Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Aβ42-induced neurodegeneration
Version of Record online: 21 FEB 2014
© 2014 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 13, Issue 3, pages 507–518, June 2014
How to Cite
Lin, R., Angelin, A., Da Settimo, F., Martini, C., Taliani, S., Zhu, S. and Wallace, D. C. (2014), Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Aβ42-induced neurodegeneration. Aging Cell, 13: 507–518. doi: 10.1111/acel.12200
- Issue online: 23 MAY 2014
- Version of Record online: 21 FEB 2014
- Manuscript Accepted: 19 DEC 2013
- NIH. Grant Numbers: NS21328, DK73691
- Simons Foundation. Grant Number: 205844
|acel12200-sup-0001-FigS1.tiff||TIFF image||4418K||Fig. S1 Multiple sequences alignment of TSPO from various species aligned using CLUSTW. Amino acids on a black background are identical. Those on a gray background are similar for side chain hydrophobicity.|
|acel12200-sup-0002-FigS2.tiff||TIFF image||839K||Fig. S2 The duration of lifecycle in wild type and tspo −/− flies. The number of days from crossing to eclosion was recorded, for each genotype, n = 10 ~ 40. Each bar = mean ± SEM.|
|acel12200-sup-0003-FigS3.tiff||TIFF image||1140K||Fig. S3 The sensitivity to starvation and heat stress in wild type and tspo −/− flies. (A) The male (upper) and female (lower) flies of 3-6 DAE were fed with water containing 4 w/v% sucrose (control group) or without sucrose (starvation group). Numbers of dead flies were counted and percent surviving calculated. N = 10–30 flies for each curve. (B) The male 3–6 DAE flies fed with regular food were incubated in 37 °C, the rate of survival was monitored after 15-h incubation. N = 6 for each group, indicating six vials of flies.|
|acel12200-sup-0004-FigS4.tiff||TIFF image||1002K||Fig. S4 The up-regulation of dTSPO expression during aging. (A) Levels of dTSPO mRNA (determined by qRT-PCR, Rp49 mRNA reference) and (B) levels of protein (assessed by densitometry of western blot, β-actin reference). In both experiments, tspo +/+ male flies were maintained on standard cornmeal medium at 22 °C. For mRNA at DAE 0 (n = 8), 25 (n = 8), 50 (n = 6), 75 (n = 3). For protein, n = 2 at each time point.|
|acel12200-sup-0005-FigS5.TIF||image/tif||90K||Fig. S5 Effect of dTSPO depletion in neurons on Aβ42 mRNA expression level. Total mRNA was extracted from heads of 1–3 DAE male Aβ42-expressing flies with or without neuronal knockdown of dTSPO. The dTSPO mRNA level was monitored by qRT-PCR. (n = 3 for each genotype)|
|acel12200-sup-0006-FigS6.tiff||TIFF image||1253K||Fig. S6 Effect of dTSPO genetic depletion on Drosophila OXPHOS complex activity and ROS production at 2–4 DAE. Mitochondria were isolated from whole body lysates of tspo +/+, +/−, and −/− flies (mixture of equal number of male and female). (A) Mitochondrial oxygen consumption rates were measured while metabolizing site I (pyuvate + malate) substrates in the absence (state 4) or presence (state 3) of ADP, N = 8 for each genotype. (B) Relative mitochondrial membrane potential assessed by TMRM uptake in mitochondria, N = 3 per genotype. (C) Mitochondrial OXPHOS complex I-IV activities normalized to citrate synthase activity, N = 4 repetitions per assay. (D) Mitochondrial aconitase activity in male and female tspo +/+ and −/− flies, expressed as the ratio of endogenous activity divided by total activity following aconitase following reactivation with Fe2+. N = 3 for each genotype. For all data above, *P < 0.05, **P < 0.01, ***P < 0.001.|
|acel12200-sup-0007-FigS7.TIF||image/tif||73K||Fig. S7 Effect of dTSPO depletion on ATP content. ATP content in whole body tissue of 7–10 DAE flies was similar in tspo −/− flies compared with tspo +/+ wild type controls. For each genotype, n = 3.|
|acel12200-sup-0008-FigS8.TIF||image/tif||156K||Fig. S8 Effect of dTSPO depletion on MDA level in TBARS assay. MDA level in whole body tissue of 20–23 DAE flies was similar in tspo −/− flies compared with tspo +/+ wild-type controls. For each genotype, n = 3.|
Table S1. Median, maximum lifespan and statistical analysis of dTSPO mutant, knockdown flies and wild-type flies treated with PK11195 and Ro5-4864.
Table S2. Median, maximum lifespan and statistical analysis of Aβ42-expressing flies with modification of dTSPO inactivation.
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