Regulation of amyloid precursor protein processing by serotonin signaling

Abstract

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-HT4) 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-HT4 receptors following agonist stimulation is considered to be a therapeutic strategy for AD treatment; however, the signaling cascade involved in 5-HT4 receptor-stimulated proteolysis of APP remains to be determined. Here we used chemical and siRNA inhibition to identify the proteins which mediate 5-HT4d 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-HT4 receptor-induced α-secretase activation.

Figure 1. 5-HT_4d receptor-stimulated APP shedding requires G protein signaling and is independent of β-arrestin recruitment.

(A) Prucalopride induced sAPPα secretion in SH-SY5Y human neuroblastoma cells is specific for the 5-HT₄ receptor. SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride and 5-HT (5-HT₄ receptor agonists) in the absence or presence of 1 µM GR113808 (5-HT₄ receptor antagonist) or PMA and secretion of sAPPα was analyzed via measuring SEAP. (B) SEAP levels were measured in supernatants of SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d (WT), pcDNA3.1-5-HT_4dDRY117/118AAY (DRY) or pcDNA3.1-5-HT_4dΔ346 (Δ346) mutants and stimulated with 1 µM prucalopride or 5-HT. (C), (D) and (F) SEAP levels were measured in supernatants of SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d and treated with 1 µM prucalopride or 5-HT in the absence or presence of 100 µM CTB (Gα_s inhibitor) (C), 100 µM NF449 (Gα_s inhibitor) (D) or 100 µM gallein (Gβγ inhibitor) (F). (E) SEAP levels were measured in SH-SY5Y cells, transfected with pEAK12-AP-APP, pcDNA3.1-5-HT_4d and pcDNAI-Amp-Gα_sDN or pcDNA3.1 at a ratio of 2∶1∶4, respectively, and treated with 1 µM prucalopride or 5-HT. Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. * P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA with Tukey-Kramer or Dunnet's post-hoc test.

Figure 2. 5-HT_4d receptor-stimulated APP shedding does not require accumulation of cAMP.

(A) and (B) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ receptor agonists) in the absence or presence of 4 µM SQ22536 (AC inhibitor) (A) or 100 µM DDA (AC inhibitor) (B) and secretion of sAPPα was analyzed via measuring SEAP. (C) Concentration of cAMP was measured in SH-SY5Y cells, transfected with pEAK12-AP-APP, pcDNA3.1-5-HT_4d and pmax-FP-Green-N at a ratio of 5∶4∶1, were treated with 1 µM prucalopride or 5-HT in the absence or presence of 4 µM SQ22536 or 100 µM DDA. Values shown are mean ± SEM of 6 individual wells and were normalized to vehicle control. *** P<0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.

Figure 3. 5-HT_4d receptor-stimulated APP shedding requires Src and phospholipase C, but not PKC or calcium.

(A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ 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_4d, were loaded with Fluo-4 NW mix and fluorescence of the calcium-sensitive dye in each well was recorded at the baseline (F₀) 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/F₀). Values shown are mean ± SEM of 2 individual wells and were normalized to vehicle control. * P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.

Figure 4. 5-HT_4d receptor-stimulated APP shedding requires inositol polyphosphates and casein kinase 2.

(A), (B) and (C) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ 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_4d 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. * P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.

Figure 5. Single knock-down of ADAM9, ADAM10, ADAM17 and MMP9 does not affect 5-HT_4d receptor-stimulated APP shedding.

(A) SH-SY5Y cells, transfected with pEAK12-AP-APP and pcDNA3.1-5-HT_4d, were treated with 1 µM prucalopride or 5-HT (5-HT₄ 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_4d 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. * P<0.05, ** P<0.01, *** P<0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.

Figure 6. Combined knock-down of ADAM9, ADAM10, ADAM17 and MMP9 does not affect 5-HT_4d receptor-stimulated APP shedding.

(A) and (B) SH-SY5Y cells, co-transfected with pEAK12-AP-APP, pcDNA3.1-5-HT₄ and combinations of 3 nM siRNA for knock-down of ADAM9 (A9) and ADAM10 (A10), ADAM9 (A9) and ADAM17 (A17), ADAM10 (A10) and ADAM17 (A17) in (A) or ADAM9, 10 and 17 or ADAM9, 10, 17 and MMP9 in (B), were treated with 1 µM prucalopride (5-HT₄ receptor agonist) in the absence or presence of 80 µM GM6001 (metalloproteinases inhibitor) (B) and secretion of sAPPα was analyzed via measuring SEAP. (C) Cell lysates of experiments in (A) and (B) were analyzed for protein expression of ADAM9, 10, 17 and MMP9 by western blotting. 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. ** P<0.01, *** P<0.001, one-way ANOVA with Tukey-Kramer's post-hoc test.

Figure 7. Schematic representation of the proposed 5-HT_4d receptor-stimulated signaling pathway leading to increased sAPPα production.

The proteins involved in 5-HT_4d 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_4d 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_4d receptor-mediated sAPPα release .