Fig 1.
Distribution of Atg8a positive punctae in the adult Drosophila CNS.
(A) Representative confocal image (1.0 μm optical section, top left) of adult male fly brains following a 4-hour fast. Adult brains were co-stained with the anti-Elav neuronal (green) and the anti-Atg8a autophagy (red) markers. (B) Higher magnification images (see Fig 1A inset) highlight areas enriched with Atg8a positive punctae, which primarily include neuronal soma (cell bodies) and regions of neuropil (blue arrows). Yellow boxes (20 μm2) show the location of regions in the CNS that primarily contain neuronal soma that were used to count and establish autophagosome punctae profiles that occur in the adult fly brain. Additional, higher magnification images are included in S1A and S1B Fig (regions highlighted by yellow arrows). (C) Magnified images from similar brain locations of non-fasted adult male flies stained with anti-Elav and anti-Atg8a antibodies. Yellow boxes indicate regions containing neuronal soma (green) that were used to count Atg8a positive punctae (red). (D) Average number of Atg8a positive punctae or autophagosomes in control (n = 53 fields) neural tissues and following a brief 4-hour fast (n = 66 fields). P*** ≤ 0.001.
Fig 2.
Influence of gender and fasting on autophagic responses and longevity profiles.
Young WT female and male flies (w1118/+, 1-week) were subjected to 0, 4, 8 or 24 hours of fasting (1% agar). (A) Total protein extracts from adult heads were prepared for each condition (n = 3) and used to generate Western blots that were sequentially probed for the Atg8a, Ref(2)P, and Actin proteins. Quantification of the (B) Ref(2)P and (C) total Atg8a (I+II), and (D) Atg8a-II values normalized to Actin loading controls. (E) The relative ratio between the Atg8a-II and Atg8a-I proteins. (◆) represents significant differences between genders and (*) represents a difference within a gender specific group. *, ◆P≤ 0.05, **, ◆◆P ≤ 0.01, ***P ≤ 0.001.
Fig 3.
The impact of aging and IF treatment on longevity, neural autophagy, aggregates and behaviors.
WT flies (w1118/+) were maintained using standard ad libitum or IF treatment conditions beginning at 1-week of age. (A) The average lifespan profiles obtained from WT female and male flies. (B) Western blots of total protein extracts from control (0h) or fasted (4h) male fly heads at 1-week, 3-week or 3-weeks IF-treated of age (25 per condition, n = 3), were probed for Atg8a, Ref(2)P, and Actin proteins. Quantification of (C) Ref(2)P and (D) Atg8a-II that were normalized using Actin. (E) The relative ratio of the Atg8a-II to Atg8a-I proteins. (F) Triton X-100 insoluble protein extracts were prepared from WT male fly at 1-week, 3-week or IF-treated 3-weeks and Western blots probed for Ref(2)P, ubiquitin (UB), and Actin proteins. (G) Quantification of Ref(2)P and UB-proteins, normalized using Actin. (H) WT (w1118/+) male flies were maintained on ad libitum or IF conditions and allowed 2 days of recovery on standard food before the initiation of the NGR assay. The climbing indexes (5-sec) of freely responding ad libitum or IF treated flies were performed at weekly intervals starting at 1-week and continuing until 4-weeks of age. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Fig 4.
The influence of genetics on basal autophagy profiles occurring in the fly CNS.
Age-matched WT (w1118/+), Atg8a1 and chico1/+ male flies (1-week) were collected and heads used to prepare total protein extracts. (A) Western blots were probed for Atg8a, Ref(2)P, ubiquitin (UB), and Actin proteins (n = 3). Quantification of total (B) Ref(2)P, (C) UB-proteins, (D) total Atg8a (I+II), and (E) Atg8a-II proteins, normalized using Actin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.
Fig 5.
Changes to the lifespan and autophagy profiles of chico1/+ male flies.
(A) The average lifespan profiles of chico1/+ mutant male flies exposed to ad libitum or IF treatment conditions, starting at 1-week of age. (B) Total head protein extracts from control (0h) or fasted (4h) male flies at 1-week, 3-week or IF-treated 3-week of age (n = 3), were used for Western blot analysis of the Atg8a, Ref(2)P, and Actin proteins. (C) The relative ratio of Atg8a-II to Atg8a-I proteins. *P ≤ 0.05.
Fig 6.
IF-dependent changes to the lifespan and autophagy profiles of Atg8a1 mutant flies.
(A) The average lifespan profiles of Atg8a1 mutant male flies were exposed to ad libitum or IF treatment conditions starting at 1-week of age. (B) The climbing indexes (5-sec) of freely responding ad libitum or IF treated Atg8a1 male flies, which were performed at weekly intervals starting at 1-week and continuing until 4-weeks of age. (C) Western blots containing total protein extracts prepared from control (0h) or fasted (4h) Atg8a1 male fly heads taken at 1-week, 3-week or IF-treated 3-week of age (n = 3) were sequentially probed for Atg8a, Ref(2)P, and Actin proteins. (D) Quantification of Ref(2)P protein levels, normalized using Actin. (E) The relative ratio of Atg8a-II to Atg8a-I protein levels. (F) Western blots of Triton X-100 insoluble head extracts from 1-week, 3-week or IF-treated 3-week old of Atg8a1 male flies that were sequentially probed for the Ref(2)P, ubiquitin (UB), and Actin proteins. (G) Quantification of Ref(2)P and UB-proteins in the Triton X-100 insoluble fraction, normalized using Actin. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.