Figure 1.
Effect of NaHS on Aβ42 production and cell viability in SH-SY5Y cells expressing APPswe.
A: Concentration-dependent effect of HENECA (10–200 nM, 24 hours) on Aβ42 production. B–C: Dose-dependent effect of NaHS (10–200 µM, 12 hours) on Aβ42 formation in the presence (B) and absence (C) of HENECA (100 nM, 24 hours). D–E: MTT assay showing the effect of NaHS alone at 10–200 µM (D) or HENECA alone at 10–200 nM (E) on cell viability of SH-SY5Y cells. Aβ42 levels in conditioned media were measured by sandwich ELISA kit. Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 6. ##p<0.01, ###p<0.001 vs Con group; **p<0.01, ***p<0.001 vs HEN group. Con, control; HEN, HENECA.
Figure 2.
Effect of NaHS on Aβ42 production involved cAMP signaling pathway.
A: Dose-dependent effect of HENECA on cAMP production in SH-SY5Y cells expressing APPswe. B: Concentration-dependent effect of NaHS (10–200 µM, 12 hours) on HENECA (100 nM, 24 hours)-stimulated cAMP upregulation. C–D: Effects of NaHS (100 µM) on cAMP (C) and Aβ42 production (D) in cells treated with forskolin (20 µM) and/or IBMX (100 µM). The intracellular cAMP and Aβ42 levels in conditioned media were measured by sandwich ELISA kits. Control values were adjusted to 100% for Aβ42 levels measurement. Data are given as means ± S.E.M, n = 6. ### p<0.001 vs Con group, **p<0.01, ***p<0.001 vs HEN group,+++p<0.001 vs forsk group, ???p<0.001 vs forsk + IBMX group. Con, control; HEN, HENECA; Forsk, forskolin.
Figure 3.
Effect of NaHS on mRNA expression of AC isoforms and AC activity.
A–B: Representative gels (A) and histogram (B) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) attenuated the effects of HENECA (100 nM, 24 hours) on mRNA expressions of AC isoforms. C: Effect of NaHS (100 µM) on AC activity stimulated by forskolin (20 µM). D: Effect of NaHS (100 µM) on Aβ42 production in SH-SY5Y cells preincubated with AC antagonist, SQ 22536 (300 µM). Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ##p<0.01, ###p<0.001 vs Con group; +++p<0.001 vs Forsk group, *p<0.05, **p<0.01 vs HEN group. Con, control; HEN, HENECA; Forsk, forskolin.
Figure 4.
Effect of NaHS on Aβ42 production involved PKA and CREB.
A: Effect of HENECA (100 nM) on Aβ42 formation was abolished by a PKA inhibitor, H89 (5, 10 and 15 µM). B–C: Representative gel (B) and histogram (C) depicting that pretreatment with NaHS (100 µM, 12 hours) attenuated the effects of HENECA (100 nM, 24 hours) on phosphorylation of CREB. Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ###p<0.001 vs Con group; *p<0.05, **p<0.01, ***p<0.001 vs HEN group. Con, control; HEN, HENECA.
Figure 5.
Effect of NaHS on expression of A2A receptors.
A–B: Representative gels (A) and histogram (B) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) did not attenuate the effects of HENECA (100 nM, 24 hours) on protein expression of A2A receptor. C: Effect of NaHS (100 µM) on production of Aβ42 in cells pre-treated with A2A receptor antagonist, ZM 241385 (50 nM). Control values were adjusted to 100%. Data are given as means ± S.E.M, n = 4–6. ##p<0.01; ###p<0.001 vs Con group. Con, control; HEN, HENECA.
Figure 6.
Effect of NaHS on production and maturation of APP.
Representative gel (A) and quantitative analysis (B–C) showing the effects of NaHS (100 µM, 12 hours) on HENECA (100 nM, 24 hours) stimulated production (B) and maturation (C) of APP. The cell lysates were analysed by western blot technique with antibody against N-terminus of APP or β-actin. The extent of maturation of APP is shown as the ratio between mAPP to imAPP. mAPP and imAPP are represented by upper and lower bands in a blot respectively. β-actin was used as a loading control. Data are given as means ± S.E.M, n = 4. ###p<0.001 vs Con group; ***p<0.001 vs HEN group. Con, control; HEN, HENECA.
Figure 7.
Effect of NaHS on activities of β- and γ-secretases.
A–B: Representative gel (A) and quantitative analysis (B) showing NaHS (25–100 µM, 12 hours) and HENECA (100 nM, 24 hours) failed to affect β-CTF (C99) expression in SH-SY5Y cells. The membrane fractions were analysed by western blot with antibody against c-terminal fragment of APP (C99) or β-actin. C–D:Effect of NaHS (100 µM) and a γ-secretase inhibitor, DAPT (1 µM, 1 hour) on HENECA (100 nM)-stimulated γ-secretase activity (C) and Aβ42 formation (D) in SH-SY5Y cells expressing APPswe. E: Effect of NaHS (100 µM) on γ-secretase activity in SH-SY5Y cells preincubated with AC antagonist, SQ 22536 (300 µM). Data are given as means ± S.E.M, n = 4–6. ###p<0.001 vs Con group; ***p<0.001 vs HEN group. Con, control; HEN, HENECA.
Figure 8.
Effect of NaHS on mRNA expressions of presenilins 1 and 2.
Representative gels (A) and histograms (B–C) demonstrating the effect of pretreatment with NaHS (100 µM, 12 hours) on HENECA (100 nM, 24 hours) stimulated mRNA expression of presenilins 1 and 2 respectively. Control values were adjusted to 100% for mRNA expression. Data are given as means ± S.E.M, n = 4. #p<0.05 vs Con group; *p<0.05 vs HEN group. Con, control; HEN, HENECA.
Figure 9.
Schematic diagram showing the inhibitory effect of H2S on HENECA induced Aβ generation in SH-SY5Y cells.
APP is an integral membrane protein which undergoes post-translational modification such as glycosylation during its transfer through intracellular secretory pathway. The mature isoform of APP (i.e. APP holoprotein) is then acted upon by β- and γ-secretases to generate Aβ. The A2A receptor agonist, HENECA, induces production of Aβ42 in SH-SY5Y cells via cAMP/PKA/CREB pathway. It enhances both synthesis and maturation processes of APP increasing total APP production. It also stimulates γ-secretase activity in mAPP cleavage resulting in Aβ generation. H2S not only interferes with the step of APP maturation, but also attenuates the production of APP holoprotein. By inhibiting AC (and subsequent cAMP production), H2S also inhibits γ-secretase activity. It ultimately leads to decreased production in Aβ.