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TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila.

Lin R, Rittenhouse D, Sweeney K, Potluri P, Wallace DC - PLoS Genet. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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 Drosophila 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.

No MeSH data available.


Related in: MedlinePlus

High male brain expression of TSPO associated with increased ethanol sensitivity in male neuronal dTSPO knockdown flies.(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.
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pgen.1005366.g002: High male brain expression of TSPO associated with increased ethanol sensitivity in male neuronal dTSPO knockdown flies.(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.

Mentions: To determine whether the increased ethanol sensitivity was attributable to dTSPO function in neurons, we depleted dTSPO in neurons by inducing dsRNA (RNAi) to knockdown dTSPO mRNA in adult flies following eclosion (days after eclosion, dae). We used the Gal4-GeneSwitch/UAS system [25] in which Gal4 is activated within the flies when fed with mifepristone (RU486). The activated Gal4 binds to the UAS of the UAS-dTSPO-RNAi which induces the dsRNA expression and inhibition of the dTSPO mRNA. Since the Gal4 element is expressed under the neuronal specific elav promoter (elav-GeneSwitch), this switch was restricted to neurons. In this way, the flies were permitted to progress through larval and pupal development with normal TSPO activity, and following eclosion, the dTSPO RNAi was induced in neurons by exposure to RU486. Male flies harboring both elav-GeneSwitch and UAS-dTSPO-RNAi (elav-GS/+; TSPO-IR/+)(GS means ‘Gene Switch’ and IR means ‘Inverted Repeats’) cassettes that were exposed to RU486 post eclosion had reduced head dTSPO mRNA as quantified by RT-PCR (Fig 2A). Therefore, activation of the elav-GS/+; TSPO-IR/+ system with RU486 specifically depletes dTSPO mRNAs in the neurons.


TSPO, a Mitochondrial Outer Membrane Protein, Controls Ethanol-Related Behaviors in Drosophila.

Lin R, Rittenhouse D, Sweeney K, Potluri P, Wallace DC - PLoS Genet. (2015)

High male brain expression of TSPO associated with increased ethanol sensitivity in male neuronal dTSPO knockdown flies.(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.
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pgen.1005366.g002: High male brain expression of TSPO associated with increased ethanol sensitivity in male neuronal dTSPO knockdown flies.(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.
Mentions: To determine whether the increased ethanol sensitivity was attributable to dTSPO function in neurons, we depleted dTSPO in neurons by inducing dsRNA (RNAi) to knockdown dTSPO mRNA in adult flies following eclosion (days after eclosion, dae). We used the Gal4-GeneSwitch/UAS system [25] in which Gal4 is activated within the flies when fed with mifepristone (RU486). The activated Gal4 binds to the UAS of the UAS-dTSPO-RNAi which induces the dsRNA expression and inhibition of the dTSPO mRNA. Since the Gal4 element is expressed under the neuronal specific elav promoter (elav-GeneSwitch), this switch was restricted to neurons. In this way, the flies were permitted to progress through larval and pupal development with normal TSPO activity, and following eclosion, the dTSPO RNAi was induced in neurons by exposure to RU486. Male flies harboring both elav-GeneSwitch and UAS-dTSPO-RNAi (elav-GS/+; TSPO-IR/+)(GS means ‘Gene Switch’ and IR means ‘Inverted Repeats’) cassettes that were exposed to RU486 post eclosion had reduced head dTSPO mRNA as quantified by RT-PCR (Fig 2A). Therefore, activation of the elav-GS/+; TSPO-IR/+ system with RU486 specifically depletes dTSPO mRNAs in the neurons.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Center for Mitochondrial and Epigenomic Medicine, Children's Hospital of Philadelphia Research Institute, Philadelphia, Pennsylvania, United States of America.

ABSTRACT
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 Drosophila 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.

No MeSH data available.


Related in: MedlinePlus