Rsu1 regulates ethanol consumption in Drosophila and humans

Abstract

Alcohol abuse is highly prevalent, but little is understood about the molecular causes. Here, we report that Ras suppressor 1 (Rsu1) affects ethanol consumption in flies and humans. Drosophila lacking Rsu1 show reduced sensitivity to ethanol-induced sedation. We show that Rsu1 is required in the adult nervous system for normal sensitivity and that it acts downstream of the integrin cell adhesion molecule and upstream of the Ras-related C3 botulinum toxin substrate 1 (Rac1) GTPase to regulate the actin cytoskeleton. In an ethanol preference assay, global loss of Rsu1 causes high naïve preference. In contrast, flies lacking Rsu1 only in the mushroom bodies of the brain show normal naïve preference but then fail to acquire ethanol preference like normal flies. Rsu1 is, thus, required in distinct neurons to modulate naïve and acquired ethanol preference. In humans, we find that polymorphisms in RSU1 are associated with brain activation in the ventral striatum during reward anticipation in adolescents and alcohol consumption in both adolescents and adults. Together, these data suggest a conserved role for integrin/Rsu1/Rac1/actin signaling in modulating reward-related phenotypes, including ethanol consumption, across phyla.

Keywords: actin; addiction; alcohol; genetics.

Fig. 1.

ics, Encoding Rsu1, is required for normal ethanol responses. Here, flies were exposed to a 130:20 ethanol:airflow rate, and bars represent means ± SEM. ST50 stands for the median sedation time; increased ST50 indicates reduced ethanol sensitivity. (A) Mutant ics^G4 flies show reduced sensitivity to ethanol-induced sedation. This phenotype and (Inset) the loss of Rsu1 protein are rescued with expression of Rsu1 cDNA (UAS-Rsu1; transgene presence indicated by √; n = 8). C, control; M, mutant; R, rescue. ***P < 0.001. (B) Brain expression pattern of ics^G4 revealed by a membrane-bound GFP reporter (UAS-mCD8-GFP; green). B shows (Upper) anterior (ant.) and (Lower) posterior (post.) confocal stacks of ics^G4 (Left) heterozygous WT and (Right) homozygous mutant flies. Expression includes neurosecretory cells in the pars intercerebralis (PI) as well as the MBs. Neuropil is counterstained with anti-Brp nc82 antibody (red).

Fig. 2.

Rsu1 is required in the adult nervous system for normal ethanol responses. (A) Suppression of Gal4 and UAS-Rsu1 expression in the nervous system with elav-Gal80 abrogates the behavioral rescue (n = 6–7). Df, genetic deficiency Df(2L)BSC147 completely removing the ics gene locus; ns, not significant (P > 0.91). (B) Rsu1 expression exclusively in the nervous system through elav^c155-Gal4 completely rescues the reduced ethanol sensitivity phenotype of ics^x5 mutant flies. ***P < 0.001 (n = 7–9). (C and D) Adult expression (D) posteclosion but (C) not throughout development rescues the reduced ethanol sensitivity phenotype of ics^G4 mutant flies. UAS-Rsu1 expression was suppressed using ubiquitously expressed Gal80^ts, which inhibits Gal4 (and therefore, Rsu1 expression) at (Inset) 18 °C (gray) but not 29 °C (green). Flies were kept for 3 d at the test temperature before ethanol exposure. ns, Not significant (P > 0.29); ST50, median sedation time. ***P < 0.001 (n = 6–9).

Fig. 3.

Rsu1 links β-integrin to Rac1 signaling. (A) ics^G4 homozygous mutants combined with heterozygous β-integrin loss-of-function mutants (mys^ts2) are as resistant to ethanol-induced sedation as ics^G4 mutants alone, indicating that Rsu1 functions downstream of β-integrin. Females were grown at 29 °C for maximum mys^ts2 effect. ns, Not significant (P > 0.69); ST-50, median sedation time. **P < 0.01 (n = 7–9). (B) ics^G4 homozygous mutants combined with dominant negative Rac1 (UAS-Rac1^DN) are as sensitive as Rac1^DN mutants alone, suggesting that Rac1 functions downstream of Rsu1. Unexpressed UAS-Rac^DN/+ lacking a Gal4 driver served as a control. ns, Not significant (P > 0.92). **P < 0.001 (n = 8–10).

Fig. 4.

Rsu1 binds to Rac1 and affects actin dynamics in Drosophila S2 cells. (A and B) Rsu1 binds to (A) both the GTP-locked forms of Rac1 (Rac1^CA) and GDP-locked forms of Rac1 (Rac1^DN) but (B) not to Rho1 GTP- (Rho1^CA) or GDP-locked (Rho1^DN) forms. (C) Rac1.GTP pull-down experiments shows that RNAi-mediated knockdown of Rsu1 leads to increased Rac1.GTP loading. (D and E) Globular (G) to filamentous (F) actin assay measuring the ratio of actin in free globular to assembled filamentous form showing that RNAi-mediated knockdown of Rsu1 causes an approximately threefold decrease in G/F actin ratio, whereas overexpression of constitutive active Rac1^CA causes an approximately ninefold decrease in G/F actin ratios compared with controls. The actin stabilizer phalloidin also decreases the G/F ratio and served as a positive control. IP, immunoprecipitation. ***P < 0.001 (n = 4–9).

Fig. 5.

Alcohol consumption preference phenotypes in flies lacking Rsu1. (A) ics Mutant flies show increased naïve ethanol preference compared with control in the two-bottle choice CAFÉ assay. This phenotype is rescued by expression of UAS-Rsu1 in all icsG4-expressing cells. (B and D) ics Rescue flies lacking Rsu1 expression in the MBs only (ics^G4 UAS-Rsu1 MB-Gal80) do not develop acquired ethanol preference but (B) have normal naïve preference on day 1 and (D) ethanol-induced sedation. (C) Adult MB-specific knockdown of Rsu1 or overexpression of Rac1^CA causes loss of acquired ethanol preference. The transgenes were expressed in adults using a mifepristone-inducible MB-GeneSwitch driver. ns, Not significant; ST-50, median sedation time. *P < 0.05; **P < 0.01 (ics^G4 vs. control); ***P < 0.001.

Fig. 6.

Genetic studies in humans. (A) Whole-brain analysis of reward anticipation large win vs. no win during the MID task shows positive BOLD response (PBR) during reward anticipation (family-wise error P < 0.05). The location of the VS (±15, 9, −9; 9-mm radius) is depicted in blue. The results of association analyses between VS and RSU1 gene are summarized. ROI, region of interest. (B) Exon/intron schematic of the RSU1 gene. The first SNP of haplotype block 6 (rs7921941; red), the last SNP of haplotype block 5 (rs12572686; green), and the main SNP (rs7078011; asterisk and blue) are highlighted. The eighth exon is indicated with an arrow. (C) Summary of genetic analyses of alcohol drinking in the human datasets IMAGEN, SAGE, and NFBC1966.