Paul R. Albert

Impaired Repression at a 5-Hydroxytryptamine 1A Receptor Gene Polymorphism Associated with Major Depression and Suicide

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.

5-HT1A Receptors, Gene Repression, and Depression: Guilt by Association

The serotonin system is implicated in major depression and suicide and is negatively regulated by somatodendritic 5-HT1A autoreceptors. Desensitization of 5-HT1A autoreceptors is implicated in the 2- to 3-week latency for antidepressant treatments. Alterations in 5-HT1A receptor levels are reported in depression and suicide, and gene knockout of the 5-HT1A receptor results in an anxiety phenotype, suggesting that abnormal transcriptional regulation of this receptor gene may underlie these disorders. The 5-HT1A receptor gene is negatively regulated in neurons by repressors including REST/NRSF, Freud-1, NUDR/Deaf-1, and Hes5. The association with major depression, suicide, and panic disorder of a new functional 5-HT1A polymorphism at C(-1019)G that selectively blocks repression of the 5-HT1A autoreceptor by NUDR further suggests a causative role for altered regulation of this receptor in predisposition to mental illness. The authors review evidence that altered transcription of the 5-HT1A receptor can affect the serotonin system and limbic and cortical areas, leading to predisposition to depression. NEUROSCIENTIST 10(6):575-593, 2004. DOI:

Association of the C(−1019)G 5-HT1A functional promoter polymorphism with antidepressant response

Antidepressants, such as serotonin or noradrenaline reuptake inhibitors (e.g. fluoxetine, nefadozone) or 5-HT1A agonists (flibanserin), desensitize the 5-HT1A autoreceptor, which may contribute to their clinical efficacy. The 5-HT1A receptor gene is repressed by NUDR/DEAF-1 in raphe cells at the C-, but not at the G-allele of the C(-1019)G polymorphism that is associated with major depression and suicide. Depressed patients (n=118) were treated with antidepressants including fluoxetine or nefadozone combined with pindolol or flibanserin alone. The severity of depression was assesssed using the Hamilton Rating Scale for Depression. Although patients had similar severity initially, those with the homozygous G(-1019) genotype responded significantly less to flibanserin (p=0.039) and in pooled antidepressant treatment groups (p=0.0497) and were approximately twice as likely to be non-responders as those with the C(-1019)C genotype. These results implicate the C(-1019)G 5-HT1A gene polymorphism as a potential marker for antidepressant response, suggesting a role for repression of the 5-HT1A gene.

Role of Cdk5-Mediated Phosphorylation of Prx2 in MPTP Toxicity and Parkinson's Disease

We reported previously that calpain-mediated Cdk5 activation is critical for mitochondrial toxin-induced dopaminergic death. Here, we report a target that mediates this loss. Prx2, an antioxidant enzyme, binds Cdk5/p35. Prx2 is phosphorylated at T89 in neurons treated with MPP+ and/or MPTP in animals in a calpain/Cdk5/p35-dependent manner. This phosphorylation reduces Prx2 peroxidase activity. Consistent with this, p35-/- neurons show reduced oxidative stress upon MPP+ treatment. Expression of Prx2 and Prx2T89A, but not the phosphorylation mimic Prx2T89E, protects cultured and adult neurons following mitochondrial insult. Finally, downregulation of Prx2 increases oxidative stress and sensitivity to MPP+. We propose a mechanistic model by which mitochondrial toxin leads to calpain-mediated Cdk5 activation, reduced Prx2 activity, and decreased capacity to eliminate ROS. Importantly, increased Prx2 phosphorylation also occurs in nigral neurons from postmortem tissue from Parkinsons disease patients when compared to control, suggesting the relevance of this pathway in the human condition.

APAF1 is a key transcriptional target for p53 in the regulation of neuronal cell death

p53 is a transcriptional activator which has been implicated as a key regulator of neuronal cell death after acute injury. We have shown previously that p53-mediated neuronal cell death involves a Bax-dependent activation of caspase 3; however, the transcriptional targets involved in the regulation of this process have not been identified. In the present study, we demonstrate that p53 directly upregulates Apaf1 transcription as a critical step in the induction of neuronal cell death. Using DNA microarray analysis of total RNA isolated from neurons undergoing p53-induced apoptosis a 5–6-fold upregulation of Apaf1 mRNA was detected. Induction of neuronal cell death by camptothecin, a DNA-damaging agent that functions through a p53-dependent mechanism, resulted in increased Apaf1 mRNA in p53-positive, but not p53-deficient neurons. In both in vitro and in vivo neuronal cell death processes of p53-induced cell death, Apaf1 protein levels were increased. We addressed whether p53 directly regulates Apaf1 transcription via the two p53 consensus binding sites in the Apaf1 promoter. Electrophoretic mobility shift assays demonstrated p53–DNA binding activity at both p53 consensus binding sequences in extracts obtained from neurons undergoing p53-induced cell death, but not in healthy control cultures or when p53 or the p53 binding sites were inactivated by mutation. In transient transfections in a neuronal cell line with p53 and Apaf1 promoter–luciferase constructs, p53 directly activated the Apaf1 promoter via both p53 sites. The importance of Apaf1 as a p53 target gene in neuronal cell death was evaluated by examining p53-induced apoptotic pathways in primary cultures of Apaf1-deficient neurons. Neurons treated with camptothecin were significantly protected in the absence of Apaf1 relative to those derived from wild-type littermates. Together, these results demonstrate that Apaf1 is a key transcriptional target for p53 that plays a pivotal role in the regulation of apoptosis after neuronal injury.

G protein specificity: Traffic direction required

This review focuses on the coupling specificity of the Galpha and Gbetagamma subunits of pertussis toxin (PTX)-sensitive G(i/o) proteins that mediate diverse signaling pathways, including regulation of ion channels and other effectors. Several lines of evidence indicate that specific combinations of G protein alpha, beta and gamma subunits are required for different receptors or receptor-effector networks, and that a higher degree of specificity for Galpha and Gbetagamma is observed in intact systems than reported in vitro. The structural determinants of receptor-G protein specificity remain incompletely understood, and involve receptor-G protein interaction domains, and perhaps other scaffolding processes. By identifying G protein specificity for individual receptor signaling pathways, ligands targeted to disrupt individual pathways of a given receptor could be developed.

Heterodimerization of mineralocorticoid and glucocorticoid receptors at a novel negative response element of the 5-HT1A receptor gene.

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.

Differential Roles of Nuclear and Cytoplasmic Cyclin-Dependent Kinase 5 in Apoptotic and Excitotoxic Neuronal Death

Cyclin-dependent kinase 5 (cdk5) is a member of the cyclin-dependent kinase family whose activity is localized mainly to postmitotic neurons attributable to the selective expression of its activating partners p35 and p39. Deregulation of cdk5, as a result of calpain cleavage of p35 to a smaller p25 form, has been suggested to be a central component of neuronal death underlying numerous neurodegenerative diseases. However, the relevance of cdk5 in apoptotic death that relies on the mitochondrial pathway is unknown. Furthermore, evidence that cdk5 can also promote neuronal survival has necessitated a more complex understanding of cdk5 in the control of neuronal fate. Here we explore each of these issues using apoptotic and excitotoxic death models. We find that apoptotic death induced by the DNA-damaging agent camptothecin is associated with early transcription-mediated loss of p35 and with late production of p25 that is dependent on Bax, Apaf1, and caspases. In contrast, during excitotoxic death induced by glutamate, neurons rapidly produce p25 independent of the mitochondrial pathway. Analysis of the localization of p35 and p25 revealed that p35 is mainly cytoplasmic, whereas p25 accumulates selectively in the nucleus. By targeting a dominant-negative cdk5 to either the cytoplasm or nucleus, we show that cdk5 has a death-promoting activity within the nucleus and that this activity is required in excitotoxic death but not apoptotic death. Moreover, we also find that cdk5 contributes to pro-survival signaling selectively within the cytoplasm, and manipulation of this signal can modify death induced by both excitotoxicity and DNA damage.

Transcriptional regulation at a HTR1A polymorphism associated with mental illness

The serotonin-1A (5-HT1A) receptor serves as a hub to regulate the activity and actions of the serotonin system, and is expressed both as a presynaptic autoreceptor on raphe neurons, and as a major postsynaptic receptor in hippocampal, cortical, and hypothalamic regions involved in mood, emotion and stress response. As such, the level of expression of 5-HT1A receptors is implicated in the development of anxiety and depression phenotypes. This review focuses on the C(-1019)G (rs6295) promoter polymorphism of the 5-HT1A receptor gene (HTR1A) and its effect on the activity of transcription factors that recognize the C-allele, including Deaf-1, Hes1 and Hes5; its effects on 5-HT1A receptor expression in pre- and postsynaptic areas; as well as its implication in early postnatal development and adult neurogenesis in the hippocampus and cortex. Although several studies have now replicated the association of the G-allele with depression, panic disorder, neuroticism, and reduced response to antidepressant or antipsychotic treatment, ethnic, disease and genetic heterogeneity among subjects in different studies may obscure such associations. Gene-gene interaction studies suggest that the 5-HT1A receptor G(-1019) allele is a risk allele which could be used as a marker for depression and related mood disorders. Finally, association of the G(-1019) allele with increased raphe 5-HT1A binding potential, increased amygdala reactivity to emotional stimuli, and reduced amygdala volume, particularly in disease states, suggests a functional role for the C(-1019)G site in 5-HT1A receptor dys-regulation and predisposition to mental illness.

The 5-HT1A receptor : signaling, desensitization, and gene transcription

The hypothesis that antianxiety or antidepressant agents (e.g., 5-HT1A agonists, 5-HT uptake blockers) exert their clinical action via enhancement of serotonergic neurotransmission due to desensitization of 5-HT1A autoreceptors predicts that regulation of this receptor plays a crucial role in the therapeutic actions of these agents. A multidisciplinary strategy is described for the characterization of the 5-HT1A receptor at the level of cellular signaling mechanisms and genetic regulation, using heterologous expression of the cloned receptor in cell lines, site-directed mutagenesis, isolation of receptor-positive neuronal cell lines, and promoter analysis of the 5-HT1A receptor gene. These analyses will yield new insights into the possible mechanisms down-regulation of 5-HT1A receptor signaling, and may suggest novel sties of inherent defect involved in anxiety syndromes or major depression.