C. elegans growth conditions and strains

Nematodes were cultured using standard techniques at 20 °C on nematode growth medium (NGM) agar plates with E. coli strain OP50, unless otherwise noted. The following worm strains were used in the study; N2(WT); DA2123, adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)]; AM140, rmIs132 [unc-54p::Q35::YFP]; TU3401, sid-1(pk3321) V; uIs69 [pCFJ90 (myo-2p::mCherry) + unc-119p::sid-1]; VP303, rde-1(ne219) V; kbIs7 [nhx-2p::rde-1 + rol-6(su1006)]. NR350, rde-1(ne219) V; kzIs20 [hlh-1p::rde-1 + sur-5p::NLS::GFP]; NR222, rde-1(ne219) V; kzIs9 [(pKK1260) lin-26p::NLS::GFP + (pKK1253) lin-26p::rde-1 + rol-6(su1006)]; CB1370, daf-2(e1370)III; DA465, eat-2(ad465) II; CB4037, glp-1(e2141ts)III; MQ887, isp-1(qm150)IV; MAH215, sqIs11 [lgg-1p::mCherry::GFP::lgg-1 + rol-6]; MAH44, glp-1(e2141ts) III; adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)]; MAH14, daf-2(e1370) III; adIs2122 [lgg-1p::GFP::lgg-1 + rol-6(su1006)]; eat-2(ad465); adIs2122[lgg-1p::GFP::lgg-1 + rol-6(su1006)]. MAH242, sqIs24 [rgef-1p::GFP::lgg-1 + unc-122p::RFP] was crossed with TU3401 to generate the neuron specific sensitive strain expressing neuron specific GFP::LGG-1. hTFR::GFP is a kind gift from Prof. Grant (Rutgers University)49. Strains used in this study were summarised in Supplementary Table 2.

Plasmid construction and transgenesis

For rub-1:EGFP translational fusion constructs, rub-1 4 kb endogenous promoter plus coding sequence were cloned into pPD95.75 vector which contain EGFP tag Microinjection of the construct was carried with the co-injection marker, myo-2p::mCherry to generate rub-1::EGFP.

Mouse

CAG-Cre mice and Nestin-Cre mice were imported from Jackson Laboratory and Dr. Jun-ichi Miyazaki’s laboratory (Osaka University), respectively. CAG-Cre mice were crossed with Rubiconflox mice17 to produce mice with systemic Rubicon deletion. Resultant mice with the Rubicon- allele were backcrossed into the C57BL/6 J wild-type strain five times, followed by intercrossing between Rubicon+/− mice to generate Rubicon−/− mice and wild-type controls. The Nestin-Cre mice were crossed with Rubiconflox mice to produce mice harbouring homozygous deletion of Rubicon specifically in the brain. All mice used in this study were maintained on a C57BL/6 J background, with the exception of the calorie restricted mice. For those experiments, we used an adult-onset 40% calorie restriction protocol developed by Turturro et al50. Female BDF1 mice were reared individually in cages. The CR protocol was continued until 9 months of age. Water was provided ad libitum. Sequence information of primer pairs used for genotyping is available upon request. Experimental procedures using mice were approved by the Institutional Committee of Osaka University. All relevant ethical guidelines were complied with. All mice were maintained under specific pathogen‐free conditions and treated with humane care under approval from the Animal Care and Use Committee of Osaka University Medical School (Osaka, Japan).

Fly stocks and culture conditions

Flies were grown on standard cornmeal–agar–yeast-based medium at 25 °C. Transgenic fly lines bearing UAS-MJDtrQ27 (#8149), UAS-MJDtrQ78s (#8150), UAS-MJDtr-Q78w (#8141), UAS-GFP-IR (#9330), UAS-dRubicon (CG12772)-IR (#43276), UAS-GFP-mCherry-Atg8a (#37749), da-GAL4 (#55849) and elav-GAL4c155 (#458) were obtained from the Bloomington Stock Center. Transgenic fly lines harbouring GMR-GAL4 were described previously51.

Lifespan analysis

For worms, synchronised eggs were obtained from 4–6 h egg lays on RNAi plates. Starting with day 1 adults, lifespan experiments were set up at a density of 15–20 animals per plate, and carried out at 20 °C. Worms were transferred to new plates every other day. Survivorship was also counted every other day. In case of glp-1 background, the egg lay was conducted at 15 °C and shifted to 25 °C to induce germline-less phenotype for 2days. The lifespan experiment of glp-1 animals was carried out at 20 °C from day1 adult stage. All of RNAi lifespan was carried out from egg onward. Death was scored as the absence of any movement after stimulation with a platinum wire. Worms that underwent internal hatching or bursting vulva, or that crawled off the plates, were censored. For TOR RNAi experiments, knockdown was performed from adult onward. For statistics, we used in house excel data sheet that can perform log-rank test52. Experimental flies were raised at a standard medium, allowed to mate for 48 h after emerging, then sorted using CO 2 anaesthesia. Vials were changed without anaesthesia to fresh food every 2–3 days, and deaths were scored until all flies were dead. More than one hundred animals per each experimental group were used in the analyses. All lifespan experiments including repeats in worms are shown in Supplementary Data 1.

RNA interference

RNA interference (RNAi) was conducted by feeding HT115 (DE3) bacteria transformed with vector L4440, which produces dsRNA against the targeted gene. Synchronised eggs were placed on the indicated RNAi plates containing IPTG and ampicillin. RNAi clones were obtained from the Ahringer or Vidal RNAi library. The let-363/TOR RNAi clone was a gift from Dr. Hansen (Sanford-Burnham Medical Research Institute). For double knockdown experiments, equal volumes of bacteria for respective RNAi are mixed and seeded on plates. Luciferase (L4440::Luc) RNAi were used as non-targeting controls. In case of glp-1 background, the egg lay was conducted at 15 °C and shifted to 25 °C to induce germline-less phenotype for 2days. For all RNAi experiments, except for TOR RNAi lifespan, RNAi knockdown was conducted from egg onward (whole-life knockdown).

RNA extraction and qRT-PCR

Worms, flies and mouse tissue samples were harvested in TRIzol (Invitrogen) or QIAZOL (QIAGEN) at respective time points. For worms, about 200 worms per condition were collected. Total RNA was extracted using RNeasy kit (QIAGEN). cDNA was generated using iScript (Bio-Rad) or PrimeScript RT reagent kit (Takara). qRT-PCR was performed with Power SYBR Green (Applied Biosystems) on a ViiA 7 Real-Time PCR System (Applied Biosystems) or with the KAPA SYBR Fast qPCR kit (KAPA Biosystems) on a CFX96 Real-Time PCR Detection System (Bio-Rad). Four technical replicates were performed for each reaction. ama-1 or cdc-42 (worms), Hsc70 (flies), 18 s rRNA (mice), and GAPDH (human) were used as internal controls. Sequences of qRT-PCR primers are shown in Supplementary Table 1.

Autophagic flux assay in worms

Autophagy flux assay in C. elegans was performed as described previously1. Briefly, After control or rub-1 RNAi knockdown, 50 μM of BafA in 0.005% DMSO or 0.005% DMSO only was co-injected with 2.5 μg/ml Cascade Blue Dextran into the intestine or head region of day1 adult worms. For whole body knockdown, DA2123 (GFP:LGG-1 transgenic animals) were used, while for neuron specific knockdown newly generated strains created by crossing MAH242 with TU3401 were used. After the injection, animals were allowed to recover on respective RNAi plates for two hours. Recovered worms were then anaesthetised by 0.1% NaN 3 and mounted on 2% agar pad. GFP::LGG-1 fluorescence was captured using a FV3000 confocal microscopy (Olympus). Six to ten worms were imaged for each condition and the experiments were repeated three times. No. of GFP::LGG-1 punctae of DMSO injected and BafA injected worms in the anterior intestines and neuros were quantified using Image J.

Multi-worm tracking analysis

A 13 cm × 10 cm agar-filled plate was divided into four regions of equal area. To keep animals from moving between regions, the regions were surrounded with glycerol, an aversive stimulus for C. elegans. Worms from a given experimental group were placed in one of the four regions, and multiple experimental groups were tested simultaneously. An adapted version of the Multi-Worm Tracker26 was used to record the locomotion of C. elegans on the agar plates. Locomotion was recorded for 10 min and the recording was analysed using Choreography (part of the Multi-Worm Tracker software) and custom-written scripts to organise and summarise the data. Animal tracks were collected as time series of centroid position for each frame of the final 2 min of the recording. The initial 8 min were ignored to allow animal recognition by the tracker to stabilise. The following Choreography filters were used to avoid image artefacts: –shadowless and -t 10. The speed of an individual was calculated as the sum of distances between sequential centroids, divided by the duration of the track. Experimental groups were summarised using mean and standard error of the mean, weighted by the duration of an animal’s track.

dRubicon antibody

To generate an affinity purified antibody against dRubicon, C + EVPEEVHEKLQQAS peptide corresponding to amino acids 317–330 of dRubicon was used to immunise rabbits. The antibody was purified from the antiserum by using antigen peptide–conjugated resin. The peptides and antibody were prepared by Eurofins Genomics Inc. (Japan).

Climbing assay

The climbing assay was performed according to a published protocol53 with slight modifications. Ten to twenty files were placed in a conical glass tube (length, 15 cm; diameter, 2.5 cm) without anaesthesia. Ten seconds after tapping the flies to the bottom of the tube, the numbers of flies in each vertical area were counted and scored as follows: score 0 (0–1.9 cm), 1 (2–3.9 cm), 2 (4–5.9 cm), 3 (6–7.9 cm), 4 (8–9.9 cm), 5 (10–15 cm). Three trials were performed on each group at 20 s intervals, and the climbing score was calculated as follows: each score multiplied by the number of flies was divided by the total number of flies, and the mean score of the 3 trials was calculated. Results are presented as the mean ± S.E. of the scores obtained in 3–9 independent experiments.

Microscopy and quantification

To monitor autophagic activity, GFP::LGG-1 and mCherry::GFP::LGG-1 animals at the L4 stage or day1 adult stage were anaesthetised in 0.1% sodium azide, and images were acquired using an Olympus FV1000 or FV3000 confocal microscope. Using these images, GFP and/or mCherry puncta in the anterior intestinal region or in the pharyngeal region were counted and quantified. In each experiment, images from six to ten worms per condition were captured and the experiments were repeated three times. For Rub-1::EGFP and Q35::YFP worms, fluorescent images of the aligned whole worms on ice cold empty plates were captured by stereomicroscope SZX16 equipped with DP80 CCD camera (Olympus) and the number of polyQ aggregates per worms was counted.

Fly eye imaging

Light microscopic images of 1-day-old adult female flies were taken using a stereoscopic microscope model SZX16 (Olympus) with a CCD camera (DP22, Olympus). Numbers of necrotic patches per compound eye were counted for each genotype.

Autophagic flux assay in Drosophila

To match the feeding status, 4-day-old flies expressing GFP-mCherry-Atg8a either with or without dRubicon-IR under the da-GAL4 driver were starved by 0.75% agar for 15 h and refed for 4 h. The brains were dissected, fixed in 4% formalin in PBS, incubated with 80% glycerol overnight, and then mounted with DAPI fluoromount-G (SouthernBiotech). Fluorescent images of KC layer were taken by confocal microscope (Leica TCS SP8) and the number of GFP and/or mCherry puncta in 25 × 50 μm ROI containing 150–200 cells were counted. Three un-overlapping images were used for quantification of each KC layers.

Stress resistance assay

Day1 adult worms were subjected to oxidative stress (4.4 mM H 2 O 2 in the empty plate) or heat stress (35 °C) for 3 h or 7 h, respectively and the survived worms were counted. 30 worms were used per condition and the experiments were repeated three times.

Western blotting

Worms or mouse tissues were lysed in lysis buffer (50 mM Tris/HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, protease and phosphatase inhibitor cocktail [Roche]) using a homogeniser. After centrifugation, the resultant supernatants were subjected to protein quantification and western blotting. Protein lysates of worm and mouse tissues were separated by SDS-PAGE and transferred to PVDF membranes, which were then blocked and incubated with specific primary antibodies. Primary antibodies and dilutions used for mouse tissues western blotting were as follows: Rubicon (Cell Signalling Technology, #8465, 1:500), LC3 (Cell Signalling Technology, #2755, 1:1000), p62 (MBL, PM045, 1:1000), and β-actin (Sigma Aldrich, A5316, 1:8000). In fly, to assess the total amount of dRubicon, two fly bodies were lysed in 100 μl of 2 × SDS buffer (2% SDS, 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% Glycerol, 0.01% bromophenol blue, and 10% 2-Mercaptoethanol). The lysates were heated at 99 °C for 10 min followed by centrifugation at 15,000 × g for 10 min and the supernatants were collected. The protein lysates of 0.75 bodies per lane were separated by 5–12% polyacrylamide gels, transferred to PVDF membranes. Blocking with 5% skim milk in PBS containing 0.1% Tween 20. The antibodies used in this study were as follows: anti-dRubicon (1:20,000), anti-actin (JLA20, 1:2,000, Developmental studies Hybridoma Bank). Signal intensities were quantified by densitometry using ImageJ. Uncropped images were provided in Supplementary Fig. 10.

Immunohistochemistry

For flies, adult female brains (3 days after eclosion) were dissected, fixed in 4% formalin in PBS, blocked with 50% Block Ace (Dainippon Sumitomo Pharmaceuticals) in PBS/T (0.5% Triton X-100 in PBS) and incubated with an anti-HA antibody (3F10, 1:500, Sigma) to stain MJDtrQ78 protein with HA-tag. The immunostainings were visualised with an Alexa 488-conjugated anti-rat antibody and nuclei were stained with DAPI. Images of Kenyon cell layer (KC layer) were taken by confocal microscopy (LSM780, Zeiss). Signal intensities of HA staining were quantified using Image J 1.51 s software. Ten bilateral KC layers from five animals were analysed for each group. For mice, immunohistochemical staining for collagen I was performed on paraffin-embedded sections. After antigen retrieval by autoclaving in 0.01 mmol/L citrate buffer (pH 6.0) for 10 min at 120 °C, the sections were blocked with 3% BSA in PBS for 60 min. The blocked sections were incubated with primary antibodies, anti-Collagen I (abcam, ab34710, 1:400) at 4 °C overnight, incubated for 30 min at room temperature, followed by detection using a HRP-diaminobenzidine compound (Nichirei Corp.). Sections were counterstained with hematoxylin.

Injection and detection of recombinant α-Syn Fibrils

Injection of fibrils and their detection were described previously38,39. In brief, prepared fibrils were diluted in sterile PBS and sonicated before intracerebral injection. Mice between 8 and 10 weeks of age were anaesthetised with chloral hydrate (250 mg/kg, i.p.) and stereotaxically injected in both hemispheres (co-ordinates: + 0.2 mm relative to Bregma, + 2.0 mm from midline) with recombinant α-Syn fibrils (1 µl of 1 mg/ml). Animals were killed at 10 months after birth. Frozen brain sections including the fibril-injected area were obtained using a CM3050S cryostat (Leica Biosystems). Sections were incubated with mouse monoclonal antibody pSyn#64 (dilution, 1:1000, Wako Pure Chemical Industries), followed by Alexa Fluor 488–conjugated secondary antibodies (dilution, 1:1000, Invitrogen) for detection. Images were acquired and quantified on a Biorevo BZ-9000 (Keyence).

Statistical analysis

Results are presented as means ± s.e.m. Statistical tests were performed with either one- or two- way ANOVA with Tukey’s test or t-test using GraphPad Prism (GraphPad Software) or Excel (Microsoft Office 2011).

Reporting summary

Further information on experimental design is available in the Nature Research Reporting Summary linked to this article.