Therapeutic Effects Of Apicidin On Alzheimer's Disease And The Related Regulation Mechanisms On ADAM10

Objective:Histone deacetylase inhibitor apicidin could significantly increase the luciferase activity of pGL4.17- ADAM10 plasmid using small molecular and high throughput drug screening of traditional Chinese medicine monomers.This study aims to(1) verify that apicidin could promote the expression of ADAM10 in cultured cells;(2) research the regulation elements and signaling pathways of the effects apicidin on ADAM10;(3) provide new experimental evidence for developing proprietary and traditional Chinese medicine monomer drugs exploited for the treatment of Alzheimer's disease.Methods and Results:1. Drug screening and cell level studies found apicidin could increase the expression of ADAM10.Through small molecular and high throughput drug screening of traditional Chinese medicine monomers,we found the luciferase activity of pGL4.17-ADAM10 in SH-SY5 Y cells, which stably express pGL4.17- ADAM10 luciferase reporter gene built by molecular cloning, could be increased by the histone deacetylase inhibitor apicidin. Cell viability assay, real-time quantitative PCR and western blotting were conducted respectively in human embryonic kidney cells HEK293, in order to research the the cytotoxicity of apicidin and the influence of apicidin on the transcription and translation of ADAM10. The results confirmed that apicidin could increase ADAM10 expression.2. Researching the regulation elements of ADAM10 affected by apicidin. After analyzing ADAM10 core promoter region, five truncated fragments of ADAM10 were connected with pGL4.17 respectively to develop the p GL4.17-ADAM10-C/D/E/E1/E2 luciferase report gene plasmids using molecular cloning. These plasmids were transfected into HEK293 cells for 24 h, and then processed with 0.25 u M apicidin for 24 h. Afterwards, the luciferase activity of HEK293 cells transfected with different truncated segments of ADAM10 was detected by chemiluminescence reagents. The results showed that: the luciferase activity of the cells transfected with pGL4.17-ADAM10-E fragment was the strongest after 24 h treatment with 0.25 uM apicidin, compared to cells with other truncated segments. Analysing ADAM10-E fragment found that the binding sites mainly existed in it were of SP1 and USF1. The interaction between SP1 or USF1 with ADAM10 promoter was verified using chromatin immunoprecipitation assay(CHIP). Decreasing SP1 or USF1 in neuroblastoma cells SH-SY5 Y using RNAi technology to detect whether the effect on ADAM10 by apicidin existed. It's found that after down-regulating USF1, the influence on ADAM10 of apicidin was nearly disappeared, while when decreaseing SP1, apicidin still mildly increased the expression of ADAM10. Further to validate the promoting effect on ADAM10 by apicidin may be affected mainly by the activity of USF1.3. Researching the signaling pathways in which apicidin regulates the expression of ADAM10 in SH-SY5 Y cells. Western blotting was used to detect the impact of protein kinase C(PKC) inhibitor Calphostin C, protein kinase A(PKA) inhibitor H89, extracellular regulated protein kinases(ERK)pathway inhibitor U0126, PI3 K inhibitor LY294002, SP1 inhibitor Mithramycin A and insulin on the expression of ADAM10 promoted by apicidin. The results indicated that: when the activity of ERK pathway and SP1 was inhibited separately, the influence of apicidin to increase ADAM10 expression was significantly lowered.4.The effect of apicidin on the expression of BACE1 and the degradation of APP. Using real-time quantitative PCR and western blotting analysis, the mRNA and protein expression levels of BACE1 in SH-SY5 Y cells were analysed, after treating with 0.25 uM apicidin for 48 h. The results demonstrated that: the expression of BACE1 on mRNA and protein levels were not influenced by apicidin compared with the control group(treated with isovolumetric DMSO in SH-SY5 Y cells for 48h) ?The influence of apicidin on APP degradation was tested after processing HEK-APP cells with 0.25 uM apicidin for 48 h, using western blotting analysis. The results showed that: compared with control group, after apicidin treatment, the expression of APP was not changed, while alpha CTF protein was increased and beta CTF protein was decreased.Conclusions:In short, the expression of ADAM10 could be improved by histone deacetylase inhibitor apicidin, and the increase might be through the regulation of the activity of USF1 by ERK signaling pathway. Meanwhile the increased ADAM10 reduces the generation of toxic A? by protecting APP from degrading to A? beta pathway. Therefore, apicidin, as a new molecular drug for regulating the expression of ADAM10, is likely expected to become an effective strategy for AD treatment.