AD Model: Amyloid Precursor Protein (APP) Secretase Activity Assay Cell Line
Alzheimer?s Disease (AD) is characterized by brain depositions of the beta amyloid (bA). The bA is the amyloid precursor protein (APP) digestion product, which is released from the cell after b-secretase and g-secretase proteolysis. A novel fluorescence-based assay cell line for secretases activity screening of new inhibitors has been developed.
Innoprot has developed an APP-tGFP MDCK stable cell line which allows to perform assays to oversee the endogenous secretase proteolytic process in living cells, allowing the identification of ?-secretase and ?-secretase inhibitors in your screening campaings. We offer this cell line as a stable cell line but we also offer vials of division-arrested cells (DA cells) in order to perform a small number of assays. Each vial of these DA cells contains 2 million cells, an enough number of cells to perform a complete experiment in a 96 well-plate.
Assay Details: Gamma and Beta secretase commercial inhibitors and a siRNA for BACE1 have been used to perform a High content analysis using this model. Results indicated that the pharmacological inhibition of secretases implicated in AD remains a valid strategy for drug screening.
Therefore, this cellular model has been used to monitor the secretases activity in vivo treated with a library compounds in an automatic epifluorescent imaging system to acquire and analyze how robust the model is. After treatments, images obtained were analyzed using Attovision software from Becton Dickinson and fluorescent vesicles into the citoplasm were quantified.
Results indicate that the detecting dynamyc range is dependent on the inhibitor biophysics and biochemical characteristics and the treatment time. This retention assay was validated with an average Z prime of 0.71+/- 0.01 for High Content Screening with a 72 hours treatment.
- This model permits evaluate a lybrary of compounds, candidates to inhibitors, in living cells studying the vesicles retention.
Fig 1: MDCK stably expressing human APP-tGFPcells were treated with 6 log dilution series(n=4) of inhibitors during 72 hours. Inhibitorsused were DAPT and L-685,458 as gamma-secretase inhibitors, beta-secretase inhibitor IV and Batimastat as alpha-secretase inhitor.
- This model provide a strategy to evaluate drug against secretases activity without the necessity to be permeable.
- This model allows to analyse in the space and time the compund effect in a multiparametric manner.
Fig 2: After that, the nucleus was stained with DAPIand cells with retained APP spots were detected by fluorescence using image analysisalgorithms. Percent Activity was calculated relative to positive (10 uM). This assay was alsoperformed with a 96 hours tratment.
Fig 3:The results indicate that the detecting dynamyc range is dependent on the inhibitor biophysics and biochemical characteristics andthe treatment time. This retention assay wasvalidated with an average Z prime of 0.71+/- 0.01 for High Content Screening with a 72 hourstreatment.
Fig 4: IC50 value for LP226A1 was determined bytreating of MDCK APP model cells withinhibitor concentrations 30uM, 25uM, 20uM,15uM, 10uM, 3 uM and 1uM during 72h.Followed this incubation, the intracellularvesicles retented are quantified. Error barsrepresent the standard deviation among 5replicate wells. Ic50 for LP226A1 was 17.8 uMand z prime for this experiment was 0.90 +/-0,01.