Uncovering multiple molecular targets for caffeine using a drug target validation strategy combining A 2A receptor knockout mice with microarray profiling

Abstract

Caffeine is the most widely consumed psychoactive substance and has complex pharmacological actions in brain. In this study, we employed a novel drug target validation strategy to uncover the multiple molecular targets of caffeine using combined A(2A) receptor (A(2A)R) knockouts (KO) and microarray profiling. Caffeine (10 mg/kg) elicited a distinct profile of striatal gene expression in WT mice compared with that by A(2A)R gene deletion or by administering caffeine into A(2A)R KO mice. Thus, A(2A)Rs are required but not sufficient to elicit the striatal gene expression by caffeine (10 mg/kg). Caffeine (50 mg/kg) induced complex expression patterns with three distinct sets of striatal genes: 1) one subset overlapped with those elicited by genetic deletion of A(2A)Rs; 2) the second subset elicited by caffeine in WT as well as A(2A)R KO mice; and 3) the third subset elicited by caffeine only in A(2A)R KO mice. Furthermore, striatal gene sets elicited by the phosphodiesterase (PDE) inhibitor rolipram and the GABA(A) receptor antagonist bicucullin, overlapped with the distinct subsets of striatal genes elicited by caffeine (50 mg/kg) administered to A(2A)R KO mice. Finally, Gene Set Enrichment Analysis reveals that adipocyte differentiation/insulin signaling is highly enriched in the striatal gene sets elicited by both low and high doses of caffeine. The identification of these distinct striatal gene populations and their corresponding multiple molecular targets, including A(2A)R, non-A(2A)R (possibly A(1)Rs and pathways associated with PDE and GABA(A)R) and their interactions, and the cellular pathways affected by low and high doses of caffeine, provides molecular insights into the acute pharmacological effects of caffeine in the brain.

Fig. 1.

Unsupervised hierarchical clustering analysis of striatal gene expression by low and high doses of caffeine in wild-type (wt) and A_2A receptor (R) knockout (ko) mice. Using whole normalized datasets without any gene filtering (i.e., entire 45,000 probe sets), we performed unsupervised hierarchical clustering analysis for striatal gene expression profiles in all mice after treated with low (10 mg/kg, caf10) and high (50 mg/kg, caf50) doses of caffeine in WT and A_2AR KO mice (3 mice/group). Large majority of microarray profiles are clustered with their corresponding groups perfectly, indicating the high quality of the microarray data. sal, Saline.

Fig. 2.

Distinct patterns of striatal gene expression by caffeine (10 mg/kg) in WT and A_2AR KO mice. Total RNA was isolated, quantified using Affymetrix Mu11kSubB oligonucleotide chips, and analyzed by permutation test as described in methods. The genes that passed the threshold for significant expression change (P value ≤0.05, fold-change ≥1.5) for each comparison are shown as points on the chart. The y-axis represents log₂ fold-change, and the x-axis represents expression (Affy fluorescent) intensity. Red dots represent those genes common to the A_2AR KO-Veh. vs. WT-Veh and the WT-Caf10 vs. WT-Veh comparisons. Green dots represent those genes common to the A_2AR KO-Veh vs. WT-Veh and A_2AR KO-Caf10 vs. WT-Caf10. Blue dots represent those genes common to the A_2AR KO-Caf10 vs. WT-Caf10 and A_2AR KO-Caf10 vs. A_2AR KO-Caf10. Black dots indicate the striatal genes that were distinct from each other in the 2 comparisons. Veh, vehicle.

Fig. 3.

Distinct patterns of striatal gene expression by caffeine (50 mg/kg) in WT and A_2AR KO mice. The microarray gene expression was processed as described in Fig. 2. The y-axis represents log₂ fold-change, and the x-axis represents expression (Affy fluorescent) intensity. Red dots represent those genes common to the A_2AR KO-Veh vs. WT-Veh and the WT-Caf50 vs. WT-Veh comparisons. Green dots represent those genes common to the A_2AR KO-Veh vs. WT-Veh and A_2AR KO-Caf50 vs. WT-Caf50. Blue dots represent those genes common to the A_2AR KO-Caf50 vs. WT-Caf50 and A_2AR KO-Caf50 vs. A_2AR KO-Caf50. Black dots indicate the striatal genes that were distinct from each other in the 2 comparisons.