Search

Selective serotonin (5-HT) reuptake inhibitors are only 30% effective for remission in subjects with major depression, and the best treatments for SSRI-resistant patients remain unclear. To model SSRI resistance, we used cF1ko mice with conditional deletion of the repressor Freud-1/CC2D1A in adult 5-HT neurons. Within weeks, this deletion leads to overexpression of 5-HT1A autoreceptors, reduced serotonergic activity, and fluoxetine-resistant anxiety--depression phenotype. We hypothesized that desipramine (DES), which targets norepinephrine (NE), may be effective in cF1ko mice. The actions of chronic DES treatment on behavior, chronic cellular activation, and NE projections were examined in both sexes of cF1ko and WT mice. In contrast to fluoxetine, chronic DES reversed the behavioral phenotypes in cF1ko mice, while in WT littermates DES slightly increased anxiety and depressionlike behaviors. Deficits in FosB+ cell counts were seen in the entorhinal cortex, hippocampal CA2/3 layer, and BLA of cF1ko mice and were reversed by chronic DES treatment, especially in GABAergic neurons. In cF1ko mice, widespread reductions were seen in NE axons, varicosities, and especially 30-60% reductions in NE synaptic and triadic contacts, particularly to inhibitory gephyrin-positive sites. DES treatment also reversed these reductions in NE innervation. These results indicate the dynamic plasticity of the adult noradrenergic system within weeks of altering serotonergic function that can be normalized by DES treatment. Accompanying these changes, DES but not fluoxetine reversed the behavioral alterations in cF1ko mice, suggesting a key role for noradrenergic plasticity in antidepressant response in this model of reduced serotonin activity. [ABSTRACT FROM AUTHOR]

Selective serotonin (5-HT) reuptake inhibitors (SSRIs) are first-line antidepressants but require several weeks to elicit their actions. Chronic SSR1 treatment induces desensitization of 5-HT 1A autoreceptors to enhance 5-HT neurotransmission. Mice (both sexes) with gene deletion of 5-HT1A autoreceptors in adult 5-HT neurons (lAcKO) were tested for response to SSRIs. Tamoxifen-induced recombination in adult lAcKO mice specifically reduced 5-HT1A autoreceptor levels. The lAcKO mice showed a loss of 5-HT1A autoreceptormediated hypothermia and electrophysiological responses, but no changes in anxiety- or depression-like behavior. Subchronic fluoxetine (FLX) treatment induced an unexpected anxiogenic effect in lAcKO mice in the novelty suppressed feeding and elevated plus maze tests, as did escitalopram in the novelty suppressed feeding test. No effect was seen in wild-type (WT) mice. Subchronic FLX increased 5-HT metabolism in prefrontal cortex, hippocampus, and raphe of lAcKO but not WT mice, suggesting hyperactivation of 5-HT release. To detect chronic cellular activation, FosB+ cells were quantified. FosB+ cells were reduced in entorhinal cortex and hippocampus (CA2/3) and increased in dorsal raphe 5-HT cells of lAcKO mice, suggesting increased raphe activation. In WTbut not lAcKO mice, FLX reduced FosB+ cells in the median raphe, hippocampus, entorhinal cortex, and median septum, which receive rich 5-HT projections. Thus, in the absence of 5-HT 1A autoreceptors, SSRIs induce a paradoxical anxiogenic response. This may involve imbalance in activation of dorsal and median raphe to regulate septohippocampal or fimbria-fornix pathways. These results suggest that markedly reduced 5-HT1A autoreceptors may provide a marker for aberrant response to SSRI treatment. [ABSTRACT FROM AUTHOR]

Freud-1/Cc2d1a represses the gene transcription of serotonin-1A (5-HT1A) autoreceptors, which negatively regulate 5-HT tone. To test the roleof Freud-1 in vivo, we generated mice with adulthood conditional knock-out ofFreud-1 in 5-HT neurons (cF1ko). In cF1ko mice, 5-HT1A autoreceptor protein, binding and hypothermia response were increased, with reduced 5-HT content and neuronal activity in the dorsal raphe. The cF1ko mice displayed increased anxiety- and depression-like behavior that was resistant to chronic antidepressant (fluoxetine) treatment. Using conditional Freud-1/5-HT1 A double knock-out (cF1/1A dko) to disrupt both Freud-1 and 5-HT1A genes in 5-HT neurons, no increase in anxiety- or depression-like behavior was seen upon knock-out ofFreud-1 on the 5-HT1A autoreceptornegative background; rather, a reduction in depression-like behavior emerged. These studies implicate transcriptional dysregulation of 5-HT 1A auto receptors by the repressor Freud-1 in anxiety and depression and provide a clinically relevant genetic model of antidepressant resistance. Targeting specific transcription factors, such as Freud-1, to restore transcriptional balance may augment response to antidepressant treatment. [ABSTRACT FROM AUTHOR]

The dopamine D2S receptor (short isoform) couples to inhibitory Gαi/o proteins to inhibit thyrotropin-releasing hormone (TRH)-stimulated p42/p44 mitogen-activated protein kinase (ERK1/2) phosphorylation in GH4ZR7 rat pituitary cells, consistent with its actions to inhibit prolactin gene transcription and cell proliferation. However, the underlying mechanism is unclear. To identify novel Gαi effectors, yeast two-hybrid screening of a GH4ZR7 cDNA library was done using constitutively active Gαi3-Q204L, and multiple clones of the RasGAP cDNA GAP1IP4BP/RASA3 were identified. In yeast mating assay, RASA3 preferentially interacted with activated forms of Gαi/o/2 proteins, but not with Gas. A direct interaction was indicated by in vitro pull-down assay, in which S-His-RASA3 preferentially bound guanosine 5'-O-(γ-thio)triphosphate-activated Gαi3 and Gαi2 compared with guanosme 5'-O-(β-thio)diphosphate-inactivated proteins. Similarly, in co-immunoprecipitation studies in HEK-293 cells, FLAG-tagged RASA3 preferentially interacted with activated mutants of Gαi3 and Gαi2 compared with wild type proteins. In GH4ZR7 cells, co-immunoprecipitation studies of endogenous proteins demonstrated a Gαi3-RASA3 complex that was induced upon TRH/D2S receptor co-activation. To address RASA3 function in dopamine D2S receptor-induced inhibition of ERK1/2 activity, endogenous RASA3 protein expression was suppressed (70% knockdown) in GH4ZR7 cells stably transfected with full-length antisense cDNA of RASA3. The selected antisense clones had similar levels of dopamine D2S receptor binding and D2S-induced inhibition of cAMP formation compared with parental GH4ZR7 cells. In these clones, D2S-mediated inhibition of TRH-induced phospho-ERK1/2 was reversed by 70-80% compared with parental GH4ZR7 cells. Our results provide a novel mechanism for dopamine D2S-induced inhibition of ERK1/2 and indicate that RASA3 links Gαi proteins to inhibit Gq-induced Ras/ ERK1/2 activation. [ABSTRACT FROM AUTHOR]

Abstract: Altered regulation of dopamine D2 receptors is implicated in addiction, schizophrenia and movement disorders, as well as lactotroph growth and regulation. Dopamine D2S and dopamine D2L receptors are alternately-spliced variants that differ by 29 amino acids in the third intracellular (i3) domain and display different sensitivity to desensitization by protein kinase C (PKC). In the present studies we determined the specific phosphorylation sites on the dopamine D2S receptor that confer PKC-mediated desensitization. In dopamine D2L receptors, we identified a PKC pseudosubstrate site responsible for the relative insensitivity of the receptor to PKC-induced uncoupling. In transiently transfected Ltk− fibroblast cells, 2-min preactivation of PKC with 12-O-tetradecanoyl 4β-phorbol 13α-acetate (TPA) completely inhibited calcium mobilization induced by the dopamine D2S receptor, but not the dopamine D2L variant. Point mutation of i3 PKC sites Ser228/229Gly rendered the dopamine D2S receptor resistant to PKC action, with lesser effects of other Ser and Thr mutations. Inactivation of the PKC pseudosubstrate motif in the dopamine D2L receptor sensitized the receptor to PKC, and this was reversed by mutation of i3 PKC sites Ser228/229. A phospho-specific antibody generated against phospho-Ser228/229 demonstrated PKC-induced phosphorylation at these sites of dopamine D2S, but not D2L receptors, in Ltk− cells. Conversely, the pseudosubstrate dopamine D2L receptor mutant displayed PKC-induced phosphorylation at Ser228/229, which was abolished when these sites were mutated. Similar phosphorylation results were observed using GH4 cells stably transfected with dopamine D2 receptors and mutants. Thus the relative location of phosphorylation and pseudosubstrate sites provides an important determinant substrate sensitivity to PKC. [Copyright &y& Elsevier]

The dopamine D2S receptor (short isoform) couples to inhibitory Galphai/o proteins to inhibit thyrotropin-releasing hormone (TRH)-stimulated p42/p44 mitogen-activated protein kinase (ERK1/2) phosphorylation in GH4ZR7 rat pituitary cells, consistent with its actions to inhibit prolactin gene transcription and cell proliferation. However, the underlying mechanism is unclear. To identify novel Galphai effectors, yeast two-hybrid screening of a GH4ZR7 cDNA library was done using constitutively active Galphai3-Q204L, and multiple clones of the RasGAP cDNA GAP1(IP4BP)/RASA3 were identified. In yeast mating assay, RASA3 preferentially interacted with activated forms of Galphai/o/z proteins, but not with Galphas. A direct interaction was indicated by in vitro pull-down assay, in which S-His-RASA3 preferentially bound guanosine 5'-O-(gamma-thio)triphosphate-activated Galphai3 and Galphai2 compared with guanosine 5'-O-(beta-thio)diphosphate-inactivated proteins. Similarly, in co-immunoprecipitation studies in HEK-293 cells, FLAG-tagged RASA3 preferentially interacted with activated mutants of Galphai3 and Galphai2 compared with wild type proteins. In GH4ZR7 cells, co-immunoprecipitation studies of endogenous proteins demonstrated a Galphai3-RASA3 complex that was induced upon TRH/D2S receptor co-activation. To address RASA3 function in dopamine D2S receptor-induced inhibition of ERK1/2 activity, endogenous RASA3 protein expression was suppressed (70% knockdown) in GH4ZR7 cells stably transfected with full-length antisense cDNA of RASA3. The selected antisense clones had similar levels of dopamine D2S receptor binding and D2S-induced inhibition of cAMP formation compared with parental GH4ZR7 cells. In these clones, D2S-mediated inhibition of TRH-induced phospho-ERK1/2 was reversed by 70-80% compared with parental GH4ZR7 cells. Our results provide a novel mechanism for dopamine D2S-induced inhibition of ERK1/2 and indicate that RASA3 links Galphai proteins to inhibit Gq-induced Ras/ERK1/2 activation.