Research On The Mechanism And Effect Of MTERF4 On Metabolism Of Amyloid Peptide Precursor

Background:Alzheimer's disease(AD)is one of the most common neurodegenerative diseases.AD is characterized by the deposition of ?-amyloid peptide(A?)in the brain,which is produced by the proteolysis of amyloid precursor protein(APP).Mitochondrial dysfunction induced by various factors has been strongly implicated in the pathogenesis of AD.Recently,the mitochondrial transcription termination factor 4(MTERF4),a member of the MTERF family,was implicated in regulating mitochondrial DNA transcription and directly in controlling mitochondrial ribosomal translation.It is plausible that abnormal MTERF4 activity could contribute to the pathogenesis of AD.Objective:This study was designed to investigate the effects of MTERF4 on APP processing and the possible mechanism in regulation of APP expression,to further explore the role of MTERF4 in AD pathogenesis and hope to provide a new idea for prevention and treatment of AD.Methods:The expression of MTERF4 in APP/PS1 mice hippocampus compared with WT control were estimated by Western blot.MTERF4 eukaryotic expression vector pcDNA-MTERF4,knockdown vector pGPH1/GFP/Neo-MTERF4 and vector control was generated with molecular cloning and PCR techniques.HEK293-APPswe cells were transfected with pcDNA-MTERF4?pGPH1/GFP/Neo-MTERF4 and vector control for 24 and 48 h and then total protein was extracted from HEK293-APPswe cells.The effects of MTERF4 overexpression or knockdown on APP,C99,C83,ADAM10 and BACE1 expression were estimated by Western blot.Total RNA was extracted from transfected cells using RNAiso Plus and then total RNA was reverse transcribed into cDNA.Quantitative PCR was performed to evaluate the effects of MTERF4 overexpression or knockdown on APP,ADAM10,and BACE1 gene expression.The effects of MTERF4 overexpression or knockdown on A?42 levels in the culture medium were assayed using a human A?42-specific sandwich ELISA kit.We co-transfected the pcDNA-MTERF4,together with luciferase reporter constructs of ADAM10 promoter pGL3-ADAM10 into the HEK293 cells to verify the effects of MTERF4 overexpression on ADAM10 promoter activities by luciferase assay.Cell cycle was quantified on a FACSCalibur flow cytometer and cell proliferation was measured using the Cell Counting Kit-8(CCK-8)assays.Results:The levels of MTERF4 protein were significantly increased in the hippocampus of 6-month-old APP/PS1 double transgenic mice compared with age-matched nontransgenic wile-type(WT)C57BL/6J mice.The MTERF4 eukaryotic expression vector was successfully constructed.The overexpression of MTERF4 induced a significant increase in the levels of APP protein and secreted A?42 in HEK293-APPswe cells compared with control cells.Further,MTERF4 overexpression shifted APP processing from ?-to ?-cleavage,as indicated by decreased C83 levels and elevated C99 levels.ADAM10 expression is suppressed by MTERF4 overexpression.The results of luciferase assay demonstrated that MTERF4 could down-regulate ADAM10 promoter activities and MTERF4-induced inhibition of ADAM10 promoter activities is mediated by the 2095 to 866 bp sequence of the promoter.MTERF4 overexpression does not alter cell cycle distribution and proliferation in HEK293-APPswe cells.MTERF4 knockdown induced a decrease in the levels of APP protein and secreted A?42 in HEK293-APPswe cells compared with control cells.MTERF4 knockdown decreased ?-and ?-cleavage,as indicated by decreased C83 and C99 levels.Western blot evealed a decrease in ADAM10 levels in MTERF4 knock down cells compared to control.The results of Q-PCR confirmed that MTERF4 could decrease APP and ADAM10 gene expressions in transcriptional level.Flow cytometry results showed that the percentage of cells in the G0/G1 phase decreased after transfecting with knockdown vector and the percentage of cells in the S phase was obviously increased.MTERF4 knockdown caused a decrease of cell proliferation at 48 h.Conclusion:The present study identified a novel role for MTERF4 in regulation of APP processing.These results suggest that MTERF4 overexpression promotes the amyloidogenic processing of APP by inhibiting ADAM10 via a transcriptional mechanism.The down-regulated MTERF4 leads to a reduction in APP expression and A?42 levels.Therefore,MTERF4 may play an important role in the pathogenesis of AD.This work provides new insights in the pathological pathway of AD and suggests a major potential for the pharmaceutical development.