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Direct imaging of APP proteolysis in living cells

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ABSTRACT

Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the β-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the β-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

No MeSH data available.


Correct targeting of mChAPPmGFP and colocalization of mCherry and mGFP.(A) Maximum intensity projection of a confocal z-stack of fixed human SH-SY5Y cells transfected with mChAPPmGFP and surface labeled with anti-mCherry coupled to secondary Alexa 405 antibody (blue). Maximum intensity projections of confocal z-stacks of living human SH-SY5Y cells (B) and rat hippocampal neurons (E) transfected with mChAPPmGFP. (H) Maximum intensity projection of a confocal z-stack of fixed and permeabilized SH-SY5Y cells transfected with apAPPha and labeled with streptavidin Alexa 568 (red), which binds to the bionitylated AP tag, and anti-HA coupled to secondary Alexa 488 antibody (green). The high degree of co-localisation of the red and green signals evident from the linear correlation of scatterplots (C, F, I) and the exponential shape of the Li’s intensity correlation analysis (D, G, J) is confirmed by the Pearson’s (Pc) and Manders’coefficients (M1 and M2) close to 1, and by the intensity correlation quotients (ICQ) close to 0.5. The probability of obtaining the observed Pcs by chance is inversely correlated with the Costes’ randomization P value, which is 100% in all cases. Scalebars, 10 µm.
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fig-2: Correct targeting of mChAPPmGFP and colocalization of mCherry and mGFP.(A) Maximum intensity projection of a confocal z-stack of fixed human SH-SY5Y cells transfected with mChAPPmGFP and surface labeled with anti-mCherry coupled to secondary Alexa 405 antibody (blue). Maximum intensity projections of confocal z-stacks of living human SH-SY5Y cells (B) and rat hippocampal neurons (E) transfected with mChAPPmGFP. (H) Maximum intensity projection of a confocal z-stack of fixed and permeabilized SH-SY5Y cells transfected with apAPPha and labeled with streptavidin Alexa 568 (red), which binds to the bionitylated AP tag, and anti-HA coupled to secondary Alexa 488 antibody (green). The high degree of co-localisation of the red and green signals evident from the linear correlation of scatterplots (C, F, I) and the exponential shape of the Li’s intensity correlation analysis (D, G, J) is confirmed by the Pearson’s (Pc) and Manders’coefficients (M1 and M2) close to 1, and by the intensity correlation quotients (ICQ) close to 0.5. The probability of obtaining the observed Pcs by chance is inversely correlated with the Costes’ randomization P value, which is 100% in all cases. Scalebars, 10 µm.

Mentions: mCherry was fused immediately after the signal peptide, which is necessary to target APP to the plasma membrane, and away from the site recognized and cleaved by the β-secretase. In order to test if mChAPPmGFP is correctly targeted to the plasma membrane, we performed a surface immunolabelling of transfected human SH-SY5Y neuroblastoma cells with a primary anti-mCherry antibody (Fig. 2A). Confocal laser scanning microscopy (CLSM) imaging reveals a good degree of labeling only in cells expressing mChAPPmGFP, confirming that mCherry is correctly folded and present on the surface of the cell. We observed red clusters not colocalising with anti-mCherry, thus indicating their intracellular localization.


Direct imaging of APP proteolysis in living cells
Correct targeting of mChAPPmGFP and colocalization of mCherry and mGFP.(A) Maximum intensity projection of a confocal z-stack of fixed human SH-SY5Y cells transfected with mChAPPmGFP and surface labeled with anti-mCherry coupled to secondary Alexa 405 antibody (blue). Maximum intensity projections of confocal z-stacks of living human SH-SY5Y cells (B) and rat hippocampal neurons (E) transfected with mChAPPmGFP. (H) Maximum intensity projection of a confocal z-stack of fixed and permeabilized SH-SY5Y cells transfected with apAPPha and labeled with streptavidin Alexa 568 (red), which binds to the bionitylated AP tag, and anti-HA coupled to secondary Alexa 488 antibody (green). The high degree of co-localisation of the red and green signals evident from the linear correlation of scatterplots (C, F, I) and the exponential shape of the Li’s intensity correlation analysis (D, G, J) is confirmed by the Pearson’s (Pc) and Manders’coefficients (M1 and M2) close to 1, and by the intensity correlation quotients (ICQ) close to 0.5. The probability of obtaining the observed Pcs by chance is inversely correlated with the Costes’ randomization P value, which is 100% in all cases. Scalebars, 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5391788&req=5

fig-2: Correct targeting of mChAPPmGFP and colocalization of mCherry and mGFP.(A) Maximum intensity projection of a confocal z-stack of fixed human SH-SY5Y cells transfected with mChAPPmGFP and surface labeled with anti-mCherry coupled to secondary Alexa 405 antibody (blue). Maximum intensity projections of confocal z-stacks of living human SH-SY5Y cells (B) and rat hippocampal neurons (E) transfected with mChAPPmGFP. (H) Maximum intensity projection of a confocal z-stack of fixed and permeabilized SH-SY5Y cells transfected with apAPPha and labeled with streptavidin Alexa 568 (red), which binds to the bionitylated AP tag, and anti-HA coupled to secondary Alexa 488 antibody (green). The high degree of co-localisation of the red and green signals evident from the linear correlation of scatterplots (C, F, I) and the exponential shape of the Li’s intensity correlation analysis (D, G, J) is confirmed by the Pearson’s (Pc) and Manders’coefficients (M1 and M2) close to 1, and by the intensity correlation quotients (ICQ) close to 0.5. The probability of obtaining the observed Pcs by chance is inversely correlated with the Costes’ randomization P value, which is 100% in all cases. Scalebars, 10 µm.
Mentions: mCherry was fused immediately after the signal peptide, which is necessary to target APP to the plasma membrane, and away from the site recognized and cleaved by the β-secretase. In order to test if mChAPPmGFP is correctly targeted to the plasma membrane, we performed a surface immunolabelling of transfected human SH-SY5Y neuroblastoma cells with a primary anti-mCherry antibody (Fig. 2A). Confocal laser scanning microscopy (CLSM) imaging reveals a good degree of labeling only in cells expressing mChAPPmGFP, confirming that mCherry is correctly folded and present on the surface of the cell. We observed red clusters not colocalising with anti-mCherry, thus indicating their intracellular localization.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Alzheimer’s disease is a multifactorial disorder caused by the interaction of genetic, epigenetic and environmental factors. The formation of cytotoxic oligomers consisting of Aβ peptide is widely accepted as being one of the main key events triggering the development of Alzheimer’s disease. Aβ peptide production results from the specific proteolytic processing of the amyloid precursor protein (APP). Deciphering the factors governing the activity of the secretases responsible for the cleavage of APP is still a critical issue. Kits available commercially measure the enzymatic activity of the secretases from cells lysates, in vitro. By contrast, we have developed a prototypal rapid bioassay that provides visible information on the proteolytic processing of APP directly in living cells. APP was fused to a monomeric variant of the green fluorescent protein and a monomeric variant of the red fluorescent protein at the C-terminal and N-terminal (mChAPPmGFP), respectively. Changes in the proteolytic processing rate in transfected human neuroblastoma and rat neuronal cells were imaged with confocal microscopy as changes in the red/green fluorescence intensity ratio. The significant decrease in the mean red/green ratio observed in cells over-expressing the β-secretase BACE1, or the α-secretase ADAM10, fused to a monomeric blue fluorescent protein confirms that the proteolytic site is still accessible. Specific siRNA was used to evaluate the contribution of endogenous BACE1. Interestingly, we found that the degree of proteolytic processing of APP is not completely homogeneous within the same single cell, and that there is a high degree of variability between cells of the same type. We were also able to follow with a fluorescence spectrometer the changes in the red emission intensity of the extracellular medium when BACE1 was overexpressed. This represents a complementary approach to fluorescence microscopy for rapidly detecting changes in the proteolytic processing of APP in real time. In order to allow the discrimination between the α- and the β-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the α-secretase cleavage without perturbing the β-secretase processing. Moreover, we obtained a quantitatively robust estimate of the changes in the red/green ratio for the above conditions by using a flow cytometer able to simultaneously excite and measure the red and green fluorescence. Our novel approach lay the foundation for a bioassay suitable to study the effect of drugs or particular conditions, to investigate in an unbiased way the the proteolytic processing of APP in single living cells in order, and to elucidate the causes of the variability and the factors driving the processing of APP.

No MeSH data available.