Mechanisms of Life Span Extension by Rapamycin in the Fruit Fly Drosophila melanogaster

Summary The target of rapamycin (TOR) pathway is a major nutrient-sensing pathway that, when genetically downregulated, increases life span in evolutionarily diverse organisms including mammals. The central component of this pathway, TOR kinase, is the target of the inhibitory drug rapamycin, a highly specific and well-described drug approved for human use. We show here that feeding rapamycin to adult Drosophila produces the life span extension seen in some TOR mutants. Increase in life span by rapamycin was associated with increased resistance to both starvation and paraquat. Analysis of the underlying mechanisms revealed that rapamycin increased longevity specifically through the TORC1 branch of the TOR pathway, through alterations to both autophagy and translation. Rapamycin could increase life span of weak insulin/Igf signaling (IIS) pathway mutants and of flies with life span maximized by dietary restriction, indicating additional mechanisms.

. Feeding behaviour of w Dah flies in the absence and presence of 200 µM rapamycin.
Flies were reared under standard density, as for lifespan experiments, and placed five per vial on standard food (see Materials and Methods). Feeding observations, using 7 day old females, were carried out on blue-dye-containing standard food that was supplemented or not with 200 µM rapamycin.
A) Figure represents feeding observations during a 30 minute period. Data are presented as proportion feeding (proportion of flies which were feeding / possible feeding events) ± SEM. There was no significant difference in the feeding behavior of flies on rapamycin food compared to flies on food not containing rapamycin (p=0.95, chi-square test). B) Figure shows the relationship between the amount of food consumed by flies (as determined spectrophotometrically) and observed feeding behavior. There was a strong positive linear correlation between the volume of blue-dyed food consumed and the proportion of feeding events observed (p<0.001, linear model) that was unaffected by addition of 200 µM rapamycin to the food (p=0.69, linear model). There is no effect of interaction between the food type (standard food compared to standard food supplemented with 200 µM rapamycin) and the way blue dye accumulates in response to observed feeding events.    Western blot analysis of phospho-T398-S6K, total S6K, and tubulin levels of whole fly protein extracts prepared from w Dah control flies and IIS mutants (chico 1 /+ heterozygotes, chico 1 /chico 1 null, and mNSC-ablated flies). Flies were sampled after 18 days of 200 μM rapamycin treatment. For all western blots, relative band intensity was estimated using Image J.

Figure S5. Complete survival curves and analysis of the effect of rapamycin lifespan extension of different food concentration.
A) Complete survival curves for the dietary restriction (DR) experiment that is presented in Figure 6. Plotted are survival curves for w Dah females against yeast concentration (0.1x, 0.5x, 1.0x, 1.5x, and 2.0x yeast) in SYA food (dashed line) and on the same food concentrations but supplemented with 200 µM rapamycin (solid line). Figure S5A and
Other stocks used in this study (chico 1 /Cyo (Bohni et al., 1999); UAS-reaper (UAS-rpr) and dilp2-GAL4 (Ikeya et al., 2002) were kind gifts from Ernst Hafen. Mutants and transgenes were backcrossed into w Dah Wolbachia positive strain for at least eight generations. All stocks were maintained and all experiments were conducted at 25ºC on a 12h:12h light:dark cycle at constant humidity using standard sugar/yeast/agar (SYA) media (Bass et al., 2007). For all experiments, including lifespan experiments, flies were reared at standard larval density and eclosing adults were collected over a 12 hour period. Flies were mated for 48 hours before sorting into single sexes.
Wolbachia status of the flies was determined by PCR, as described in Toivonen et al. (Toivonen et al., 2007). While laboratory strains w 1118 and yw were Wolbachia negative, w Dah strain was Wolbachia positive. We have removed Wolbachia from w Dah by tetracyclin treatment and at least five successive generations without tetracycline prior to lifespan experiments (Toivonen et al., 2007). As Wolbachia had no affect on rapamycinmediated lifespan extention or egg-laying under our conditions, we have used w Dah Wolbachia positive strains for our experiments.

Lifespan experiments
Flies were maintained in vials at a density of ten flies per vial.  Figure   S2F).

Lipid assays
For triacylglyceride (TAG) content quantification, batches of two flies, or heads and thoraces from 5-6 flies, were homogenized in 0.05% Tween according to Gronke et al. 2003. TAG content was quantified using the Triglyceride Infinity Reagent (ThermoScientific) using triglyceride standards and normalised to total protein content as determined using the BCA protein assay reagent (Pierce).

Quantitative RT-PCR
Live flies were snap frozen in liquid nitrogen and stored at -80°C . Total RNA from heads and thoraxes of 10 flies was extracted using TRIzol (GIBCO) according to the manufacturer's instructions. mRNA was reverse transcribed using oligo(dT) primer and the Superscript II system (Invitrogen). Quantitative PCR was performed using the Prism 7000 sequence-detection system (Applied Biosystems), SYBR Green (Molecular Probes), ROX ReferenceDye (Invitrogen), and Hot StarTaq (Qiagen, Valencia, CA) by following the manufacturers' instructions. Primers for atg5 and actin5C used were as follows: act5C-R AATCCGGCCTTGCACATG. Primers were optimized (Advanced Biosystems procedure), and relative quantities determined (relative standard curve method) and normalized to actin5C.

Paraquat injections
Injection pipettes were drawn out of 10mm glass capillaries using a Flaming-Brown micropipette puller (Programme 2). A home-built microinjection machine was used to deliver paraquat at a final dose of 50ng/mg. Both paraquat solution and mock control solution contained blue dye for visualization of injections (FD&C Blue No.1).

Feeding assay
Flies were reared as for the survival analysis and placed at 5 per vial with no anaesthesia 24 hours prior to observation. Feeding assays were carried out by direct observation on undisturbed flies (Wong et al., 2008). The number of feeding events, as measured by proboscis extension into solid medium, was recorded. Data is presented as the proportion of flies feeding during the observation period.
In order to measure the amount of food that was consumed by flies and to determine if there was a correlation between observed feeding behavior and food intake, we have performed dye-calibrated feeding observation as described in (Wong et al., 2009).
Briefly, 7 day-old female flies were put on standard SYA food containing 2.5% blue dye (w/w; FD&C Blue No.1) and 200 µM rapamycin or ethanol as a control. Feeding was observed for 30 minutes, and flies were then frozen in liquid nitrogen. The amount of blue dye was determined spectrophotometrically. The relationship between observed feeding events and blue-dye consumption was analyzed as previously described (Wong et al., 2009).

Lysotracker staining, imaging and image analysis
Lysotracker staining is based on selective visualisation of lysosomes and autophagosomes due to their low pH and is a reliable steady state method that gives an indication of autophagy induction. For Lysotracker staining, complete guts were removed from flies that had been maintained on 200 µM rapamycin or control food for 5 days (N = 5 flies in each group). Dissections were performed in PBS. Each gut was mounted into a custom made imaging chamber and stained with Lysotracker DS Red DND-99 (Invitrogen, Molecular Probes,) 1 µM for 3min. Each preparation was then washed three times with PBS and mounted in mounting medium (Mounting medium, Vectashield, H1200) containing DAPI (1.5µg/ml). Imaging was performed using a Zeiss LSM 700 confocal microscope with a 20x objective. In each preparation an area of midgut proximal to the proventriculus was imaged to control for variation along the gut. Three separate, adjacent 100 mm 2 images were obtained from each preparation. Laser power (at 555 nm), digital gain and optical settings were kept constant between the images.
Each image was then background subtracted and passed through a high pass digital filter (Lucida Image Analysis, Kinetic Imaging, Liverpool, UK) to enhance punctate structures. Images were then thresholded to binarise and quantify pixels of high value.
The mean sum of these pixels for each condition is presented.

Mass Spectroscopy analysis for quantification of rapamycin in flies
Mass Spectrometry measurements were performed in order to determine the concentration of rapamycin in flies, using methods adapted from (Serkova et al., 1999).

S methionine incorporation.
Standard sugar-yeast-agar based food was supplemented with 100 µCi 35 Smethionine/ml of food (American Radiolabeled Chemicals 1mCi/37MBq ARS 0104A). 15 flies were transferred to each vial containing radioactive food. After 3h of feeding flies were transferred to non-radioactive food for 30min in order to purge undigested 35 Smethionine radioactive food out of the intestines. Flies that were in contact with the radioactive food for 1 minute were used as a background control. Flies were then homogenized in 200 µM 1% SDS and heated for 5 minutes at 95˚C. Samples were centrifuged for 2x5 minutes at maximum speed and supernatant retained. Proteins were precipitated by the addition of the same volume of 20% cold TCA (10% TCA final concentration) and incubated for 15 minutes on ice. Samples were then centrifuged at 16,000 g for 15 minutes, the pellet washed twice with acetone and then resuspended in 200 µl of 4M guanidine-HCl. Samples (100 µl) were mixed with 3 ml of scintillant (Fluoran-Safe 2, BDH) and radioactivity counted in a liquid scintillation analyzer (Tri-Carb 2800TR, Perkin Elmer), with appropriate quench corrections. Measurements were normalized to total protein for each sample, as determined using Bradford reagent (BioRad).