Zinc Regulates Amyloid Precursor Protein Processing And Aβ Production In APPsw SH-SY5Y Cells

PrefaceAlzheimer's disease (AD) is a disease clinically characterized by progressive intellectual deterioration. Senile plaque (SP), the major pathological hallmark of AD, is caused by the pathological deposition of P-amyloid (Aβ). AP is generated from the amyloid precursor protein (APP) by a proteolytic activity ofβ-and y-secretase. Alternatively, APP can be cleaved byα-secretase at a position between theβ-andγ-cleavage points, resulting in the preclusion of Aβproducton. Under physiological condition, there is nanomolar AP in the extracellular space, which acts as a neurotrophic factor. In AD, the abnormal cleavage of APP, excessive production of Aβ, and the transition of Aβfrom soluble status to insoluble status are the key factors in AD progression. In the recent years, multiple studies have indicated the correlation between mental ions (zinc, iron and copper) and secretase in APP processing. In this study, we aimed at investigating the potential effect of zinc on APP processing and Aβproduction, using zinc ion and TPEN (a zinc chelator) in an AD cell model.MethodsSH-SY5Y cells stable transfected APPsw gene were used for the present study. The expression levels of ADAM 10, BACE 1, Presenilin 1, sAPPa, sAPPβ, C99, C83 and Aβwere detected by western blot analysis after treated by zinc ion and TPEN. Zinquin fluorescence was used to evaluate the intracellular change of zinc ion. MTT was used to detect the change of cell viability after treated by different concentrations of zinc ion and TPEN. Results1.The effect of ZnSO4 and TPEN on the viability of APPsw SH-SY5Y cellThe cell viability decreased as the concentrations of zinc ion and TPEN increasing compared with the control group. We selected 1μM ZnSO4,70μM ZnSO4 and 1μM TPEN as our treatment factors with cell viability decreasing by 1.5%,43.9% and 16.6%, respectively.2. Altered intracellular concentration of Zn2+ after treated by ZnSO4 and TPENZinquin fluorescence results showed that the 1μM ZnSO4 slightly increased the intracellular fluorescence and 70μM ZnSO4 led to a significant increase of fluorescence compared to the control group. While 1μM TPEN treatment resulted in a significant decrease of the fluorescence.3. Altered expression levels of ADAM 10, BACE 1 and Presenilin 1 after treated by ZnSO4 and TPENWestern blot results showed that 1μM ZnSO4 caused a significant increase of ADAM 10. (P<0.01); 70μM ZnSO4 led to a significant increase of BACE 1 and Presenilin 1, compared to the control group (P<0.05); 1μM TPEN resulted in a significant decrease of BACE 1 and Presenilin 1 (P<0.05); And using 70μM ZnSO4 and 1μM TPEN in combination led to no statistical change of ADAM 10, BACE1 and Presenilin 1, compared to the control group.4. The effect of ZnSO4 and TPEN on APP processing and AβsecretionWestern blot results showed that 1μM ZnSO4 caused a significant increase of sAPPa and C83 (P>0.05), and a significant decrease of sAPPP, C99 and Aβ, compared to the control group. (P<0.05); 70μM ZnSO4 lead to a significant increase of sAPPβ, C99 and Aβ(P<0.01), and a decreased expression of sAPPa and C83 (P<0.05); 1μM TPEN resulted in a significant decrease of sAPPP and Aβ(P<0.05); And using 70μM ZnSO4 and 1μM TPEN in combination led to no statistical change of sAPPa, sAPPP, C99,C83andAβ.Conclusion1. The viability of APPsw SH-SY5Y cell decreased as the concentration of ZnSO4 and TPEN increased in culture medium.2. The ZnSO4 treatment led to an increased intracellular concentration of Zn2+, whereas TPEN treatment resulted in a decreased concentration of Zn2+3. Low concentration (1μM) of Zn2+ could increase the expression level and proteolytic activity of a-secretase, inhibit proteolytic activity ofβ-secrestase, and consequently inhibit the Aβsecretion.4. High concentration (70μM) of Zn2+ could increase the expression level and proteolytic activity ofβ-secrestase andγ-secrestase, inhibit proteolytic activity of a-secretase, and consequently enhance the Aβsecretion.5. TPEN suppressed the expression level and proteolytic activity of P-secrestase andγ-secrestase, and consequently inhibited the Aβsecretion.