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. 2021 Oct 31;30(5):341–355. doi: 10.5607/en21021

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Copyright © Experimental Neurobiology 2021

This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fig. 4 — Sirt2 knockdown enhances rotenone-induced neuronal toxicity in the Drosophila dopamine system. (A) Levels of dSirt2 mRNA from fly head lysates of 12 h starved wild-type flies were analyzed by real-time PCR. Gapdh was used for normalization. Data are presented as the mean±SD. ***p<0.001 (unpaired Student’s t-test). (B) Levels of oxidative stresses-related genes (dPuc, dGstD1, dHsp22, and dMrp4) mRNA from fly head lysates of 12 h starved wild-type flies. mRNA levels were analyzed by real-time PCR. Gapdh was used to normalize changes in specific gene expression. Genotypes: UAS-Egfp RNAi^{VALIUM20-EGFP.shRNA.4}/+. Data are presented as the mean±SD. *p<0.05; **p<0.005; ***p<0.001 (unpaired Student’s t-test). (C) Levels of Sirt2 mRNA from fly head lysates of ple-Gal4/Gfp RNAi and ple-Gal4/Sirt2 RNAi flies were analyzed by real-time PCR. RNAi-mediated knockdown of Sirt2 in Drosophila reduced the Sirt2 transcription levels. Gapdh was used to normalize changes in specific gene expression. Data are presented as the mean±SD. *p<0.05 (unpaired Student’s t-test). (D) Levels of Sirt2 mRNA from fly head lysates of Ple-Gal4/Gfp RNAi flies fed rotenone (500 μM) were analyzed by real-time PCR. The Sirt2 level was significantly upregulated in the rotenone exposed flies. Gapdh was used to normalize changes in specific gene expression. Data are presented as the mean±SD. *p<0.05 (unpaired Student’s t-test). (E) Survival rate of ple-Gal4/UAS-Gfp RNAi and ple-Gal4/UAS-Sirt2 RNAi flies fed rotenone (500 μM) at the indicated time points. The lifespan of rotenone-treated flies was significantly reduced by the downregulation of Sirt2. The percentage of flies that survived was quantified. Genotypes: Gfp RNAi is ple-Gal4/UAS-Egfp RNAi^{VALIUM20-EGFP.shRNA.4}, and Sirt2 RNAi is ple-Gal4/UAS-Sirt2 RNAi^HMS00485. Data are presented as the mean±SEM of 4 independent experiments. *p<0.05; ***p<0.001 (unpaired Student’s t-test). (F, G) Dopaminergic neurons were visualized via expression of UAS-mCD8-GFP under control of the ple-Gal4. The number of dopaminergic neurons in PPL1 cluster was analyzed in ple>mCD8::GFP/Gfp RNAi and ple>mCD8::GFP/Sirt2 RNAi flies after 48 h of exposure to rotenone (500 μM). Arrowheads indicate dopaminergic neurons in PPL1 cluster (left). Genotypes: ple>mCD8::GFP/Gfp RNAi is ple-Gal4,USA-mCD8-GFP/UAS-Egfp RNAi^{VALIUM20-EGFP.shRNA.4}, and ple>mCD8::GFP/Sirt2 RNAi is ple-Gal4,USA-mCD8-GFP/UAS-Sirt2 RNAi^HMS00485. Data are presented as the mean±SEM. *p<0.05 (n=5, unpaired Student’s t-test). Scale bars, 50 µm.