Observation of amyloid precursor protein cleavage and A beta generation in living cells by using multiphoton laser scanning microscopy.
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.


CONCLUSIONS
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.
Li
Xiao-Qing
XQ
Department of Neurology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Zhang
Su-Ming
SM
Yang
Hua-Jing
HJ
Zhang
Zhi-Hong
ZH
D016229:Amyloid beta-Peptides / Q000235:genetics / Q000378:metabolism*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal; D009474:Neurons / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014162:Transfection
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D010446:Peptide Fragments