TOR-mediated autophagy regulates cell death in Drosophila neurodegenerative disease

Abstract

Target of rapamycin (TOR) signaling is a regulator of cell growth. TOR activity can also enhance cell death, and the TOR inhibitor rapamycin protects cells against proapoptotic stimuli. Autophagy, which can protect against cell death, is negatively regulated by TOR, and disruption of autophagy by mutation of Atg5 or Atg7 can lead to neurodegeneration. However, the implied functional connection between TOR signaling, autophagy, and cell death or degeneration has not been rigorously tested. Using the Drosophila melanogaster visual system, we show in this study that hyperactivation of TOR leads to photoreceptor cell death in an age- and light-dependent manner and that this is because of TOR's ability to suppress autophagy. We also find that genetically inhibiting TOR or inducing autophagy suppresses cell death in Drosophila models of Huntington's disease and phospholipase C (norpA)-mediated retinal degeneration. Thus, our data indicate that TOR induces cell death by suppressing autophagy and provide direct genetic evidence that autophagy alleviates cell death in several common types of neurodegenerative disease.

Figure 1.

Light-dependent photoreceptor degeneration in flies with overexpression of Rheb. (A–F) Examination of the morphology by transmission EM. Cross sections were obtained from 2 (D2)- or 20-d-old (D20) flies maintained under 12-h light/12-h dark cycle, with the exception of the dark-reared GMR>rheb. 20-d-old control (GMR-Gal4/+; A), 2-d-old GMR>rheb (B), 20-d-old GMR>rheb (C), 20-d-old GMR>rheb reared in the dark (D), 2-d-old tsc1²⁹ (E), and 20-d-old tsc1²⁹ (F) are shown. (G) The time course of retinal degeneration, as determined by examining the number of rhabdomeres per ommatidium. Error bars indicate SD. R, rhabdomere. Bar, 2 µm.

Figure 2.

Rheb induced cell death independent of cell growth. (A–E) Examination of the morphology of 2-d-old (D2) ninaE>rheb (A), 30-d-old (D30) ninaE>rheb (B), 30-d-old GMR>s6k (C), 30-d-old thor² (D), and 30-d-old GMR>pten ommatidia (E) by transmission EM. Cross sections of retinas were obtained from 30-d-old flies kept under a 12-h light/12-h dark cycle except D2 ninaE>rheb flies (A), which were <2 d old. (F) Mean number of rhabdomeres per ommatidium from 30-d-old flies. Error bars indicate SD. Bar, 2 µm.

Figure 3.

Rheb/TOR signaling drives photoreceptor cell death by suppressing autophagy. (A–C) Transmission EM images of cross sections from wild type (w¹¹¹⁸; A), atg7^d77 (B), and atg7^d77 dark reared (C). The sections were obtained from 30-d-old flies maintained under 12-h light/12-h dark cycle, with the exception of dark-reared atg7^d77 (C). (D) Histogram of the mean number of rhabdomeres per ommatidium from 30-d-old flies. (E–G) Retinal morphology of ninaE>rheb (E), ninaE-atg1 (F), and ninaE>rheb/ninaE-atg1 (G). The sections were obtained from 30-d-old flies maintained under 12-h light/12-h dark cycle. (H) Histogram of the mean number of rhabdomeres per ommatidium from 30-d-old flies. (D and H) Error bars indicate SD. Bar, 2 µm.

Figure 4.

Neuronal degeneration in HQ120 flies was suppressed by overexpression of TSC1/2 or Atg1. (A) Time course of photoreceptor degeneration was determined by the optical neutralization technique. The flies were reared under a 12-h light/12-h dark cycle. Each data point was based on the examination of ≥60 ommatidia from at least four flies. Error bars indicate SD. (B–D) Retinal morphology of 10-d-old flies examined by transmission EM. HQ120 (B), HQ120;GMR>Tsc1/2 (C), and HQ120;ninaE-atg1 (D) are shown. Bar, 2 µm.

Figure 5.

Suppression of retinal degeneration in norpA flies by overexpression of either TSC1/2 or Atg1. (A) The time course of retinal degeneration, as determined by optical neutralization. (B–D) Examination of retinal morphology by transmission EM. norpA^P24 (B), norpA^P24;GMR>Tsc1/2 (C), and norpA^P24;ninaE-atg1 (D) are shown. 10-d-old flies under light/dark cycle were used. (E and F) Stable rhodopsin–arrestin complexes in norpA^P24 flies were reduced by overexpression of either TSC1/2 or Atg1. (E) Western blot of fractions from fly heads exposed to light for 1 h. Pellet (P) and supernatant (S) fractions are indicated. Dark-reared flies <2 d old were used. (F) Histogram quantifying the percent of Arr2 bound to rhodopsin after treatment with 1 h of light. The asterisk indicates a statistically significant difference relative to wild type (Student's unpaired t test; *, P < 0.05). (A and F) Error bars indicate SD. Bar, 2 µm.