Reactive oxygen species are required for 5-HT-induced transactivation of neuronal platelet-derived growth factor and TrkB receptors, but not for ERK1/2 activation.

High concentrations of reactive oxygen species (ROS) induce cellular damage, however at lower concentrations ROS act as intracellular second messengers. In this study, we demonstrate that serotonin (5-HT) transactivates the platelet-derived growth factor (PDGF) type β receptor as well as the TrkB receptor in neuronal cultures and SH-SY5Y cells, and that the transactivation of both receptors is ROS-dependent. Exogenous application of H₂O₂ induced the phosphorylation of these receptors in a dose-dependent fashion, similar to that observed with 5-HT. However the same concentrations of H₂O₂ failed to increase ERK1/2 phosphorylation. Yet, the NADPH oxidase inhibitors diphenyleneiodonium chloride and apocynin blocked both 5-HT-induced PDGFβ receptor phosphorylation and ERK1/2 phosphorylation. The increases in PDGFβ receptor and ERK1/2 phosphorylation were also dependent on protein kinase C activity, likely acting upstream of NADPH oxidase. Additionally, although the ROS scavenger N-acetyl-l-cysteine abrogated 5-HT-induced PDGFβ and TrkB receptor transactivation, it was unable to prevent 5-HT-induced ERK1/2 phosphorylation. Thus, the divergence point for 5-HT-induced receptor tyrosine kinase (RTK) transactivation and ERK1/2 phosphorylation occurs at the level of NADPH oxidase in this system. The ability of 5-HT to induce the production of ROS resulting in transactivation of both PDGFβ and TrkB receptors may suggest that instead of a single GPCR to single RTK pathway, a less selective, more global RTK response to GPCR activation is occurring

Mechanism of PDGFβ and TrkB receptor transactivation.

<p>Gα_i-coupled GPCRs such as 5-HT_1A initiate transactivation signaling, which gets relayed through Gα or Gβγ subunits. PLC activation results in intracellular calcium release and activation of PKC. The NADPH oxidase subunits subsequently assemble and produce ROS. Active NADPH oxidase is required for both 5-HT-induced RTK and ERK1/2 phosphorylation but only endogenous ROS (or exogenous H₂O₂) is involved in RTK transactivation.</p

5-HT-induced PDGFβ receptor transactivation requires PKC and NADPH oxidase.

<p>(A) SH-SY5Y cell cultures were pretreated with vehicle or 0.1, 1 or 10 µM of the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Following drug treatments, cell lysates were evaluated by immunoblot analysis as described in Materials and Methods. Data were normalized to total PDGFRβ protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-1021 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-PDGFRβ 1021 (pY1021) and PDGFRβ at 180 kDa are shown. (B) Cell cultures were pretreated with vehicle or 1, 10 or 100 µM of the NADPH oxidase inhibitor apocynin for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-Y1021 as described in “A”. (C) Cultures were pretreated with vehicle or 0.1 µM of the PKC inhibitor Go 6983 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-Y1021 as described in “A”. (Data are representative of 3-5 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).</p

5-HT induced ERK1/2 phosphorylation diverges from the transactivation pathway at or after NADPH oxidase.

<p>(A) SH-SY5Y cells were treated with 0.01 to 100 µM H₂O₂ for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total ERK1/2 protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-ERK immunoreactivity compared to vehicle-treated cells. (B) SH-SY5Y cell cultures were pretreated with vehicle or 10, 100 or 1000 µM of the ROS scavenger <i>N</i>-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min and lysates were evaluated as in “A”. Cell cultures were also pretreated with vehicle or the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) (C) or apocynin (D) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-ERK1/2 as described in “A”. (E) Cultures were pretreated with vehicle or 0.1 µM of the PKC inhibitor Go 6983 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min, and results were analyzed for phospho-ERK1/2 as described above. Representative blots of phospho-ERK1/2 and total ERK1/2 at 42 and 44 kDa are shown. (Data are representative of 4-8 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).</p

H₂O₂ concentrations sufficient for inducing PDGFβ receptor phosphorylation do not result in cell death.

<p>SH-SY5Y cells were treated with 0, 0.1, 1, 10, 100, or 1000 µM H₂O₂ for (A) 30 min, or (B) overnight. Following treatment with MTT reagents and lysis, cell viability was measured and compared to control (VEH) values. (Data are representative of 4 independent experiments. * = p < 0.05 compared to vehicle-treated cells, one-way ANOVA, Tukey post-test).</p

5-HT can transactivate TrkB receptors via ROS.

<p>(A) SH-SY5Y cells were treated with vehicle (VEH) or 0.01 to 10 µM H₂O₂ for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total TrkB protein expression and are expressed as the fold change (average ± S.E.M.) in TrkB phospho-816 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-TrkB Y816 (pY816) and TrkB at 145 kDa are shown. (B) Cell cultures were incubated with 0.1 µM 5-HT for 0, 1, 2, 5, 10, or 15 min, and fold change in TrkB Y816 phosphorylation was measured with respect to vehicle. (C) Cultures were pretreated with vehicle or 1000 µM of the ROS scavenger <i>N</i>-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Normalized data was analyzed for phospho-TrkB Y816. (D) Cells were incubated overnight with 0.01 or 0.1 µg/mL pertussis toxin (Ptx) followed by 5 min treatment with 0.1 µM 5-HT. (E) Cell cultures were pretreated with vehicle or 1 or 10 µM of the PDGF receptor kinase inhibitor AG 1296 for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. Western blots were evaluated for changes in phospho-TrkB Y816. (Data are representative of 5-6 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test).</p

H₂O₂ increases PDGFβ receptor phosphorylation in SH-SY5Y cells and primary neuron cultures.

<p>(A) SH-SY5Y cells were treated with vehicle (VEH) or 0.01 to 100 µM H₂O₂ for 5 min. Following drug treatments, cell lysates were evaluated by Western blot analysis as described in Materials and Methods. Data were normalized to total PDGFRβ protein expression and are expressed as the fold change (average ± S.E.M.) in phospho-1021 immunoreactivity compared to vehicle-treated cells. Representative blots for phospho-PDGFRβ 1021 (pY1021) and PDGFRβ at 180 kDa are shown. (B) Primary mouse cortical neuron cultures were treated with 0.1 µM H₂O₂ for 5 min. Lysates were evaluated for phospho-Y1021 as described in “A”. (C) SH-SY5Y cell cultures were pretreated with vehicle or 1000 µM of the ROS scavenger <i>N</i>-acetyl-l-cysteine (NAC) for 45 min followed by treatment with vehicle or 100 nM 5-HT for 5 min. (Data are representative of 4-6 independent experiments. * = p < 0.05 compared to vehicle-treated cells; # = p < 0.05 compared to 5-HT-treated cells, one-way ANOVA, Tukey post-test, or Student’s t-test).</p

5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including cortical neurons. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins towards the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands

Acute 5-HT7 receptor activation increases NMDA-evoked currents and differentially alters NMDA receptor subunit phosphorylation and trafficking in hippocampal neurons

Abstract Background N-methyl-D-aspartate (NMDA) receptors are regulated by several G protein-coupled receptors (GPCRs) as well as receptor tyrosine kinases. Serotonin (5-HT) type 7 receptors are expressed throughout the brain including the thalamus and hippocampus. Long-term (2–24 h) activation of 5-HT7 receptors promotes the expression of neuroprotective growth factor receptors, including the platelet-derived growth factor (PDGF) β receptors which can protect neurons against NMDA-induced neurotoxicity. Results In contrast to long-term activation of 5-HT7 receptors, acute (5 min) treatment of isolated hippocampal neurons with the 5-HT7 receptor agonist 5-carboxamidotryptamine (5-CT) enhances NMDA-evoked peak currents and this increase in peak currents is blocked by the 5-HT7 receptor antagonist, SB 269970. In hippocampal slices, acute 5-HT7 receptor activation increases NR1 NMDA receptor subunit phosphorylation and differentially alters the phosphorylation state of the NR2B and NR2A subunits. NMDA receptor subunit cell surface expression is also differentially altered by 5-HT7 receptor agonists: NR2B cell surface expression is decreased whereas NR1 and NR2A surface expression are not significantly altered. Conclusions In contrast to the negative regulatory effects of long-term activation of 5-HT7 receptors on NMDA receptor signaling, acute activation of 5-HT7 receptors promotes NMDA receptor activity. These findings highlight the potential for temporally differential regulation of NMDA receptors by the 5-HT7 receptor

Data on acylglycerophosphate acyltransferase 4 (AGPAT4) during murine embryogenesis and in embryo-derived cultured primary neurons and glia

Whole mouse embryos at three developmental timepoints, embryonic (E) day E10.5, E14.5, and E18.5, were analyzed for Agpat4 mRNA expression. Primary cortical mouse cultures prepared from E18.5 mouse brains were used for immunohistochemistry. Our data show that Agpat4 is differentially expressed at three timepoints in murine embryogenesis and is immunodetectable in both neurons and glial cells derived from the developing mouse brain. This paper contains data related to research concurrently published in Bradley et al. (2015) [1]