Inhibition of serotonergic raphe neurons is mediated by somatodendritic 5-HT1A autoreceptors, which may be increased in depressed patients. We report an association of the C(-1019)G 5-HT1A promoter polymorphism with major depression and suicide in separate cohorts. In depressed patients, the homozygous G(-1019) allele was enriched twofold versus controls (p = 0.0017 and 0.0006 for G/G genotype and G allele distribution, respectively), and in completed suicide cases the G(-1019) allele was enriched fourfold (p = 0.002 and 0.00008 for G/G genotype and G allele distribution, respectively). The C(-1019) allele was part of a 26 bp imperfect palindrome that bound transcription factors nuclear NUDR [nuclear deformed epidermal autoregulatory factor (DEAF-1)]/suppressin and Hairy/Enhancer-of-split-5 (Drosophila) (Hes5) to repress 5-HT1A or heterologous promoters, whereas the G(-1019) allele abolished repression by NUDR, but only partially impaired Hes5-mediated repression. Recombinant NUDR bound specifically to the 26 bp palindrome, and endogenous NUDR was present in the major protein-DNA complex from raphe nuclear extracts. Stable expression of NUDR in raphe cells reduced levels of endogenous 5-HT1A protein and binding. NUDR protein was colocalized with 5-HT1A receptors in serotonergic raphe cells, hippocampal and cortical neurons, and adult brain regions including raphe nuclei, indicating a role in regulating 5-HT1A autoreceptor expression. Our data indicate that NUDR is a repressor of the 5-HT1A receptor in raphe cells the function of which is abrogated by a promoter polymorphism. We suggest a novel transcriptional model in which the G(-1019) allele derepresses 5-HT1A autoreceptor expression to reduce serotonergic neurotransmission, predisposing to depression and suicide.

Altered regulation of 5-HT1A receptors is implicated in mood disorders such as anxiety and major depression. To provide insight into its transcriptional regulation, we previously identified a novel DNA element [14 bp 5'-repressor element (FRE)] of the 5-HT1A receptor gene that mediates repression in neuronal and non-neuronal cells (Ou et al., 2000). We have now cloned a novel DNA binding protein [five' repressor element under dual repression binding protein-1 (Freud-1)] that binds to FRE to mediate repression of the 5-HT1A receptor or heterologous promoters. Freud-1 is evolutionarily conserved and contains two DM-14 basic repeats, a predicted helix-loop-helix DNA binding domain, and a protein kinase C conserved region 2 (C2)/calcium-dependent lipid binding (CalB) calcium/phospholipid binding domain. An intact CalB domain was required for Freud-1-mediated repression. In serotonergic raphe cells, overexpression of Freud-1 repressed the 5-HT1A promoter and decreased 5-HT1A receptor protein levels, whereas transfection of antisense to Freud-1 derepressed the 5-HT1A gene and increased 5-HT1A receptor protein expression. Calcium-dependent signaling blocked Freud-1-FRE binding and derepressed the 5-HT1A promoter. Treatment with inhibitors of calmodulin or CAM-dependent protein kinase reversed calcium-mediated inhibition of Freud-1. Freud-1 RNA and protein were present in raphe nuclei, hippocampus, cortex, and hypothalamus, and Freud-1 protein was colocalized with 5-HT1A receptors, suggesting its importance in regulating 5-HT1A receptors in vivo. Thus, Freud-1 represents a novel calcium-regulated repressor that negatively regulates basal 5-HT1A receptor expression in neurons and may play a role in the altered regulation of 5-HT1A receptors associated with anxiety or major depression.

Monoamine oxidase A (MAO A) degrades serotonin, norepinephrine, and dopamine and produces reactive oxygen that may cause neuronal cell death. We have previously reported that a novel transcription factor R1 (RAM2͞CDCA7L͞JPO2) inhibits the MAO A promoter and enzymatic activities. This study reports the roles of MAO A and R1 in apoptosis and proliferation. We have found that in serum starvation-induced apoptosis, p38 kinase, MAO A, and caspase-3 were increased, whereas Bcl-2 and R1 were reduced. Using a p38 kinase inhibitor, R1 overexpression, and MAO A inhibitor, we have shown that MAO A and R1 are downstream of p38 kinase and Bcl-2, but upstream of caspase-3. Inhibition of MAO A prevents cell apoptosis. This notion was further supported by the finding that serum starvation-induced apoptosis is reduced in cortical brain cells from MAO A-deficient mice compared with WT. In addition, we found that MAO A and R1 are involved in the c-Myc-induced proliferative signaling pathway in the presence of serum. Immunoprecipitation and immunohistochemistry experiments indicate that the oncogene c-Myc colocalizes with R1 and induces R1 gene expression. Using R1 overexpression, R1 small interfering RNA, and a MAO A inhibitor, we found that R1 and MAO A act upstream of cyclin D1 and E2F1. In summary, this study demonstrates the functions of MAO A and its repressor R1 in apoptotic signaling pathways.c-Myc ͉ caspase-3 ͉ p38 kinase ͉ proliferation ͉ transcription factor M onoamine oxidases (MAOs) A and B, located on the outer mitochondria membrane with a 70% amino acid sequence identity (1, 2), catalyze the oxidative deamination of biogenic and dietary amines including monoamine neurotransmitters serotonin, norepinephrine, dopamine, and phenylethylamine. MAO plays important roles in several psychiatric and neurological disorders (3). MAO exists in two forms, MAO A and MAO B. Their catalytic activity generates H 2 O 2 and nitrogen species, which are toxic products and may cause oxidative damage to mtDNA and have potential implications for apoptosis, aging, and neurodegenerative processes (4).The in vivo function of MAO A and MAO B has been studied extensively in MAO A and MAO B knockout (KO) mice (5-7). The regulation of MAO A and MAO B gene expression by extracellular stimulation, phorbol 12-myristate 13-acetate (PMA), has also been examined. PMA selectively increases MAO B but not MAO A gene expression by activation of protein kinase C and mitogen-activated protein kinase (MAPK) signaling pathways (8).Ample evidence has indicated an important role for MAO A in apoptosis. MAO A expression has been shown to increase after the depletion of neurotrophic factor mediated by p38 kinase in PC12 cells (9). A MAO A inhibitor, clorgyline, was able to protect cells from apoptosis induced by serum starvation in human melanoma m14 cells (10). More recently, MAO A has been found to be a target of a dopaminergic neurotoxin, N-methyl-(R)-salsolinol, which leads to apoptosis in a human neuroblastoma SH-SY5Y cell line (11). This apoptotic activity co...

Negative regulation of neuronal serotonin (5-HT1A) receptor levels by glucocorticoids in vivo may contribute to depression. Both types I (mineralocorticoid) and II (glucocorticoid) receptors (MR and GR, respectively) participate in corticosteroid-induced transcriptional repression of the 5-HT1A gene; however, the precise mechanism is unclear. A direct repeat 6-base pair glucocorticoid response element (GRE) half-site 5-TGTCCT separated by 6 nucleotides was conserved in human, mouse, and rat 5-HT1A receptor promoters. In SN-48 neuronal cells that express MR, GR, and 5-HT1A receptors, deletion or inactivation of the nGRE (negative GRE) eliminated negative regulation of the rat 5-HT1A or heterologous promoters by corticosteroids, whereas its inclusion conferred corticosteroid-induced inhibition to a heterologous promoter. Bacterially expressed recombinant MR and GR preferentially bound to the nGRE as a heterodimer, as identified in nuclear extracts of MR/GR-transfected COS-7 cells, and with higher affinity than MR or GR homodimers. In SN48 and COS-7 cells, concentration-dependent coactivation of MR and GR was required for maximal inhibitory action by corticosteroids and was abrogated in the L501P-GR mutant lacking DNA binding activity. Corticosteroid-mediated transcriptional inhibition was greater for MR/GR in combination than for MR or GR alone. These data represent the first identification of an nMRE/GRE and indicate that heterodimerization of MR and GR mediates direct corticosteroid-induced transrepression of the 5-HT1A receptor promoter.

Monoamine oxidase (MAO)A Monoamine oxidase (MAO)2 is located on outer membranes of mitochondria in neuronal, glial, and other cells. It catalyzes the oxidative deamination of monoamine neurotransmitters such as serotonin, norepinephrine, dopamine, and phenylethylamine (1). MAO exists in two isoforms, MAO A and MAO B. MAO A preferentially oxidizes serotonin (5-hydroxytryptamine), norepinephrine, and dopamine and is irreversibly inhibited by low concentration of clorgyline (2). MAO B preferentially oxidizes phenylethylamine and benzylamine and is irreversibly inactivated by low concentrations of pargyline and deprenyl (3). MAO A and B are coded by different genes (4) closely linked on the X chromosome, Xp11.2-11.4 (5). Both genes consist of 15 exons with identical exonintron organization (6) and both are regulated by Sp1 factors; however, there are some differences in their cis-elements (7-9). The core promoter of MAO A consists of Sp1-binding sites that are activated by Sp1 (7) and repressed by a novel repressor R1 (RAM2/CDCA7L) (8). This core promoter has bi-directional activity (7). In addition, a functional polymorphism of a 30-bp repeat sequence has been identified and located ϳ1.2 kb upstream of the MAO A translation start site (10). The human MAO B promoter contains two clusters of overlapping Sp1 sites separated by a CACCC element (11). An Sp1-like transcription factor, transforming growth factor-␤-inducible early gene, also activates the MAO B promoter via overlapping Sp1 sites (12). The expression of MAO B is regulated by protein kinase C and the MAPK signaling pathway (13).A mutation in the human MAO A gene, which results in no enzymatic activity, is associated with aggressive behavior in males in a Dutch family (14). MAO A knock-out (KO) mice showed aggressive behavior in males (15), but MAO B KO mice did not show aggressive behavior (16). Interestingly, a spontaneous mutation of the MAO A gene in MAO B KO mice resulted in MAO A/B double KO mice, which showed hyperreactivity and anxiety-like behavior (12).Steroid hormones are involved in the regulation of many functions in which MAO also plays an important roles such as response to stress, behavioral adaptation and mood (17). Significant hypersecretion of glucocorticoids has been shown to be associated with depression (18). The synthetic glucocorticoid, dexamethasone, increases MAO A activity in human fibroblasts (19) and rat brain frontal cortex (20). Both anti-glucocorticoid agents (21) and MAO A inhibitors (22, 23) have been used in the treatment of depression. Androgens have also been shown to increase MAO activity in the rat brain (24).The biological action of glucocorticoids and androgens are mediated through their respective receptors, glucocorticoid receptor (GR) and androgen receptor (AR). GR and AR bind to derivatives of a common response element, i.e. the consensus glucocorticoid response element (GRE), 5Ј-GGTACAnnn-

The serotonergic neurons of the raphe nuclei are the primary site of serotonin synthesis in the brain. They send projections to a wide variety of brain regions, including the hippocampus, cortex, limbic system, and hypothalamus (1). Activation of postsynaptic receptors in the above regions is associated with serotonergic regulation of memory, motivation, emotion, neuroendocrine stress response, etc. (2-4). The activity of serotonergic neurons of the raphe nuclei is regulated in part by presynaptic autoreceptors. The 5-HT1A autoreceptor is located at the cell body and dendrites of raphe serotonergic neurons (5, 6) and mediates negative feedback inhibition of the firing rate through recurrent activation of potassium channels via pertussis toxin-sensitive G proteins (7) to decrease serotonin release. Thus the 5-HT1A receptor plays a major role in controlling serotonergic outflow to the wide variety of brain regions that are innervated by the raphe nuclei.Abnormal regulation of 5-HT1A receptor expression is implicated in depression and anxiety disorders. 5-HT1A receptor knockout mice display increased anxiety-related behaviors (8 -10), suggesting that a loss of the 5-HT1A autoreceptors is correlated with symptoms of anxiety (3). On the other hand, 5-HT1A receptor levels are increased in the midbrain of suicide victims with major depression compared with nondepressed suicides (11). Down-regulation of the 5-HT1A autoreceptor by antidepressants (12, 13) disinhibits action potential firing of the raphe neuron, thereby enhancing serotonergic neurotransmission (14 -17). The prolonged (2-3 week) time course required for antidepressant action suggests an alteration in transcriptional activity of the 5-HT1A receptor.To investigate the mechanisms that regulate cell-specific and basal regulation of the 5-HT1A receptor, we have identified the transcriptional start site and examined the regulation of the transcriptional activity of a 2.719-kb 1 fragment of the rat 5-HT1A receptor gene in several cell lines, including 5-HT1A receptor-positive RN46A raphe and SN48 septal cells (18 -20) and receptor-negative L6 myoblast and C6 glioblastoma cells (21). We identified a region of the rat 5-HT1A receptor gene located upstream of an ubiquitously active promoter region that reduces transcriptional activity. In the present study, we have identified a 14-bp element in the 5Ј-flanking region of the 5-HT1A gene that mediates transcriptional repression in raphe cells, but is dispensable in receptor-negative cells where an adjacent 12-bp element maintains repression of the gene. In contrast to the single repressor-DNA complex present in 5-HT1A receptor-expressing cells that may modulate basal levels of receptor expression, the presence of two protein-DNA complexes in receptor-negative cell lines provides a dual mechanism to repress 5-HT1A receptor expression.

Genetic studies of delinquent and criminal behavior are rare in spite of the wide recognition that individuals may differ in their propensity for delinquency and criminality. Using 2524 participants in Add Health in the United States, the present study demonstrates a link between the rare 2 repeat of the 30-bp VNTR in the MAOA gene and much higher levels of self-reported serious and violent delinquency. The evidence is based on a statistical association analysis and a functional analysis of MAOA promoter activity using two human brain-derived cell lines: neuroblastoma SH-SY5Y and human glioblastoma 1242-MG. The association analysis shows that men with a 2R report a level of serious delinquency and violent delinquency in adolescence and young adulthood that were about twice (CI: (0.21, 3.24), P ¼ 0.025; and CI: (0.37, 2.5), P ¼ 0.008 for serious and violent delinquency, respectively) as high as those for participants with the other variants. The results for women are similar, but weaker. In the functional analysis, the 2 repeat exhibits much lower levels of promoter activity than the 3 or 4 repeat.

Background:The function of KLF11/TIEG2 under stressful conditions is undefined. Results: KLF11 increases brain MAO expression through its promoter and a chromatin partner, which can be enhanced by stress. Conclusion: This is the first elucidation of mechanisms underlying stress-induced KLF11-MAO up-regulation. Significance: This novel KLF11-MAO pathway may play an important role in stress-related brain disorders.