Neuro2a细胞中淀粉样前体蛋白（APP）的内吞回收测定

Florent Ubelmann; Tatiana Burrinha; Claudia Guimas Almeida

doi:10.21769/BioProtoc.2635

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Peer-reviewed

Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells

Neuro2a细胞中淀粉样前体蛋白（APP）的内吞回收测定

FU Florent Ubelmann

Tatiana Burrinha

Claudia Guimas Almeida email

发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2635 浏览次数: 7422

评审: Anonymous reviewer(s)

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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 (APP)

Recycling (回收)

Alzheimer’s (阿尔茨海默病)

Immunofluorescence (免疫荧光)

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

Circular glass coverslips, 13 mm (VWR, Marienfeld, catalog number: 630-1597 )
Note: Autoclaved, pre-washed with 40% ethanol/60% HCl 1 h at RT and washed 4 times, 15 min each, with Milli-Q water at RT.
Superfrost glass slides (MENZEL GERHARD, catalog number: 2586E )
24-well dishes (SARSTEDT, catalog number: 83.1836 ) for mammalian cell culture
Plastic Pasteur pipette (SARSTEDT, catalog number: 86.1171 )
Parafilm (Fisher Scientific, catalog number: 11782644)
Manufacturer: Bemis, Parafilm, catalog number: PM999 .
Neuroblastoma Neuro2a cells (ATCC, catalog number: CCL-131 )
APP-RFP plasmid (Szodorai et al., 2009) (S. Kins, University of Kaiserslautern)
Culture medium: Dulbecco’s modified Eagle medium (DMEM)-GlutaMAX (Thermo Fisher Scientific, Gibco^TM, catalog number: 10566016 ) with 10% fetal bovine serum (FBS) (Sigma-Aldrich, catalog number: F0804 ); 100 U/ml penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11668019 )
Opti-MEM (Thermo Fisher Scientific, Gibco^TM, catalog number: 31985062 )
Lipofectamine RNAiMax (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 13778150 )
Murine Anti-APP N-terminal monoclonal antibody (22C11) (Merck, catalog number: MAB348 )
HEPES (1 M) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15630080 )
Phosphate buffer saline (PBS) (Thermo Fisher Scientific, Gibco^TM, catalog number: 10010031 )
Donkey anti-mouse Alexa 488 (Thermo Fisher Scientific, catalog number: A-21202 )
Coverslip-slide Mounting solution (FluoroMount-G) (Southern Biotech, catalog number: 0100-01 )
Saponin (Sigma-Aldrich, catalog number: 47036 )
Sodium chloride (NaCl) (NZYTech, catalog number: MB15901 )
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
Acetic acid (Merck, catalog number: 1.00063.2511 )
Acid-stripping buffer (see Recipes)
Fixative solution (see Recipes)

Equipment

CO₂ incubator for primary cell culture (BINDER, model: CB 160 )
Counting chamber (Belden, Hirschmann, catalog number: 8100103 )
Epifluorescence upright microscope Z2 (Carl Zeiss, model: Axio Imager Z2 ) equipped a 60x NA-1.4 oil immersion objective and an AxioCam MRm CCD camera (Carl Zeiss)

Software

ImageJ 2.0 v1.51n software (free download from http://rsb.info.nih.gov/ij/)
GraphPad Prism 6 (https://www.graphpad.com/scientific-software/prism/)

Procedure

English

中文翻译

文章信息

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如何引用

Ubelmann, F., Burrinha, T. and Guimas Almeida, C. (2017). Measuring the Endocytic Recycling of Amyloid Precursor Protein (APP) in Neuro2a Cells. Bio-protocol 7(23): e2635. DOI: 10.21769/BioProtoc.2635.