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The relationship between serotonin (5-HT) and major depressive disorder (MDD) has been extensively studied but certain aspects are still ambiguous. Given the evidence that 5-HT neurotransmission is reduced in depressed subjects, it is possible that one or more of the 5-HT regulators may be altered in the dorsal raphe nucleus (DR) of depressed subjects. Candidates that regulate 5-HT synthesis and neuronal activity of 5-HT neurons include intrinsic regulators such as tryptophan hydroxylase 2, 5-HT autoreceptors, 5-HT transporter and transcription factors, as well as afferent regulators such as estrogen and brain-derived neurotrophic factor. The present study was designed to quantify mRNA concentrations of the above 5-HT regulators in an isolated population of 5-HT-containing DR neurons of MDD subjects and gender-matched psychiatrically normal control subjects. We found that mRNA concentrations of the 5-HT1D receptor and the transcription factors, NUDR and REST, were significantly increased in DR-captured neurons of female MDD subjects compared to female control subjects. No significant differences were found for the transcripts in male MDD subjects compared to male controls. This study reveals sex-specific alterations in gene expression of the pre-synaptic 5-HT1D autoreceptors and 5-HT-related transcription factors, NUDR and REST, in DR neurons of women with MDD.
Major depressive disorder (MDD) is a common, recurring and debilitating mood disorder. Considerable evidence from clinical in vivo imaging and pharmacological challenge studies as well as human postmortem studies has accumulated to suggest that the serotonin (5-HT) system is disrupted in subjects with MDD. The dorsal raphe nucleus (DR) in the midbrain contains about 50% of the brain’s serotonin neurons and the majority of forebrain 5-HT axons and terminals arise from the midbrain DR (Tork 1990). Several key regulators of 5-HT neurotransmission reside in the DR including the rate-limiting enzyme, tryptophan hydroxylase 2 (TPH2; EC188.8.131.52), the serotonin transporter (SERT) and the 5-HT1A autoreceptor and these targets have been the focus of investigations aimed at elucidating the alterations in serotonin neurotransmission in MDD.
Several studies have examined TPH2 in the DR of depressed or suicide subjects. A number of reports from the same group found that TPH2 mRNA levels are increased and the number of TPH-immunoreactive neurons are increased in the DR in depressed suicide subjects (Underwood et al. 1999; Boldrini et al. 2005; Bach-Mizrachi et al. 2006, 2008). However, Bonkale and colleagues reported no change in TPH-immunoreactivity in individual subnuclei of the DR between depressed suicide and control subjects, which indicates no alteration in the TPH protein in depressed suicide subjects (Bonkale et al. 2004). Similarly, a study of Nissl-stained sections of the DR found that the number of neurons and volumes in the entire DR were not significantly different between the mood disorder group and control group, and only the ventrolateral subnucleus of the DR showed a 31% reduction in the number of neurons in the mood disorder group relative to controls (Baumann et al. 2002). These studies reveal conflicting results regarding the role of TPH2 in DR neurons of subjects with depression.
The SERT has been another serotonin substrate investigated in depressed subjects. Although a previous report found that the cellular levels of SERT mRNA in the DR was greater in suicide victims (Arango et al. 2001), there are several studies which show opposite results such as a radioligand binding assay of SERT which revealed no changes in SERT distribution and binding sites in the DR between suicide victims with depression and control subjects (Bligh-Glover et al. 2000). Two other studies reported that SERT mRNA expression or the number of SERT binding sites in the DR was not different between those who committed suicide and control subjects (Little et al. 1997; Anisman et al. 2008).
Serotonin 1A receptors located in the DR represent somatodendritic autoreceptors which regulate the activity of DR neurons; hence a dysfunction of these receptors has been intensely investigated in subjects with MDD. Stockmeier et al. (1998) reported that 5-HT1A agonist binding was significantly increased in specific subnuclei of the DR of subjects with MDD (Stockmeier et al. 1998). In contrast, Arango et al. (2001) subsequently reported a decrease in 5-HT1A receptor ‘binding capacity’ in the dorsal raphe of depressed suicide victims relative to controls (Arango et al. 2001). However, a more recent report from these investigators contradicts their earlier finding and reveals an increase in 5-HT1A autoreceptor binding sites in the rostral dorsal raphe of suicide subjects (Boldrini et al. 2008) which is consistent with the original Stockmeier et al. (1998) report. Other findings include an in vivo imaging study that reported decreased [11C]WAY 100635 binding in the brainstem region of the DR in elderly depressed patients which provides further evidence of altered 5-HT1A autoreceptor function in depression (Meltzer et al. 2004).
More recent studies have identified novel regulators of serotonin function. These include a nuclear protein complex, nuclear deformed epidermal autoregulatory factor-1 (Deaf-1) or the human homolog, NUDR, FRE under dual repression binding protein-1 (Freud-1) and repressor element-1 (RE-1) silencing transcription factor (REST) also known as neuron-restrictive silencing factor. These transcription factors bind to the specific repressor sequence in the 5-HT1A promoter and lead to reduction in 5-HT1A receptor transcriptional activity as well as protein expression. A recent study also found that the RE-1 sequence is present within the promoter region of the TPH2 gene (Patel et al. 2007) which can bind REST and repress transcriptional activity of TPH2. In addition, there are several afferent regulators whose terminals innervate the DR and regulate DR neuronal activity. For example, brain-derived neurotrophic factor by its action on tropomyosine-related kinase B receptors (TrkB) and estrogen by its action on estrogen receptors (ERα and ERβ) have been reported to exert profound effects on serotonin DR neurons and serotonin neurotransmission (Joffe and Cohen 1998; Mattson et al. 2004; Shively and Bethea 2004; Lasiuk and Hegadoren 2007; Martinowich and Lu 2008).
The aforementioned regulators all have been shown to regulate various parameters of serotonin function and therefore may be involved in the pathophysiology of major depressive disorder. There have been no previous studies which have measured the cellular expression of all of the above 5-HT-related regulators in isolated DR neurons of both male and female subjects with MDD using a laser-capture microdissection (LCM) approach. The present study was designed to quantify the cellular concentration of mRNA transcripts of several 5-HT-related genes in a pure population of serotonin-containing DR neurons in female and male subjects diagnosed with MDD and in psychiatrically-normal control subjects matched for gender using LCM of immunofluorescently-stained neurons and quantitative real time PCR.
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- Materials and methods
The present study represents the first report using laser-capture microdissection and Q-PCR to quantify mRNA transcripts in individual TPH2-immunofluorescent DR neurons of female and male MDD subjects and gender-matched control subjects. The results revealed that mRNA expression of NUDR, REST and the 5-HT1D receptor was significantly increased in DR serotonin-containing neurons of female subjects diagnosed with MDD relative to matched female control subjects, but the mRNA transcripts measured were unaltered in midbrain DR neurons of male depressed subjects compared to male controls.
NUDR is the human homolog of Drosophila deformed epidermal autoregulatory factor-1, which has been identified as a DNA-binding protein and gene regulator (Huggenvik et al. 1998). Recent investigations have shown that NUDR represses both human and rat 5-HT1A receptor promoter luciferase constructs and is colocalized with 5-HT1A receptors in the prefrontal cortex, hippocampus and raphe nuclei (Lemonde et al. 2003). While stable expression of NUDR reduced the expression and binding of 5-HT1A receptors and mRNA levels in raphe cells suggesting that NUDR negatively regulates 5-HT1A receptor (Lemonde et al. 2003), a subsequent study revealed that NUDR functions as a transcriptional enhancer in non-serotonergic cells (Czesak et al. 2006). Therefore, NUDR may have a dual function depending on the synaptic localization of 5-HT1A receptors. A recent study using a gene knockout strategy reported that NUDR−/− embryos display neural tube exencephaly at the mid-hindbrain region which caused death at birth (Hahm et al. 2004), this may suggest that NUDR may play an important neurodevelopment role in the early formation of midbrain structures.
We previously documented a gender-specific reduction in protein levels of NUDR and 5-HT1A receptors in the prefrontal cortex of women with depression, but not in depressed men (Szewczyk et al. 2009). The present observation is consistent with this previous report of alterations in NUDR only in depressed females, but an increase in NUDR mRNA was found in the DR compared to a decrease in NUDR protein in the prefrontal cortex. As NUDR is believed to function as a 5-HT1A receptor gene repressor in DR neurons, elevated NUDR mRNA in the depressed female subjects may reflect an adaptive change to increase NUDR synthesis and activity to down-regulate over-expression of somatodendritic 5-HT1A autoreceptors in the DR of depressed subjects. Increased 5-HT1A receptor binding sites has been previously reported in the DR of MDD or depressed suicide subjects (Stockmeier et al. 1998; Boldrini et al. 2008). Similarly, the mean level of 5-HT1A receptor mRNA in DR neurons was increased by 3.73-fold in the depressed female subjects, however the data did not reach statistical significance (p = 0.0619). The trend towards an increase in 5-HT1A receptor mRNA in depressed female DR neurons is in agreement with a 5-HT1A receptor binding assay where increased 5-HT1A receptor binding was found in the DR of both male and female depressed suicides as compared to gender-matched control subjects (Boldrini et al. 2008), likewise an earlier study reported 5-HT1A receptor binding sites were increased in the subnuclei of the DR of suicide victims with MDD compared with controls (Stockmeier et al. 1998).
Repressor element-1 silencing transcription factor (REST) is another negative transcriptional regulator of 5-HT1A receptors. It binds to the RE-1 site adjacent to the dual repressor element upstream of the promoter region in the 5-HT1A receptor gene sequence of rat, mouse and human (Lemonde et al. 2004). Hence REST binding contributes to silencing of 5-HT1A receptors in some neuronal and non-neuronal cells (Schoenherr and Anderson 1995; Lemonde et al. 2004). We found a gender-specific increase in REST mRNA expression in DR neurons of female MDD subjects as compared to their matched control subjects but there were no changes in the male subject groups. In theory, the increase in REST in DR neurons may result in increased binding to the 5-HT1A RE-1 promoter site and lead to a decrease in 5-HT1A autoreceptor expression. The decrease in 5-HT1A autoreceptors may reduce inhibitory tone and increase the synaptic concentrations of serotonin. However, it is interesting that both REST and NUDR are two transcriptional repressors of 5-HT1A receptors that are elevated in DR neurons of depressed women, but yet 5-HT1A receptor mRNA remains elevated in depressed women although not statistically significant. Perhaps these changes reflect an adaptive molecular response triggering transcriptional repressor mechanisms to suppress 5-HT1A autoreceptor expression in DR neurons of depressed women and facilitate 5-HT neurotransmission, but the elevation in the transcriptional repressors is not sufficient or functional to completely abolish the enhanced inhibition via increased expression of somatodendritic 5-HT1A receptors.
In addition to 5-HT1A somatodendritic autoreceptors, 5-HT1B and 5-HT1D pre-synaptic terminal autoreceptors also play a key role in regulating serotonin neurotransmission. The 5-HT1B and 5-HT1D receptors are highly expressed in dorsal raphe nuclei in rats (Davidson and Stamford 1995; Bonaventure et al. 1998) and in human (Bidmon et al. 2001). The finding that 5-HT1B and 5-HT1D receptors can form homo- and heterodimers with each other (Xie et al. 1999) further add to the complexity of the 5-HT1B and 1D autoreceptors action. The increase in 5-HT1D receptor mRNA in DR neurons of depressed women is supported by previous rodent studies measuring the 5-HT1B receptor mRNA (rodent equivalent of the human 5-HT1D). For example, 5-HT1B receptor mRNA was elevated (25%) in the DR of learned helplessness rats compared to controls (Neumaier et al. 1997) albeit the magnitude of change was less compared to our observations. It is also interesting that rats treated for 21 days with fluoxetine reduced 5-HT1B receptor mRNA expression in the DR by 83% of control, whereas rats treated for 7 days and then allowed 7 days of drug washout, had 5-HT1B receptor mRNA levels in the DR return to control levels (Neumaier et al. 1996a). It is important to note that methodological differences between Neumaier et al. (1997) using in situ hybridization histochemistry and our study using RT-PCR must be considered when comparing fold-changes in mRNA expression. The gender-specific increase in the mean levels of 5-HT1D receptor mRNA in DR neurons of female MDD subjects as compared to their matched control subjects is potentially a very important observation that may further elucidate the molecular mechanisms associated with diminished serotonin neurotransmission in depression. Over-expression of pre-synaptic 5-HT1D autoreceptors in depressed women may reflect excessive inhibitory tone on serotonin neurons leading to reduced synaptic levels of 5-HT. Such a hypothesis is supported by our observations of elevated NUDR and REST mRNA in DR neurons of depressed women to dampen the inhibitory tone on DR neurons by acting to repress 5-HT1A autoreceptors.
There has been only one previous report published on midbrain 5-HT1D receptors in suicide victims where a significant decrease was found in 5-HT1D binding affinity in the depressed suicides (Arranz et al. 1994). Another study reported increased 5-HT1D receptor binding sites in globus pallidus, but these alterations where restricted to those suicides who died by violent means (Lowther et al. 1997). It will be important for future studies to confirm and further investigate the over-expression of 5-HT1D receptors in depression.
The TPH2 gene promoter sequence also contains an RE-1 element and REST has been shown to regulate transcription of TPH2. Patel et al. (2007) showed that in transfected C6-glioma cells dominant-negative inhibition of REST produced a 4.4-fold up-regulation of TPH2 mRNA compared to a reference gene which demonstrated the silencing effect of Neuron-restrictive silencing element (NRSE)/REST on TPH2 transcription. Increased REST repression of TPH2 in depression may be maladaptive, but we did not observe any significant changes in any of the TPH isoforms in DR neurons of depressed subjects nor is there any evidence in the literature to support reduced transcription of TPH2 in depressed subjects. Our findings on TPH isoforms in DR neurons of MDD subjects are in contrast to those of Arango and colleagues (Bach-Mizrachi et al. 2008) that have reported an increase in TPH2 mRNA expression using in situ hybridization in depressed suicide subjects in the caudal level of the DR and median raphe nuclei. The only DR subnucleus that exhibited an increase in TPH2 mRNA expression was the caudal subnucleus. The same group also found a higher number and density of TPH-immunoreactive neurons in the mid-rostrocaudal DR region and elevated TPH immunoreactivity in DR region at the level of trochlear decussation in depressed suicides (Underwood et al. 1999; Boldrini et al. 2005). In the context of elevated REST (a TPH2 repressor) mRNA transcripts in women with depression and the lack of change in TPH2 mRNA in the DR of depressed women and men, the increase in TPH2 mRNA reported in DR of depressed suicides by Bach-Mizrachi et al. (2008) is difficult to interpret mechanistically. It is conceivable that based on the functional heterogeneity of the DR reported in rodents (Abrams et al. 2004; Lowry et al. 2008) that alterations in TPH2 mRNA are restricted to the caudal DR in depressed suicide subjects. However, given the considerable evidence of alterations in prefrontal cortical serotonergic markers in major depressed subjects (Austin et al. 2002; Stockmeier et al. 2009; Szewczyk et al. 2009) and that the majority of forebrain 5-HT terminals arise from the midbrain dorsal raphe nucleus which contains more than 90% of the DR 5-HT neurons (Baker et al. 1990, 1991; Tork 1990), the rationale for choosing to examine the mid-rostral DR in the present study is strongly justified.
TPH2-T is a truncated form of TPH2 and is a functional single nucleotide polymorphism in the TPH2 gene (Haghighi et al. 2008). This polymorphism results in low efficiency of normal splicing and produces a truncated TPH2-T isoform by alternative splicing. TPH2-T lacks the catalytic domain and hence TPH2 enzyme activity that may reduce serotonin production. Therefore, to understand the involvement of truncated TPH2-T, we measured the TPH2-T mRNA and found there is no difference in mRNA expression of TPH2-T in depressed subjects suggesting that TPH2-T does not play a major role in the pathophysiology of depression. This is the first study which measured TPH2-T mRNA transcripts in human DR neurons and established that the TPH2 truncated form does not appear to play a role in major depressive disorder.
The previous study by Bonkale et al. 2004 reporting no changes in TPH2 protein combined with the present report of no changes in TPH2 mRNAs in the DR of depressed subjects represent two independent studies conducted on two separate cohorts of MDD and control subjects which consistently demonstrates a lack of change in TPH2 biosynthesis in the dorsal raphe nuclei of subjects diagnosed with major depressive disorder. These studies are at odds with those of Arango and colleagues (Underwood et al. 1999; Boldrini et al. 2005; Bach-Mizrachi et al. 2008) reporting increased TPH2 expression in the raphe of depressed suicides and therefore raise questions regarding the subject characteristics of their depressed cases or perhaps methodological differences.
Of the remaining transcripts measured in the DR of depressed subjects (Freud-1, SERT, TREK-1, TrkB, ERα and ERβ), none of these mRNAs were significantly altered in depressed subjects within or across genders. With the exception of SERT, no previous studies have examined mRNA expression of these transcripts in DR neurons of depressed subjects. For example, postmortem studies of depressed suicides revealed a greater percentage of DR neurons expressing higher grain density for SERT mRNA (Arango et al. 2001). In contrast, a recent study found no difference in SERT mRNA in the DR between those who committed suicide and control subjects (Anisman et al. 2008). Similarly, earlier studies found no difference in SERT binding sites as well as SERT mRNA levels in the DR of depressed subjects as compared to matched control subjects (Little et al. 1997; Bligh-Glover et al. 2000; Klimek et al. 2003). Our finding of no change in SERT mRNA in DR neurons of MDD subjects is consistent with these previous studies.
One potentially important variable to consider in human postmortem mood disorder studies is the confounding affect of antidepressant medication on the dependent measures. While no published studies to date have documented effects of antidepressants on the transcription factors, NUDR and REST, several rodent studies have reported alterations in gene expression of various serotonergic markers in the dorsal raphe. For example, repeated fluoxetine treatment significantly decreased (−38%) 5-HT1A receptor mRNA levels in the anterior raphe area (Le Poul et al. 2000), paroxetine and fluoxetine both reduced 5-HT1B receptor mRNA in the DR by 36% and 27%, respectively (Anthony et al. 2000), whereas fluoxetine significantly reduced expression of TPH2 and SERT mRNAs as determined by RT-PCR in the brainstem (Dygalo et al. 2006). These studies illustrate the importance to consider the patients’ medication history and obtain, when possible, accurate clinical records and toxicological reports for all subjects diagnosed with MDD.
As toxicology screens were negative for antidepressants for all depressed subjects, it is unlikely that the alterations in the mRNA transcripts in the MDD subjects were influenced by antidepressant medication. However, the potential effects of antidepressants can not be entirely ruled out since several of the MDD subjects had a prescription for an antidepressant at the time of death, and this may raise questions regarding the compliance of these patients and the possible long-term effects of antidepressants on these specific serotonin regulators. It is important to note that having a prescription for a medication does not necessarily imply compliance. In fact, only 25–50% of patients with MDD adhere to their antidepressant routine for the length of time recommended by depression treatment guidelines, and close to 50% of depressed patients referred from primary care to specialty care treatment fail to complete the referral (Trivedi et al. 2007).
Gender factor and hormonal effect
The increase in mRNA expression of 5-HT1D receptor, NUDR and REST in female depressed subjects raises the issue of a biochemical alteration specific to women. These results emphasize the facts that women have different biochemical profiles compared to men with respect to the pathophysiology of depression (Carey et al. 1995; Kessler 2003; Baca et al. 2004) and raise the possibility of ovarian hormones playing a role. Currently, there is no available data on the direct effects of estrogen or progesterone on 5-HT1D receptor, NUDR or REST. In a previous rodent study, estrogen selectively decreased 5-HT1B (which is human 5-HT1D counterpart) mRNA in the mid-ventromedial subregion of the DR neurons, where 5-HT1B mRNA was associated with higher anxiety-like behavior and inversely correlated with TPH2 mRNA levels (Hiroi et al. 2006; Hiroi and Neumaier 2009). These results suggest that estrogen may reduce 5-HT1B autoreceptor and increase TPH2 synthesis in a coordinated fashion, thereby increasing the capacity for 5-HT synthesis and release in distinct forebrain regions that modulate specific components of anxiety behavior (Hiroi et al. 2006). On the other hand increased baseline hippocampal 5-HT levels in only female 5-HT1B knockout mice mirrors and supports our study demonstrating increased 5-HT1D receptor expression and possible diminished serotonin neurotransmission only in depressed women (Jones and Lucki 2005).
Although the precise hormonal stage of the women involved in the study is difficult to ascertain, we have evidence from toxicology screens and from structural interviews that none of the female subjects were receiving hormonal therapy at the time of death. Furthermore, all of the female subjects in our study were over 45 years of age, suggesting that the majority of the women were likely perimenopausal or post-menopausal, so it is unlikely that circulating ovarian hormones had a confounding effect on 5-HT1D receptor, NUDR and REST mRNA expression in our female subjects. But this does not exclude the possibility that long-term fluctuations in ovarian hormones may have played a role in the mechanisms regulating gene expression of 5-HT1D receptor, NUDR and REST in depressed women.
In summary, this report presents evidence of increased gene expression of two novel transcription factors, NUDR and REST, and of 5-HT1D receptors in an isolated population of serotonin-containing dorsal raphe neurons of women with MDD. These findings reveal alterations in specific regulators of serotonin function that may provide clues for understanding the mechanisms associated with diminished serotonin neurotransmission and the higher incidence of depression in women.