Direct imaging of APP proteolysis in living cells
Authors: Parenti N., Grosso A.D., Antoni C., Cecchini M., Corradetti R., Pavone F.S., Calamai M.
Autors Affiliation: European Laboratory for Non-linear Spectroscopy (LENS), University of Florence, Sesto Fiorentino, Florence, Italy; Department of Neuroscience, Psychology, Drug Research and Child Health, University of Florence, Florence, Italy; NEST, Istituto Nanoscienze-CNR and Scuola Normale Superiore, Pisa, Italy; National Institute of Optics, National Research Council of Italy (CNR), Florence, Italy
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 Aß peptide is widely accepted as being one of the main key events triggering the development of Alzheimer\’s disease. Aß 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 ß-secretase BACE1, or the a-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 ß- and the a-secretase activity, we have created a variant of mChAPPmGFP with a mutation that inhibits the ß-secretase cleavage without perturbing the ß-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.
Volume: 2017 (4) Pages from: to:
More Information: The research leading to these results has received funding from the European Union\’s Horizon 2020 research and innovation programme under grant agreement no 654148 Laserlab-Europe (EU-H2020 654148), from the Italian Ministry of Health in the framework of the project \”Automated digital scanning and diagnosis of tissues using multimodal non-linear optical microscopy\” (GR-2011-02349626), and from the Italian Ministry for Education, University and Research in the framework of the Flagship Project NANOMAX. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.KeyWords: ADAM10 endopeptidase; amyloid beta protein; amyloid precursor protein; beta secretase 1; green fluorescent protein; red fluorescent protein; secretase; small interfering RNA, Alzheimer disease; animal cell; Article; bioassay; cell membrane; confocal laser scanning microscopy; controlled study; enzyme activity; flow cytometry; fluorescence activated cell sorting; fluorescence microscopy; fluorescence spectroscopy; gene mutation; gene overexpression; genetic transfection; hippocampal neuronal culture; human; human cell; molecular cloning; neuroblastoma cell line; nonhuman; polymerase chain reaction; protein degradation; protein expression; rat; Western blottingDOI: 10.7717/peerj.3086