Observation of amyloid precursor protein cleavage and A beta generation in living cells by using multiphoton laser
Xiao-Qing Li1, Su-Ming Zhang, Hua-Jing Yang
1Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
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.
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