Schizophrenia is one of the most devastating of psychiatric disorders [1]. The treatment of schizophrenia requires the suppression of hallucinations, delusions, agitation and the behavioural problems that accompany these symptoms [2]. Psychotherapy and rehabilitation can be undertaken when the acute symptoms start to subside through antipsychotic drug treatment.

The first antipsychotic drug, chlorpromazine, introduced in the early 1950s, was a major breakthrough because, unlike previously used sedative drugs, it could ameliorate hallucinations and delusions without overly sedating the patient [3]. Many other antipsychotic drugs were subsequently introduced [4], but these have not significantly advanced the treatment of schizophrenia. The early promise of the second-generation antipsychotics (atypical antipsychotics), such as clozapine and olanzapine, has been replaced by an acceptance that they are no more effective than the first-generation drugs [5]. However, second-generation antipsychotics have recently shown positive effects on verbal cognition [6]. Second-generation drugs have fewer neurological side effects but, unfortunately, many induce weight gain and the metabolic syndrome [7–10].

Our current understanding of the cause of schizophrenia is based on the pharmacological effects of the antipsychotic drugs used to treat the illness: they all bind to post-synaptic dopamine receptors especially D2 and the affinity at D2 receptors is both necessary and sufficient for the antipsychotic effects [11]. This, coupled with the observation that drugs that release dopamine into the synaptic cleft can induce the positive symptoms of schizophrenia (behaviours and feelings that are not real but imaginary), led to the hypothesis that excessive dopamine transmission in certain brain regions may cause the symptoms of schizophrenia [12]. A post-synaptic blockade occurs rapidly after a person ingests an antipsychotic drug. In contrast, the therapeutic effects of antipsychotics take days or weeks to accrue [13]. This suggests that downstream effects are important. One possibility is that the post-synaptic dopamine blockade causes a downstream cascade that has a therapeutic effect through altered gene transcription [14, 15]. A downstream effect, such as altered transcription, would explain the delay in the onset of therapeutic action. Other clinical observations also demonstrate the need for a more complex model than a post-synaptic dopamine blockade. Patients frequently fail to respond to an antipsychotic but subsequently show a robust response to a different drug despite the fact that both block the D2 receptor [16]. Moreover, many patients with schizophrenia show only a partial response to antipsychotics or fail to respond at all [17]. A refinement of the dopamine hypothesis proposes that an increase of D2 levels in the striatum may cause hallucinations and delusions and reduced D1 levels in the frontal lobes may cause cognitive deficits [18, 19]. This model is compatible with the delayed treatment effect but cannot explain the individual responsiveness to antipsychotics.

Epigenetic changes are another mechanism used to explain these clinical observations. They also offer an alternative therapeutic target for this serious disease: after 50 years of frustration we need to move our focus beyond post-synaptic dopamine receptors. Epigenetic changes associated with a drug can alter the expression of a single or variable number of genes without altering the gene sequence(s) [20]. Specifically, DNA methylation is a core epigenetic mechanism that involves the covalent binding of a methyl group to the 5-carbon position of cytosine leading to altered gene expression [21]. It is influenced by stochastic events including exposure to a variety of environments such as drug treatment [22, 23]. If DNA methylation plays a role in drug response, the drug or its metabolite must modify the methylation profile of the genome [20].

Limited research demonstrates that antipsychotic drugs can alter DNA methylation and gene expression [24]. However, most of this research has been conducted using variable post-mortem human brain tissues [25] and inappropriate non-brain cell types [26] that are not always ideal. Therefore, we have assessed the effects of a therapeutic equivalent dose of olanzapine (2.5 mg/kg per day for 21 days), a commonly used antipsychotic, on DNA methylation in rat brains using rat methylation arrays. The results demonstrate for the first time that the effect of olanzapine on DNA methylation is widespread and tissue specific, which may account for its efficacy and adverse effects.

First, we assessed the locomotor activity of rats split into olanzapine-treated and vehicle-treated groups. Activity was significantly decreased (P = 0.001) in the olanzapine-treated group compared to the vehicle-treated group (Additional file 1: Figure S1a). Further, olanzapine-treated rats significantly increased in weight (P = 0.004) compared to the control group (Additional file 1: Figure S1b). Second, we assessed gene-specific DNA methylation across (almost) all genes in response to olanzapine treatment on two brain regions (hippocampus and cerebellum) and the liver, as detailed below.

Olanzapine causes widespread and tissue-specific changes in genome-wide methylation in rats in vivo

Widespread changes in gene-specific DNA methylation were apparent in all three tissues studied (hippocampus, cerebellum and liver), as shown by the heat map for the hippocampus (Figure 1A). The results identified genes where there was an increase or a decrease in methylation in drug-treated rats compared to controls. Specifically, almost twice as many genes showed an increase compared to the genes that showed a decrease in methylation, in response to olanzapine in each of the three tissues (Additional file 2: Table S1A, Additional file 3: Table S1B, Additional file 4: Table S2A, Additional file 5: Table S2B, Additional file 6: Table S3A and Additional file 7: Table S3B). Also, the set of genes affected differs across the three tissues. Approximately 75% of genes with an increase (Figure 1B) and over 90% of the genes with a decrease (Figure 1C) in methylation, following olanzapine treatment, were specific to a given tissue. Further, there was a small number of genes with a similar pattern of increase (total 164) or decrease (total 24) in the two brain regions and a smaller number with an increase (41) in all three tissues. The tissue-specific results are novel and were further assessed as follows.

Pathways and associated network functions of genes that had changes in methylation in the cerebellum following olanzapine treatment

We also analysed genes that had increased methylation in the cerebellum (Additional file 4: Table S2A and Additional file 5: Table S2B). The most significant pathway identified for the cerebellum was for ephrin receptor signalling (P = 5.23 × 10^–4) (Table 2a; Figure 2). Synaptic long-term potentiation (P = 2.94 × 10^–3), which is implicated in learning and plasticity, was among the most significant pathways identified (Additional file 1: Figure S4). Moreover, pathways involved in signalling (Erk/Mapk, circadian rhythm and protein kinase A) were identified (Table 2a). Interestingly, genes with reduced methylation were also involved in pathways such as ephrin B signalling (Table 2b).

(a) Top canonical pathways (Genes with increased methylation)	P- value	No of moleculesa
Ephrin receptor signalling	5.23 × 10–4	24/176 (0.136)
Erk/Mapk signalling	1.59 × 10–3	24/184 (0.130)
Circadian rhythm signalling	1.94 × 10–3	8/33 (0.242)
Protein kinase A signalling	2.61 × 10–3	41/372 (0.110)
Synaptic long-term potentiation	2.94 × 10–3	17/113 (0.150)
Associated network functions
Cardiovascular disease, cell signalling, small molecule biochemistry		25
Cellular development, tissue morphology, cardiac dilation		23
Molecular transport, protein synthesis, protein trafficking		12
Behaviour, nervous system development and function		11
Neurological disease, psychological disorders, cell-to-cell signalling		10
(b) Top canonical pathways (Genes with decreased methylation)	P- value	No of molecules
Ephrin B signalling	4.0 × 10–3	7/72 (0.097)
G beta gamma signalling	4.1 × 10–3	8/99 (0.081)
Germ cell-Sertoli cell junction signalling	5.0 × 10–3	11/148 (0.074)
tRNA splicing	8.3 × 10–3	4/32 (0.125)
Tetrahydrofolate salvage from 5, 10 methenyltetrahydrofolate	8.6 × 10–3	2/6 (0.333)
Associated network functions
Cell death and survival, cellular development		14
Energy production, lipid metabolism, small molecule biochemistry		14
DNA replication and repair, development, carbohydrate metabolism		14
Neurological disease, cellular function and maintenance, molecular transport		11

^aFor the top canonical pathways, the ratio is the number of molecules in a given pathway that meet the cut-off (P ≤ 0.01) divided by the total number of molecules in the pathway.

There are a number of methods to test the effects of antipsychotic drugs, including the locomotor activity test [27] and the prepulse inhibition test [28]. In this study, we used the locomotor activity test. Significantly reduced locomotor activity in olanzapine-treated rats in this experiment (Figure 1A,B) has suggested that the drug administration paradigm employed was sufficient to cause therapeutically relevant effects in rats. Comparable therapeutic doses in rats were effective in previous studies and resulted in locomotor-suppressive effects [27]. Moreover, the significant increase in weight of olanzapine-treated rats in this and previous studies [29] indicated that the paradigm adapted might also be capable of causing metabolic disturbances, as seen in patients taking olanzapine for a long time [30]. Interestingly, the molecular results showed that olanzapine treatment caused genome-wide DNA methylation changes (Figure 1). Further, the results showed that most genes affected were tissue specific (hippocampus, cerebellum or liver). Also, the gene-specific methylation changes affected a number of networks that were tissue-specific, as expected. More importantly, the identified networks support two known effects of olanzapine, discussed in the following sections. The first is the recovery from psychosis [31] and the second is the adverse effects [32] of olanzapine. Olanzapine-induced DNA methylation changes in genes involved in canonical pathways may alter the associated network functions. However, further study is required to analyse the effects (on a protein level) of, specifically, the gene-specific methylation changes on each identified network.

We argue that the two manifestations could be attributed to tissue-specific alterations that disturb the coordinated expression of genes critical in the identified networks (Tables 1 and 2). This model is backed by a number of observations. First, the phenotypic effect of olanzapine is not immediate; rather it takes days or weeks after the initiation of treatment [13]. This may be the time that is needed for gene-specific methylation to alter the expression of the specific genes [33, 34]. Also, patients may not respond to this drug, depending on their CYP 1A2 genotype, which can metabolize this drug [35] or acquire resistance. Patients may need to take a different type of antipsychotic drug [16]. During this time, a patient may be affected by metabolic disorders, weight gain and related adverse effects [30]. We will discuss the specific mechanisms of the effects of olanzapine in the following section.

Olanzapine-based psychosis recovery may involve changes in gene methylation

We argue that an increase or a decrease in methylation of specific gene promoters, following olanzapine treatment, may decrease or increase their transcriptional efficiency [36, 37], specifically for the hippocampus, which is one of the primary sites responsible for psychotic symptoms [15, 38, 39]. Further, the pattern of transcriptional efficiency may also be modulated by other factors such as chromatin structure and elongation efficiency [38, 40, 41]. We acknowledge that the prefrontal cortex and nucleus accumbens, which are also implicated in psychosis [40, 42, 43], may need to be investigated in future studies.

We note that in the hippocampus, dopamine-DARPP32 feedback in the cAMP signalling pathway (P = 1.6 × 10^–3) was the most significant pathway identified. Neurons in the midbrain release dopamine, which modulates cAMP (cyclic adenosine 3,5-monophosphate) production by activating dopamine receptors [44]. These results suggest that the antipsychotic effects of olanzapine may involve alterations in gene-specific methylation that leads to the dysregulation of genes involved in dopamine-DARPP32 feedback in the cAMP signalling pathway (Figure 3). This includes several differentially methylated genes such as Drd1/5 and Nos1. The dopamine blockade leads to the progressive reduction of psychosis while its disturbance leads to psychosis [45]. All antipsychotics block post-synaptic D2 receptors [11]. A serotonin-dopamine antagonist was formulated following the synthesis of second-generation antipsychotics [11]. However, patients frequently fail to respond to one antipsychotic but respond to a different drug even if both block the D2 receptor [16]. Also, schizophrenia patients may partially respond to an antipsychotic or do not respond at all [17]. This may be due to several factors, and one possibility would be the delay in the onset of therapeutic actions partly or fully influenced by the downstream effects, such as altered transcription [14, 33]. As such, the differentially methylated genes involved in the dopamine-signalling pathway may stop or reduce transcription and gene expression [14, 21, 33]. In fact, decreased expression of DARPP32 in the prefrontal cortex has been reported in schizophrenia patients [43, 46, 47]. Also, DNA methylation differences have been observed in the dopamine D2 receptor gene within and between pairs of monozygotic twins discordant for psychoses [48] and there is an overwhelming evidence for the involvement of dopamine in psychosis including schizophrenia [19].

Further studies on the effects of drugs may help to identify the genes and pathways that underlie psychosis. For example, a decreased expression of CDC42 was reported in the cerebral cortex of schizophrenic patients in post-mortem studies, and this has been implicated in defects in dendritic spines in cortical neurons in the patients [49]. CDC42 can reorganize septin fibre formation, which is thought to stabilize actin filaments needed for a normal spine shape and synaptic plasticity, as reviewed in Ide and Lewis [49]. However, cautious interpretation of the results is important because actual epigenetic changes in schizophrenic patients may represent changes in methylation status [50].

Our results show that olanzapine caused an increase or a decrease in the methylation of genes previously implicated in schizophrenia (Table 3), which may reflect the fact that olanzapine could alleviate psychiatric symptoms via mechanisms involving DNA methylation. Among the genes that decreased in methylation in the hippocampus is Map6, which is implicated in schizophrenia [51] and is involved in molecular transport, nervous system development and function (Additional file 1: Figure S6). This emphasizes that methylation may serve an intermediary role whose actual effect is realized through gene expression.

Genea	Reference (PubMedID)	Affected in	Peak length (bp)	Peak to transcription start site (bp)	Promoter class
Nos1	20645313	Hippocampus	334	-3,719	LCP
Map6	16624526	Hippocampus	169	-2,382	ICP
Drd5	11304828	Hippocampus	844	41	HCP
Cacna1c	23183239	Hippocampus	425	-3,692	LCP
Bdnf	16818862	Hippocampus	329	-492	ICP
Gsksb	18500637	Hippocampus	573	-1,042	HCP
Tf	18045615	Hippocampus	159	-3,020	LCP
Cnp	16389193	Hippocampus	464	1,198	HCP
Amacr	20875727	Hippocampus	558	164	HCP
Psen2	19232479	Hippocampus	249	620	LCP
Pax6	10376119	Hippocampus	341	326	ICP
Dlx1	18384059	Hippocampus	431	-2,043	HCP
Dlx1	18384059	Cerebellum	1,330	257	HCP
Cnp	16389193	Cerebellum	769	940	HCP
Psen2	19232479	Cerebellum	455	442	LCP
Pax6	10376119	Cerebellum	349	-3,481	ICP
Stx1a	15219469	Cerebellum	149	72	HCP
Drd5	11304828	Cerebellum	264	-169	HCP
Mmp9	20037727	Cerebellum	138	-325	LCP
Tnf	11244489	Cerebellum	269	49	LCP
Grin1	12679240	Cerebellum	264	-1,595	HCP
Tf	18045615	Cerebellum	749	-1,704	LCP
Dlg4	21151988	Cerebellum	134	-1,459	LCP
Amacr	20875727	Cerebellum	138	-48	HCP
Dao	19685198	Cerebellum	287	-2,385	LCP
Dlx1	18384059	Liver	171	-2,993	HCP
Cnp	16389193	Liver	644	-2,644	HCP
Sod2	15193990	Liver	550	594	HCP
Drd5	11304828	Liver	369	-316	HCP
Il6	20393813	Liver	789	-3,030	LCP
Comt	11381111	Liver	638	-3,542	HCP
Apoe	14674716	Liver	2,158	-1,223	LCP
Drd2	18829695	Liver	257	-91	HCP
Nos1	20645313	Liver	348	172	LCP
Drd1	20127886	Liver	540	739	LCP

^aAll genes except Nos1, Map6, Il6 and Comt had increased methylation in olanzapine-treated rats.

HCP, high CpG contents; ICP, intermediate CpG contents; LCP, low CpG contents.

Among the genes that showed an increase in methylation in hippocampus was Bdnf, which has been previously implicated in schizophrenia [52]. This corroborates previous findings that showed its regulatory role in the expression of the dopamine D3 receptor gene (DRD3) [53]. The relatively lower methylation and higher expression of BDNF was also observed in schizophrenia patients compared to healthy controls [54].

Our results suggested that the efficacy of olanzapine might be achieved by changes in gene-specific methylation of relevant genes that take part in psychosis-related pathways. A list of 123 such genes that increased in methylation in both brain regions is given in Additional file 9: Table S5. That methylation may serve an intermediary role in modulating gene expression is apparent in the cerebellum, which is dominated by a number of signalling pathways including ephrin receptors and synaptic long-term potentiation (Table 2). Ephrin ligands and receptors guide axons during neural development and regulate neuronal plasticity in adults [55, 56]. Specifically, ephrin plays an important role in the regulation of neuronal migration, which is essential for the development of the nervous system and the proper functioning of the brain [57]. Neuronal cells have ahigher variation in DNA methylation than non-neuronal cells, supporting the idea that the epigenetic status of neuronal cells changes in response to the environment in the brain [58]. Interestingly, DNA methylation was found to be highly heritable and significantly correlated with gene expression in the human brain [59].

Furthermore, the synaptic long-term potentiation (LTP) pathway was one of the top canonical pathways in the cerebellum. Synaptic activity can persistently modify the way a neuron reacts to subsequent inputs by affecting either its intrinsic excitability or its synaptic efficacy, which is enhanced during long-term potentiation [60]. Specifically, in a rat cerebellum, synaptic transmission and granule cell intrinsic excitability are enhanced during LTP [47]. LTP is a well-known model for synaptic plasticity and it is typically induced by high-frequency activation of NMDA receptors at glutamatergic synapses [61]. Such results allow us to postulate that the efficacy of olanzapine may be due to its effect on the regulation of dopamine- DARPP32 feedback in the cAMP signalling pathway in the hippocampus, via DNA methylation.

Further, an atypical antipsychotic induced a restrictive chromatin state in the present study and previous reports [62]. On the other hand, clozapine was found to induce MII1, a mediator of open chromatin [63]. A restrictive chromatin state through DNA methylation has been implicated in psychiatric disorders [64]. Also, olanzapine, unlike clozapine and sulpiride, did not activate brain DNA demethylation in mice [65]. Moreover, atypical antipsychotics might regulate the transcription and function of genes that are related to histone post-transcriptional modifications [62]. Therefore, the mechanisms of actions of olanzapine on the chromatin structure and on any epigenetic machinery need to be studied further.

The known functions of affected genes suggest that the observed epigenetic changes may underlie the amelioration of symptoms and account for certain adverse effects including the metabolic syndrome. The results give a novel insight into the mechanism of action of olanzapine, therapeutic effects and the side effects of antipsychotics.