Regional Distribution and Relative Abundance of Serotonin2c Receptors in Human Brain: Effect of Suicide

Abnormalities in serotonin receptor subtypes have been observed in the postmortem brain of suicide victims. We examined the regional distribution of serotonin (5HT)(2C) receptor mRNA in several areas of the human brain and also compared its protein and mRNA expression in the prefrontal cortex (PFC), hippocampus, and choroid plexus between suicide victims and normal control subjects. 5HT(2C) receptors were found to be distributed in several areas of the human brain (in order of abundance): highly concentrated and richest in choroid plexus; hypothalamus; nucleus accumbens; with the lowest abundance in PFC and cerebellum. Comparison of 5HT(2C) receptors between suicide victims and control subjects showed higher protein levels in the PFC but not the hippocampus or choroid plexus of suicide victims. However, there were no significant differences in mRNA levels between suicide victims and control subjects in these brain areas. These results suggest that 5HT(2C) receptors are richly distributed throughout the brain with the highest level in the choroid plexus and that abnormalities in protein expression of 5HT(2C) receptors in the PFC may be associated with suicide.     

Attenuated 5-HT1A receptor signaling in brains of suicide victims: involvement of adenylyl cyclase,phosphatidylinositol 3-kinase,Akt and mitogen-activated protein kinase

Positron emission tomography studies in major depression show reduced serotonin (5-HT)1A receptor antagonist-binding potentials in many brain regions including occipital cortex. The functional meaning of this observation in terms of signal transduction is unknown. We used postmortem brain samples from depressed suicide victims to examine the downstream effectors of 5-HT1A receptor activation. The diagnosis was established by means of psychological autopsy using Diagnostic and Statistical Manual of Mental Disorders (DSM) III-R criteria. Measurements of [35S]GTPgammaS binding to Galphai/o in the occipital cortex of suicide victims and matched controls revealed a blunted response in suicide subjects and a decrease in the coupling of 5-HT1A receptor to adenylyl cyclase. No significant group differences were detected in the expression levels of Galphai/o, Galphaq/11 or Galphas proteins, or in the activity of cAMP-dependent protein kinase A. Studies of a parallel transduction pathway downstream from 5-HT1A receptor activation demonstrated a decrease in the activity of phosphatidylinositol 3-kinase and its downstream effector Akt, as well as an increase in PTEN (phosphatase and tensin homolog deleted on chromosome 10), the phosphatase that hydrolyzes phosphatidylinositol 3,4,5-triphosphate. Finally, the activation of extracellular signal-regulated kinases 1 and 2 was attenuated in suicide victims. These data suggest that the alterations in agonist-stimulated 5-HT1A receptor activation in depressed suicide victims are also manifest downstream from the associated G protein, affecting the activity of second messengers in two 5-HT1A receptor transduction pathways that may have implications for cell survival.     

Up-Regulation of Immunolabeled α2A-Adrenoceptors, Gi Coupling Proteins, and Regulatory Receptor Kinases in the Prefrontal Cortex of Depressed Suicides

Abstract : Suicide and depression are associated with an increased density of α₂-adrenoceptors (radioligand receptor binding) in specific regions of the human brain. The function of these inhibitory receptors involves various regulatory proteins (G_i coupling proteins and G protein-coupled receptor kinases, GRKs), which work in concert with the receptors. In this study we quantitated in parallel the levels of immunolabeled α_2A-adrenoceptors and associated regulatory proteins in brains of suicide and depressed suicide victims. Specimens of the prefrontal cortex (Brodmann area 9) were collected from 51 suicide victims and 31 control subjects. Levels of α_2A-adrenoceptors, Gα_1/2 proteins, and GRK 2/3 were assessed by immunoblotting techniques by using specific polyclonal antisera and the immunoreactive proteins were quantitated by densitometry. Increased levels of α_2A-adrenoceptors (31-40%), Gα_1/2 proteins (42-63%), and membrane-associated GRK 2/3 (24-32%) were found in the prefrontal cortex of suicide victims and antidepressantfree depressed suicide victims. There were significant correlations between the levels of GRK 2/3 (dependent variable) and those of α_2A-adrenoceptors and Gα_1/2 proteins (independent variables) in the same brain samples of suicide victims (r = 0.56, p = 0.008) and depressed suicide victims (r = 0.54, p = 0.041). Antemortem antidepressant treatment was associated with a significant reduction in the levels of Gα_1/2 proteins (32%), but with modest decreases in the levels of α_2A-adrenoceptors (6%) and GRK 2/3 (18%) in brains of depressed suicide victims. The increased levels in concert of α_2A-adrenoceptors, Gα_1/2 proteins, and GRK 2/3 in brains of depressed suicide victims support the existence of supersensitive α_2A-adrenoceptors in subjects with major depression.     

Reduced activation and expression of ERK1/2 MAP kinase in the post-mortem brain of depressed suicide subjects

The extracellular regulated kinases (ERK) 1 and ERK2 are members of mitogen-activated protein (MAP) kinase family that play an important role in transducing extracellular signals to the nucleus and have been implicated in a broad spectrum of biological responses. To test the hypothesis that MAP kinases may be involved in depression, we examined the activation of p44/42 MAP kinase and expression of ERK1 and ERK2 in the post-mortem brain tissue obtained from non-psychiatric control subjects (n = 11) and age- and the post-mortem interval-matched depressed suicide subjects (n = 11). We observed that p44/42 MAP kinase activity was significantly decreased in the prefrontal cortical areas (Brodmann's areas 8, 9 and 10) and the hippocampus of depressed suicide subjects without any change in the cerebellum. This decrease was associated with a decrease in mRNA and protein levels of ERK1 and ERK2. In addition, the expression of MAP kinase phosphatase (MKP)2, a 'dual function' ERK1/2 phosphatase, was increased in the prefrontal cortex and hippocampus. These studies suggest that p44/42 MAP kinases are less activated in the post-mortem brain of depressed suicide subjects and this may be because of reduced expression of ERK1/2 and increased expression of MKP2. Given the role of MAP kinases in various physiological functions and gene expression, alterations in p44/42 MAP kinase activation and expression of ERK1/2 may contribute significantly to the pathophysiology of depressive disorders.     

Autoradiographic Demonstration of Increased α2-Adrenoceptor Agonist Binding Sites in the Hippocampus and Frontal Cortex of Depressed Suicide Victims

Abstract: To examine directly in the brain the status of α₂-adrenoceptors in major depression, the specific binding of the agonist [³H]UK 14304 was measured by quantitative receptor autoradiography in the hippocampus and frontal cortex of suicide victims (n = 17) with a retrospective diagnosis of depression (n = 7) or other psychiatric disorders (n = 10) as well as of matched control subjects (n = 9). In suicide victims, a significant increase in the number of α₂-adrenoceptors was found in the CA1 field (40%) and dentate gyrus (20%) of the hippocampus and in the external layers I (33%) and II (31%) of the frontal cortex, compared with that in matched controls. In depressed suicide victims, the increase in α₂-adrenoceptors in the CA1 field (57%) was significantly greater (24%, p < 0.05) than that observed in the group of suicide victims with other diagnoses (26%). In the same depressed suicide victims, the increase in cortical α₂-adrenoceptors was restricted to layer I (34%) and it was equivalent to that found in layer I (33%) of suicide victims with other diagnoses. The results indicate that suicide is associated with increases in the high-affinity state of brain α₂-adrenoceptors and that there is a pronounced localized increase of this inhibitory receptor in the hippocampus of depressed suicide victims.     

The interaction between TPH2 promoter haplotypes and clinical-demographic risk factors in suicide victims with major psychoses

Tryptophan hydroxylase isoform 2 (TPH2) is a rate-limiting enzyme in the biosynthesis of serotonin (5-HT) and is predominantly localized in the brain. Previous studies have suggested that there is an association between serotonergic dysfunction in the brain and suicidality. This study was designed to examine whether the -473T > A and -8396G > C polymorphisms of the TPH2 gene may be associated with completed suicide in subjects with major psychoses from the Stanley Foundation Brain Bank sample. TPH2 genotypes were determined in 69 subjects with a diagnosis of schizophrenia or bipolar disorder, among which 22 died by suicide. Genomic DNA was amplified by polymerase chain reaction and typed by automated methods. Both markers were found to be in Hardy-Weinberg equilibrium and in strong linkage disequilibrium. No association with history of suicide was found for either polymorphism. Haplotype analysis with EHAP showed no association between completed suicide and haplotype distribution (chi2 = 1.877; 3 df; P = 0.598). Nor was there any association between suicide and these genetic markers even when clinical-demographic factors were considered as covariates in the haplotype analysis. These findings suggest that these 5' marker haplotypes in the TPH2 gene do not influence suicidal behaviour.     

Enhanced serum cortisol response to 5-hydroxytryptophan in depression and mania

The serum cortisol responses to D, L-5-hydroxytryptophan (5-HTP), 200 mg per oral, in unmedicated depressed and manic patients, were both significantly greater than that of normal controls. The cortisol response to 5-HTP in depressed patients was significantly correlated with ratings of specific symptoms of depression. It was also greater in non-psychotic than in psychotic depressed patients as well as in those manic or depressed patients who attempted suicide compared to those who had not. In view of evidence for decreased brain serotonergic activity in depression and perhaps mania, the results suggest at least some serotonin receptors may be supersensitive in some patients with affective disorders.     

Loss-of-function mutation in tryptophan hydroxylase-2 identified in unipolar major depression

Dysregulation of central serotonin neurotransmission has been widely suspected as an important contributor to major depression. Here, we identify a (G1463A) single nucleotide polymorphism (SNP) in the rate-limiting enzyme of neuronal serotonin synthesis, human tryptophan hydroxylase-2 (hTPH2). The functional SNP in hTPH2 replaces the highly conserved Arg441 with His, which results in approximately 80% loss of function in serotonin production when hTPH2 is expressed in PC12 cells. Strikingly, SNP analysis in a cohort of 87 patients with unipolar major depression revealed that nine patients carried the mutant (1463A) allele, while among 219 controls, three subjects carried this mutation. In addition, this functional SNP was not found in a cohort of 60 bipolar disorder patients. Identification of a loss-of-function mutation in hTPH2 suggests that defect in brain serotonin synthesis may represent an important risk factor for unipolar major depression.     

Neuropeptide Y in Frontal Cortex Is Not Altered in Major Depression

Abstract: Previously, we reported a modest but significant reduction in the concentration of neuropeptide Y in frontal cortices from victims of suicide relative to age-matched natural or accidental death control subjects. The reduction in neuropeptide Y appeared to be greatest in a subgroup of victims of suicide for which there was indirect evidence of histories of depression. We pursued these initial findings in the present study by measuring neuropeptide Y concentrations in frontal cortices from natural or accidental death control subjects and from suicide victims in whom a firm diagnosis of major depression was established by psychiatric autopsy. Because several subjects with major depression had a comorbid diagnosis of alcoholism, a group of victims of suicide that had an Axis I diagnosis of alcohol dependence was also studied. No significant differences in neuropeptide Y concentrations were observed between control subjects and victims of suicide with major depression or victims of suicide with alcohol dependence. These findings do not support a role for neuropeptide Y in major depression.     

Reduced Neuropeptide Y Concentrations in Suicide Brain

Neuropeptide Y (NPY) was measured in postmortem brain tissue from victims of suicide and from individuals dying a sudden natural or accidental death (controls). Concentrations of NPY-immunoreactivity were measured by radioimmunoassay in frontal cortex (BA 10), temporal cortex (BA 22), caudate nucleus, and cerebellum. Concentrations of NPY-immunoreactivity were significantly lower in postmortem frontal cortex (-14%) and caudate nucleus (-27%) from suicide victims compared with age-matched controls. A subgroup of suicides with evidence of a history of depression revealed more robust reductions in concentrations of NPY-immunoreactivity in frontal cortex and caudate nucleus, as did four individuals who died from natural causes and also were described as having a possible history of depression. Concentrations of NPY-immunoreactivity in temporal cortex and cerebellum from victims of suicide or from the subgroup of subjects with a possible history of depression were not significantly different from those of age-matched controls. We suggest there is a deficit in the brain NPY system leading to region-specific reductions in peptide concentrations in subjects who have a history of depression.     

Altered editing of serotonin 2C receptor pre-mRNA in the prefrontal cortex of depressed suicide victims

Five adenosines within the coding sequence of the serotonin 2C receptor (5-HT2C) pre-mRNA are converted to inosines by RNA editing (named A, B, C' (E), C, and D sites). In human prefrontal cortex (PFC), the most abundant 5-HT2C mRNA sequences result from editing at the A site, or from the editing combinations AC'C, ABCD, and ABD. In suicide victims with a history of major depression, C' site editing is significantly increased, D site editing is significantly decreased, and the C site shows a trend toward increased editing. Treatment of mice with the antidepressant drug fluoxetine (Prozac) causes changes in C', C, and D site editing that are exactly opposite to those seen in suicide victims. Thus, one outcome of fluoxetine treatment may be to reverse the abnormalities in 5-HT2C pre-mRNA editing seen in depressed suicide victims.     

Substituted 3-(4-(1,3,5-triazin-2-yl)-phenyl)-2-aminopropanoic acids as novel tryptophan hydroxylase inhibitors

Tryptophan hydroxylase (TPH) is a key enzyme in the synthesis of serotonin. As a neurotransmitter, serotonin plays important physiological roles both peripherally and centrally. Here we describe the discovery of substituted triazines as a novel class of tryptophan hydroxylase inhibitors. This class of TPH inhibitors can selectively reduce serotonin levels in murine intestine after oral administration without affecting levels in the brain. These TPH inhibitors may provide novel treatments for gastrointestinal disorders associated with dysregulation of the serotonergic system, such as chemotherapy-induced emesis and irritable bowel syndrome.     

Reserpine modulates serotonin transporter mRNA levels in the rat brain

Despite recent advances in determining central serotonin (5-HT) function, the basic aspects by which serotonin neurotransmission is controlled and regulated are still not understood. Since the serotonin transporter (5-HTT) is involved in terminating the action of 5-HT that is released from the presynaptic nerve terminal, the regulation of 5-HTT may be an important step in controlling 5-HT neurotransmission at the synaptic cleft. The present study investigated the effects of reserpine administration on 5-HTT gene expression as well as on tryptophan hydroxylase (TPH) expression in the rat brain. Male Sprague-Dawley rats were injected with reserpine (10 mg/kg, i.p.) and sacrificed at 8 h, 3 days, 7 days or 21 days after the injection. Control rats were injected with saline and sacrificed either at 8 h or 21 days after the injection. The midbrain region was dissected, RNA was isolated and probed for 5-HTT expression using Northern Blotting. Data were analyzed using Super-Anova followed by post-hoc Dunnett's test. While mRNA levels for 5-HTT were unchanged at 8 h after reserpine, a significant decrease was noted at 3 days and 7 days (F=10; p<0.0001). 5-HTT mRNA levels returned to control levels by 21 days. In contrast, TPH expression was unaltered at all time points examined. The results of this study provide useful information regarding the role that the 5-HTT may be playing in the homeostatic control of 5-HT neurotransmission at the synapse.     

Optimal vitamin D spurs serotonin: 1,25-dihydroxyvitamin D represses serotonin reuptake transport (<Emphasis Type="Italic">SERT</Emphasis>) and degradation (<Emphasis Type="Italic">MAO-A</Emphasis>) gene expression in cultured rat serotonergic neuronal cell lines

Background

Diminished brain levels of two neurohormones, 5-hydroxytryptamine (5-HT; serotonin) and 1,25-dihydroxyvitamin D₃ (1,25D; active vitamin D metabolite), are proposed to play a role in the atypical social behaviors associated with psychological conditions including autism spectrum disorders and depression. We reported previously that 1,25D induces expression of tryptophan hydroxylase-2 (TPH2), the initial and rate-limiting enzyme in the biosynthetic pathway to 5-HT, in cultured rat serotonergic neuronal cells. However, other enzymes and transporters in the pathway of tryptophan metabolism had yet to be examined with respect to the actions of vitamin D. Herein, we probed the response of neuronal cells to 1,25D by quantifying mRNA expression of serotonin synthesis isozymes, TPH1 and TPH2, as well as expression of the serotonin reuptake transporter (SERT), and the enzyme responsible for serotonin catabolism, monoamine oxidase-A (MAO-A). We also assessed the direct production of serotonin in cell culture in response to 1,25D.

Results

Employing quantitative real-time PCR, we demonstrate that TPH-1/-2 mRNAs are 28- to 33-fold induced by 10 nM 1,25D treatment of cultured rat serotonergic neuronal cells (RN46A-B14), and the enhancement of TPH2 mRNA by 1,25D is dependent on the degree of neuron-like character of the cells. In contrast, examination of SERT, the gene product of which is a target for the SSRI-class of antidepressants, and MAO-A, which encodes the predominant catabolic enzyme in the serotonin pathway, reveals that their mRNAs are 51–59% repressed by 10 nM 1,25D treatment of RN46A-B14 cells. Finally, serotonin concentrations are significantly enhanced (2.9-fold) by 10 nM 1,25D in this system.

Conclusions

These results are consistent with the concept that vitamin D maintains extracellular fluid serotonin concentrations in the brain, thereby offering an explanation for how vitamin D could influence the trajectory and development of neuropsychiatric disorders. Given the profile of gene regulation in cultured RN46A-B14 serotonergic neurons, we conclude that 1,25D acts not only to induce serotonin synthesis, but also functions at an indirect, molecular-genomic stage to mimic SSRIs and MAO inhibitors, likely elevating serotonin in the CNS. These data suggest that optimal vitamin D status may contribute to improving behavioral pathophysiologies resulting from dysregulation of serotonergic neurotransmission.

     

Effect of a new potential psychotropic drug, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride,on the expression of serotonin-related genes in the mouse brain

Investigation of molecular mechanisms underlying psychotropic drug action is the main aim of molecular psychopharmacology. Previously, a new synthetic varacin analog, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine (TC-2153) was shown to produce anxiolytic and anticonvulsant effects in mice. This study investigated the effects of chronic TC-2153 administration on the expression of some serotonin-related genes in the mouse brain. The drug was administered (10 mg/kg, per os, 16 days) to adult male mice of the ASC (Antidepressant Sensitive Catalepsy) strain characterized by altered behavior and hereditary impairment of the brain serotonin system. Expression of genes encoding tryptophan hydroxylase 2 (TPH2), the key enzyme of serotonin synthesis, monoamine oxydase A (MAOA), the major serotonin-degrading enzyme, 5-HT transporter (SERT), and 5-HT_1A receptor was studied using quantitative RT-PCR. TC-2153 significantly reduced the 5-HT_1A receptor and MAOA mRNA levels in the midbrain, but did not have any effect on the expression of these genes in the frontal cortex and the hippocampus. The drug did not affect the expression of TPH2 and SERT in the midbrain. The results indicate that the brain 5-HT system is involved in the molecular basis of TC-2153 action.     

Impact of the tryptophan hydroxylase 1 gene A218C polymorphism on amygdala activity in response to affective facial stimuli in patients with major depressive disorder

Tryptophan hydroxylase-1 (TPH1) is the rate-limiting enzyme in serotonin biosynthesis, and allelic variations at the TPH1 locus have been implicated in the pathophysiology of depression. Using 1.5-Tesla functional magnetic resonance imaging, we investigated the possible relationship between TPH1 A218C polymorphism and amygdala response to negative facial stimuli in 26 right-handed female subjects with major depressive disorder (MDD). Genotyping was performed with the polymerase chain reaction. We found a significant association between A allele of the TPH1 A218C polymorphism and neural activations in response to negative facial stimuli. Subjects with the A allele of the TPH1 A218C polymorphism showed greater brain activity in the bilateral amygdala under the sad vs. the neutral condition compared with subjects homozygous for the C allele. Our results suggest that the A218C polymorphism of the TPH1 gene serves as a modulator of amygdala activity in patients with MDD.     

Brain 5-HT synthesis in the Flinders Sensitive Line rat model of depression: an autoradiographic study

Alterations of serotonin (5-HT) levels and serotonergic transmission have been associated with depression. 5-HT synthesis is an important factor of serotonergic neurotransmission that may also be altered in depression. Many studies of the relationships between brain serotonergic functions and affective disorders have been performed in different animal models. In this study, brain regional 5-HT synthesis was examined using the alpha-[(14)C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method in a genetic rat model of depression, Flinders Sensitive Line (FSL) rats, and was compared to both the Flinders Resistant Line (FRL) rats and the control Sprague-Dawley (SD) rats. The plasma concentration of free tryptophan in the FSL rats was not significantly different (p > 0.05; ANOVA and post-hoc Bonferroni correction) when compared to that of the FRL and SD rats. The FSL rats had significantly lower 5-HT synthesis (one sample two-tailed t-test on the ratio) than both the FRL and SD rats (the mean ratios were 0.78 +/- 0.12 and 0.73 +/- 0.15, respectively). Overall, the 5-HT synthesis in the FRL rats was not significantly different (p > 0.05) from that in the SD rats (one sample two-tailed t-test on the ratio and the mean ratio was 0.93 +/- 0.13). Studies of individual brain structures, such as the raphe nuclei and their many terminal areas, including the nucleus accumbens, cingulate and frontal cortex, hippocampus, amygdala, and thalamus revealed significant reductions (typically 25-50%) in 5-HT synthesis in the FSL rats compared to the non-depressive FRL and SD rats. These results suggest that significantly reduced 5-HT synthesis in the raphe nuclei and limbic areas in FSL rats may contribute to their depressive features.