Demonstration By FRET Of Dynamic Proteolysis Process Of APP In Vivo

ObjectiveTo investigate the mechanism of the APP cleavage progress and Aβgeneration, the construction of recombinant eukaryotic expression plasmid was made encoding Swedish and APP717 mutations of amyloid precursor protein (APP) and fluorescent protein.MethodsThe cyan and yellow fluorescence protein sequences (which were named as CFP and YFP, respectively.) were obtained by polymerase chain reaction (PCR) with the template of BglII-digestion product of the plasmid pcDNA3.0-CFP-CaM-YFP. The last 300 bases of APP sequence (which was named as C99 containing APP717 mutation) were amplified by PCR with the template of the plasmid pcDNA3.0-APP harboring APP717 mutation. The 54 bases in the middle of APP sequence were synthesized (which was named as 54bp containing Swedish mutation) by biological company. The four fragments, mentioned above (which were CFP, YFP, C99 as well as 54bp.) were inserted into the vector pcDNA3.0. By genetic engineering the recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were constructed and identified by enzyme digestion and sequencing assay. The recombinant plasmids pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP were transfected into SH-SY5Y cells respectively. The mRNA expression of the fusion gene was examined by reverse transcript polymerase chain reaction assay (RT-PCR). The protein expression of fusion gene and fluorescence change was detected by multi-photon con-focal microscopy at 12h, 24h, 48h, 72h and 96h after transfection. At 12h, 18h, 24h, 36h, 48h and 72h after transfection of pcDNA3.0-CFP-54bp-YFP-C99 or pcDNA3.0-CFP-54bp-YFP, as soon as the cells were excited at 820nm of CFP excitation wavelength, the CFP images were collected at the CFP emission wavelength of 473nm and YFP images were also collected at the YFP emission wavelength of 525nm. FRET phenomenon was observed to get an understanding ofβ-cleavage of APP. Calculate the intensities of CFP (ICFP) and YFP (IYFP) and then turn them into the ratio Fret ( Fret=IYFP : ICFP). At 48h after pcDNA3.0-CFP-54bp-YFP-C99 transfection, multi-photon con-focal microscope was used to detect the yellow fluorescence, to observe its distribution and trace Aβlabeled by YFP. Aβgeneration was confirmed by immunocytochemistry. The viability of transfection cells was measured via MTT assay to find the Aβeffect on the cells at specific time point.Results1. The length of PCR-product of CFP,YFP and C99 was 818bp,738bp and 325bp. The length of product of CFP,YFP and C99 via different double-digestion was 702bp,723bp and 310bp. The electrophoresis result is identical to our expectation. The different double-digestion of the plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP resulted in different fragments and confirmed the accomplishment of the two recombinant plasmids. Finally sequencing assay showed that the fusion genes of CFP-54bp-YFP-C99 and CFP-54bp-YFP were totally same to that base information in gene bank.2. The purpose fragment was amplified in transfected cells while it was absent in control cells.3. Cyan and yellow fluorescence could be detected in cells at 12h after transfection. At 24h the intensity of fluorescence strengthened. At 48h the fluorescence increased further. But at 72h the fluorescence became to decrease and by 96h it diminished.4. FRET occurred in pcDNA3.0-CFP-54bp-YFP transfection cells and the cell shape was clear. FRET was present in pcDNA3.0-CFP-54bp-YFP-C99 transfected-cells at 12h after transfection, but absent thereafter. Much intracellular fluorescence accumulation spread within the cells.5. In pcDNA3.0-CFP-54bp-YFP transfected-cells, Fret unchanged. In pcDNA3.0-CFP-54bp-YFP-C99 transfected-cells, Fret declined.6. At 48h in transfection cells, the fusion gene product of CFP-54bp-YFP-C99 could be cleaved byβ- andγ-secretase and liberate Aβ.7. Aβwas firstly generated within the cell. It accumulated and deposited widespread in the cell and membrane. Very little Aβwas secreted outside of the cell but did not form obvious deposition.8. The cell shape was abnormal due to Aβgeneration and deposition.9. Aβwas found by immunocytochemistry.10. Aβintracellular accumulation resulted in the decrease of transfected-cell viability examined by MTT assay and the difference was significant in comparison with the control group.Conclusion1. The construction of recombinant plasmid pcDNA3.0-CFP-54bp-YFP-C99 and pcDNA3.0-CFP-54bp-YFP containing Swedish (and APP717) mutation of APP and two types of fluorescent protein sequences was accomplished.2. The fusion gene could be expressed correctly at mRNA and protein levels and become to be a strong tool for investigating the mechanism of APP cleavage and Aβgeneration.3. FRET could be used to detect the occurrence ofβ-cleavage sensitively.4. CFP-54bp-YFP-C99 could be cleaved byβ-secretase and CFP-54bp-YFP could not which indicated that C99 would be significant forβ-cleavage of APP and might function as signal-peptide for directing the cleavage. If C99 function was disturbed, then the initiation of Aβmight be blocked. It may provide a new idea for the early therapy of AD.5. The fusion gene could be translated into correct protein. The expression product of fusion gene could be sequentially cleaved byβ- andγ-secretase and generate Aβlabeled by YFP.6. Aβwas produced mainly and firstly within the cell and little was liberated into the cell space.7. Before Aβdeposited outside of the cell, Aβaggregated within the cell and induced the secondary damage to the cell which leaded to the cell viability decrease and shape abnormality.