Abstract
The established primary trigger of Alzheimer’s disease’s is β-amyloid (Aβ) (Mucke and Selkoe, 2012). Amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes also depends on its recycling back to the plasma membrane (Koo et al., 1996; Ubelmann et al., 2017). The following recycling assay has been optimized to assess APP recycling by live murine Neuro2a cells, a neuroblastoma cell line (Ubelmann et al., 2017).
Keywords: APP, Recycling, Alzheimer’s, Immunofluorescence
Background
Aβ42 accumulation is a primary trigger of Alzheimer’s disease. APP endocytosis is required for Aβ42 generation (Koo and Squazzo, 1994; Grbovic et al., 2003; Cirrito et al., 2008; Rajendran et al., 2008). Upon endocytosis, APP can recycle back to the plasma membrane likely escaping processing at endosomes. While many studies have characterized APP endocytosis, mechanisms that regulate APP recycling need to be established. A robust, sensitive and quantitative assay is thus necessary. APP recycling has been assessed qualitatively by immunofluorescence and quantitatively using bulk biotinylation of surface proteins followed by a chase for endocytosis and a chase for recycling after stripping or blocking of surface proteins (Koo et al., 1996; Yamazaki et al., 1996; Chaufty et al., 2012). We developed a methodology for following and measuring APP recycling in Neuro2a cells using classical immunofluorescence and semi-quantitative cell biology methods. Our assay relies on the transient expression of APP tagged with the red fluorescent protein (RFP), to detect and quantify the cellular pool of APP and on using an antibody against the ectodomain of APP, to selectively detect and quantify the trafficking of the surface pool of APP.
Materials and Reagents
Equipment
Software
Procedure
Note: The APP recycling assay is performed on Neuro2a cells grown on glass coverslips.
Data analysis
Note: Data analysis is done using Microsoft Excel.
Notes
Recipes
Acknowledgments
We thank for the gift of APP-RFP plasmids from Dr. S. Kins (University of Kaiserslautern) and for Neuro2a cells from S. Miserey-Lenkei (Institut Curie). This work has been supported by CEDOC and by a Marie Curie Integration Grant (334366 TrafficInAD FP7-PEOPLE-2012-CIG; Marie Curie Actions, EC), iNOVA4Health–UID/Multi/04462/2013, a program financially supported by Fundação para a Ciência e Tecnologia (FCT)/Ministério da Educação e Ciência, through national funds and co-funded by FEDER under the PT2020 Partnership Agreement. CGA is Investigator FCT (IF/00998/2012, FCT). FU was the recipient of an FRM postdoctoral fellowship (SPE20130326599) and an FCT post-doctoral fellowship (SFRH/BPD/94186/2013). This protocol was adapted from our recent publication in EMBO reports (Ubelmann et al., 2017). The authors declare no conflict of interest.
References
If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.