Role of Autophagy in Glycogen Breakdown and Its Relevance to Chloroquine Myopathy
Figure 5
Glycogen synthase knockdown inhibits autophagosome growth and improves CQ-induced myopathy phenotype.
(A–D) Glycogen synthase (GlyS) is required for glycogen synthesis in D. melanogaster muscles. PAS staining for glycogen was absent in Dmef2–Gal4/UAS–GlySi muscles (B) compared to control Dmef2–Gal4/UAS–whitei muscles (A). Antiglycogen immunostaining for glycogen was absent in Dmef2–Gal4/UAS–GlySi muscles (D) compared to Dmef2–Gal4/UAS–whitei control muscles (C). (E–K) GlyS is required for the formation of large CQ-induced autophagosomes. Vesicles are much smaller in Dmef2–Gal4, UAS–GFP–Atg8/UAS–GlySi larval muscle starved 6 h in low-nutrient food +2.5 mg/ml CQ (G) than in control Dmef2–Gal4, UAS–GFP–Atg8/UAS–whitei larval muscle (E). (F, H) The difference in autophagosome size is clearly evident at high magnification. (I–K) Quantification of autophagy changes due to GlyS gene knockdown in Dmef2–Gal4, UAS–GFP–Atg8 larvae starved on low-nutrient food +2.5 mg/ml CQ for 6 h. SEM is indicated, with n = 5 (I) or n = 10 (J–K) ventral longitudinal muscles from individual animals (*p<.05, **p<.01). (I) Each of the four UAS-GlyS RNAi transgenes tested caused a significant decrease in the total area of GFP–Atg8 vesicles in the muscle compared to the UAS–whitei control. (J) Vesicle number was unchanged by GlyS knockdown. (K) UAS–GlyS RNAi caused a highly significant decrease in the mean vesicle size (area) compared to the control. (L–M) GlyS gene expression, monitored using a MiMIC transposon insertion (MI01490), showed expression in the larval muscle (L) but not the fat body (M) (green, GFP; blue, DAPI). (N–O) Larvae were starved on low-nutrient food for 6 h prior to dissection of the fat bodies. Autophagy in Cg-Gal4/+; UAS–GFP–Atg8/UAS–GlySi (O) was not substantially different from autophagy in Cg-Gal4/+; UAS–GFP–Atg8/UAS–whitei control fat bodies (GFP, green; DAPI, blue). (P) EM of muscle from Dmef2–Gal4/UAS–GlySi animal starved on low-nutrient food +CQ. Note that the intermyofibril spaces (red asterisk) and sarcomere structure are not distorted. (Q) GlyS or Atg1 knockdown significantly improved the crawling time of larvae treated with CQ and starved for 6 h. SEM is indicated for n = 10 larvae. The p values were calculated relative to white RNAi control larvae (*p<.05, **p<.01).