42 research outputs found

    5-HT<sub>4d</sub> receptor-stimulated APP shedding requires inositol polyphosphates and casein kinase 2.

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    <p>(A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT<sub>4d</sub>, were treated with 1 µM prucalopride or 5-HT (5-HT<sub>4</sub> receptor agonists) in the absence or presence of 20 µM IP3K inhibitor (A), 80 µM CGA (IPMK inhibitor) (B) or 2.5 µM TBB (CK2 inhibitor) (C) and secretion of sAPPα was analyzed via measuring SEAP. (D) SEAP levels were measured in supernatants of SH-SY5Y cells, co-transfected with pEAK12-AP-APP, pcDNA3.1-5-HT<sub>4d</sub> and 3 nM siRNA for knock-down of CK2 and treated with 1 µM prucalopride. (E) Cell lysates of (D) were analyzed for CK2 expression levels by western blotting. (F) Quantification of experiments in (E). Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.</p

    Schematic representation of the proposed 5-HT<sub>4d</sub> receptor-stimulated signaling pathway leading to increased sAPPα production.

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    <p>The proteins involved in 5-HT<sub>4d</sub> receptor-mediated non-amyloidogenic APP shedding are shown with green circles while orange circles and red characters indicate proteins or second messengers that were tested but were ineffective in modulating 5-HT<sub>4d</sub> receptor-stimulated sAPPα release. The dotted lines with the question marks indicate remaining areas of investigation for further elucidation of the molecular mechanism of α-secretase activation. cAMP-dependent pathway of α-secretase induction was previously reported and is depicted as a plausible way for 5-HT<sub>4d</sub> receptor-mediated sAPPα release <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087014#pone.0087014-Maillet1" target="_blank">[15]</a>.</p

    Regulation of Amyloid Precursor Protein Processing by Serotonin Signaling

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    <div><p>Proteolytic processing of the amyloid precursor protein (APP) by the β- and γ-secretases releases the amyloid-β peptide (Aβ), which deposits in senile plaques and contributes to the etiology of Alzheimer's disease (AD). The α-secretase cleaves APP in the Aβ peptide sequence to generate soluble APPα (sAPPα). Upregulation of α-secretase activity through the 5-hydroxytryptamine 4 (5-HT<sub>4</sub>) receptor has been shown to reduce Aβ production, amyloid plaque load and to improve cognitive impairment in transgenic mouse models of AD. Consequently, activation of 5-HT<sub>4</sub> receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT<sub>4</sub> receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT<sub>4d</sub> receptor-stimulated α-secretase activity in the SH-SY5Y human neuronal cell line. We show that G protein and Src dependent activation of phospholipase C are required for α-secretase activity, while, unexpectedly, adenylyl cyclase and cAMP are not involved. Further elucidation of the signaling pathway indicates that inositol triphosphate phosphorylation and casein kinase 2 activation is also a prerequisite for α-secretase activity. Our findings provide a novel route to explore the treatment of AD through 5-HT<sub>4</sub> receptor-induced α-secretase activation.</p></div

    TcPINK1 phosphorylates Histone H1 and the Ubl domain of Parkin.

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    <p>(A) Purity of Histone H1 and immunoprecipitated GST-tagged Ubl Parkin was evaluated by Coomassie staining. (B) <i>In vitro</i> phosphorylation assay using [γ-32P]-ATP, purified TcPINK1, and Ubl Parkin or Histone H1 shows that both are specifically phosphorylated by WT and not KI TcPINK1. WT TcPINK1 also displays autophosphorylation activity.</p

    Single knock-down of ADAM9, ADAM10, ADAM17 and MMP9 does not affect 5-HT<sub>4d</sub> receptor-stimulated APP shedding.

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    <p>(A) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT<sub>4d</sub>, were treated with 1 µM prucalopride or 5-HT (5-HT<sub>4</sub> receptor agonists) in the absence or presence of 80 µM GM6001 (metalloproteinases inhibitor) and secretion of sAPPα was analyzed via measuring SEAP. (B) SEAP levels were measured in supernatants of SH-SY5Y cells, co-transfected with pEAK12-AP-APP, pcDNA3.1-5-HT<sub>4d</sub> and 3 nM siRNA for knock-down of ADAM9 (A9), ADAM10 (A10), ADAM17 (A17) and MMP9 and treated with 1 µM prucalopride. (C) Cell lysates of (B) were analyzed for protein expression of ADAM9, 10, 17 and MMP9 by western blotting. The ADAM10 and ADAM17 immature precursor proteins are indicated by an x, whereas the mature catalytically active forms are indicated by an xx for ADAM9, 17 and MMP9. The immature ADAM9 and the mature ADAM10 proteins were not visible. (D) Quantification of experiments in (C). Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.</p

    Human PINK1 phosphorylates Parkin and Ubiquitin, but not PINKtide and Histone H1 <i>in vitro</i>.

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    <p>(A) Coomassie staining of the different substrates tested for <i>in vitro</i> phosphorylation by human PINK1. (B) Quantification of <i>in vitro</i> [γ-32P]-ATP PINKtide phosphorylation by purified human PINK1-FLAG. Human PINK1 did not specifically phosphorylate the PINKtide <i>in vitro</i> (n = 2 technical replicates, cpm: counts per minute). (C) An <i>in vitro</i> phosphorylation assay using [γ-32P]-ATP was performed with purified WT and KI human PINK1-FLAG and different putative PINK1 substrates. While WT PINK1 specifically phosphorylates both Parkin and Ubiquitin, Histone H1 was not found to be phosphorylated <i>in vitro</i>. Anti-FLAG WB shows equal loading of WT and KI human PINK1 (the full-length and 2 processed PINK1 forms are shown; note that the samples for Ubiquitin were run on a different gel type, causing a different migration pattern for PINK1).</p

    Human, mouse and <i>T</i>. <i>castaneum</i> PINK1 alignment.

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    <p>(A) Sequence alignment of human, mouse, and <i>T</i>. <i>castaneum</i> PINK1. Mouse and human PINK1 are 82% identical; Tc and human PINK1 are 36% identical. Polar, non-charged amino acids (green), basic (blue), and negatively charged hydrophilic (red) amino acids are indicated, as well as the start and end of the kinase domain (black arrowhead), and the 3 unique insertions (I1, I2, and I3). (B) Schematic representation of human, mouse, and Tc PINK1 indicating one of the 3 unique insertion regions in the kinase domain of PINK1 is lacking in TcPINK1 (MTS, mitochondrial targeting sequence; CTD, C-terminal domain).</p

    5-HT<sub>4d</sub> receptor-stimulated APP shedding requires Src and phospholipase C, but not PKC or calcium.

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    <p>(A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT<sub>4d</sub>, were treated with 1 µM prucalopride or 5-HT (5-HT<sub>4</sub> receptor agonists) and PMA in the absence or presence of 50 µM Bosutinib (Src inhibitor) (A), 30 µM D609 (PLC inhibitor) (B) or 2 µM GF109203X (PKC inhibitor) (C) and secretion of sAPPα was analyzed via measuring SEAP. Values shown are mean ± SEM of 6 individual wells and were normalized towards vehicle control. (D) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT<sub>4d</sub>, were loaded with Fluo-4 NW mix and fluorescence of the calcium-sensitive dye in each well was recorded at the baseline (F<sub>0</sub>) and after stimulation with 1 µM prucalopride, 30 µM ATP (purinergic ionotropic receptors agonist), 20 µM Ionomycin (calcium ionophore) or DMSO (F). Calcium response shown is a ratio of maximum fluorescence intensity at 40 sec to baseline fluorescence (F/F<sub>0</sub>). Values shown are mean ± SEM of 2 individual wells and were normalized to vehicle control. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.</p

    TcPINK1 autophosphorylates and phosphorylates PINKtide.

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    <p>(A) Purity of <i>E</i>. <i>coli</i>-expressed WT and KI TcPINK1 was evaluated by Coomassie staining. Both forms of TcPINK1 are equally enriched. (B) Quantification of [γ-32P]-ATP <i>in vitro</i> autophosphorylation of purified WT or KI TcPINK1. (C) Quantification of [γ-32P]-ATP <i>in vitro</i> phosphorylation of PINKtide by purified WT or KI TcPINK1 (mean ± SEM, n = 4 independent experiments). Statistical significance was calculated between WT and KI TcPINK1 using Student’s <i>t</i>-test (*: p-value < 0.05; **: p-value < 0.01).</p
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