M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

DRD4 gene and human behavior

Abstract

DRD4 gene, encoding receptor type 4, is one of most popular candidates in genetic association studies of human behavior. Variable Number of Tandem Repeats (VNTR) region in exon III is associated with attention deficit hyperactivity disorder (ADHD), opioid dependence, and probably with many other phenotypes. In order to evaluate validity of these findings it is essential to understand the mechanism whereby DRD4 exon III VNTR may affect behavior. The mechanism necessarily includes several levels, starting from characteristics of receptor and leading, via intracellular and physiological processes, toward complex behavioral phenotypes. In vitro studies suggest that 7R allele of exon III VNTR is hypofunctional, relative to 2R and 4R alleles. D4 receptor regulates synthesis of melatonin in pineal gland. The pattern of changing concentration of melatonin in blood is associated with diurnal preference, and diurnal preference is associated with multiple forms of behavior. Elevated blood melatonin decreases cognitive performance. We hypothesize that DRD4 affects shape of blood melatonin profile which, in turn, modulates behavior. Hence, exon III VNTR may contribute to behavioral variability via it’s effect on blood melatonin levels. We propose series of experiments for testing this hypothesis.

Introduction

DRD4 gene

The DRD4 gene encodes type 4 (D4), one of five dopamine receptors in humans. The D4 belong to family of G protein coupled receptors. It is localized in plasma membrane. Upon activation with dopamine the receptor triggers various intracellular signaling processes, including inhibition of adenylate cyclase activity (Oak, Oldenhof et al. 2000).

D4 receptor is found in various regions of the brain, including cerebral cortex, hippocampus, hypothalamus and striatum, and in other organs such as adrenal gland and testes (reviewed in (Tarazi and Baldessarini 1999)). Highest levels of DRD4 expression are detected in retina and pineal gland (Missale, Nash et al. 1998, Su, Wiltshire et al. 2004, Kim, Bailey et al. 2010). M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

The gene contains several polymorphic loci, including 48bp Variable Number of Tandem Repeats (VNTR) in exon III. The VNTR leads to variation in length of 3rd cytoplasmic loop of the protein. Eleven alleles of the VNTR were found worldwide – from 1 repeat (Li, Liu et al. 1999) to 11 repeats. Most common alleles are 2R, 4R and 7R (Chang, Kidd et al. 1996). Interestingly, the region is polymorphic in some other mammalian species such as dogs (Wan, Hejjas et al. 2013), cetaceans, but not mice and rats (Larsen, Mogensen et al. 2005).

In vitro studies have demonstrated functional differences between the alleles. In reporter gene assay lowest transcriptional activity was shown for 7R, highest – for 2R, and intermediate – for 4R (Schoots and Van Tol 2003). There was no significant association between the VNTR and DRD4 expression in human post-mortem samples of frontal cortex (p = 0.1), however, carriers of 7R allele had lower expression values (Simpson, Vetuz et al. 2010).

Number of repeats in this locus affects pharmacological profile of receptor: affinity to spiperone is increasing with length of repeat region, but only in presence of NaCl (Van Tol, Wu et al. 1992, Asghari, Schoots et al. 1994). Although, authors of the study noted that the differences are small and unlikely to have physiological or clinical relevance. The VNTR sequence can be deleted from the receptor without consequence to its binding profile (Asghari, Schoots et al. 1994), coupling to G proteins, and effect on calcium flux (Kazmi, Snyder et al. 2000).

In cultured cells, transfected with DRD4 gene, potency of dopamine to inhibit cAMP formation was reduced for 7R variant, compared with 2R and 4R (Asghari, Sanyal et al. 1995). 10R variant was shown to be 2- to 3-fold more potent in dopamine-mediated coupling to than 2R (Jovanovic, Guan et al. 1999). In transfected frog oocytes 2R and 7R variants were more active than 4R in respect to opening potassium channels (Wedemeyer, Goutman et al. 2007).

The 7R variant is more sensitive to chaperone-like effect of quinpirole, than 2R and 4R (Van Craenenbroeck, Clark et al. 2005).

Studies of receptor dimerization demonstrated that 2R and 4R, but not 7R, variants can form heterodimers with dopamine receptor D2S (short splicing form of D2). Accordingly, intracellular signalling mediated by D4 receptor was potentiated by activation of D2 receptor only when 2R or 4R variants, but not 7R, of D4 were used (González, Rangel-Barajas et al. 2011). Similar effect was observed in heterodimerization of D4 with D2L (long form of D2): binding of M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

D4 7R with D2L was less efficient, and functional consequences of the dimerization were less pronounced, than for 2R and 4R variants (Borroto-Escuela, Van Craenenbroeck et al. 2011).

In case of homodimerization of D4 receptors the 2R and 4R formed more dimers than 7R, although 7R receptors had higher affinity to each other (but not toward 2R and 4R) (Van Craenenbroeck, Borroto-Escuela et al. 2011).

One study reported that 7R variant, compared to 2R and 4R, has higher affinity to proteins Nck and Grb2, and the binding was mediated by SH3-binding motifs in 3rd cytoplasmic loop of D4 (Oldenhof, Vickery et al. 1998).

Despite mixed evidence it appears that 7R variant corresponds to diminished receptor function, compared to 2R and 4R.

Several other polymorphic markers were found in DRD4 gene, including 120bp VNTR in region, 12bp and 13bp VNTRs in 1st exon, poly(G) STR in intron 1 and multiple SNP (Wong, Buckle et al. 2000). Functional analysis of these markers was conducted as well, although much less thoroughly than in case of exon III VNTR, which is considered so far the most important polymorphism of the gene, due to it’s large size and location in coding sequence.

Association studies of DRD4 gene

Since it’s discovery in 1992 (Van Tol, Wu et al. 1992) the DRD4 exon III VNTR became a popular object of genetic association studies. Initial interest was caused by observations of high affinity of atypical antipsychotic drug clozapine to D4 receptor, compared to D2 receptor (Van Tol, Bunzow et al. 1991), and findings of elevated DRD4 expression in (Seeman, Guan et al. 1993). Naturally, it was hypothesized that genetic variation in DRD4 may explain variations in individual susceptibility to schizophrenia and in response to clozapine. The results of early association studies of schizophrenia (Barr, Kennedy et al. 1993, Nanko, Hattori et al. 1993, Sommer, Lind et al. 1993) and clozapine response (Shaikh, Collier et al. 1993) were not significant. Subsequent research demonstrated that clozapine binds to multiple targets in the brain besides D4 receptors (Roth, Sheffler et al. 2004), and specific D4 antagonist is not effective in treatment of schizophrenia (Bristow, Kramer et al. 1997, Kramer, Last et al. 1997). Moreover, initial finding of elevated DRD4 expression in schizophrenic brains was not replicated (Oak, Oldenhof et al. 2000). M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Meanwhile, in the middle 90-s researchers started to test associations between DRD4 and other mental disorders (e.g., alcoholism (George, Cheng et al. 1993), Parkinson disease (Nanko, Hattori et al. 1993)), as well as other behavioral phenotypes such as personality traits (Ebstein, Novick et al. 1996). The justification for selection of DRD4 gene for those tests was usually based on role of dopamine in reward and reinforcement. Since then significant number of association studies was published, implicating multiple phenotypes as diverse as creativity (Mayseless, Uzefovsky et al. 2013), pre-school (Farbiash, Berger et al. 2013), incarceration rate (Schwartz and Beaver 2013), (Gervasini, Gordillo et al. 2013), longevity (Grady, Thanos et al. 2013), political attitude (Settle, Dawes et al. 2010), skiing and snowboarding (Thomson, Hanna et al. 2013). Many of these results were not replicated (e.g., see (Kluger, Siegfried et al. 2002) for meta-analysis of personality trait Novelty Seeking) and for many more no replication studies were published. However, associations of two phenotypes – ADHD (Wu, Xiao et al. 2012) and opioid dependence (Chen, Liu et al. 2011) - were supported by meta-analyses.

What can we do to reveal which phenotypes are truly associated with DRD4 exon III VNTR?

First, the mechanism (if any) of DRD4 role in behavior should be understood.

Second, genetic association studies should be based on the understanding of this mechanism. A hypothesis to be tested in genetic association study should be clearly defined. Without such hypothesis one may justify virtually any number of statistical tests, and it will decrease statistical power. For instance, using broad hypothesis like “dopamine is implicated in reward” suggests testing all polymorphic markers in all dopamine-related genes, with all behaviors contingent on reward processing. The list of statistical tests in such case can be any long.

Complex behavioral phenotypes, such as AHDH and substance abuse, are dependent on many factors, probably including genetic variants. All those factors would interact with each other and create picture that might be too complex to unravel in any study with reasonable sample size. But more proximal traits (“endophenotypes”), including characteristics of tissues and organs, could be relatively easier to tackle. Prediction of complex behaviors based on few genetic variants might be unlikely. But prediction of cellular and physiological phenotypes, based on genes implicated in cellular and tissue function, could be plausible. M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Following sections describe how DRD4 gene polymorphism might be linked to such physiological phenotypes and ultimately to behavioral phenotypes.

D4 and melatonin

There are at least two lines of evidence that may elucidate function of D4 receptors in the brain. One bears on D4 activity in prefrontal cortex, role of D4 in regulation of neuronal excitability (Yuen, Zhong et al. 2010) and in associative learning (Laviolette, Lipski et al. 2005, Lauzon, Bishop et al. 2009). The second line of evidence concerns a role of D4 in regulation of melatonin synthesis in pineal gland. We believe that this second model has better potential in generating testable hypotheses.

In rats DRD4 is expressed in pineal gland at levels much higher than in any other tissue (except of retina). DRD4 expression in pineal is much higher at night than at daytime (Kim, Bailey et al. 2010). Specifically, the expression is low in first half of a night, but it rises later on and then decreases in the morning (Bai, Zimmer et al. 2008).

In humans DRD4 expression in pineal is also higher than in any other organ (BioGPS). The only report of DRD4 expression in human pineal presents data from 10 postmortem samples (Andrew Su, personal communication). Although representation of the data on BioGPS website suggests that the expression is higher at day than at night, a closer look at times of death of study subjects reveals that it is impossible to judge whether the expression profile is significantly different from expression profile in rats. Several samples included into “Pineal night” group were collected from subjects who died at 0.00-2.00 (12am-2am), i.e. in first half of night, when DRD4 expression is expected to be low (by analogy with rat studies).

The role of D4 in pineal was investigated in rat pinealocytes culture and extracted pineal glands (Gonzalez, Moreno-Delgado et al. 2012). The synthesis and secretion of melatonin in pineal are triggered by noradrenaline, released from sympathetic nerves. The noradrenaline

1 1B , which initiate intracellular signalling cascade that leads to

synthesisactivates and receptors secretion β andof melatonin. α D4 receptors, synthesized in the end of night, are activated by dopamine, form heterodimers with norepinephrine receptors and suppress synthesis and release of melatonin. Presumably same mechanism operates in human pineal gland. M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Melatonin and diurnal preferences

Both in rats (Barassin, Saboureau et al. 1999) and in humans (Brzezinski 1997)1 melatonin secretion is maintained at very low level at daytime and peaks in the middle of a night. If blood is sampled during the night at 30 min – 1 hour intervals, and melatonin concentration is measured, a characteristic melatonin concentration profile with peak at around 2am-4am can be plotted (Figure 1).

Figure 1 Daily rhythm of melatonin concentration in human blood plasma. Dotted line indicates 24am time point. Taken from (Cajochen, Krauchi et al. 2003).

The shape of such profile varies by individual, but displays considerable intra-individual stability both in rats (Barassin, Saboureau et al. 1999) and humans (Lerchl and Partsch 1994). Melatonin profiles are routinely measured in sleep research (e.g., (Duffy, Dijk et al. 1999, Dijk, Duffy et al. 2012)).

Lets assume that in humans D4 receptors regulate melatonin secretion via same mechanism as discovered in rats: blockade of norepinephrine receptors in second half of a night. Then the downward slope of melatonin profile is determined by, among other factors, activity of D4 receptors. Hence, variation of D4 activity may contribute to variation in slope of melatonin profile (Figure 2).

1 As well as in many other species Reiter, R. (1983). The Role of Light and Age in Determining Melatonin Production in the Pineal Gland. The Pineal Gland and its Endocrine Role. J. Axelrod, F. Fraschini and G. P. Velo, Springer US. 65: 227-241.. M. Monakhov. DRD4 gene and human behavior. March 15, 2015. Melatonin

Time

benchmark profile more efficient D4 less efficient D4

Figure 2 Hypothesized effect of D4 receptor efficacy on melatonin profile.

One important characteristic of sleep behavior is diurnal preference (Adan, Archer et al. 2012). Namely, people differ in preferred time of waking up and going to bed, and in preferred time for engagement in demanding activities. Morning types wake up early, prefer to do demanding work in the morning, and go to bed early. Evening types wake up late, prefer to work in evening and go to bed late. The Morning and Evening types represent extremes of continuous distribution of Morningness-Eveningness trait, while most people can be classified as Intermediate type. The chronotype can be determined using self-report questionnaires, such as Composite Scale of Morningness (Smith et al., 1989).

Results from several studies indicate that shape of melatonin profile, and in particular, the slope of melatonin decscension after peak, is associated with diurnal preference. Gibertini et al demonstrated that decline of melatonin concentration is fastest in Morning types, slowest in Evening types, with Intermediate types in between. They noticed “extended peak for the evening types or the steep decline in melatonin levels in the morning types”. They also suggested that “duration of the melatonin signal or it’s strength as waking approaches may be related to circadian type” (Gibertini, Graham et al. 1999). M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Duffy et al have shown that in Evening types the decrease of melatonin concentration is delayed, relative to Morning types, even when timescale is adjusted for habitual wake time. Another interesting aspect of melatonin profile was also different between chronotypes in that study: in Morning types the profile was distinguished with sharp peak at around 3am, while Evening types had a flat plateau at 3am to 7am (Duffy, Dijk et al. 1999).

Morera-Fumero et al measured melatonin three times (9am, 12pm, 12am) and demonstrated that at 9am blood melatonin concentration is significantly lower in Morning types, compared to Evening types (Morera-Fumero, Abreu-Gonzalez et al. 2013).

Griefahn at al measured melatonin concentrations in saliva during nighttime. Their report concerns mostly with large differences between chronotypes in terms of melatonin levels in first half of night. However, delayed decrease of melatonin level in Evening types in second half of night was also detected (Griefahn 2002).

Liu et al reported that intervals between peak of melatonin concentration and midpoint of habitual sleep were significantly different between Morning and Evening types. The data suggest that the difference between chronotypes might be caused by slower decrease of melatonin level after peak in Evening types (Liu, Uchiyama et al. 2000).

It should be noted, that melatonin is not the only factor that mediates circadian rhythms. Pinealectomy (surgical removal of pineal gland) doesn’t disrupt sleep-wake cycle in rats (Fisher and Sugden 2010). In humans absence of blood melatonin doesn’t always disrupt sleep behavior (Zeitzer, Ayas et al. 2000). However, even if melatonin is not necessary for maintenance of circadian rhythms, it doesn’t mean that melatonin can’t alter circadian behavior. Being present in blood, it may affect various physiological processes, and variation in melatonin levels (as well as in shape of melatonin profiles) may contribute to variation in behavior.

Chronotype and behavior

The Eveningness-Morningness dimension is associated with various forms of human behavior. Eveningness correlates with higher Sensation Seeking (personality trait), alcohol use, smoking, eating disorders (Muro, Goma-i-Freixanet et al. 2012), drug use, lower Harm Avoidance (personality trait), with apathetic, volatile, and disinhibited temperaments (Ottoni, M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Antoniolli et al. 2011), higher number of sexual partners (Piffer 2010), with anxiety disorder, disorder and personality disorder (Lemoine, Zawieja et al. 2013), depressive symptoms and trait anxiety (Pabst, Negriff et al. 2009), insomnia (Fernandez-Mendoza, Vela- Bueno et al. 2009).

Notably, sleep behaviors, partially reflected in scores of Eveningness-Morningness, are implicated in ADHD and opioid dependence (phenotypes associated with DRD4 gene according to meta-analyses). ADHD is associated with sleep onset latency, difficulty in waking up, delayed onset of rising melatonin levels (Kooij 2013). Sleep problems often accompany opioid abuse (Mahfoud, Talih et al. 2009, Nettleton, Neale et al. 2011). For instance, intention “to improve sleep” is common motive for non-medical use of opioids (Barth, Maria et al. 2013). However, without prospective and experimental studies it is difficult to tell whether sleep disturbance cause ADHD and opioid dependence, or vice versa.

Melatonin and cognitive performance

It is suggested that rise of sleepiness in the evening is caused by rising melatonin levels (Cajochen, Krauchi et al. 2003). Melatonin decreases body temperature (Reid, Van den Heuvel et al. 1996, Krauchi, Cajochen et al. 1997, Mazzoccoli, Giuliani et al. 2004), while decreased body temperature is related to diminished activity of muscles, nerves, cardiovascular and respiratory systems (Drust, Waterhouse et al. 2005).

Most people experience slow build up of energy after awakening in the morning. Sleep inertia causes low cognitive performance and may persist for up to 2 hours (Wright, Lowry et al. 2012, Thompson, Jones et al. 2014). Could it be that this inertia is mediated by residual levels of melatonin in the blood? Melatonin, given to healthy human subjects at 7am, impairs attention (Graw, Werth et al. 2001). So, individual differences in morning melatonin levels may mediate differences in behavior in the hours that follow the awakening. Such effect may potentially lead to variation in multiple behavioral constructs, including personality traits, preferences and susceptibility to mental disorders.

M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

The hypothesis

We hypothesize that polymorphism of DRD4 gene influences human behavior via regulation of melatonin production. In particular, hypofunctional variants of the gene (such as exon III VNTR 7R-allele), can make downregulation of melatonin secretion in second half of a night less efficient. Less efficient downregulation would translate into slower decline of melatonin level, typical for Evening types, and higher blood melatonin in morning hours. If such shape of melatonin profile increases person’s Eveningness, then hypofunctional DRD4 variant may increase susceptibility to behaviors associated with Eveningness. Also, higher morning melatonin may impair cognitive performance without effect on Eveningness.

Proposed experimental tests We need to understand multi-level mechanism of DRD4 effects (if any) on behavior: how does VNTR affect function of D4 receptor, how does the receptor affect melatonin production, how does melatonin production affect melatonin profile, and how does melatonin profile affect human behavior.

So far we know that DRD4 VNTR affects and dimerization (D4:D4 and D4:D2), with 7R allele associated with less efficient expression and dimerization. We don’t know whether similar effect is present for heteromers of D4 with norepinephrine receptors. However, dopamine and norepinephrine receptors share similar structure (just as dopamine and norepinephrine molecules themselves). It makes good sense to presume that DRD4 exon III VNTR affects D4 binding to norepinephrine receptor in similar manner.

We know that D4 activity decreases melatonin production. We know also that particular shape of melatonin profile is associated with Eveningness, and Eveningness is associated with several other behavioral phenotypes (although it is not clear whether the shape of melatonin profile is a cause of Eveningness phenotype or not). We know that administration of melatonin in the morning decreases attention.

What we don’t know is how DRD4 VNTR affects melatonin production, and how D4 activity affects melatonin profile.

M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Effect of DRD4 exon III VNTR on melatonin secretion by pineal gland

Pinealocytes extracted from rat brain can be maintained in culture as long as 15 days (Isobe, Fujioi et al. 2001). Cultured pinealocytes produce melatonin after stimulation with norepinephrine (Tosini, Doyle et al. 2000)(Isobe, Fujioi et al. 2001). The pinealocytes can be transformed using adenoviral vector (McTague, Ferguson et al. 2013).

Effects of DRD4 VNTR on melatonin production may be tested by transforming rat pinealocytes with different variants of human D4 receptor and then measuring effects of dopamine on norepinephrine-stimulated synthesis of melatonin. 7R variant of DRD4 may confer slower decline of melatonin level. One has to make sure that human DRD4 gene variants work properly in rat cells (it’s activation decreases norepinephrine-induced melatonin), and that endogenous rat DRD4 doesn’t hinder the experiment.

One study reported circadian rhythm of melatonin production in cultured pinealocytes (Isobe, Fujioi et al. 2001). This model potentially can be used to study effects of DRD4 variation on melatonin decline after peak.

If hypofunctional variants of DRD4 decrease rate of suppression of melatonin production, then further experiments can be done to investigate the underlying mechanism. For example, parameters of heterodimerization between D4 and norepinephrine receptors can be measured, effects of D4 polymorphism on norepinephrine-induced cAMP levels and on AANAT expression can be tested.

Effects of D4 on melatonin profile Does activation of D4 receptor cause faster decline of blood melatonin after peak? Does blockade cause slower decline?

D4 antagonist can be administered to either rats or humans after melatonin peak. The blockade of receptor my decrease rate of melatonin decline. D4 may increase the rate of melatonin decline.

Ideally, rat pinealocytes can be transduced in vivo by adenoviral vectors carrying different DRD4 variants, and effects on melatonin profiles can be measured. The transduction may require targeting of viral vector toward pinealocytes via specific antigens such as D4 receptor itself.

M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

Effect of shape of melatonin profile on human behavior

Blood melatonin profile in humans can be manipulated by administration of timed slow release melatonin, so as to increase melatonin only in second half of a night. Then effects of such treatment on emotional states and cognition can be tested on the following day.

Genetic association study of DRD4 exon III VNTR and shape of melatonin profile

We can test if rate of melatonin decline after peak is associated with DRD4 exon III VNTR in sufficiently large human sample. Hypofunctional variants, such as 7R allele, can be associated with slower melatonin decline. This effect may manifest in different ways. For example, activity of D4 receptor may increase over time, gradually suppressing rate of melatonin production. In this case changes in D4 activity would lead to changes in rate of melatonin level decline. Alternatively, activity of D4 receptor may switch off melatonin synthesis relatively fast, and subsequent melatonin level decline will be determined by it’s clearance from blood. It is not clear which mechanism takes place, but in any case activity of D4 receptor would change area under curve (AUC) of melatonin after peak.

Peak values of melatonin concentration are highly variable, and since in this study we are interested in shape of melatonin profile, the concentration values should be normalized. Measures of melatonin concentration are often somewhat noisy, and these value may require some smoothening prior to analysis (e.g., by means of LOWESS regression or running mean smoothening).

For a statistical test we can use linear regression of AUC value on dummy genotype variable that equals number of copy of 7R allele in subject’s genotype (additive genetic model), controlling for appropriate covariates such as sex, age and race. Since variants other than 2R, 4R and 7R were less thoroughly tested in functional studies, the analysis of association should be restricted to carriers of genotypes composed only by these three alleles.

M. Monakhov. DRD4 gene and human behavior. March 15, 2015.

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