Genetic analysis of dTSPO, an outer mitochondrial membrane protein, reveals its functions in apoptosis, longevity, and Ab42-induced neurodegeneration

Abstract

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P-element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5-4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ-irradiation or H2O2 exposure, as well as extended male fly lifespan and inhibited Aβ42-induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.

Figure 1

The localization of dTSPO and its depletion in tspo −/− and dsRNA knockdown flies. (A) The mitochondrial localization of dTSPO in S2 cells. S2 cells were stained with MitoTracker Deep Red (red) and TSPO fluorescent probe (green) sequentially. (B) Western blot of dTSPO, COX-I (IMM), and Porin (VDAC) (OMM) in isolated mitochondria from whole bodies of wild type flies treated with increasing concentrations of digitonin which dissolves the OMM releasing its proteins. dTSPO is lost along with porin. For (1)–(5), the concentrations of digitonin are 0, 0.25, 0.5, 1, 2 mg mL⁻¹. (C) Western blot showing depletion of dTSPO in tspo −/− and dsRNA (RNAi) whole-body knockdown flies (mixture of equal numbers of male and female flies). Porin (VDAC) provides the OMM mitochondrial control and α-tubulin provides the total protein loading control. The densitometry of western films was shown (N = 2). Bars report mean ± SEM.

Figure 2

Inhibition of apoptosis in wing disks by dTSPO inactivation. (A) Wing disc apoptosis of male (upper) and female (lower) 3rd-instar larvae of wild type or tspo −/− flies irradiated with 30 Gray of γ-ray and TUNEL stained. (B) Quantification of apoptosis in (A) by measuring the area of the TUNEL positive pixels divided by total disk pixels, n = 3 to 10 wing disks quantified. (C) and (D) The same method but comparing control versus dTSPO dsRNA knockdown flies, n = 6–9. Bars report mean ± SEM. ***P < 0.001.

Figure 3

Inhibition of apoptosis in isolated larval brain cells by dTSPO inactivation. Effect of H₂O₂ on induction of apoptosis in 3rd-larvae brain cells from tsps +/+ or tspo −/− flies (mixture of equal numbers of male and female flies). (A) Flow cytometry quantification of FITC-Annexin V positive cells (dead cells) versus FITC-Annexin V negative and Propidium Iodide negative cells (healthy) cells (each bar, n = 3–6). (B) Assay of caspase 3/7 activity (n = 3). Bars report mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Effects of dTSPO inhibition on Drosophila lifespan and oxidative stress resistance. (A) Left, exposure of wild type (tspo +/+) male flies to 5 or 50 μm PK11195 (n = 78–88 counted for each group). Right, exposure of tspo +/+ flies to 0.1 or 1 μm Ro5-4864 (n = 62–72 counted for each group). Drugs administered in 4% sucrose at 25 °C. (B) Extension of lifespan of tspo −/− males (left) though not female (right) relative to tspo +/+ flies maintained in standard cornmeal medium at 25 °C, n = 250–350.(C) Extension of lifespan of whole body dTSPO dsRNA knockdown males (left) though not female (right) relative to control flies maintained on cornmeal medium at 25 °C, n = 50–100. (D) Protection of males (left) but not females (right) tspo −/− flies to exposure to 5% H₂O₂ in 5% sucrose/PBS at 25 °C relative to tspo +/+ flies, n = 50–75.

Figure 5

Effect of dTSPO reduction on Aβ42-induced neurodegeneration. Systemic reduction (tspo +/−) or neuron-specific knockdown of dTSPO restored neuronal human Aβ42-induced lifespan reduction in male and female flies. (A) Survival curves of male (left) and female (right) flies in response to neuronal expression of human Aβ42 (elav>Aβ42) relative to control flies (elavGal4 control) lacking the UAS-Aβ42 target gene and amelioration of the lifespan reduction induced by Aβ42 expression by partial systemic inactivation of tspo +/− or neuronal dsRNA inactivation of dTSPO expression (elav>Aβ42, dTSPO-RNAi). (males, n ≈ 200 and females, n ≈ 100). (B, C) Reduced neuronal human Aβ42 induced tissue loss in response to systemic (tspo +/−) or neuronal depletion (dTSPO-RNAi). (B) Representative histological sections for only male brains were displayed. Arrows indicate regions of neuronal loss (vacuole-like), DAE = 45. (C) Quantification of number of vacuole-like regions per single head of male (left, n = 3–6 for each genotype, DAE = 45) or female (right, n = 3–4 for each genotype, DAE = 60) flies. (D) Effect of dTSPO depletion on neuronal Aβ42-induced male fly head caspase 3/7 activation (n = 3), DAE = 20. Each bar reports mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Effect of dTSPO depletion on 7–10 DAE Drosophila mitochondrial OXPHOS. Mitochondria were isolated from whole body homogenates of tspo +/+, tspo +/−, and tspo −/− flies (mixture of equal number of males and females). (A) Mitochondrial oxygen consumption rate when metabolizing site I (pyruvate + malate) substrates in the absence (state 4) or presence (State 3) of ADP. N = 3 for each genotype. (B) Mitochondrial OXPHOS complexes I to V specific activities normalized using citrate synthase activity. N = 3 for each enzyme value. (C) Mitochondrial aconitase activity in male and female tspo +/+ versus −/− flies expressed as the percentage ratio of the endogenous activity divided by the Fe²⁺-recovered activity. N = 3 for each assay. *P < 0.05, ** P < 0.01, *** P < 0.001.