AKT kinase activity is required for the effect of lithium on TCF/LEF reporter activity. (a) HEK293 (black bars) and HT-22 (grey bars) expressing firefly luciferase driven by a TCF/LEF promoter were treated overnight with LiCl (10 mM), or NaCl (10 mM). TCF/LEF reporter activity was measured by Dual-Glo (Promega), and normalized to renilla luciferase activity under the control of ubiquitous EF1α promoter. TCF/LEF reporter activity is shown as the fold change relative to the control NaCl treatments. (b–c) Densitometric western blot analysis of phosphorylated and total AKT1 expression in whole cell lysate from HEK293 and HT-22 cells treated overnight with LiCl (10 mM) or NaCl (10 mM). β-Actin was used as the loading control for densitometry. Levels of p-AKT(308) and p-AKT(473) are shown as fold change relative to total AKT1 within each treatment in HEK293 and HT-22 cells. For all results, data are mean±SEM from at least three independent experiments. (d) Schematic of PTEN, PI3K inhibitor, and AKTI-17 interference with AKT signaling examined in three experiments (Ex.1, 2, 3). (e–h) TCF/LEF luciferase reporter assays of cells treated overnight with LiCl (10 mM) or NaCl (10 mM). Reporter activity was measured by Dual-Glo assay (Promega), and normalized to EF1α renilla luciferase activity. Data represent mean±SEM from at least three independent experiments. (e) HEK293 cells were transiently transfected with a plasmid expressing PTEN, or an empty plasmid (control), and treated with LiCl (dark grey bars) or NaCl (white bars). Relative TCF/LEF activity is shown as fold change relative to control (empty plasmid transfection and NaCl treatment). (f) HEK293 cells were treated with the PI3K inhibitors wortmannin (0.1, 1, and 10 μM) or LY294002 (1, 10, and 20 μM) in the presence of LiCl. Relative TCF/LEF activity is shown as the fold change relative to NaCl treatment. (g) HEK293 cells were treated with AKTI-17 (0.0003–3 μM) overnight in the presence of LiCl. The fraction of TCF/LEF reporter activity relative to the control (inhibitor absent) is plotted against the concentrations of AKTI-17 (IC₅₀=0.008 μM). (h) HT-22 cells were transfected with constitutively active myristoylated AKT1 (CA-AKT1), wild-type AKT1 (WT-AKT1), or reagents only (control), and treated with LiCl or NaCl overnight. TCF/LEF activity is shown relative to the control condition (control transfection and NaCl treatment). (i) HT-22 cells expressing firefly luciferase controlled by a mutated TCF/LEF promoter (mutation marked as black triangles) were transfected with Myr-AKT1, WT-AKT1, or reagents (control), and treated with LiCl or NaCl overnight. For all results, data are mean ± SEM. ^***p<0.001 compared to NaCl control.

Lithium induces distinct functional responses in vivo in inbred mouse strains that vary in AKT activation. (a, b) Densitometric western blot analysis of relative expression of p-AKT(308) in total cell lysate from striatum of C57BL/6J (black) and DBA/2J (grey) mice that were injected with saline vehicle or LiCl (85 mg/kg, i.p.) 60 min before saline vehicle or -amphetamine (3.5 mg/kg, i.p.) challenge. Striatal tissues were taken 30 min after -amphetamine (or vehicle) administration. Expression of p-AKT (308) was normalized to a β-actin loading control, and fold change in p-AKT(308) compared with vehicle treatment in each mouse strain. Data represent the average of three independent western blot analyses for each animal and four animals in each treatment group (n=12). (c–e) Densitometric western blot analysis of relative expression of p-AKT(308) and p-GSK-3β(9) in striatal lysate of C57BL/6J (black) and DBA/2J (grey) mice injected with saline vehicle or LiCl (85 mg/kg, i.p.) 90 min before tissue harvest. Expression of p-AKT(308) and p-GSK-3β(9) was normalized to the level of total AKT1 or GSK-3β in each sample, and fold change in phosphorylated protein compared with vehicle treatment in each mouse strain is quantified. Data represent the average of three independent western blot analyses for each animal and three animals in each treatment group. (f) DBA/2J or C57BL/6J mice were injected with saline vehicle or LiCl (85 mg/kg, i.p.) 60 min before -amphetamine (3.5 mg/kg, i.p.) challenge. Total distance traveled (cm) over 80 min after -amphetamine injection was quantified. Number of animals per group (n) is indicated. (g) DBA/2J or C57BL/6J mice were treated with saline vehicle or LiCl (85 mg/kg, i.p.) 45 min before the FST. Time spent immobile (%) is relative to the vehicle group immobility time. Number of animals per group (n) is indicated. For all results, data are mean±SEM. ^*p<0.05 and ^**p<0.01 compared with vehicle-treated mice; NS, p>0.05.

AKT kinase activity is required for lithium to attenuate AIH in C57BL/6J mice. (a) C57BL/6J mice were pretreated with AKTI-17 (0.1, 1 or 10 μM in 100% DMSO, i.c.v.) or 100% DMSO vehicle (Veh) 60 min before -amphetamine challenge (3.5 mg/kg, i.p.). Total distance traveled was quantified for 80 min after -amphetamine injection. (b, c) C57BL/6J mice were pretreated with AKTI-17 (10 μM in 100% DMSO, i.c.v.) or 100% DMSO vehicle (Veh) 45 min before administration of lithium (85 mg/kg, i.p.) or saline vehicle (Veh), which occurred 60 min before -amphetamine challenge (time 0; 3.5 mg/kg, i.p.). Distance (cm) traveled within 5-min bins is plotted against the total 80 min recording time (b), and total distance (cm) traveled was quantified in the 80-min period after -amphetamine challenge (c). Number of animals per group (n) is indicated. For all results, data are mean ± SEM. ^*p<0.05 and ^**p<0.01 compared with vehicle-treated mice; NS, p>0.05.

Active AKT restores lithium sensitivity to DBA/2J mice in the AIH paradigm. (a) Schematic of viral-mediated delivery of AKT forms and testing of lithium sensitivity of DBA/2J mice in the AIH assay. (b–d) DBA/2J mice were striatally injected with HSV viruses (∼1 × 10⁸ infection particles per ml) expressing (b) β-galactosidase (HSV-β-gal), (c) a constitutively active form of AKT (HSV-CA-AKT1), or (d) wild-type AKT (HSV-WT-AKT1) 3 days before behavioral testing to allow expression of the delivered genes. On the test day, mice were administrated lithium (85 mg/kg, i.p.) or saline vehicle (Veh) 60 min before -amphetamine challenge (time 0; 3.5 mg/kg, i.p.), and activity was recorded for an additional 80 min. Distance (cm) traveled is shown for each 5-min bin across the total recording time (left panels), and for the 80-min period after -amphetamine challenge (right panels). Representative histology sections (4 × and 20 × ) are shown for each viral delivery. Insets, arrows point to AKT-immunoreactivity. Quantification of AKT-immunoreactive neurons is shown in Supplementary Figure S5A. Representative histology sections (4 × and 20 × ) for HSV-β-gal expression are shown in Supplementary Figure S5B. Data shown are mean±SEM. ^**p<0.005, as compared with vehicle control mice. Number of animals per group (n) is indicated. Ac, anterior commissure, Acb, nucleus accumbens, Lv, lateral ventricle.

CHIR99021 is a highly selective inhibitor of GSK-3. (a) Profiles of BIO, SB-216763, AR-A014418, and CHIR99021 using KINOMEscan profiling technology (Ambit). Compounds were screened against 359 kinases for competitive binding with active site binders. Competitive binding relative to positive controls (% of control) is shown, with larger circles indicate higher-affinity binding. The complete list of kinases screened, as well as competitive binding data (% control), are available in Supplementary Table 2. (b) Summary of functional properties of CHIR99021. GSK-3β enzymatic activity determined by KinaseGlo assay (Promega) and concentrations of each compound that produce 50% inhibition (IC₅₀) are shown. TCF/LEF reporter activity was measured in HEK293 cells and concentrations of each compound that produce 50% activation (EC₅₀) are shown. (c) Activity of CHIR99021 in the TCF/LEF reporter assay in HEK293 and HT-22 cells. TCF/LEF luciferase reporter activity was measured by Dual-Glo assay (Promega), and normalized to EF1α renilla luciferase activity. Data represent mean±SEM from at least three independent experiments. ^***p<0.001.

CHIR99021 attenuates mood-related behaviors in lithium-responsive and non-responsive mouse strains. (a) Selected pharmacokinetic properties of CHIR99021 systemic administration (12.5 mg/kg, i.p) in C57BL/6J plasma and brain tissue. AUC (area under the curve from the time of dosing to the time of the last observation), T_1/2 (terminal half-life) and C_max (maximum concentration) were determined one hour after CHIR99021 administration. The full pharmacokinetic report of CHIR99021 is available on request. For all results, data are mean±SEM. ^*p<0.05. Number of animals per group (n) is indicated. (b) C57BL/6J (black bars) and DBA/2J (grey bars) mice were treated with vehicle (45% PEG400, 45% saline, 10% DMSO) or CHIR99021 (12.5 mg/kg, i.p.) 60 min before -amphetamine challenge (time 0; 3.5 mg/kg, i.p.), and the total distance (cm) traveled in the following 80 min was quantified. (c) C57BL/6J (black bars) and DBA/2J (grey bars) mice were treated with vehicle (45% PEG400, 45% saline, 10% DMSO) or CHIR99021 (25 mg/kg, i.p.) 45 min before the FST. Time spent immobile is shown as the percentage relative to the vehicle group. (d) HEK293 cells were treated with AKTI-17 (0.01–25 μM) overnight in the presence of CHIR99021 (10 μM). Percent (%) TCF/LEF activity relative to the CHIR99021 response (AKTI-17 absent) is plotted against the concentrations of AKTI-17. (e) C57BL/6J mice were pretreated with AKTI-17 (10 μM in 100% DMSO, i.c.v.) or 100% DMSO vehicle (Veh) 45 min before administration of CHIR99021 (12.5 mg/kg, i.p.) or vehicle (45% PEG400, 45% saline, 10% DMSO) 60 min before -amphetamine administration (time 0; 3.5 mg/kg, i.p.). Total distance traveled (cm) was quantified for 80 min after -amphetamine challenge. Post hoc comparison, p=0.003 (CHIR99021 vs Veh), p=0.57 (CHIR99021 vs AKTI-17). For all results, data are mean ± SEM. ^*p<0.05 and ^**p<0.01 compared with vehicle-treated mice; NS, p>0.05.