Selective chemical genetic inhibition of protein kinase C epsilon reduces ethanol consumption in mice

Abstract

Reducing expression or inhibiting translocation of protein kinase C epsilon (PKCε) prolongs ethanol intoxication and decreases ethanol consumption in mice. However, we do not know if this phenotype is due to reduced PKCε kinase activity or to impairment of kinase-independent functions. In this study, we used a chemical-genetic strategy to determine whether a potent and highly selective inhibitor of PKCε catalytic activity reduces ethanol consumption. We generated ATP analog-specific PKCε (AS-PKCε) knock-in mice harboring a point mutation in the ATP binding site of PKCε that renders the mutant kinase highly sensitive to inhibition by 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (1-NA-PP1). Systemically administered 1-NA-PP1 readily crossed the blood brain barrier and inhibited PKCε-mediated phosphorylation. 1-NA-PP1 reversibly reduced ethanol consumption by AS-PKCε mice but not by wild type mice lacking the AS-PKCε mutation. These results support the development of inhibitors of PKCε catalytic activity as a strategy to reduce ethanol consumption, and they demonstrate that the AS- PKCε mouse is a useful tool to study the role of PKCε in behavior.

Keywords: 1-NA-PP1; 1-tert-butyl-3-naphthalen-1-ylpyrazolo[3,4-d]pyrimidin-4-amine (PubChem CID: 4877); AS-Kinase; Alcohol; Ethanol; Protein kinase C.

Fig. 1

Generation of AS-PKCε knock-in mice. (A) Schematic showing targeting strategy for generating the M486A mutation (red asterisk) in exon 11 of the mouse Prkce gene. DTA = diphtheria toxin A expression cassette for negative selection; Neo = neomycin expression cassette for positive selection. Triangles represent loxP sites for Cre-recombinase mediated excision of the Neo cassette in embryonic stem cell clones. (B) PCR of tail DNA demonstrated presence of mutant (A) and wild type (+) alleles. (C) Western blot analysis showed similar levels of PKCε immunoreactivity in AS-PKCε (A/A) and wild type (+/+) hippocampus. (D) The distribution of brain PKCε immunoreactivity was similar in AS-PKCε (A/A) and wild type (+/+) mice.

Fig. 2

1-NA-PP1 pharmacokinetics after intraperitoneal injection of 30mg/kg 1-NA-PP1. Data shown are (A) plasma and (B) brain concentrations of 1-NA-PP1, with n = 3 for each data point.

Fig. 3

GABA_A γ2 receptor subunit phosphorylation. Intraperitoneal injection of 25mg/kg 1-NA-PP1 decreased GABA_A γ2-S(P)327 immunoreactivity compared with vehicle. Left panel shows representative western blots for anti GABA_A γ2S(P)327 immunoreactivity (top) and total GABA_A γ2 immunoreactivity (bottom) from the same vehicle (Veh)- and 1-NA-PP1 (1-NA)-treated samples. Right panel shows mean ± S.E.M. results from all animals. *P = 0.0175, t(8) = 2.98, two-tailed, unpaired t-test; n = 5 per group.

Fig. 4

Ethanol consumption by AS-PKCε mice. (A) AS-PKCε mice were habituated to vehicle injections and allowed to achieve a stable baseline level of drinking. Arrows point to days when animals received vehicle injections. 1-NA-PP1 reduced ethanol consumption (B) and this effect was reversible since it was no longer present 48 hours after administration of 1-NA-PP1 (C). (D) 1-NA-PP1 did not alter preference for ethanol over water or water intake (E) 1-NA-PP1 (30 mg/kg) reduced saccharin intake (F), but not quinine (G) intake.(H) 1-NA-PP1 (30 mg/kg) did not alter ethanol clearance. *P < 0.05, Dunnett’s test; n = 18 per group (A-E), n = 14 per group (F and G), n = 7 per group (H).

Fig. 5

Ethanol consumption by wild type mice. (A) C57BL/6NTac mice were habituated to vehicle injections and stable baseline drinking was attained prior to administration of 1-NA-PP1. Arrows point to days when animal received vehicle injections. (B) Administration of 1-NA-PP1 did not significantly alter drinking by C57BL/6NTac mice (n = 7 per group). (A) C57BL/6J mice were habituated to vehicle injections and stable baseline drinking was attained prior to administration of 1-NA-PP1. Arrows point to sessions when animals received vehicle injections. (B) 1-NA-PP1 produced a small but significant increase in ethanol consumption in C57BL/6J mice at 20mg/kg, but not at 30mg/kg (n = 10 per group). *P < 0.05, Dunnett’s test.

Fig. 6

Ethanol-induced ataxia and LORR. In AS-PKCε mice, 30mg/kg 1-NA-PP1 prolonged recovery from ataxia induced by 1.5g/kg ethanol; n = 11 (vehicle), n = 13 (1-NA-PP1). (B) In AS-PKCε mice, 1-NA-PP1 also increased the duration of LORR induced by 3.6g/kg ethanol; n = 25 (vehicle), n = 26 (1-NA-PP1). (C) In wild type mice, 1-NA-PP1 did not alter recovery from ataxia induced by 1.5g/kg ethanol; n = 8 (vehicle), n = 7 (1-NA-PP1). (D) In wild type mice, 1-NA-PP1 also did not alter the duration of the LORR induced by 3.6g/kg ethanol (n = 8 per group). * P = 0.0014, t₄₉ = 3.392, two-tailed, unpaired t-test.