The significant increase in weight gain of olanzapine treated animals in the present study and in previously reported studies [18] has suggested that the paradigm adapted was capable of causing the metabolic disturbances observed in patients taking chronic olanzapine [19]. Furthermore, the significantly reduced locomotor activity of olanzapine treated rats indicated the therapeutic efficacy of the drug administered, which was comparable to the dosage applied in previous studies [20]. This argument was further supported by the observed changes in methylation of DA pathway genes.

Of special interest to this report are the genes involved in dopamine synthesis, transport, receptor, metabolism, interaction, and function [6]. The rationale for this focus stemmed from the fact that although antipsychotics interact with some dopamine receptors (D2), the actual mechanism of clinical effect behind antipsychotic efficacy in the treatment of psychosis is not fully understood. What is missing from the dopamine hypothesis of psychosis is the understanding of the underlying molecular mechanism(s) that may begin to reveal the full spectrum of the antipsychotics effects. Specifically, the results of the present study suggest an involvement of DNA methylation in genes of the dopamine pathway as an essential epigenetic mechanism in treating psychosis [21]. Our results showed that olanzapine causes an increase in DNA methylation in a significant (~40%) number of genes with an important role in dopamine neurotransmission. These include genes involved in the dopamine synthesis, transport, receptor, and metabolism (Figure 2). Further, the majority of DA pathway genes affected by olanzapine treatment were found to be hippocampus-specific, which is viewed as one of the primary sites for schizophrenia symptoms [21–23]. Also, a number of genes identified in the current study have been previously implicated in schizophrenia [24–27]. This increase in methylation in the DA pathway candidate genes is expected to interfere with transcription and suppress the functional gene product [28]. We also assessed if the methylated regions of individual genes have the potential to interfere with transcription. The results argue that gene-specific differentially methylated regions (DMRs) have necessary features of active promoters. All the affected DA pathway genes were differentially methylated in their promoter regions and therefore could result in altered gene expression [29]. For example, the promoter region of the DRD5 gene is differentially methylated in all three tissues. This gene has been implicated in cognitive functions that include working memory [30]. Furthermore, DRD1 and DRD5 have been reported to have distinct regulatory roles on synaptic plasticity, spontaneous motor activity, memory and the information being processed by the hippocampus [31, 32]. Here we show that DRD5, which encodes the D5 subtype DA receptor, and has been previously described as a susceptibility gene for schizophrenia [33, 34], may result in diminished D5 subtype by increase in methylation following olanzapine treatment. This is expected in all three tissues studied. It is also known that DRD5 interacts with DRD2 in the process of augmenting or suppressing cellular functions [35]. Further, the DRD5 gene region differentially methylated in response to olanzapine is compatible with methylation specific interference of transcription, as we have identified in silico predicted CTCF transcription factor binding sites (Figure 3) that warrant further confirmation. Similarly, the expression of DRD2 could be regulated through methylation or demethylation of cytosines at the “putative” promoter region of DRD2 [36–38] . Thus, the results included in this report offer a unifying mechanism of DNA methylation, which may represent the molecular basis for response to olanzapine, in a manner where the drug affects transcription of candidate genes from the dopamine pathway in addition to its effect on D2 blockade. This suggests that alterations to DNA methylation, in particular, and epigenetic changes, in general, may be used to develop novel strategies for the treatment of psychosis. We acknowledge the added value of confirming the methylation changes in the promoter regions of DA genes using an additional technique possibly involving a larger number of rats in our future study. Further, we intend to explore the expression of mRNA and proteins of relevant genes that are affected by DNA methylation so as to investigate the efficacy of olanzapine in treating psychosis via altering methylation status. Various confounding factors such as gene-diet/drug interactions could affect methylation changes [12, 39, 40]. Therefore, in the present study, proper caution was taken to exclude confounding factors that could possibly lead to methylation changes. For example, all experimental animals were kept in a uniform environment and were not exposed to other drugs or environments including diets, which may contribute to differences in methylation status of the treatment and control groups. All rats were of the same breed, sex, age and comparable body weight. They showed similar significant effects from olanzapine on weight gain and locomotor activity.