Essential role of D1R in the regulation of mTOR complex1 signaling induced by cocaine

Abstract

The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that is involved in neuronal adaptions that underlie cocaine-induced sensitization and reward. mTOR exists in two functionally distinct multi-component complexes known as mTORC1 and mTORC2. In this study, we show that increased mTORC1 activity induced by cocaine is mediated by the dopamine D1 receptor (D1R). Specifically, cocaine treatment increased the phosphorylation on residues Thr2446 and Ser2481 but not on Ser2448 in the nucleus accumbens (NAc) and that this increase in phosphorylated mTOR levels was also apparent when complexed with its binding partner Raptor. Furthermore, the increase in phosphorylated mTOR levels, as well as phosphorylated 4E-BP1 and S6K, downstream targets of mTORC1 were blocked with SCH23390 treatment. Similar results were also observed in the dopamine-transporter knockout mice as the increase in phosphorylated mTOR Thr2446 and Ser2481 was blocked by SCH23390 but not with raclopride. To further validate D1R role in mTORC1 signaling, decrease in phosphorylated mTOR levels were observed in D1R knockout mice, whereas administration of SKF81297 elevated phosphorylated mTOR in the NAc. Lastly deletion of mTOR or Raptor in D1R expressing neurons reduced cocaine-induced locomotor activity. Together, our data supports a mechanism whereby mTORC1 signaling is activated by cocaine administration through the stimulation of D1R.

Keywords: Cell signaling; Cocaine; Dopamine; Nucleus accumbens; Raptor; mTOR.

Figure 1

Cocaine activates mTORC1 signaling in the NAc of mice. Representative western blots and graphs of densitometry values for phosphorylated and total mTOR (Thr2446, Ser2448 and Ser2481) in the NAc of mice treated with (a) acute (15mg/kg) and (b) repeated cocaine (CO, 15mg/kg, 5 day) or vehicle (veh). (c) Representative images and graph of densitometry values for co-IPs showing an increased interaction between Raptor and phosphorylated mTOR in the NAc of mice administered with repeated cocaine. Samples were immunoprecipitated (IP) with Raptor conjugated sepharose beads and then subjected to western blotting (WB) using antibodies for phosphorylated mTOR Thr2446, Ser2448, Ser2481, mTOR and Raptor (Left panel, n = 4), whereas WB was performed using the same total lysate as the co-IPs (Right panel). Representative western blots and graphs of densitometry values for phosphorylated and total (d) 4E-BP1, elF4E (e) S6K and S6 in the NAc of mice treated with repeated cocaine (CO, 15mg/kg, 5 day) or vehicle (Veh). Data presented as mean ± SEM and expressed as a percentage of control (** p < 0.01, *** p < 0.001, **** p < 0.0001, two-tailed unpaired t test).

Figure 2

DAT KO mice show increased mTORC1-mediated signaling in the NAc. Representative western blots and graphs of densitometry values for phosphorylated and total (a) mTOR (Thr2446, Ser2448 and Ser2481) (b) 4E-BP1, elF4E (c) S6K and S6 in the NAc of DAT KO or wild type (WT) mice. Data presented as mean ± SEM and expressed as a percentage of control (n = 8; two-tailed unpaired t test). (d) Locomotor activity for DAT KO and WT mice treated with rapamycin (Rap; 2mg/kg, 5mg/kg, 10mg/kg) or vehicle (n = 9-12mice/group, two-way ANOVA). (e) Time course of locomotor activity for DAT KO mice treated with rapamycin or vehicle (n = 9-12mice/group, two-way ANOVA). Mice were administered vehicle or rapamycin following a 30min habituation to locomotor chambers. Data represented as mean ± SEM (** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to WT_Vehicle; # p < 0.05 compared to DAT KO_Vehicle).

Figure 3

D1R regulates phosphorylated mTOR levels in the NAc. (a) Representative western blots and graphs of densitometry values for total and phosphorylated mTOR Thr2446, Ser2448 and Ser2481 levels in DAT KO mice treated with the D1R antagonist, SCH23390 (SCH, 0.5mg/kg), D2R antagonist, raclopride (Rac, 3mg/kg) or vehicle (n = 4mice/group, two-way ANOVA). (b) Representative western blots and graphs of densitometry values for total and phosphorylated mTOR levels in the NAc of D1R KO or WT mice (n = 4mice/group, two-tailed t-test). Representative western blots and graphs of densitometry values for total and phosphorylated mTOR levels in the NAc of C57BL/6J mice treated with (c) SKF81297 (n = 4-6mice/group) or (d) quinpirole (n = 4mice/group). Data presented as mean ± SEM and expressed as percentage of control (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to WT/vehicle; # p < 0.05, ### p < 0.001 compared to DAT KO_Veh).

Figure 4

Inhibition of D1R blocks activation of the mTORC1 signaling pathway by repeated cocaine treatment. Following 5 days of cocaine treatment, C57BL/6J mice were pretreated with SCH23390 (0.5mg/kg) or vehicle prior to the last cocaine injection. Representative western blots and graphs of densitometry values for phosphorylated and total (a) mTOR (Thr2446, Ser2448 and Ser2481) (b) 4E-BP1, elF4E (c) S6K and S6. Data presented as mean ± SEM and expressed as percentage of control (n = 4 mice/group; *** p < 0.001, **** p < 0.0001 compared to Veh_Veh; # p < 0.05, ## p < 0.001, ### p < 0.001 compared to Cocaine_Veh; two-way ANOVA).

Figure 5

mTOR or Raptor deletion in D1 neurons display lower locomotor response upon cocaine administration. (a) Representative western blots and graphs of densitometry values of total mTOR levels in the NAc of D1mTOR^+/+ and D1mTOR^-/- mice (n = 4mice/group; * p < 0.05, two-tailed unpaired t test). (b) Total locomotor activity for D1mTOR^+/+ and D1mTOR^-/- mice treated with cocaine (Co, 15mg/kg) or vehicle (Veh; n = 11/group, **** p < 0.0001 compared to Veh_D1mTOR^+/+, # p < 0.05 Co_D1mTOR^+/+ vs Co_D1mTOR^-/-; Two-way ANOVA) (c) Time course of locomotor activity for D1mTOR^+/+ and D1mTOR^-/- mice treated with cocaine (# p < 0.05 cocaine_ D1mTOR^+/+ vs cocaine_ D1mTOR^-/-, Three-way ANOVA with repeated measures). (d) Representative western blots and graphs of densitometry values of total Raptor levels in the NAc of D1Raptor^+/+ and D1Raptor^-/- mice (n = 4mice/group; * p < 0.05, two-tailed unpaired t test). (e) Total locomotor activity for D1Raptor^+/+ and D1Raptor^-/- mice treated with cocaine (15mg/kg) or vehicle. (Veh; n = 5-9mice/group, * p < 0.05, **** p < 0.0001 compared to Veh_D1Raptor^+/+, # p < 0.05 Co_D1Raptor^+/+ vs Co_D1Raptor^-/-; Two-way ANOVA). (f) Time course of locomotor activity for D1Raptor^+/+ and D1Raptor^-/- mice treated with cocaine. (# p < 0.05 cocaine_ D1Raptor^+/+ vs cocaine_ D1Raptor^-/-, Three-way ANOVA with repeated measures).