our data indicate that global autophagy becomes dysfunctional with age and is blocked at the later steps of autophagosomal degradation. We demonstrate that post-reproductive inhibition of the VPS-34/BEC-1/EPG-8 autophagic nucleation complex as well as its upstream regulators strongly extend C. elegans life span. Furthermore, we show that post-reproductive inhibition of bec-1 mediates longevity specifically through the neurons. Thus, in contrast to previous studies that suggest positive roles of autophagy during aging, our data indicate that suppression of early autophagy in aged worms results in improved neuronal integrity, contributing to enhanced global health and culminating in increased longevity.

Previous Section Next Section Materials and methods C. elegans strains C. elegans strains were maintained at 20°C using standard procedures (Brenner 1974) unless indicated differently. A complete list of the strains used in this study is in the Supplemental Material. RNAi screen Eight-hundred dsRNA-expressing HT115 Escherichia coli bacteria specific to C. elegans gene regulatory factors was prepared from the Ahringer and Vidal libraries (Supplemental Table 1). All clones were sequence-validated using the M13 forward primer. The library was grown overnight in 2× YT medium in deep 96-well plates and seeded onto 24-well NGM agar plates with 100 µg/mL ampicillin and 1 mM β-D-isothiogalactopyranoside (IPTG) at 2× native density. A minimum of four replicate wells per RNAi clone was seeded. Nontargeting controls were comprised of four plates of both EV and gfp RNAi. gfp RNAi was a kind gift from Scott Kennedy. rrf-3 mutant worms were synchronized via liquid culture until day 9, cleaned, and sorted with the COPAS Biosorter with 20 worms per well. Live/dead scoring of all wells was performed on day 32, when most control animals were dead. Scoring was performed manually by flushing each well with M9 buffer and immediately counting moving and dead worms. Only worms that could be identified as live or dead were scored; missing worms were not scored. Contaminated wells or offspring-containing wells were not scored; combined, these conditions represented ∼10% of all wells. Life span assays Worms were synchronized using liquid culture sedimentation. dsRNA-expressing bacteria were grown overnight, seeded on NGM agar plates with 100 µg/mL ampicillin and 1 mM IPTG, and incubated overnight at room temperature. Day 0 was determined by the appearance of first internal eggs. A total of 105–140 animals was placed on three to four replicate plates, with 35 worms per 6-cm plate. Worms were picked to new plates and scored every 2 d. For double-RNAi treatment, bacteria were diluted in a 1:1 ratio before being plated at 1× native density. All assays were performed at 20°C. For the temperature-sensitive glp-1 mutant, worms were maintained at the restrictive temperature of 25°C until day 0, when they were transferred to 20°C. In the life span assays indicated in Supplemental Table S4 as “plate only,” worms were maintained and synchronized only on plates. Worms were scored as alive until there was no movement after repeated prodding with an eyelash. Worms were censored when they crawled off the plate, were bagged, burst, were dropped on transfer, were contaminated, and burrowed. All RNAi clones used for life span assays were from the Ahringer libary and were sequence-validated using the M13 forward primer. LGG-1::GFP microscopy Worms were grown as described and transferred to the respective RNAi treatments at day 9. On the day of analysis, 100 worms from each knockdown condition were paralyzed using 0.5% NaAz and mounted on a 2% agar pad on a glass microscope slide. GFP signals were acquired using a STED superresolution microscope (Leica) at 100× magnifications. Images were taken of the hypodermis, which was identified by locating the plane between the muscle and cuticle. At least 60 total regions were imaged from 50 different worms per replicate with two biological replicates. All images used for comparative quantifications were taken on the same day with the same settings and by the same user. Pharynx imaging and analysis Worms were grown as described and transferred to the respective RNAi treatments at day 9. On day 20, 50 age-synchronized worms from each knockdown condition were paralyzed using 0.5% NaAz and mounted on a 2% agar pad on a glass microscope slide. The images of the pharynges were acquired on a SP5 microscope (Leica) using the differential intense contrast (DIC) filter and a 63×/1.4 NA oil immersion objective. At least 30 whole worms per condition were imaged in each replicate with three technical replicates. Images were scored blind for pharynx degradation based on a three-point scale of damage related to changes in the structure of the corpus, isthmus, or terminal bulb. Worms with no obvious damage to any of the three parts were scored as 1, worms with damage to only one or two parts were scored as 2, and worms with damage to all three parts were scored as 3. For comparative analysis, all worms were imaged at the same time with the same settings and by the same user. This experiment was carried out in two independent biological replicates. Muscle cell imaging and analysis Worms were grown as described and transferred to the respective RNAi treatments at day 9. On day 20, 30 age-synchronized worms from each knockdown condition were incubated in fixation buffer (160 mM KCl, 100 mM Tris HCl at pH 7.4, 40 mM NaCl, 20 mM Na 2 EGTA, 1 mM EDTA, 10 mM spermidine HCl, 30 mM Pipes at pH 7.4, 1% Triton X-100, 50% methanol) for 1 h at room temperature with rotation. Worms were washed twice with PBS and incubated in a 1:200 dilution of Phalloidin–Atto 565 (Sigma) in PBS–0.5% Triton X-100 for 4 h at room temperature with rotation. Worms were then mounted on a 2% agar pad on a glass microscope slide. The 561-nm signals were acquired using a STED CW superresolution microscope (Leica) and a 63×/1.4 NA oil immersion objective. At least 40 images comprising two to four cells from 20 different worms were imaged per replicate with three technical replicates. Images were scored blind based on a five-point scale of muscle fiber degradation. Cells showing no obvious degradation were scored as 1; cells with kinks or striations were scored as 2; cells with small lesions combined with striations or other damage were scored as 3; cells with muscle fiber breaks, gross striations, and lesions were scored as 4; and cells in which the muscle fibers were no longer intact were scored as 5. For comparative analysis, all worms were imaged at the same time with the same settings and by the same user. This experiment was carried out in two independent biological replicates. Pharynx pumping assay Worms were grown in liquid culture and transferred to the respective RNAi treatments at day 9. On day 20, 50 age-synchronized worms from each knockdown condition were transferred individually to an agar plate seeded with a bacterial lawn. The number of contractions in the terminal bulb of the pharynx was scored during a 30-sec period immediately upon transfer using a stereomicroscope. This experiment was carried out in two independent biological replicates. Movement scoring/thrashing assay Worms were grown as described and transferred to the respective RNAi treatments at day 9. On day 20, 50 age-synchronized worms from each knockdown condition were transferred individually in a 20-µL drop M9 buffer on a Petri dish. After a 30-sec recovery period, the numbers of body bends were scored during a 30-sec period using a stereomicroscope. A body bend was defined as a change in the reciprocating motion of bending at the mid-body. This experiment was carried out in two independent biological replicates. Imaging and analysis of the axonal network Worms were grown as described and transferred to the respective RNAi treatments at day 9. On day 20, >100 age-synchronized worms from each knockdown condition were paralyzed using 0.5% NaAz and mounted on a 2% agar pad on a glass microscope slide. GFP signal was acquired using a STED superresolution microscope (Leica) at magnifications of 40×/1.2 NA oil immersion objective. Head regions of at least 50 different worms were imaged per knockdown condition and replicate. Axonal degeneration was scored on a three-point scale based on the amount of visible bubbling and neuron integrity. An intact axon without visible bubbling, kinks, or gaps was type 1; a slightly damaged axon with a medium amount of bubbling, occasional kinks, or very few to no gaps was type 2; and a largely disrupted axon with a high amount of bubbling and frequent gaps was type 3. All images used for comparative quantifications were taken on the same day with the same settings and by the same user. This experiment was carried out in two independent biological replicates. Western blotting Worms were grown as described and transferred to the respective RNAi treatments at day 9. For each time point or knockdown condition, samples from 200 synchronized animals were harvested, washed three times with M9 buffer and once with ddH 2 O, and suspended in 2× standard Laemmli buffer via 10 min of boiling. The samples were subjected to standard SDS-PAGE and Western blotting. The antibodies used were monoclonal mouse anti-GFP (1:10,000; Roche), mouse monoclonal (6G6) anti-RFP (1:1000; Chromotek), mouse monoclonal anti-α-Tubulin (1:10,000; Sigma), and rabbit monoclonal anti-ubiquitin (Lys48-specific Apu2 clone; 1:1000; Millipore). Antibodies were diluted in PBST (5% milk powder, 0.1% Tween20), which was also used as a blocking agent. For the time-course analysis of cleaved GFP, worms were maintained in liquid culture as described, and a portion of the culture was harvested at each time point following a 40% Percoll wash. This wash was repeated in the event that the cleaned worms contained <95% living worms. Each Western blot is representative of similar results obtained in two independent biological replicates. Chloroquine treatment To pharmacologically inhibit the lysosome, animals were treated with chloroquine diphosphate salt (Sigma-Aldrich). Briefly, worms were grown as described in liquid culture until day 0 or day 14 and subsequently incubated with 20 mM chloroquine or DMSO vehicle control for 24 h. The drug treatment was performed in M9 liquid medium supplemented with EV HT115 bacteria at a density of 3 × 109 cells per milliliter at 20°C while shaking. The worms were harvested at day 15 following a 40% Percoll wash. This wash was repeated in the event that the cleaned worms contained <95% living worms. Statistics All statistical analysis was performed with Graph Prism 6. P-values for life span curves were calculated using the log-rank (Mantel-Cox) test. MTL was calculated as the mean remaining life span of the worms from the day of first treatment. P-values for quantification of GFP::LGG-1 foci were calculated using the nonparametric Mann-Whitney U-test. P-values for muscle health, pharynx integrity, and neuronal integrity were calculated using a χ2 test with two tails and 95% confidence interval. P-values for quantitative PCRs (qPCRs), body bend counts, pharynx pumping, and chymotrypsin assay were calculated using a two-tailed t-test. Significance was scored as follows for all experiments: P < 0.0001 (****), P < 0.001 (***), P < 0.01 (**), and P < 0.05 (*).

Previous Section Next Section Acknowledgments We thank the Institute of Molecular Biology Core Facilities for their support, especially the Media Laborarory and the Genomics, the Microscopy, and the Bioinformatics Core Facilities. Particular thanks to Kolja Becker for designing the worm count program for life span assays. We thank Alicia Meléndez (Queens College, City University of New York) for providing us with the BEC-1::RFP reporter strain, and Scott Kennedy (Harvard Medical School) for providing bacteria expressing dsRNA constructs targeting GFP. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by National Institutes of Health Office of Research Infrastructure Programs (P40 OD010440). We thank BioGraphix (http://www.biographix.cz) for the graphical abstract in Figure 6. This work was supported by the Boehringer Ingelheim Foundation and a Marie Curie Reintegration grant (321683, call FP7-PEOPLE-2012-CIG). The Large Particle Sorter (Biosorter, Union Biometrica) used for our large-scale RNAi longevity screen was financed through the Deutsche Forschungsgemeinschaft Major Research Instrumentation Program (INST 247/768-1 FUGG).

Previous Section Next Section Footnotes Supplemental material is available for this article.

Received May 8, 2017.

May 8, 2017. Accepted August 9, 2017.