TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in <i>Drosophila</i>

<div><p>The heavy consumption of ethanol can lead to alcohol use disorders (AUDs) which impact patients, their families, and societies. Yet the genetic and physiological factors that predispose humans to AUDs remain unclear. One hypothesis is that alterations in mitochondrial function modulate neuronal sensitivity to ethanol exposure. Using <i>Drosophila</i> genetics we report that inactivation of the mitochondrial outer membrane translocator protein 18kDa (TSPO), also known as the peripheral benzodiazepine receptor, affects ethanol sedation and tolerance in male flies. Knockdown of dTSPO in adult male neurons results in increased sensitivity to ethanol sedation, and this effect requires the dTSPO depletion-mediated increase in reactive oxygen species (ROS) production and inhibition of caspase activity in fly heads. Systemic loss of dTSPO in male flies blocks the development of tolerance to repeated ethanol exposures, an effect that is not seen when dTSPO is only inactivated in neurons. Female flies are naturally more sensitive to ethanol than males, and female fly heads have strikingly lower levels of dTSPO mRNA than males. Hence, mitochondrial TSPO function plays an important role in ethanol sensitivity and tolerance. Since a large array of benzodiazepine analogues have been developed that interact with the peripheral benzodiazepine receptor, the mitochondrial TSPO might provide an important new target for treating AUDs.</p></div

Comparable ethanol sensitivity in female <i>tspo</i>-/- and <i>tspo</i> +/+ flies.

<p>(A) With 34% ethanol vapor, half sedation time for <i>tspo</i>+/+ was 23.0±2.5 min and for <i>tspo</i>-/- was 20.0±1.7 min, p > 0.05, n = 14 vials tested. (B) With 44% ethanol vapor half sedation time for <i>tspo</i>+/+ was 11.0±0.5 min and for <i>tspo</i>-/- was 11.0±0.5 min, p > 0.05, n = 10. (C) Recovery after withdraw from exposure to 44% ethanol vapor, half recovery rate for <i>tspo</i>+/+ was 16.0±1.2 min and for <i>tspo</i>-/- was 18.0±1.2 min, p > 0.05, n = 8. Data presented as mean ± SEM.</p

Depletion of dTSPO in neurons suppresses caspase activity which is sufficient to increase ethanol sensitivity.

<p>Gene switch was accomplished as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005366#pgen.1005366.g002" target="_blank">Fig 2</a>. (A) Caspase 3/7 activity was moderately reduced in heads of elav-GS/+; TSPO-IR/+ or elav-GS/+; p35/+ (elav-GeneSwitch plus UAS-p35) flies when the dTSPO dsRNA or p35 were induced with RU486. All groups were measured twice, and data presented as mean. Since the TSPO-IR and P53 products are only expressed in neurons, but the caspase activity was assayed in whole heads, the ~20% decrease in caspase 3/7 underrepresents the extent of caspase reduction in neurons. This is demonstrated by whole body knockout of TSPO in which the relative whole body caspase 3/7 activity of <i>tspo</i> +/+ flies was 1.000±0.008 and of <i>tspo</i>-/- flies was 0.078±0.015, p < 0.001, n = 4. (B) Induction of caspase inhibitor p35 with RU486 significantly increased sensitivity to 34% ethanol vapor, Chi Square log rank test, p = 0.0006, n = 8 vials tested. (C) Flies harboring only UAS-p35 (p35/+) exposed to 34% ethanol vapor with or without RU486 were not different, Chi Square log rank test, p = 0.846, n = 12. Data presented as mean ± SEM.</p

High male brain expression of TSPO associated with increased ethanol sensitivity in male neuronal dTSPO knockdown flies.

<p>(A) Levels of dTSPO mRNA in the heads of elav-GS/+; TSPO-IR/+ male and female flies with or without RU486 dsRNA induction, n = 3 groups of flies tested. Data presented as mean ± SEM. *** p < 0.001. (B-D) Gene switch and control flies with or without RU486 (elav-GS/+ = elav-GeneSwitch and TSPO-IR/+ = UAS-dTSPO-RNAi). To induce gene switch, the flies were raised on regular food with 50 μl of 4 mg/ml RU486 added on the surface of the food in vials for three days. (B) Sensitivity of elav-GS/+;TSPO-IR/+ flies to 44% ethanol vapor with and without RU486, half sedation time with RU486 was 16.0±0.6 min and without RU486 was 23.3±1.5, p < 0.001, n = 10. (C) Sensitivity of flies harboring elav-GS/+ with or without RU486 exposed to 44% ethanol vapor, half sedation time with RU486 was 14.0±0.5 min and without RU486 was 15.3±0.9, p > 0.05, n = 13, vials tested. (D) Sensitivity of flies harboring only TSPO-IR/+ exposed to 44% ethanol and with and without RU486, half sedation time with RU486 was 25.0±2.0 min and without RU486 was 22.5±1.0, p > 0.05, n = 10.</p

Systemic loss of dTSPO inhibits the development of ethanol tolerance.

<p>Tolerance is revealed as a longer period required for sedation at a second exposure to 54% ethanol solution vapor. (A) Differential sedation from first versus second ethanol exposure of <i>tspo</i> +/+ versus <i>tspo</i>-/- flies: For <i>tspo</i> +/+ flies half sedation time for first exposure was 12.0±0.5 and for second exposure was 26.4±1.9, p < 0.001. For <i>tspo</i>-/- flies half sedation time for first exposure was 12.3±0.3 and for second exposure was 15.0±1.6, p = 0.11. Difference between second exposure sedation of <i>tspo</i> +/+ and <i>tspo</i>-/- flies, p < 0.001, n = 8, vials tested. (B) Differential sedation from first versus second exposure of elav-GS/+; TSPO-IR/+ flies with or without RU486 induction, n = 12. Gene switch was induced as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005366#pgen.1005366.g002" target="_blank">Fig 2</a>. (C) dTSPO mRNA levels in the heads of tspo+/+ male flies after exposure to 54% ethanol vapor showing progressive loss of dTSPO mRNA for the first 4 hours after exposure followed by partial recover to original levels, n = 3 groups of flies tested. (D) dTSPO mRNA levels in the bodies of male <i>tspo</i>+/+ flies after exposure to 54% ethanol vapor showing a progressive increase up to 4 hours after exposure followed by a decline (n = 3). Data presented as mean ± SEM.</p

Increased ethanol sensitivity in male <i>tspo</i> mutant flies.

<p>Sensitivity to acute ethanol sedation was increased in <i>tspo</i>-/- flies compared with <i>tspo</i>+/+ flies (A-C). (A) With 34% ethanol solution, half sedation time for <i>tspo</i>+/+ was 30.6±1.9 min and for <i>tspo</i>-/- was 17.8±1.5 min, p = 0.01, n = 13 vials tested. (B) With 44% ethanol solution, half sedation time for <i>tspo</i>+/+ was 14.3±2.2 min and for <i>tspo</i>-/- was 13.8±0.6 min, p > 0.05, n = 8 vials tested. (C) With 54% ethanol solution, half sedation time for <i>tspo</i>+/+ was 11.3±0.8 min and for <i>tspo</i>-/- was 11.3±0.3 min, p > 0.05, n = 8. (D). The rate for recovery after ethanol withdraw was slower in <i>tspo</i>-/- than <i>tspo</i>+/+ flies, half recovery time for <i>tspo</i>+/+ was 20.4±2.1 min and for <i>tspo</i>-/- was 28.1±3.6 min, p > 0.05, n = 8. Data presented as mean ± SEM.</p