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.

Regulation of ER stress proteins, such as the 78-kilodalton glucose regulated protein (GRP78) by chronic treatment with mood stabilizing drugs suggests that this family of proteins may be involved in the pathophysiology of mood disorders. Indeed, increased levels of GRP78, GRP94, and calreticulin, a third member of the ERCurrent theories of the pathophysiology of mood disorders suggest that prolonged increases in glucocorticoid levels, likely the result of prolonged environmental stress, lead to biochemical changes in cortical and limbic neurons that leave individuals vulnerable to depression (Duman et al. 1997). A number of studies have found evidence of neuronal atrophy and loss of hippocampal neurons in response to stress (Magarinos et al. 1996;Uno et al. 1989;Sapolsky et al. 1985), and a report of reduced hippocampal volumes in elderly patients with histories of depression suggests that analogous processes may occur in individuals with depression (Sheline et al. 1996). Some models of mood disorders (Hyman and Nestler 1996; Post 1992) have attempted to incorporate the notion of compensatory processes into an understanding of the pathophysiologic changes that occur in patients with mood disorders, but few studies have tested these notions empirically (Post 1992). Part of the difficulty has been identifying neuroprotective mechanisms that may be of relevance for mood disorders.Recently, we found that treatment with the mood stabilizing drugs, valproate and carbamazepine, increased expression of the 78-kilodalton glucose-regulated protein (GRP78) . GRP78 is a member of the ER stress protein family that includes GRP94 and calreticulin, all of which function as Ca 2 ϩ binding proteins and molecular chaperones to assist in the regulation of protein folding (Gething 1997). These proteins have neuroprotective properties, are induced by seizures and ischemic damage, and may be involved in Alzheimer's disease pathology (Yu et al. 1999;Lowenstein et al. 1994;Hamos et al. 1991).Increased expression of ER stress proteins by mood stabilizing drugs suggest that these proteins may be in- METHODS Postmortem brain tissue was obtained from the StanleyFoundation Neuropathology Consortium (4 groups of n ϭ 15 age-and sex-matched subjects, i.e., subjects with bipolar disorder (BD), subjects with Major Depressive Disorder (MDD), subjects with schizophrenia (SCZ), and non-psychiatric, non-neurologic comparison subjects) (Johnston et al. 1997). Details on dissection and clinical characteristics have been previously published (Dowlatshahi et al. 1998(Dowlatshahi et al. , 1999. Briefly, all medical records for psychiatric cases were reviewed by two psychiatrists. Diagnosis was determined according to DSM-IV criteria. If there was disagreement about the diagnosis, a third psychiatrist read the records. If a diagnosis could not be established, a family member was then interviewed. All interviewed relatives were first degree except for two (one aunt and one father-in-law). Similarly, a clinical interview was performed with a family member...