Keywords

1 Impact of Genetic and Epigenetic Regulation on the Function of Organic Cation Transporters: A Short Overview

The interindividual variability of expression and function of genes involved in drug absorption, distribution, metabolism, and excretion (ADME) accounts for various interindividual differences regarding disease development, drug–drug interaction (DDI), drug response, and treatment outcome. In order to gain a better understanding of these differences and their consequences, it is necessary to unravel the interplay of genetic and non-genetic factors as well as epigenetic modifications affecting the expression and function of these ADME genes (Meyer et al. 2013). This aims at revealing disease mechanisms and to treat patients with a more individualized drug therapy, in which the individual genetic makeup is taken into consideration (Fisel et al. 2017). Drug transporters belong to the group of ADME genes and are considered important players in normal physiological processes and homeostasis as well as in drug response and safety (César-Razquin et al. 2015; Giacomini et al. 2010).

Among the drug transporters, the organic cation transporters (OCT) 1, OCT2, and OCT3 (encoded by SLC22A1, SLC22A2, and SLC22A3) have been intensively studied because they mediate the transport of organic cations and zwitterions, including many clinically-relevant drugs such as metformin, tramadol, and sumatriptan (Koepsell 2020; Nies et al. 2011; Tzvetkov et al. 2016; Yee et al. 2018). OCT1 is mainly expressed in the liver whereas OCT2 shows a high renal expression (Schaeffeler et al. 2011; Winter et al. 2016). In liver and kidney, OCT1 and OCT2 mediate the uptake of substances from the blood into hepatocytes and proximal tubule epithelial cells, respectively. OCT3 shows a more broad distribution and is expressed in a variety of tissues. There is a growing amount of studies addressing the impact of genetic and epigenetic regulation of OCTs on hepatic and renal physiological processes, disease susceptibility, and response to drug substrates (Yee et al. 2018). Even though OCTs have been shown to be expressed in the blood–brain barrier (BBB) and other brain areas (Lin et al. 2010), information on the effect of genetic and epigenetic regulation of these transporters on brain physiological function and drug response is very limited.

The aim of this chapter is to give a brief overview of the impact of genetic and epigenetic regulation of OCTs in general and to summarize the work about the role of interindividual OCT variation in the central nervous system (CNS).

1.1 Genetic Variants of OCTs

After the initial cloning of SLC22A1, SLC22A2, and SLC22A3 (Gorboulev et al. 1997; Gründemann et al. 1998), systematic resequencing of the SLC22A1 gene identified 14 coding variants that lead to amino acid substitutions and decreased or abolished function of OCT1 in vitro with pharmacodynamic consequences in vivo (Kerb et al. 2002; Shu et al. 2003, 2007; Tzvetkov et al. 2016). Five of these variants are common among different populations and are therefore of particular pharmacological interest: rs12208357 (p.R61C), rs55918055 (p.C88R), rs34130495 (p.G401S), rs72552763 (p.M420del), and rs34059508 (p.G465R). Similarly, variant rs316019 in SLC22A2 (p.S270A) has been identified as a common variant of OCT2. The impact of these common OCT1 and OCT2 variants has been intensively investigated in vitro, in pharmacokinetic studies and in studies on drug response considering established drug substrates of these transporters, e.g. the antidiabetic drug metformin, some opioids, antiviral medication, and anticancer drugs such as oxaliplatin (Koepsell 2020; Tzvetkov et al. 2016; Yee et al. 2018). The p.S270A variant of OCT2, for example, reduces the risk for cisplatin-induced nephrotoxicity without changing drug disposition (Iwata et al. 2012). SLC22A2 might also be a susceptibility gene for aspirin intolerance in asthmatics (Park et al. 2011). Loss of function variants of SLC22A1 seem to play a role in effectiveness of analgesic treatment with opioids. This effect might be of special relevance in children resulting from their lower OCT1 expression in comparison with adults or in CYP2D6 ultrarapid metabolizers, because this genetic makeup also increases the plasma concentrations of opioids and thereby the toxicity risk (Matic et al. 2017). Based on these and many other studies, the International Transporter Consortium considers genetic variants of OCT1 as highly important for drug disposition, response, and toxicity while the in vitro and in vivo effects of the OCT2 variant are less conclusive (Yee et al. 2018).

Large-scale systematic sequencing projects including thousands of individuals from different geographical regions identified additional variants and confirmed OCT1 as highly polymorphic (Nies et al. 2011; Seitz et al. 2015; Yee et al. 2018). Recent exome/whole genome next generation sequencing efforts of >100,000 individuals have revealed a multitude of novel missense variants, many of which only occur in single individuals (Karczewski et al. 2020). These variants are so rare that they cannot be detected in clinical trials, yet they may contribute to lack of efficacy or adverse drug reactions (Kozyra et al. 2017; Schärfe et al. 2017).

Genetic variants of OCTs leading to an altered protein expression and/or function may not only affect drug response, but may also alter susceptibility for certain diseases (Goswami et al. 2014; Koepsell 2020; Nies et al. 2011). For example, two SLC22A1 genetic variants correlate with an increased risk for type 2 diabetes mellitus in Chinese patients (Long et al. 2018). Genetic variants of SLC22A1 were further associated with the development of primary biliary cirrhosis (Ohishi et al. 2014). A genetic variant of SLC22A3 was shown to lead to reduced OCT3 expression and thereby to a decreased risk of colorectal cancer in a Japanese population (Ren et al. 2019). The SLC22A3 gene was furthermore identified as risk factor for coronary artery disease as well as prostate cancer (Eeles et al. 2008; Trégouët et al. 2009).

1.2 Epigenetic Regulation of OCTs

Epigenetics is defined as heritable variation of the expression of a gene that is not caused by changes in the DNA sequence (Holliday 2006). This variation can be achieved by noncoding RNAs, histone modification, or DNA methylation. Regulation of ADME genes by genetic variants is studied widely and the resulting pharmacogenomics knowledge is already used in clinical practice (van der Wouden et al. 2019). In contrast, even though epigenetics are getting more and more into focus and are acknowledged to play important roles in not only the normal cell type- and developmental stage-specific gene expression, but also in pathophysiological processes or drug response (pharmacoepigenetics), this field is much less studied and understood in comparison with pharmacogenomics (Fisel et al. 2016). One reason for this might be that it is very difficult to study epigenetic regulation of genes because they are highly dynamic in contrast to actual variations in the DNA sequence. For example, the methylation pattern of renal cell carcinoma (RCC) cell lines differs significantly from RCC tumors and metastases regarding ADME and drug target genes. SLC22A2, for example, is hypermethylated in RCC cell lines, but not in tumors and metastases translating into reduced expression of OCT2 in RCC cell lines in comparison with tumors and metastases. These findings indicate that the use of cell lines to study epigenetic regulation is difficult and might often not lead to reliable results in respect of the real physiological situation (Winter et al. 2016, 2017).

There is some information on epigenetic regulation of OCTs in normal and pathophysiological states. For example, the relatively restricted expression of OCT1 to the liver and OCT2 to the kidney seems to be a result of methylation patterns. Accordingly, SLC22A1 is strongly methylated in the kidney compared to liver tissue, whereas SLC22A2 is hypermethylated in the liver and hypomethylated in the kidney (Aoki et al. 2008; Fisel et al. 2016).

The SLC22A1 gene was shown to be hypermethylated in human hepatocellular carcinoma (HCC) leading to decreased protein expression (Schaeffeler et al. 2011). This knowledge is especially valuable regarding the fact that platinum drugs used as anticancer agents are substrates of OCT1. Reduced OCT1 expression therefore probably leads to reduced treatment success that might be restored by pretreatment of patients with demethylating agents, e.g. decitabine. Furthermore, the methylation state of SLC22A1 could be used in risk assessment and improvement of early diagnosis of HCC in patients at risk of developing HCC. Similar to the finding of SLC22A1 hypermethylation in HCC, SLC22A3 promoter methylation is significantly increased in prostate cancer resulting in reduced expression of OCT3 (Chen et al. 2013).

Epigenetic regulation of OCTs by noncoding microRNAs (miRNAs) has also been described. Li et al. identified genetic variant rs3088442 in the 3′ untranslated region of the SLC22A3 gene to be associated with coronary heart disease (Li et al. 2015). Mechanistically, the A allele of rs3088442 generates a binding site for miR-147, which inhibits OCT3 expression. The deficiency of OCT3 in turn inhibits lipopolysaccharide induced monocytic inflammatory response explaining the athero-protective role of this variant. Another example is the regulation of OCT expression by miR-21, which is upregulated in clear cell renal cell carcinoma (Gaudelot et al. 2017) and also inversely correlated with OCT1 expression in human liver (Rieger et al. 2013). Silencing of miR-21 in human kidney cells in vitro resulted in increased expression of OCT1 and OCT2, which could be another strategy to restore treatment success with anticancer agents that are transported by OCTs.

The ontogeny of OCT1 and OCT2 is another process, in which dynamic epigenetic regulation of the OCTs might play a role indicated by a strong increase in OCT1 and OCT2 protein expression from neonates to adults (Cheung et al. 2019; Hahn et al. 2017; Prasad et al. 2016). In contrast to drug-metabolizing enzymes, there is not much known about the ontogeny of drug transporters and its underlying mechanisms. Since it was shown for cytochrome P450 3A4 (CYP3A4) that hypermethylation of transcription factor binding sites leads to decreased protein expression before birth, a similar role of methylation or other epigenetic processes may occur in the ontogeny of drug transporter genes including SLC22A1 and SLC22A2 (Brouwer et al. 2015; Mooij et al. 2016).

2 Implications of OCT Regulation in the CNS

In contrast to the variety of studies regarding the genetic and epigenetic regulation of OCTs in liver and kidney, not much has been reported on the role of genetic variants or epigenetic makeup in physiological and pathophysiological processes in the brain. Due to the lack of information on the role of genetic regulation of OCT1 and on epigenetic regulation of OCTs in the CNS in general, this section will focus on genetic regulation of OCT2 and OCT3 and its impact on the CNS.

However, regarding OCT1, it is important that this highly polymorphic transporter transports various opioids, e.g. the active compound of tramadol, O-desmethyltramadol, morphine, and methylnaltrexone, as well as the antimigraine agent sumatriptan (Matthaei et al. 2016; Meyer et al. 2019; Tzvetkov 2017). The common genetic variants of OCT1 leading to decreased or loss of function of OCT1 alter the pharmacokinetics and the effectiveness of opioid treatment probably by decreasing hepatic reuptake of the active substances and thereby increasing their plasma levels and availability (Tzvetkov 2017). A finding underlining this hypothesis is that patients with two nonfunctional alleles showed significantly lower tramadol consumption in order to treat postoperative pain via patient-controlled analgesia compared with carriers of at least one active OCT1 allele (Stamer et al. 2016; Tzvetkov 2017). These findings impressively show that even though the expression of OCT1 in the brain is under debate, interindividual differences in OCT1 function in the liver can still affect the efficacy as well as the toxicity risk of drugs acting in the brain.

Pharmacogenetic studies are often difficult to perform in humans, which also affects the amount of research that has been conducted to date on the effects of OCT gene regulation on the human brain. Animal studies, especially those using knockout (KO) mouse models have proven to be valuable in identifying not only the physiological roles of transport proteins but also the pathophysiological processes caused by altered transport function in vivo (Frick et al. 2013; Nies et al. 2011; Stieger and Gao 2015). There are KO mice existing for all OCT homologs as well as Oct1/Oct2 double KO mice. All of these strains are viable and don’t show direct phenotypical aberrations (Nies et al. 2011). KO and also wild type (WT) mice have been used in various studies in order to elucidate the role of Octs on different brain processes and predict possible effects of human OCT variants. Therefore, results gained from such studies are reviewed here, next to findings gained from studies performed with human subjects.

2.1 Genetic Regulation of OCT2 in the CNS

2.1.1 Localization of OCT2 in the CNS

Even though there are studies reporting on the localization of OCT2 in mouse and human brain, the results are not always consistent and do not create a precise expression pattern.

In mice, Oct2 was reported to be expressed in the BBB and also in the blood–cerebrospinal fluid (CSF) barrier (Sweet et al. 2001; Wu et al. 2015), yet BBB expression has been challenged (Chaves et al. 2020). Expression was furthermore shown in limbic and other stress-related regions of the mouse brain as well as in the dorsal root ganglia cells (Bacq et al. 2012; Couroussé et al. 2015; Sprowl et al. 2013). Cholinergic and monoaminergic axon terminals in various mouse forebrain regions likewise showed Oct2 expression, pointing out an implication of Oct2 on presynaptic reuptake and recycling of choline and monoamines (Matsui et al. 2016).

Similar to mice, in human brain, OCT2 seems to be expressed in the BBB, indicated by Lin and colleagues who reported the OCT2 protein in the luminal membrane of brain microvessel endothelial cells (Lin et al. 2010). Yet, a recent study detected only negligible amounts of OCT2 mRNA in human brain microvessels (Chaves et al. 2020). OCT2 expression was also found in human dorsal root ganglia cells (Lin et al. 2010; Sprowl et al. 2013). Expression of the transporter was also detected in pyramidal cells of cerebral cortex and hippocampus as well as in other regions like the corpus striatum, nucleus amygdaloideus, and the thalamus in humans (Busch et al. 1998). Taubert et al. also reported OCT2 expression in human substantia nigra and furthermore in other dopaminergic regions of the brain (Busch et al. 1998; Taubert et al. 2007). The Human Protein Atlas, a freely available resource providing widespread information about the human proteome, reports low OCT2 mRNA expression levels in some brain regions including pons and medulla, midbrain, cerebellum, and olfactory region (Uhlén et al. 2015). There is no data on protein expression provided for these brain regions by this database.

2.1.2 Consequences of Disturbed OCT2 Function in the CNS: What Do We Learn from Animal Studies?

Consistent with the expression of Oct2 in limbic regions of the mouse brain, the transporter seems to be involved in mood-related behavior and stress response. Oct2 KO mice showed less anxiety and increased resignation in behavioral tests as well as higher sensitivity to norepinephrine (NE) and serotonin (5-HT) selective antidepressants in comparison with WT mice with normal Oct2 expression (Bacq et al. 2012). These findings point to the idea that OCT2 controls 5-HT and NE clearance in limbic regions of the brain and should be considered as a genetic factor influencing the response to NE- and 5-HT selective antidepressants. Another study reveals the role of Oct2 in the hormonal response to acute stress (Couroussé et al. 2015). Oct2 KO mice showed increased basal as well as stress-induced plasma corticosterone levels and were more vulnerable to repeated stressful conditions. OCT2 could therefore be a genetic factor influencing the hormonal processes occurring during acute stress implying that genetic variants or epigenetic regulations that alter OCT2 transport function could critically affect vulnerability to stress.

Other animal studies have shown an impact of Oct2 on neurotoxicity of different substances. OCTs transport 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) across the BBB, which is the prodrug of its active metabolite and neurotoxin 1-methyl-4-phenyl-pyridinium (MPP+). MPP+ is also a substrate of OCTs and leads to dopaminergic neurodegeneration and thereby mimics the symptoms of Parkinson’s disease (PD) (Koepsell 2020; Wu et al. 2015). Inhibition of Oct1 and Oct2 with amantadine significantly decreased MPTP transport in mouse and rat brain microvessel endothelial cells (Lin et al. 2010). Furthermore, after intraperitoneal application of MPTP in mice, extracellular MPTP and MPP+ levels in the brain accompanied with dopaminergic toxicity were significantly reduced in Oct1/Oct2 double KO mice (Lin et al. 2010; Wu et al. 2015). Intrastriatal infusion with MPTP, however, increased dopaminergic neurotoxicity in Oct1/Oct2 double KO and also in aged mice that show a lower Oct expression in the BBB in comparison with young mice, probably because of reduced neurotoxin clearance from the brain (Wu et al. 2015). Given these results and the fact that the two endogenous neurotoxins 1-benzyltetrahydroisoquinoline and N-methyl-(R)salsolinol, that are structurally similar to MPTP, are substrates of OCT1 and OCT2 (Wu et al. 2015), OCT2 genetic and epigenetic regulation could crucially influence dopaminergic toxicity caused by these substances and thereby even alter susceptibility for developing PD.

Another finding regarding the role of Oct2 in the effect of neurotoxins is that Oct2 KO mice are protected from neurotoxicity caused by oxaliplatin, a platinum drug that is used in the treatment of colorectal cancer and a substrate of human OCT2 (Huang et al. 2020; Koepsell 2020; Sprowl et al. 2013). Genetic and epigenetic variations of OCT2 could therefore be important factors influencing the risk of treatment-induced neurotoxicity.

Taken together, these results indicate that genetic variants and epigenetic patterns altering OCT2 function might affect mood- and stress-related behavior, the response to NE- and 5-HT selective antidepressants as well as the vulnerability to endogenous and exogenous neurotoxins.

2.1.3 Genetic Variants of OCT2 and Their Implications on CNS Function

Genetic variants of human OCT2 were shown to play a role in smoking cessation therapy (Bergen et al. 2014). The human SLC22A2 genetic variant rs316019 (p.S270A), that was shown to reduce transport of some OCT2 probe substrates in vitro, and a linked, intronic variant (rs316006) were associated with smoking abstinence in individuals randomized to smoking-cessation treatment, namely to nicotine replacement therapy (NRT) and varenicline. The underlying mechanism is unknown, but the findings suggest that smoking cessation therapy either by NRT or also by nicotinic acetylcholine receptor partial agonist pharmacotherapies like varenicline shows higher effectiveness in subjects with reduced OCT2 function. This could be due to different reasons, like increased plasma concentration or reduced BBB transport of the medications. Another reason might be that reduced OCT2 activity might lead to increased monoamine concentrations in the brain and thereby to potential mood enhancement since OCT2 is thought to be involved in monoamine clearance in limbic brain regions. This hypothesis is supported by the observation that dopamine was reported to be downregulated in the nucleus accumbens, a central component of the limbic system, after smoking cessation (Bacq et al. 2012; Bergen et al. 2014; Zhang et al. 2012).

SLC22A2 might also be a gene regulating the risk of PD, regarding the fact that OCT2 is expressed at relatively high levels in dopaminergic regions in the brain including substantia nigra, the brain area associated with PD (Taubert et al. 2007). Moreover, OCT2 transports two dopaminergic neuromodulators, histidyl-proline diketopiperazine (cyclo(his-pro)) and salsolinol, with high efficiency (Taubert et al. 2007). Balanced levels of the two substances in dopaminergic cells seem to be crucial for cell integrity, with salsolinol showing cytotoxic and cyclo(his-pro) showing neuroprotective effects. In patients with PD, increased salsolinol levels were observed and associated with apoptotic nigral cell death. The SLC22A2 variant rs8177516 (p.R400C), occurring in African-Americans with an ethnic-specific allelic frequency of 1.5%, was reported to decrease the transport of cyclo(his-pro) whereas salsolinol transport was not affected by this variant. Taken together, these findings lead to the conclusion that the rs8177516 variant might increase susceptibility to dopaminergic cell death and thereby also increase the risk for developing PD (Farthing and Sweet 2014; Grottelli et al. 2016; Taubert et al. 2007).

Another role of OCT2 – and OCT3 – in the brain might be to take part in controlling the development of speech and language competence. This hypothesis arises from the finding that the deletion of a gene cluster including SLC22A2 and SLC22A3, among other genes, was reported to lead to motor speech disorders in two affected children (Peter et al. 2017). Certainly, these findings are not sufficient evidence that OCT2 and/or OCT3 take part in development of speech competence, but it would be interesting to conduct further studies to elucidate the role of OCT2 and/or OCT3 in this process.

Taken together, the findings hint towards an impact of OCT2 on response to smoking cessation therapy and on maintaining dopaminergic cell integrity. OCT2 genetic and epigenetic regulation that alter transport function of certain substrates could therefore affect the effectiveness of smoking cessation therapy as well as the risk for developing PD.

2.2 Genetic Regulation of OCT3 in the CNS

2.2.1 Localization of OCT3 in the CNS

Similar to OCT2, information on the exact localization of OCT3 in the brain is limited. There are only few studies reporting on the region- and cell-specific expression of the transporter in rats and mice and the study situation regarding the human brain is even less satisfactory. Therefore, widespread comprehensive studies on the gene and protein expression of OCT3 in rat, mice, and human brain areas would be necessary to elucidate the expression pattern of the transporter.

In rats, Oct3 was found to be expressed in circumventricular organs and in regions of the brain involved in aminergic neurotransmission. On the cellular level, Oct3 seems to be expressed in neurons and, to a lesser extent, in glial cells in these brain regions (Haag et al. 2004; Vialou et al. 2004). mRNA and protein expression of Oct3 was also detected in rat medial hypothalamus and mRNA expression was moreover reported in choroid plexus epithelium and thereby in the blood–CSF barrier (Gasser et al. 2006; Sweet et al. 2001). Rat and mouse Oct3 expression was detected in the basolateral complex of the amygdala, which is part of the limbic system. The expression was found in glial cells and pre- and post-synaptic neurons and was localized as expected to the plasma membrane, but also to nuclear membranes and endomembrane systems. This indicates that OCT3, similar to OCT2, plays a role in the clearance of extracellular monoamines and that the transporter might furthermore mediate their intracellular distribution (Gasser et al. 2009, 2017; Hill and Gasser 2013).

In humans, OCT3 was also shown to be expressed in the brain by northern blot analysis (Wu et al. 2000). More specifically, Geier and colleagues detected OCT3 mRNA and protein expression in the BBB (Geier et al. 2013). Another study showed that OCT3 was expressed in normal human astrocytes on mRNA and protein levels (Inazu et al. 2003). The human protein atlas reports low mRNA expression of OCT3 in various brain regions as well as high protein expression in cerebellum. Consistent with the findings from Geier and colleagues, the database also shows OCT3 protein expression in the cerebral cortex and thereby the BBB. Protein expression is furthermore reported in basal ganglia and in the hippocampus, a region assigned to the limbic system. The database provides no data for other brain regions (Uhlén et al. 2015).

2.2.2 Consequences of Disturbed OCT3 Function in the CNS: What Do We Learn from Animal Studies?

Consistent with the localization of Oct3 in circumventricular organs of the rat brain, that are known to regulate fluid exchange, Oct3 seems to be crucial for neural and behavioral response to environmental variation in osmolarity in mice (Vialou et al. 2004). More precisely, Oct3 KO mice showed an increased salt-ingestion behavior under sodium depletion as well as under water deprivation conditions. Even though the underlying mechanism is unclear, this finding was the first physiological evidence discovered for the critical role of OCTs in the brain.

Oct3, similar to Oct2, further seems to be involved in regulating mood-related behavior in the mouse brain, as already indicated by the localization of the transporter in the limbic system. In line with this, Oct3 was shown to be important in aminergic neurotransmission. Increased anxiety levels to high dose psychostimulants were detected in Oct3 KO mice, but the Oct3 KO mice were less sensitive to the locomotor stimulating activity of psychostimulants (Vialou et al. 2008). With increasing dose of amphetamine or cocaine, locomotor activity decreased due to the emergence of stereotyped behavior. This is apparent in the Oct3 WT mice but not as apparent in the Oct3 KO mice (Vialou et al. 2008). On the other hand, Mayer et al. reported no difference in amphetamine stimulated locomotor activity between genotypes across a range of doses, but did show that the OCT blocker, decynium-22, attenuated the locomotor stimulant effects of amphetamine in Oct3 WT but not in Oct3 KO mice, implicating a role for Oct3 in the actions of amphetamines (Mayer et al. 2018). In contrast with these findings, Wultsch and colleagues reported that Oct3 KO mice showed decreased anxiety in comparison with WT mice, but no cognitive impairment or social-dominant behavior in a comprehensive behavioral test battery (Wultsch et al. 2009). Despite the controversial results, the studies presumed that the altered anxiety behavior of the KO mice might be due to a role of Oct3 in neuronal signaling via modulation of neurotransmitter concentrations in the synaptic cleft. In particular, the authors indicated that Oct3 might serve as an alternative transporter that removes monoamine transmitters that escape neuronal sodium-dependent high-affinity uptake 1 (Vialou et al. 2008; Wultsch et al. 2009). This hypothesis is supported by the finding that Oct3 expression and function is increased in mice deficient for the high-affinity 5-HT uptake transporter 5-HTT (SERT, serotonin transporter encoded by SLC6A4). The mechanism underlying the increased transporter expression is not known (Baganz et al. 2008). Inhibition of Oct3 further led to an increased antidepressant-like effect in those mice compared to WT mice indicating that the role of Oct3 in controlling extracellular 5-HT brain levels is notably elevated in absence of the high-affinity uptake transporter. Given the fact that human carriers of a common deletional genetic variant in the promoter region of the 5-HTT gene that receive antidepressant drugs often show increased resistance to conventional treatment with selective 5-HT reuptake inhibitors, these findings offer an attractive option in such cases (Baganz et al. 2008; Daws 2009). Another study addressing the role of Oct3 in mood-related behavior in mice found that Oct3 KO mice showed impaired social behavior, namely reduced interaction preference (Garbarino et al. 2019). Taken together, these results indicate that OCT3 might be an important modulator of mood-related behavior by influencing neurotransmitter concentrations in the synaptic cleft in limbic brain regions. Genetic and epigenetic regulation of OCT3 might thereby alter such behavior as well as the reaction to psychostimulants and antidepressants.

Further animal studies suggest that Oct3, like Oct2, is involved in stress response. Stress causes a rapid increase in 5-HT release in the dorsomedial hypothalamus, where Oct3 has been shown to be expressed in rat brain (Gasser et al. 2006). Feng and colleagues showed that rats treated with an Oct3 inhibitor had increased extracellular 5-HT concentrations in the medial hypothalamus under mild restraint, which represented a stressful condition in this study. The magnitude and duration of the inhibitor-induced increase in 5-HT levels was notably smaller in rats not exposed to mild restraint and animals not treated with the inhibitor did not show any restraint-induced elevation in extracellular 5-HT levels (Feng et al. 2010). These findings support the theory that Oct3 serves as an alternative transporter to the high-affinity monoamine transporters and that this alternative transport is of special importance under conditions when 5-HT levels are high, e.g. in stress situations. Gasser and colleagues showed that accumulation of the OCT3 substrate histamine and efflux of MPP+ from acutely prepared rat medial hypothalamic explants is inhibited by corticosterone, a corticosteroid hormone that displays a rapid increase in its levels after exposure to a stressor. They concluded that regulation of OCT3 through corticosterone might influence the acute regulation of stress response (Gasser et al. 2006). This presumption is supported by the finding that Oct3 expression and function is decreased in mice after activation of the hypothalamic-pituitary-adrenal (HPA) axis via repeated swim conditions. HPA axis activation causes the release of corticosterone and an increase in extracellular 5-HT levels in forebrain regions that is independent of 5-HTT. These results lead to the assumption that HPA axis activation caused by stressful conditions decreases Oct3 function by increasing levels of corticosterone. Furthermore, chronic exposure to stress seems to lead to reduced Oct3 expression consequently causing elevations in extracellular 5-HT levels (Baganz et al. 2010). In sum, the findings indicate that OCT3 plays a crucial role in regulating extracellular 5-HT brain levels and that corticosterone-induced inhibition of OCT3 leads to increased 5-HT levels under stressful conditions. Therefore, genetic and epigenetic variations affecting OCT3 function could have an impact on 5-HT clearance in the brain in general and could further influence stress response, e.g. if a genetic variant of the transporter leads to an altered inhibitory potential of corticosterone.

Another study analyzing the role of Oct3 in the mouse brain reported an impact of Oct3 together with the dopamine transporter (DAT, encoded by SLC6A3) in neurotoxicity mediated by paraquat, an herbicide that has been associated with increasing the risk for developing PD. After treatment with paraquat, Oct3 KO mice showed increased striatal damage in comparison with WT mice (Rappold et al. 2011).

Altogether, these findings regarding the role of Oct3 in mouse and rat brain indicate that Oct3 serves as an alternative transport mechanism to the sodium-dependent high-affinity monoamine uptake 1 and thereby regulates extracellular monoamine levels in the brain. Because of this feature, OCT3, similar to OCT2, seems to be involved in mood-related behavior and stress response. Furthermore, the findings hint to possible roles of OCT3 in response to variations in osmolarity and in paraquat-induced neurotoxicity. Genetic and epigenetic regulations of OCT3 might thereby influence anxiety levels under particular situations, reaction to environmental stress, response to antidepressants and psychostimulants as well as susceptibility to develop neurotoxin-induced PD.

2.2.3 Genetic Variants of OCT3 and Their Implications on CNS Function

Only very few studies have been conducted so far to study the effect of genetic variations of human OCT3 on CNS function.

Although OCT3 has been reported to be localized in the limbic system of the human brain (Uhlén et al. 2015) and was shown to play a role in monoaminergic neurotransmission and thereby in mood-related behavior in mice (Vialou et al. 2008), no correlation was found between the allele or genotype frequencies of seven known OCT3 genetic variants and the occurrence of depression in a Caucasian population (Hengen et al. 2011). These results of course are not sufficient evidence that OCT3 is not involved in the development of depression and that genetic variants other than the seven tested cannot have an impact on disease susceptibility.

However, OCT3 genetic variants have actually been associated with the development of polysubstance use in Japanese individuals with methamphetamine (MAP) dependence (Aoyama et al. 2006). MAP is a substrate of OCT3 and the transporter affects the disposition of MAP as well as behavioral changes caused by the substance. OCT3 knock-down mice, for example, were reported to show increased MAP-induced locomotor activity (Kitaichi et al. 2005). When genotype and allelic frequency of 5 known OCT3 genetic variants were compared between MAP users and a control group, no significant difference was found. However, when the group of MAP users was divided into single-MAP users and polysubstance users, genotype and allelic frequencies of variant rs509707 and the allelic frequency of variant rs4709426 as well as the haplotypic frequencies of both intronic variants were significantly associated with polysubstance use. This indicates that OCT3 plays a role in the development of polysubstance use in people already dependent on MAP (Aoyama et al. 2006).

Another impact of OCT3 genetic variants seems to be on the development of obsessive-compulsive disorder (OCD). Because of the role of the transporter in the termination of monoamine signaling in the CNS, SLC22A3 seems to be a candidate gene for various neuropsychiatric disorders. Two novel genetic variants were found to be present in patients with early onset OCD in a Caucasian population. One of them was a promoter variant that significantly increased OCT3 expression in vitro and was found in three unrelated male patients. The other variant, that was found in three related female patients, was localized in the coding region of SLC22A3 (p.M370I) and led to a significant decrease in norepinephrine transport function in vitro (Lazar et al. 2008).

Taken together, the results of the different studies regarding the role of OCT3 variants in CNS function indicate that such variants as well as epigenetic regulation of OCT3 could alter the susceptibility for drug abuse disorders as well as for OCD.

3 Conclusion

Genetic and epigenetic regulations of OCTs have been shown to be important players in the response to various substrate drugs, in the risk for DDI as well as in disease susceptibility and treatment outcome for certain diseases and medications (Fisel et al. 2016; Koepsell 2020). In contrast to this relatively widespread knowledge of OCT regulation in peripheral organs, not much is known about the effects of this regulation on the CNS.

The first problem regarding this topic is the lack of information on the exact region- and cell-specific localization of OCTs in the brain. Animal studies describe expression of the transporters in various brain regions, but few have comprehensively studied gene as well as protein expression in different areas of the brain and the results gained in these animal studies are sometimes contradictory. Even less is known about the region- and cell-specific localization of OCTs in the human brain. Widespread comprehensive studies on gene and protein expression of OCTs in rat, mice, and human brain areas would therefore be necessary to elucidate their exact expression pattern that in turn could indicate possible OCT functions. Findings gained from such studies could also be used to reveal species-related differences between the expression of human transporters and their homologs in mice and rat in order to align the results gained from animal studies with research performed on human subjects.

The impact of genetic variation of OCTs on CNS function is also only sparsely studied. Findings regarding the general role of the transporters in the CNS gained from animal studies that often use KO mice models can be used to form hypotheses regarding the impact of variation in transporter function in brain. However, the number of these animal studies is limited, and sometimes contradictory, demanding further investigation. Furthermore, there is often a lack of studies that examine the influence of variations in human OCTs on brain processes, in which the animal homologs are supposed to be involved.

Moreover, to our knowledge, there is no information on the role of epigenetic regulation of OCTs on processes determining CNS function. It has been shown that Oct3 expression is downregulated after repeated stressful conditions in mice and elevated in mice deficient for 5-HTT with the underlying mechanisms being unclear (Baganz et al. 2008, 2010). One could speculate that the changes in transporter expression are mediated by epigenetic processes in order to react to environmental circumstances like stress or to the increased extracellular monoamine levels in the brain caused by missing 5-HTT. However, altered expression could also be caused by other mechanisms and therefore it would be interesting to conduct further studies to elucidate the cause for these shifts in expression and function.

In conclusion, OCTs seem to be crucial players in different processes in the brain, with OCT2 and OCT3 being of special importance. These processes include the regulation of extracellular monoamine levels as well as the transport of neurotoxins across the blood–brain barrier and their distribution in the brain. Therefore, the transporters might regulate not only various mood- and stress-related behaviors and response to psychostimulants and antidepressants, but also the risk for acute neurotoxicity as well as the susceptibility for neurodegenerative diseases. Because of this emerging knowledge on the role of OCTs in CNS processes and function, comprehensive studies should be conducted in order to elucidate the effects of genetic and epigenetic regulation of these transporters on these different processes.