The prevalence of depression is increasing worldwide, as is the number of patients suffering from treatment-resistant depression; these patients constitute 30% of the patient population treated for MDD [2,49,50]. In addition to the growing number of depression cases, and the burden this condition exerts on the global economy, society, and healthcare systems, there has not been significant advances in mental health treatments offered for this disorder in the past decades. These two facts highlight the urgent need for the research and identification of new therapeutic compounds that can be incorporated into depression treatment options. One of the potential new compounds recently suggested by preclinical studies as having a positive effect on alleviating depression symptoms, as well as those of anxiety and psychotic disorders, is CBD [27,34,35,51]. However, not all cannabis-derived compounds were shown to have such a positive effect. In fact, exposure to cannabis itself was associated with an increased risk of developing depression and anxiety disorders in human subjects, and it increased the prevalence of depressive- and anxiety-like behaviors in animal models [52,53]. These findings indicate that exposure to cannabis and cannabis-derived compounds impacts depression symptomatology (positively or negatively) in different ways, and detailed knowledge on the exact molecular mechanisms responsible for mediating these effects is crucial for the development of cannabinoid-based pharmacological therapies for depression-related phenotypes.

Recent research on cannabis and cannabis-derived compounds provided strong evidence of their widespread impact on epigenetic regulation across different organisms and biological systems [54]. In this review, we explored the hypothesis that cannabis and cannabis-derived compounds (i.e., CBD and THC) change DNAm levels of genes residing in loci associated with MDD [18]. Identification of such an overlap could be a first step in understanding how cannabis and cannabis-derived compounds may, on the molecular level, contribute to changes in depression symptomatology. In this literature review, we identified 95 genes residing in MDD-associated loci that were also reported as differentially methylated upon exposure to cannabis or cannabis-derived compounds. The differential methylation of these genes was reported in both human subjects and animal models and was identified in various brain regions and peripheral tissues. The CDH13 gene was identified most consistently among the 95 genes as differentially methylated upon exposure to cannabis-derived compounds. It acts as a negative regulator of neural proliferation, is widely expressed throughout the human brain, and is reported to increase sensitivity to drug cues [55,56,57]. CDH13 expression is crucial for neurite outgrowth and motor neuron pathfinding, as well as synapse formation in neurons with monoaminergic or GABAergic specifications [58,59,60,61]. In addition to being associated with MDD, genetic polymorphism in CDH13 was also linked with other mental health phenotypes, including substance abuse, smoking cessation outcomes, ADHD, violent behavior, schizophrenia, and bipolar disorder symptoms [62,63,64,65,66,67]. Moreover, CDH13 was found to be upregulated in the amygdala of depression patients and, in parallel, was shown to play a protective role in various models exposed to environmental stressors [60,68,69,70]. Based on this evidence, it is thus conceivable that the epigenetic regulation of CHD13 by cannabis-derived compounds could modify depression symptoms by the alteration of its expression levels, which are already known to be disturbed in the brains of depression patients [70].

Other genes among the 95 were consistently identified across the reviewed epigenome-wide studies, including two genes from the voltage-gated calcium channels (VGCCs) CACNA1C and CACNA1E. VGCCs are transmembrane proteins activated in response to the depolarization of the cell membrane, and they mediate the flux of calcium ions into excitable cells [71,72]. These genes are highly associated with various mental disorder phenotypes, such as neurodevelopmental disorders, intellectual disability, bipolar disorder, anxiety, and depression [73,74], and there is increasing evidence that CACNA1C expression in the brain directly regulates depression-related behaviors [72]. Interestingly, the genetic risk variants of mental disorders in CACNA1C act as both mQTLs and eQTLs; therefore, the genotype at these risk SNPs is associated with a variation in DNAm and expression levels of the gene [56,75]. It is not known whether exposure to cannabis or cannabis-derived compounds restores the DNAm and expression levels of CACNA1C to those of non-carriers or whether the restoration is associated with an improvement in depression symptoms; these questions need further investigation.

TENM2 and TCF4 also showed changes in DNAm levels in brain tissue in animal models exposed to CBD. In addition, DMPs in sperm that varied between cannabis users and controls before and after abstinence were identified in the TENM2 and TCF4 genes [48]. The TENM2 gene, also known as latrophilin-1-associated synaptic surface organizer (Lasso), is located postsynaptic in interaction, with a presynaptic latrophilin-1 in the synaptic cleft [76]. Latrophilin-1 is an adhesion G-protein-coupled receptor that contributes to the regulation of neurotransmitter release. Furthermore, the synaptic interaction of Lasso-latrophilin-1 promotes synapse formation and calcium signaling, both mechanisms especially important for neuronal development and signaling [76,77,78]. On the other hand, the TCF4 gene encodes transcription factor 4, which is expressed during neural development. Apart from MDD, TCF4 has also been associated with other psychiatric and neurological disorders, such as bipolar disorder and post-traumatic stress disorder [79,80]. Since the TENM2 and TCF4 genes are abundantly expressed in the central nervous system and especially in brain regions important for depression-related phenotypes, such as the hippocampus and cortex [56,76,81], DNAm changes in those genes due to exposure to cannabis or cannabis-derived compounds could also be of importance in future treatments for MDD.

The molecular mechanisms behind such changes in DNAm are not yet understood. There is no evidence that THC or CBD can directly interfere with the DNAm machinery, such as DNMTs, despite evidence of changes in stress-induced DNAm activity [35]. However, recent in silico evidence indicates that CBD can bind to and inhibit TET1, potentially regulating DNA demethylation [82]. It is, however, possible that the changes in DNAm result from the regulation of neurotransmitter release by cannabinoids, which can indirectly regulate the activity or gene expression levels of enzymes involved with the DNAm process [83,84].

Overall, in this review, we identified multiple genes residing in MDD-associated loci that are epigenetically regulated by cannabis or cannabis-derived compounds. There are, however, a few limitations that need to be taken into consideration when interpreting our findings and the extent of the overlap of genes across studies. Even though 39 out of the 95 genes were identified as being differentially methylated upon exposure to cannabis or cannabis-derived compounds in more than one study, many genes were identified as having altered methylation patterns in only one study. This result could be due to the large variability in study designs in the reviewed epigenome-wide reports, given that no two studies performed the exact same research experiment; as a result, we could not perform the cross-replication of the reported findings. The sources of heterogeneity in the reviewed studies involved variations in the types of compounds tested, the doses of these compounds, the developmental stages at which exposure took place, the different mammalian organisms analyzed, the tissues used, the laboratory methods used to quantify DNAm patterns genome-wide, and the statistical methods applied to identify differences in DNAm levels between compared groups [41,42,43,44,45,46,47,48]. Some of the differences in experimental design also clustered between studies, e.g., the impact of cannabis use on DNAm levels was tested in human subjects only, while the effect of exposure to cannabis-derived compounds (CBD or THC) on DNAm patterns was tested only in animal models. Additionally, epigenome-wide studies performed in human study participants were performed only in easy-to-access peripheral tissues (blood and sperm), while animal-based studies commonly used various dissected brain regions (hippocampus, cortex, and nucleus accumbens). By their nature, DNAm patterns vary greatly across tissues, cell types, genetic backgrounds, sex, age, and due to environmental exposures [85,86,87,88,89]. Therefore, DNAm patterns are likely to be impacted to a different extent at different loci depending on the study design employed. Thus, these differences between experimental setups make it difficult to conclude whether the lack of overlap with some genes differentially methylated upon exposure to cannabis or cannabis-derived compounds is due to false positive findings or due to highly variable study design. In addition to the high variability in experimental designs, different studies used different thresholds for reporting significant findings. Most studies reported genes as differentially methylated if their DMPs or DMRs were significant either study-wide or genome-wide; however, other studies reported genes with p-value thresholds suggestive of association (a p-value < 10⁻⁵ is commonly used for epigenome-wide association studies performed with Illumina’s methylation array). Whether more overlapping differentially methylated genes can be identified across epigenome-wide studies of cannabis or cannabis-derived compounds and whether more of these differentially methylated genes can be found to overlap with genes residing in MDD-associated loci are questions that need to be tested further by performing large, well-designed studies of these exposures across multiple independent cohorts. It is also of high interest to highlight the fact that none of the studies identified DNAm changes in cannabinoid receptors. This finding emphasizes the importance of mapping not only the targets of pharmacological compounds developed and used for the treatment of complex disorders but also researching impact of these therapeutics on epigenomic and transcriptomic regulation to gain in-depth knowledge of the influence of these agents on biological systems.