Polymorphisms in the regulatory region of the human serotonin 5-HT2A receptor gene (HTR2A) influence gene expression.

BACKGROUND: Genomic variation in the regulatory region of the serotonin (5-HT) 2A receptor gene (HTR2A) may contribute to altered levels of 5-HT2A receptor and to psychiatric disease. METHODS: Frequency and linkage disequilibrium (LD) were determined for promoter single nucleotide polymorphisms (SNPs) -1438A/G, -1420C/T, and -783A/G in 156 subjects. Functional relevance of -1438A/G and -783A/G was assayed in vitro using a luciferase reporter assay and ex vivo using quantitative real time polymerase chain reaction in a set of human fibroblast cell lines. RESULTS: Significant LD was observed between SNPs -1438A/G and -783A/G. In vitro assays showed no significant differences in promoter activity between the A- and G-allele of -1438 locus when expressed with the major alleles at -1420C/T and -783A/G; however, when the minor allele G at -783 was expressed with G-allele at -1438, promoter activity was significantly decreased. 5-HT2A receptor mRNA expression in human fibroblast cell lines confirmed that -783A/G polymorphism significantly modified the effects of -1438A/G SNP. CONCLUSIONS: Our results demonstrate that SNP -783A/G modifies the effects of the major SNP -1438A/G. Future studies examining the association of -1438A/G polymorphism with diseases and 5-HT2A receptor expression analyses should account for this epistasis.

Detection of an mRNA polymorphism by differential display.

Differential display technology was utilized to compare programs of gene expression in primary cultures of human skin fibroblasts from normal volunteers and patients diagnosed with melancholic depression. Polymorphic transcripts of a single gene differing by one tandem repeat sequence of four nucleotides (TGAT) in the 3' noncoding region were detected.

Decreased serotonin 5-HT2A receptor-stimulated phosphoinositide signaling in fibroblasts from melancholic depressed patients.

Abnormalities in serotonin (5-HT) receptors and 5-HT receptor-mediated signal transduction systems have been widely reported in mood disorders. This study was intended to evaluate 5-HT(2A) receptor-coupled activation of phosphatidylinositol (PI) hydrolysis in subtypes of depression. Samples for fibroblast culture were obtained from patients with major depression with or without melancholia, and normal controls. Dose response curves were determined for 5-HT-induced PI hydrolysis. PI response was determined for bradykinin and l-alpha-lysophosphatidic acid (LPA), alternative Gq-coupled receptor agonists. [125I]LSD binding for 5-HT(2A) also was conducted. Finally, Western blot analysis was performed for phospholipase Cbeta1 (PLCbeta1) and Galpha(q/11) proteins. The maximum PI response observed with 5-HT was significantly lower in melancholics but not nonmelancholic patients relative to controls. Activation of PI hydrolysis by bradykinin and LPA was not reduced in melancholic vs melancholics and controls; responses to both agonists actually were increased in the melancholic group. [125I]LSD binding, PLCbeta1, and Galpha(q/11) protein levels did not differ between groups. The data raise the possibility that the reduced 5-HT(2A) receptor-induced PI hydrolysis is intrinsic to the receptor itself or its coupling to Gq protein, and is not related to altered availability of the 5-HT(2A) receptor, Gq or PLC.

Differential expression of pentraxin 3 in fibroblasts from patients with major depression.

In this study, differential display technology was used to compare gene expression in cultured fibroblasts from patients with major depression, melancholic subtype vs nonmelancholic depressives and normal volunteer controls. Genes differentially expressed in depressives and normals included an overexpressed 269 bp sequence tag showing approximately 95% identity with the Homo sapiens long pentraxin 3 (PTX3) gene sequence in the 3' noncoding region. The 269 bp complimentary DNA probe hybridized with the 1.9 kb PTX3 mRNA. The densitometric analysis of slot blots showed that the mean steady-state mRNA level of PTX3 was 3.5-fold higher in the melancholic group as compared to that in normal controls and in nonmelancholic depressives (n=8, all groups). Incubation experiments were then conducted: confluent fibroblasts from melancholics and normal volunteers were incubated with isoproterenol 1 microM, dexamethasone (DEX) 500 nM, and interleukin 1beta (IL-1beta) 50 ng/ml, for 0, 0.5, 1, 4, and 24 h. mRNA was isolated and quantitated using Northern blot analysis. Isoproterenol produced no significant change in PTX3 expression; DEX produced a significant increase at 4 and 24 h; IL-1beta induced an increase in both the groups that peaked at 4 h, declining to near basal levels at 24 h. There were no differences between melancholics or controls in mean change or maximum response with either DEX or IL-1beta. The results are consistent with the reported effects of IL-1beta on PTX3 expression in mouse brain. The results are of potential importance because PTX3 is a member of the long pentraxin subfamily of acute-phase proteins, which is inducible by IL-1beta, and may play roles in neuroimmunity and neuroprotection.

Pharmacological actions of the antidepressant venlafaxine beyond aminergic receptors.

The present study examines the effects of the antidepressant venlafaxine, a dual amine reuptake inhibitor, on (a) in vivo regulation of the densities of high- and low-affinity dihydroalprenolol (DHA) binding sites in the cortex of normal and reserpinized Sprague-Dawley rats and (b) targets beyond the beta adrenoceptor. While venlafaxine (30 mg/kg i.p. b.i.d.) administered for 4 d did not alter the DHA-binding parameters in the cortex of normal rats, it significantly reduced, in reserpinized animals, the number of up-regulated low-affinity sites (R(L)) which have been tentatively identified as serotonin(1B) sites. The drug did not influence the up-regulated high-affinity (R(H)) DHA-binding sites (beta-adrenoceptor sites). Venlafaxine failed to alter the up-regulated R(L) sites in brains of rats depleted of serotonin (5-HT) by p-chlorophenylalanine (PCPA) indicating that the normalization by venlafaxine of the up-regulated R(L) receptor population is mediated by increased synaptic 5-HT. Venlafaxine, given for a short period of time, thus mimicked the action of fluoxetine. While venlafaxine (20 mg/kg i.p. b.i.d.) given for 10 d did not change protein kinase A activity as assessed by the phosphorylation of kemptide in the 900 g supernatant or particulate fractions, the drug significantly reduced phosphorylated cAMP response-element binding protein (CREB-P) in nuclear lysates of cortex after chronic but not acute administration. Depletion of 5-HT by PCPA did not alter the venlafaxine-induced change in nuclear CREB-P. Lastly, analysis of reverse transcribed cortical CREB mRNA by competitive PCR indicated that the mean steady-state levels of CREB mRNA in venlafaxine vs. saline-treated animals were not significantly different. Therefore, since the phosphorylation status of CREB determines its transcriptional activity the reduction of nuclear CREB-P may be venlafaxine's most relevant action beyond the adrenoceptor.

Cyclic AMP-dependent protein kinase in subtypes of major depression and normal volunteers.

We have shown a reduction in beta adrenoceptor-linked, cyclic AMP-dependent protein kinase [protein kinase A (PKA)] activity in fibroblasts of patients with major depression with melancholic features relative to normal volunteers. We evaluated a group of 35 patients with major depression subtyped by DSM-IV criteria as melancholic, atypical, and those not meeting either subtype designation ('non-subtyped') and 21 normal volunteers to ascertain whether or not the PKA activity abnormality was specific to melancholia. The melancholics showed marked reduction in cyclic AMP-stimulated PKA activity relative to normal volunteers. Although the atypicals were statistically significantly lower, almost all fell into the range for the normals. The non-subtyped group fell between the atypicals and the melancholics. Basal activity was significantly lower in atypical and melancholic groups. The data suggest that reduced PKA activity is consistently found in melancholic major depression and may not be seen with other depressive subtypes.

Signal transduction abnormalities in melancholic depression.

Intracellular signal transduction cascades, particularly those linked to protein kinases A (PKA) and C (PKC), have been implicated in mood disorders. This study examined the activity of PKA and PKC, as well as levels of PKA regulatory (R) and catalytic (C) subunit proteins, in fibroblasts cultured from skin biopsies from patients with major depression, melancholic subtype, in contrast to non-melancholic depressives and controls (n = 12 each group). PKA activity was determined as a function of the transfer of 32P to a target polypeptide, Kemptide. R and C subunit expression was assayed in the melancholic depressed and normal control groups by Western blots. In a separate experiment, the degree of phosphorylation of the endogenous substrate cAMP response element-binding protein (CREB) was estimated in samples from melancholic and non-melancholic patients and normal controls (n = 8 each) after incubation with isoproterenol or phorbol ester, which activate PKA and PKC respectively. Melancholics had significantly reduced phosphorylation of Kemptide in contrast to non-melancholics and controls. This was associated with lower levels of PKA RII alpha, C alpha, and C beta subunit isoform proteins, but not RI alpha, RI beta, or RII beta. Furthermore, activation of both PKA and PKC was associated with reduced CREB-P in melancholics relative to normal controls. Finally, PKA activity was found to correlate positively with Hamilton depression scores after 16 weeks of treatment with serotonin reuptake inhibitor antidepressants. These data further implicate signal transduction abnormalities in melancholic major depression, particularly PKA and PKC. This suggests an abnormality of factors controlling the expression or degradation of these enzymes.