Daniel S. Cowen

Coupling of neuronal 5-HT7 receptors to activation of extracellular-regulated kinase through a protein kinase A-independent pathway that can utilize Epac

The roles of 3′,5′‐cyclic adenosine monophosphate (cAMP) and protein kinase A in 5‐hydroxytryptamine (5‐HT)7 receptor‐mediated activation of extracellular‐regulated kinase (ERK) were studied in cultured hippocampal neurons and transfected PC12 cells. Activation of ERK by neuronal Gs‐coupled receptors has been thought to proceed through a protein kinase A‐dependent pathway. In fact we identified coupling of 5‐HT7 receptors to activation of adenylyl cyclase and protein kinase A. However, no inhibition of agonist‐stimulated ERK activation was found when cells were treated with H‐89 and KT5720 at concentrations sufficient to completely inhibit activation of protein kinase A. However, activation of ERK was found to be sensitive to the adenylyl cyclase inhibitor 9‐(tetrahydrofuryl)‐adenine, suggesting a possible role for a cAMP‐guanine nucleotide exchange factor (cAMP‐GEF). Co‐treatment of cells with 8‐(4‐chlorophenylthio)‐2′‐O‐methyladenosine 3′,5′‐cyclic monophosphate, a direct activator of the cAMP‐GEFs Epac1 and 2, reversed the inhibition of agonist‐stimulated ERK activation induced by adenylyl cyclase inhibition. Additionally, over‐expression of Epac1 enhanced 5‐HT7 receptor‐mediated activation of ERK. These results demonstrate that the activation of ERK mediated by neuronal Gs‐coupled receptors can proceed through cAMP‐dependent pathways that utilize cAMP‐GEFs rather than protein kinase A.

Interaction of FGF-2 with IGF-1 and BDNF in stimulating Akt, ERK, and neuronal survival in hippocampal cultures

The significance of multiple growth factors acting on individual neurons in the central nervous system is presently unclear. Cultured hippocampal neurons were used in the present study to compare the neurotrophic actions of fibroblast growth factor-2 (FGF-2) with the better characterized growth factors, insulin-like growth factor (IGF)-1 and brain-derived neurotrophic factor (BDNF). Additionally, cultures were utilized to identify possible interactions between FGF-2 and the other growth factors. Activation of the ERK and Akt pro-survival pathways, as well as neuronal survival itself, were studied. The maximal magnitude of Akt activation stimulated by FGF-2 was found to be similar to that stimulated by IGF-1 and BDNF. In contrast, IGF-1 was less effective at inducing ERK activation than were BDNF and FGF-2. All three agents were found to promote survival of neurons cultured under serum-free, low-insulin conditions, with FGF-2 surprisingly being significantly more effective than the other two peptides. Co-treatment with maximal concentrations of either IGF-1 or BDNF enhanced FGF-2-stimulated Akt and ERK activation. However, no enhancement of survival beyond that stimulated by FGF-2 was observed with co-treatment. These findings suggest that FGF-2 may play an important role in promoting the survival of hippocampal neurons. Additionally, an interesting dissociation was identified between the positive interaction of FGF-2 with both IGF-1 and BDNF in activating Akt and ERK, and the lack of enhancement of FGF-2-induced neuroprotection.

Serotonin and neuronal growth factors – a convergence of signaling pathways

Monoamines, including serotonin (5‐HT), have traditionally been associated with short‐term signaling pathways in neurons, such as the modulation of cAMP and Ca2+ levels. In contrast, neuronal growth factors, such as neurotrophins, have been traditionally associated with signaling pathways, such as those for activation of extracellular‐regulated kinase (ERK) and Akt (protein kinase B), which are known to induce long‐term protective changes. It has therefore been unclear how antidepressants that increase serotonin (5‐HT), induce such changes as hippocampal neuroprotection and neurogenesis. It has been hypothesized, that the actions of 5‐HT may be mediated indirectly through increased synthesis of peptide growth factors. However, there is increasing evidence that some subtypes of 5‐HT receptors can directly couple to activation of the ERK and Akt pathways. Such coupling suggests a more direct potential role for 5‐HT in mediating the long‐term actions induced by antidepressants.

Age-dependent decline in hippocampal neurogenesis is not altered by chronic treatment with fluoxetine.

There has been ongoing controversy as to whether selective serotonin reuptake inhibitors (SSRIs) exhibit the same antidepressant efficacy and risk profile within different age groups. Although the etiology of such potential differences is currently not clear, age-dependent differences in the rate of hippocampal neurogenesis offer one possibility. In the current studies we have therefore examined whether fluoxetine, the prototypical selective serotonin reuptake inhibitor, differentially modulates neurogenesis in adolescent, young adult, and aged rats. Proliferation in the dentate gyrus was measured by assaying expression of the endogenous proliferative marker, Ki67. Survival of proliferating cells was assayed by staining with BrdU. We confirmed previous reports that the rate of neurogenesis, as well as the survival of proliferating cells, decreases significantly with age. Moderate decreases were found in young adult rats relative to adolescent rats, and profound decreases were found in aged rats. We additionally found that age did not alter the response to 25 days of treatment with fluoxetine. In fact, we did not observe enhancement of hippocampal neurogenesis, nor enhancement of proliferating cell survival, in any of the three age groups despite using doses of fluoxetine which have been reported to be effective. In addition to finding no age-dependent effects, our data question the general reproducibility of previously reported fluoxetine effects in animals.

5-HT1A receptors couple to activation of Akt, but not extracellular-regulated kinase (ERK), in cultured hippocampal neurons

5‐HT1A receptors have been hypothesized to mediate some of the neuronal plasticity and behavioral responses stimulated by serotonin selective reuptake inhibitors. Although the cellular signaling pathways required for inducing these actions have not yet been determined, roles for the neuroprotective extracellular‐regulated kinase (ERK) mitogen‐activated protein (MAP) kinase and Akt pathways have been suggested. In the current studies we have utilized primary cultures to directly determine whether hippocampal 5‐HT1A receptors couple to activation of Akt and ERK. We found that E18 hippocampal neurons exhibit a twofold activation of Akt when exposed to nanomolar concentrations of 5‐HT. The 5‐HT1/7 receptor‐selective agonist 5‐carboxamidotryptamine maleate (5‐CT) and the 5‐HT1A/7 receptor‐selective agonist 8‐hydroxy‐N,N‐dipropyl‐aminotetralin (8‐OH‐DPAT) maleate were found to activate Akt with equal efficacy, and similar potency, to 5‐HT. p‐MPPI and WAY‐100635, antagonists selective for 5‐HT1A receptors, completely inhibited 5‐CT‐ stimulated Akt activation. Activation of Akt was also inhibited by pretreatment with pertussis toxin as well as the phosphatidylinositol 3‐kinase inhibitors, wortmannin and LY294002. In contrast, the 5‐HT selective antagonist, SB269970, caused no inhibition. Although the density of 5‐HT1A receptors expressed by cultured neurons was sufficient to activate Akt, no activation of ERK was observed. These findings suggest that Akt, and not ERK, may be relevant to previous reports of hippocampal 5‐HT1A receptors mediating neurotrophic responses.

Cumulative Activation of Akt and Consequent Inhibition of Glycogen Synthase Kinase-3 by Brain-Derived Neurotrophic Factor and Insulin-Like Growth Factor-1 in Cultured Hippocampal Neurons

Brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) seem to play key roles in mediating neuronal plasticity in the hippocampus. In the current studies, we have used cultured hippocampal neurons to study possible interactions between the two growth factors in modulating neuronal signaling pathways. BDNF and IGF-1 were found to each effectively activate the neuroprotective Akt pathway, with the magnitude of activation being at least additive when cultures were simultaneously treated with supramaximal concentrations of peptides. Likewise, a cumulative inhibitory Akt-dependent phosphorylation of proapoptotic glycogen synthase kinase-3 was observed. Immunofluorescent studies demonstrated that a single population of neurons responded to BDNF and IGF-1. In contrast, the magnitude of BDNF-stimulated extracellular signal-regulated kinase (ERK) activation was found to be much greater than that of IGF-1-stimulated ERK, such that the difference in magnitude stimulated by BDNF in the presence and absence of IGF-1 did not reach statistical significance. Consistent with the observed agonist-stimulated activation of Akt, BDNF and IGF-1 were both found to act as neurotrophins, enhancing neuronal survival under low-insulin culture conditions. Maximal survival was achieved when both growth factors were present. These findings provide insight into the significance of multiple growth factors stimulating activation of ERK and Akt in the central nervous system. In some cases, the magnitude of activation required to elicit biological responses may be achieved only with a combination of compounds.

Differential Coupling of Serotonin 5-HT1A and 5-HT1B Receptors to Activation of ERK2 and Inhibition of Adenylyl Cyclase in Transfected CHO Cells

Abstract: Although the subtypes of serotonin 5‐HT1 receptors have distinct structure and pharmacology, it has not been clear if they also exhibit differences in coupling to cellular signals. We have sought to compare directly the coupling of 5‐HT1A and 5‐HT1B receptors to adenylyl cyclase and to the mitogen‐activated protein kinase ERK2 (extracellular signal‐regulated kinase‐2). We found that 5‐HT1B receptors couple better to activation of ERK2 and inhibition of adenylyl cyclase than do 5‐HT1A receptors. 5‐HT stimulated a maximal fourfold increase in ERK2 activity in nontransfected cells that express endogenous 5‐HT1B receptors at a very low density and a maximal 13‐fold increase in transfected cells expressing 230 fmol of 5‐HT1B receptor/mg of membrane protein. In contrast, activation of 5‐HT1A receptors stimulated only a 2.8‐fold maximal activation of ERK2 in transfected cells expressing receptors at 300 fmol/mg of membrane protein but did stimulate a 12‐fold increase in activity in cells expressing receptors at 3,000 fmol/mg of membrane protein. Similarly, 5‐HT1A, but not 5‐HT1B, receptors were found to cause significant inhibition of forskolin‐stimulated cyclic AMP accumulation only when expressed at high densities. These findings demonstrate that although both 5‐HT1A and 5‐HT1B receptors have been shown to couple to G proteins of the Gi class, they exhibit differences in coupling to ERK2 and adenylyl cyclase.

Activation of Extracellular Signal-Regulated Kinase (ERK) and Akt by Human Serotonin 5-HT1B Receptors in Transfected BE(2)-C Neuroblastoma Cells Is Inhibited by RGS4

Abstract: Regulator of G protein signaling (RGS) proteins are GTPase‐activating proteins for heterotrimeric G proteins. One of the best‐studied RGS proteins, RGS4, accelerates the rate of GTP hydrolysis by all Gi and Gqα subunits yet has been shown to exhibit receptor selectivity. Although RGS4 is expressed primarily in brain, its effect on modulating the activity of serotonergic receptors has not yet been reported. In the present study, transfected BE(2)‐C human neuroblastoma cells expressing human 5‐HT1B receptors were used to demonstrate that RGS4 can inhibit the coupling of 5‐HT1B receptors to cellular signals. Serotonin and sumatriptan were found to stimulate activation of extracellular signal‐regulated kinase. This activation was attenuated, but not completely inhibited, by RGS4. Similar inhibition by RGS4 of the protein kinase Akt was also observed. As RGS4 is expressed at high levels in brain, these results suggest that it may play a role in regulating serotonergic pathways.

Enhanced activation of Akt and extracellular-regulated kinase pathways by simultaneous occupancy of Gq-coupled 5-HT2A receptors and Gs-coupled 5-HT7A receptors in PC12 cells.

The most commonly prescribed antidepressants, the serotonin (5‐HT) selective reuptake inhibitors, increase 5‐HT without targeting specific receptors. Yet, little is known about the interaction of multiple receptor subtypes expressed by individual neurons. Specifically, the effect of increases in cAMP induced by Gs‐coupled 5‐HT receptor subtypes on the signaling pathways modulated by other receptor subtypes has not been studied. We have, therefore, examined the activation of the extracellular‐regulated kinase (ERK) and Akt pathways by Gs‐coupled 5‐HT7A receptors and Gq‐coupled 5‐HT2A receptors, which are co‐expressed in discrete brain regions. Agonists for both receptors were found to activate ERK and Akt in transfected PC12 cells. 5‐HT2A receptor‐mediated activation of the two pathways was found to be Ca2+‐dependent. In contrast, 5‐HT7A receptor‐mediated activation of Akt required increases in both [cAMP] and intracellular [Ca2+], while activation of ERK was inhibited by Ca2+. The activation of ERK and Akt stimulated by simultaneous treatment of cells with 5‐HT2A and 5‐HT7A receptor agonists was found to be at least additive. Cell‐permeable cAMP analogs mimicked 5‐HT7A receptor agonists in enhancing 5‐HT2A receptor‐mediated activation of ERK and Akt. A role was identified for the cAMP–guanine exchange factor, Epac, in this augmentation of ERK, but not Akt, activation. Our finding of enhanced activation of neuroprotective Akt and ERK pathways by simultaneous occupancy of 5‐HT2A and 5‐HT7A receptors may also be relevant to the interaction of other neuronally expressed Gq‐ and Gs‐coupled receptors.

Differential coupling of 5‐HT1 receptors to G proteins of the Gi family

Since all 5‐HT1 receptors couple to Gi–type G proteins and inhibit adenylyl cyclase, the functional significance of five distinct subtypes of 5‐HT1 receptors has been unclear. In previous studies we have used transfected cells to demonstrate that 5‐HT1B receptors can couple more efficiently than 5‐HT1A receptors to activation of extracellular signal‐regulated kinase (ERK) and to inhibition of adenylyl cyclase. These findings suggested the possibility that individual 5‐HT1 receptors differentially couple to isoforms of Giα. In the present study we utilized a model system in which pertussis toxin resistant forms of human Giα1, Giα2, and Giα3 were used to directly compare the coupling of human 5‐HT1A, 5‐HT1B, and 5‐HT1D receptors to each Giα in transfected human HeLa cells. 5‐HT1A receptors displayed a preference for Giα1 and Giα2, relative to Giα3. Pertussis toxin resistant forms of Giα1, Giα2, and Giα3 rescued 73%, 76%, and 44%, respectively, of the ERK activation stimulated by 5‐HT in the absence of pertussis toxin. In contrast, pertussis toxin resistant forms of Giα1, Giα2, and Giα3 rescued 32%, 118%, and 35% of 5‐HT1B receptor‐stimulated activity, respectively, indicating that 5‐HT1B receptors coupled primarily through Giα2. A similar preference for Giα2 was found in studies of the 5‐HT1D receptor, where toxin resistant Giα1, Giα2, and Giα3 rescued 30%, 70%, and 40% of activity, respectively. In conclusion, the observed differential coupling of 5‐HT1 receptors to isoforms of Giα, provides additional evidence for our previous findings that the subtypes of 5‐HT1 receptors exhibit similar, but distinct, functions.