Abstract
OBJECTIVE: To investigate the proteolytic mechanism of amyloid precursor protein (APP) and to explore amyloid-beta (A beta) generation in living neurons. METHODS: DNA fragments were amplified by PCR or synthesized. The four fragments, CFP, 54bp, YFP and C99 were ligated into pcDNA3.0 vector to construct the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99. The SH-SY5Y cells were transiently transfected with pcDNA3.0-CFP-54bp-YFP or pcDNA3.0-CFP-54bp-YFP-C99. The expression of fusion gene was examined under a multiphoton laser scanning microscope. Fluorescence resonance energy transfer (FRET) was used to measure the beta cleavage and gamma cleavage of APP. A beta generation was confirmed by immunocytochemistry and multiphoton laser scanning microscopy. Cell viability was tested by MTT assay at different time points. RESULTS: (1) The double restriction endonuclease digestion and sequencing analysis confirmed the authenticity of the recombinant plasmids pcDNA3.0-CFP-54bp-YFP and pcDNA3.0-CFP-54bp-YFP-C99. (2) Blue and yellow fluorescences were detected in the transfected cells. (3) FRET occurred in pcDNA3.0-CFP-54bp-YFP-transfected cells but not in pcDNA3.0-CFP-54bp-YFP-C99-transfected cells. (4) A beta was produced in the pcDNA3.0-CFP-54bp-YFP-C99 transfected cells. (5) A beta-deposition was widespread in the cell. (6) Cell viability decreased along with the intracellular A beta deposition. CONCLUSION: C99 is important for the APP beta cleavage. A beta may be generated and deposited in cells at the early stage of Alzheimeros disease. Intracellular A beta accumulation brings deleterious effects on cells.