D. Hal Manier

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.

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.

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 venlafaxines most relevant action beyond the adrenoceptor.

β-Adrenoceptor-linked protein kinase a (PKA) activity in human fibroblasts from normal subjects and from patients with major depression

Human fibroblasts from normal subjects and from patients with major depression are cultured and their β-adrenoreceptor-cyclic AMP-protein kinase A (PKA) system characterized. The results indicate that the β-adrenoreceptor-mediated activation of PKA in the 900 g supernatant fraction of human fibroblasts is mediated via β-adrenoreceptors. The activation of PKA by isoproterenol is very rapid with maximal stimulation occurring at 5 seconds. The time course of PKA activation by isoproterenol in fibroblasts from patients with major depression is identical to that in fibroblasts from normal subjects but the magnitude of activation is significantly reduced in fibroblasts from patients with major depression. Dose-response curves on cyclic AMP mediated activation of PKA confirmed the previously reported reduction in activation of PKA in patients with major depression but demonstrated that this reduction occurs without a change in the EC50 values of cyclic AMP (approximately 20 nmol/L). The blunted β-adrenoceptor-linked PKA responses in patients with major depression occur without a change in the expression of the PKA catalytic subunit Cα. The studies suggest that the β-adrenoceptor-coupled adenylate cyclase PKA system in human fibroblasts may represent a valid model to explore possible abnormalities in the fine tuning of the β-adrenergic transduction cascade in patients with affective disorders.

Protein kinases A and C in post-mortem prefrontal cortex from persons with major depression and normal controls

Major depression (MDD) is a common and potentially life-threatening condition. Widespread neurobiological abnormalities suggest abnormalities in fundamental cellular mechanisms as possible physiological mediators. Cyclic AMP-dependent protein kinase [also known as protein kinase A (PKA)] and protein kinase C (PKC) are important components of intracellular signal transduction cascades that are linked to G-coupled receptors. Previous research using both human peripheral and post-mortem brain tissue specimens suggests that a subset of depressed patients exhibit reduced PKA and PKC activity, which has been associated with reduced levels of specific protein isoforms. Prior research also suggests that specific clinical phenotypes, particularly melancholia and suicide, may be particularly associated with low activity. This study examined PKA and PKC protein levels in human post-mortem brain tissue samples from persons with MDD (n=20) and age- and sex-matched controls (n=20). Specific PKA subunits and PKC isoforms were assessed using Western blot analysis in post-mortem samples from Brodmann area 10, which has been implicated in reinforcement and reward mechanisms. The MDD sample exhibited significantly lower protein expression of PKA regulatory Ialpha (RIalpha), PKA catalytic alpha (Calpha) and Cbeta, PKCbeta1, and PKCepsilon relative to controls. The melancholic subgroup showed low PKA RIalpha and PKA Cbeta, while the portion of the MDD sample who died by suicide had low PKA RIalpha and PKA Calpha. These data continue to support the significance of abnormalities of these two key kinases, and suggest linkages between molecular endophenotypes and specific clinical phenotypes.

5,7-Dihydroxytryptamine-induced lesions of serotonergic neurons and desipramine-induced down-regulation of cortical beta adrenoceptors: A re-evaluation

Studies on mechanisms of desipramine (DMI) induced desensitization of the norepinephrine (NE) receptor coupled adenylate cyclase system in brain have shown that the endogenous agonist NE plays a pivotal role for both the development of subsensitivity and the down-regulation of beta adrenoceptors [l-4]. Moreover, an intact serotonergic neuronal input is co-required for the down-regulation of central beta adrenoceptors by tricyclic antidepressants [5-71, monoamine oxidase (MAO) inhibitors, and electroconvulsive treatment [8]. In animals with a reduced synaptic availability of serotonin (WIT), cortical beta adrenoceptors display a marked decrease in agonist affinity as determined in competition-binding studies [9, lo], with no significant changes occurring in the Kd values of antagonist binding. Since the previously reported lcso values for isoproterenol were derived from rather shallow displacement curves [9] and the density of beta adrenoceptors was determined by Scatchard analysis of antagonist binding, information on high and low agonist affinity of beta adrenoceptors is desirable but not available. Consequently, we have re-evaluated the effect of chronic treatment with DMI and of specific lesions of serotonergic neurons with 5,7dihydroxytryptamine (5,7-DHT) on beta adrenoceptors in rat cortex by using non-linear regression analysis of competition-binding curves.

The ‘serotonin/norepinephrine link’ beyond the β adrenoceptor

Abstract C6 rat glioma cells were utilized as a model system to probe the ‘serotonin/norepinephrine link’ at the level of preproenkephalin (PPE) gene expression. The β adrenoceptor mediated increase in PPE mRNA was attenuated by the selective β1 adrenoceptor antagonist metoprolol which blocked the isoproterenol induced cyclic AMP generation by 97%. The subtype nonspecific antagonist propranolol blocked both the isoproterenol induced increase in cyclic AMP and the increase in the PPE mRNA steady-state levels. Serotonin (5-HT) had no effect on the density of β adrenoceptors or their down-regulation by isoproterenol and did not alter the PPE gene expression in the absence of the β signal. However, 5-HT significantly deamplified the β signal mediated enhancement of the PPE mRNA thus indicating that the aminergic link occurs beyond the β adrenoceptor.

The serotonin/noradrenaline-link in brain

SummaryThe present studies were undertaken to assess the role of noradrenaline (NA) and serotonin (5HT) in the regulation of the NA receptor coupled adenylate cyclase system and its alteration by desipramine (DMI) in brain structures with or without noradrenergic neuronal projections. In contrast to cortex and limbic forebrain, where chronic DMI administration caused subsensitivity of the NA sensitive adenylate cyclase linked to a down-regulation of beta adrenoceptors, the drug failed to alter the NA receptor coupled adenylate cyclase system in the striatum. Selective lesions of serotonergic axons with 5,7-dihydroxytryptamine caused a significant increase in the density of beta adrenoceptors in cortex, limbic forebrain and striatum and prevented the down-regulation by DMI of beta adrenoceptors in cortex and limbic forebrain while the responsiveness of the NA sensitive adenylate cyclase was reduced to the same extent as in sham-lesioned control animals. The discrepancy between beta adrenoceptor number and NA responsiveness following lesions of 5HT axons was particularly profound in the striatum. The analysis of highand low-affinity components of agonist binding demonstrated that the increase in striatal beta adrenoceptors is due to a marked increase in receptors with low affinity while the number of receptors with high affinity is unchanged. The results lend further support to the view that the synaptic availability of NA is a prerequisite for the induction of subsensitivity of the NA sensitive adenylate cyclase and for the down-regulation of its beta adrenoceptor population by DMI and that 5HT plays a pivotal role in both the regulation of the number and the function of central beta adrenoceptors.

Dose-dependent down-regulation of ß-adrenoceptors by isoproterenol in rat C6 glioma cells

Nano- and micromolar isoproterenol concentrations were compared by studying cyclic AMP, beta-adrenoceptor density and beta 1-adrenoceptor mRNA in rat C6 glioma cells. 1 microM isoproterenol significantly changed all parameters at 15-30 min. The beta 1-antagonist metoprolol attenuated the response. No effects of nanomolar isoproterenol on these early changes were observed, although the density of beta-adrenoceptors was significantly reduced beginning at 12 h. The results indicate a different process for beta-adrenoceptor desensitization in C6 cells following physiologically low agonist concentrations.

Noradrenergic signal transfer as a target of antidepressant therapy

Neurohormones such as norepinephrine (NE), serotonin (5-HT) and dopamine (DA) are, like hormones, informational molecules that transmit specific sets of instructions through specific membrane receptors from the outside to the inside of the cell. While research on the mode of action of antidepressant drugs has centered in the past on their effects on synthesis, release, metabolism, reuptake and hence the availability of neurohormones such as NE or 5-HT at presumptive noradrenergic and/or serotonergic receptor sites, two discoveries made during the second half of the 1970s have shifted the research emphasis on the mode of action of these drugs from acute presynaptic to delayed postsynaptic receptor mediated events. First, Vetulani and Sulser (1975) discovered that various prototypes of antidepressant treatments, including ECT, if administered on a clinically relevant time basis, decreased the neurohormonal sensitivity of the NE receptor-coupled adenylate cyclase system in brain.