Prohibitin depletion extends lifespan of a TORC2/SGK‐1 mutant through autophagy and the mitochondrial UPR

Abstract Mitochondrial prohibitins (PHB) are highly conserved proteins with a peculiar effect on lifespan. While PHB depletion shortens lifespan of wild‐type animals, it enhances longevity of a plethora of metabolically compromised mutants, including target of rapamycin complex 2 (TORC2) mutants sgk‐1 and rict‐1. Here, we show that sgk‐1 mutants have impaired mitochondrial homeostasis, lipogenesis and yolk formation, plausibly due to alterations in membrane lipid and sterol homeostasis. Remarkably, all these features are suppressed by PHB depletion. Our analysis shows the requirement of SRBP1/SBP‐1 for the lifespan extension of sgk‐1 mutants and the further extension conferred by PHB depletion. Moreover, although the mitochondrial unfolded protein response (UPRmt) and autophagy are induced in sgk‐1 mutants and upon PHB depletion, they are dispensable for lifespan. However, the enhanced longevity caused by PHB depletion in sgk‐1 mutants requires both, the UPRmt and autophagy, but not mitophagy. We hypothesize that UPRmt induction upon PHB depletion extends lifespan of sgk‐1 mutants through autophagy and probably modulation of lipid metabolism.

We added IPTG (1 mM final concentration) (Sigma-Aldrich) to the bacterial culture and harvested cells by centrifugation (8 min at 6000×g, 4°C) after 2 hours at 37°C. Following, we washed the pellets with S Basal and harvested them again. Finally, we re-suspended bacterial pellets to a final concentration of 30 g/l in complete S Medium. Bacterial stocks were kept at 4°C up to 4 days before being used. For double RNAi treatments, bacterial stocks were mixed in a proportion of 1:1 before seeding the plates. A semi-synchronous embryo population was grown on plates seeded with the appropriate RNAi bacterial clone at 20°C until the desired stage (young adult, day 1, day 5 or day 10 of adulthood). During egg-laying, we transferred worms every day and every 2 days thereafter.
To pharmacologically induce a strong mitochondrial stress, we transferred L3 to fresh RNAi plates containing 0.25 mM Paraquat (Sigma-Aldrich).

Transcription Factor RNAi screen
We dispensed 60 synchronized sgk-1;Phsp-6::gfp L1 larvae per well in 96 well plates using a microplate dispenser (EL406 washer dispenser, BioTek) and added 75 μl of bacterial cultures from a Transcription-Factor RNAi sub-library. We used an empty feeding vector, pL4440, as negative control (control RNAi) and phb-1 RNAi as positive control. Two independent replicates were carried out. We incubated worms at 20ºC with shaking (120 rpm -New Brunswick™ Innova® 44/44R) until they reached the young adult stage. Worms were immobilised with levamisole and plates were washed with water to eliminate bacteria before imaging. We acquired pictures of each well using the IN Cell Analyzer 2000 (GE Healthcare) and performed the image analysis with a user-defined protocol which was developed on the Developer Toolbox software (GE Healthcare).
For the statistical analysis, outliers were removed by excluding the 5th and the 95th percentile of the distribution. Wells with less than 10 worms were removed from the analysis. A quality assay was performed in the control wells: only control wells with mean GFP intensity between 200 and 500 a.u. and a coefficient of variation < 0.5 were accepted. If less than 2 control wells remained accepted, the plate was discarded and repeated. In order to make data from different plates comparable, data are normalized by dividing the GFP value of each worm by the mean of the GFP of the four negative control wells. Finally, statistics are assessed by running an ANOVA test followed by a Dunnett's test. Candidates are defined based on the p value and the fold change (FC) (p value < 10 -4 and FC < 0.6).

Cholesterol supplementation
Nematode were grown from eggs in NGM agar plates, seeded with RNAi bacteria, and supplemented with cholesterol at 25 mg/ml (high cholesterol) or 5 mg/ml (and equivalent amount of vehicle). least two independent assays were carried out and the combined data was analysed using the GraphPad Prism software (version 5.0a).

Imaging and quantification of mitochondrial morphology
zcIs14 [Pmyo-3::GFP(mit)] and sgk-1(ok538);zcIs14[Pmyo-3::GFP(mit)] reporters were mounted on 2% agarose pads using 40 mM levamisol as anaesthetic. Mitochondria in the body wall muscle were imaged, except around the vulva and pharyngeal regions, using a confocal Nikon A1R equipped with a Plan Apo VC 60x/2 objective. We used ImageJ to remove background, to apply median filter and to convert to a binary image by auto-thresholding. Images (whole cells) were segmented and area and perimeter per segment were calculated. Mean area/perimeter ratio was calculated to represent the mitochondrial interconnectivity. Data was analyzed using the GraphPad Prism software (version 6.0c).

Quantification of HLH-30
Animals were mounted at day 1 of adulthood and anaesthetised with 10mM levamisole Given the strong effect of the anaesthetics in triggering HLH.30 nuclear localization, we imaged all the worms within 2 minutes from the time they were removed from food. The two first intestinal cells of Phlh- for sgk-1 and sgk-1 PHB-depleted animals, respectively).

Quantification of lmp-1:: RFP
Animals were mounted at the young adult stage and anaesthetised with 10mM levamisole. We analysed the first two intestinal cells of wild type and sgk-1 animals under control conditions and upon phb-1 depletion. At least 4 independent replicas between 20-30 worms per condition (n= 73 and n=90 for wild type and PHB-depleted worms, respectively, and n=70 and n=78 for sgk-1 and sgk-1 PHB-depleted animals, respectively).

Lysotracker staining
Lysotracker Green DND-26, 1 mM solution in DMSO, (Invitrogen) was diluted in M9 Buffer as working solution 100mM. Young adults were placed onto RNAi-bacteria seeded plates containing 1 mM of Lysotracker. RNAi-bacteria seeded plates containing the equivalent amount of vehicle were used as control. After 16 hours worms were transfer to fresh RNAi-bacteria seeded plate without Lysotracker to clear intestinal lumen from excess dye. Image analysis was performed using the ImageJ software. Lysotracker and DMSO-only containing plates were imaged under the same conditions. Three independent assays were carried out and the combined data was analysed using the GraphPad Prism software (version 5.0a).

Transmission electron microscopy
Adult worms were immersed in 0.8% glutaraldehyde + 0.8% osmium tetroxide in 0. oxide. Samples were infiltrated on a rotator or with an embedding machine for 2 hours in a 3:1 ratio of propylene oxide to resin and next for 2 hours in a 1:3 ratio of propylene oxide to resin. Next, samples where incubated overnight in 100% resin and the next day changed to fresh 100% resin for 4 hours. Finally, samples were arranged in a flat embedding mold and cured at 60°C for 2 days.
Worms were cut at the level of the first intestinal cells, immediately before and after the gonad turn, specifically at 175 µm, 185 µm, 250 µm and 350 µm from the mouth.

Quantification of transmission electron microscopy structures.
For mitochondrial area quantification, 2 and 4 different animals were analyzed for D1 and D5 adulthood respectively; n ≥ 92 mitochondria per background were measured. For Golgi area quantification, 2 to 3 animals were analyzed, n ≥ 20 mitochondria per background. To determine ER-mitochondria contact size, a maximal distance of 50 nm between organelles was considered as a contact; n between 22 and 107 mitochondria per background, from 2 to 3 animals. ERmitochondria contact site lengths were normalized to mitochondrial perimeters. To quantify myelinated forms we analysed 12 areas of 56 μm 2 per condition and counted complex myelinated autophagic structures and myelinated autolysosomes ( Figure S4d). Data was analyzed using the GraphPad Prism software (version 5.0a).

Oxygen consumption
OCR was measured 8 times in basal conditions as well as after each injection. Working solutions were diluted in M9 at the final concentrations: FCCP (Sigma-Aldrich) 250 μM, oligomycin (Sigma-Aldrich) 250 μM and NaN3 (Sigma-Aldrich) 400 mM. Basal OCR corresponds to the average of 8 measurements under basal conditions. After drug addition, the values derived from the average of the last 4 measurements. At least three independent assays were carried out and the combined data was analysed by t-test using GraphPad Prism software (version 5.0a).

Quantification of Autophagy
Ex[Pnhx-2::mCherry:: LGG worms were imaged with a 63x objetive. mCherry punctae within one int9 cell, per one focal plane, were quantified. [Psqst-1::sqst-1::gfp] worms were imaged with a 40x objetive. Z-stacks of the head region were acquired and maximum intensity projection was applied. GFP loci within the head region were quantified. At least 3 independent assays were carried out and the combined data was analysed by t-test using GraphPad Prism software (version 5.0a)

Quantification of autophagosomes and autolysosomes
To quantify autophagosomes (AP) and autolysosomes (AL) we used the dual reporter sqIs11[Plgg-1::mCherry::GFP:: LGG-1]. We quantified the number of punctae of AL/AP (red or yellow punctae respectively) of the two first intestinal cells in wild type and sgk-1 animals under control RNAi (n= 25, 31 respectively) and phb-1 RNAi (n= 31, 26 respectively). Three independent replicas were analysed. In OP50, AP and AL were analysed in the intestine and hypodermis (head region) of wild type and sgk-1 mutants. Two independent replicas, n= 7 for wild type and n=14 for sgk-1(ok538).
For the quantification we used ImageJ software, selecting a specific stack and a defined area occupied by the two first intestinal cells for each worm, after processing with a gaussian filter (sigma radius: 1) and selecting a fixed threshold for all images. Finally, we performed an automatic particle counting using "analize particle" plugin with a previous watershed segmentation.

Lifespan Analysis
Synchronized eggs were obtained by hypochlorite treatment of adult hermaphrodites and placed on NGM plates seeded with HT115(DE3) E. coli bacteria, carrying appropriate RNAi plasmid constructs. RNAi was applied from the L1 larval stage, except for sgk-1 mutants on sptl-1, nhr-8 or sbp-1 RNAi, where RNAi was applied from the late-L3 larval stage. Adults were transferred to fresh plates every day during the reproductive period and afterwards on alternate days. Worms were scored as dead when they no longer responded to touch. Exploded animals or those exhibiting bagging were censored. For N-Acetyl-L-Cysteine (NAC) (Sigma-Aldrich) treatments we added NAC to a final concentration of 8 mM to NGM plates. We used the GraphPad Prism software (version 5.0a) to plot survival curves and to determine significant differences in lifespans (log-rank (Mantel-Cox) test). See Table S1 for lifespan statistics.