Transcriptome profiling and pathway analysis of genes expressed differentially in participants with or without a positive response to topiramate treatment for methamphetamine addiction

Abstract

Background: Developing efficacious medications to treat methamphetamine dependence is a global challenge in public health. Topiramate (TPM) is undergoing evaluation for this indication. The molecular mechanisms underlying its effects are largely unknown. Examining the effects of TPM on genome-wide gene expression in methamphetamine addicts is a clinically and scientifically important component of understanding its therapeutic profile.

Methods: In this double-blind, placebo-controlled clinical trial, 140 individuals who met the DSM-IV criteria for methamphetamine dependence were randomized to receive either TPM or placebo, of whom 99 consented to participate in our genome-wide expression study. The RNA samples were collected from whole blood for 50 TPM- and 49 placebo-treated participants at three time points: baseline and the ends of weeks 8 and 12. Genome-wide expression profiles and pathways of the two groups were compared for the responders and non-responders at Weeks 8 and 12. To minimize individual variations, expression of all examined genes at Weeks 8 and 12 were normalized to the values at baseline prior to identification of differentially expressed genes and pathways.

Results: At the single-gene level, we identified 1054, 502, 204, and 404 genes at nominal P values < 0.01 in the responders vs. non-responders at Weeks 8 and 12 for the TPM and placebo groups, respectively. Among them, expression of 159, 38, 2, and 21 genes was still significantly different after Bonferroni corrections for multiple testing. Many of these genes, such as GRINA, PRKACA, PRKCI, SNAP23, and TRAK2, which are involved in glutamate receptor and GABA receptor signaling, are direct targets for TPM. In contrast, no TPM drug targets were identified in the 38 significant genes for the Week 8 placebo group. Pathway analyses based on nominally significant genes revealed 27 enriched pathways shared by the Weeks 8 and 12 TPM groups. These pathways are involved in relevant physiological functions such as neuronal function/synaptic plasticity, signal transduction, cardiovascular function, and inflammation/immune function.

Conclusion: Topiramate treatment of methamphetamine addicts significantly modulates the expression of genes involved in multiple biological processes underlying addiction behavior and other physiological functions.

Trial registration: ClinicalTrials.gov NCT00345371.

Figure 1

Enriched synaptic long-term potentiation canonical pathway , identified by ingenuity pathway analysis based on differentially expressed genes ( P value  <  0.05 ) with the ordinary student’s t -test. The pathway was also detected by onto-tools pathway-express. (A) Week 8 TPM group (29 genes: ATF2, CAMK2D, CAMK2G, CREB1, EP300, GNAQ, GRINA, MAP2K1, MAPK1, MAPK3, PLCB2, PPP1CA, PPP1CB, PPP1CC, PPP1R10, PPP1R12A, PPP1R14B, PPP1R7, PPP3CB, PPP3CC, PRKACA, PRKACB, PRKAR1A, PRKCD, PRKCH, PRKCI, PRKCQ, PRKCZ, and RRAS); and (B) Week 12 TPM group (10 genes; ATF4, CREB5, EP300, GNAQ, KRAS, PPP1R10, PRKACB, PRKAR2A, PRKCB, and PRKCQ). Symbols with a single border represent single genes; those with a double border represent complexes of genes or the possibility that alternative genes might act in the pathway. Red symbols represent up-regulated gene clusters and green symbols represent down-regulated clusters.

Figure 2

Integrated model of the biological pathways related to TPM treatment for methamphetamine addiction. The joint effects of TPM and methamphetamine act on multiple molecular pathways that eventually result in modulations of neuroplasticity and neurotoxicity/neurodegeneration, which have a combined effect on cognitive/behavioral function. Pathways enriched exclusively in the TPM responder groups at Weeks 8 and 12 are highlighted in gray.

Figure 3

Schematic diagram of study workflow , including probe set filtering steps and statistical test strategies for detecting significant single genes and pathways. The probe intensities measured in 209 hybridized Affymetrix HG-U133 plus 2.0 arrays were normalized by Robust Multichip Average followed by a baseline correction step. Probes marked ‘Presence’ in fewer than four arrays in each group (because for Week 12 placebo group, only two positive responders were included, probes with two valid measurements were kept) were removed. Probes corresponding to control or less well-defined genes, and duplicated probes were removed. Genes with low FCs; i.e., within 1 standard deviation (denoted by σ) for a total of L (~7500) genes also were removed, as most of them were not likely to be differentially expressed to a statistically significant extent. The remaining genes were tested by the ordinary Student’s t-test, and genes with P values < 0.05 were used for pathway analysis. In total, 3698, 3532, 3328, and 3405 genes were tested for the Week 8 TPM, Week 8 placebo, Week 12 TPM, and Week 12 placebo groups, respectively.