在亚细胞分辨率水平上定量测定极化原代神经元内淀粉样前体蛋白（APP）内吞运输的新方法

Florent Ubelmann; Tatiana Burrinha; Cláudia Guimas Almeida

doi:10.21769/BioProtoc.2629

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A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution

在亚细胞分辨率水平上定量测定极化原代神经元内淀粉样前体蛋白（APP）内吞运输的新方法

FU Florent Ubelmann

Tatiana Burrinha

Cláudia Guimas Almeida email

发布: 2017年12月05日第7卷第23期 DOI: 10.21769/BioProtoc.2629 浏览次数: 6882

评审: Yanjie LiAlessandro DidonnaAnonymous reviewer(s)

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Jan 2017

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Abstract

Alzheimer’s disease’s established primary trigger is β-amyloid (Aβ) (Mucke and Selkoe, 2012). The amyloid precursor protein (APP) endocytosis is required for Aβ generation at early endosomes (Rajendran and Annaert, 2012). APP retention at endosomes depends on its sorting for degradation in lysosomes (Haass et al., 1992; Morel et al., 2013; Edgar et al., 2015; Ubelmann et al., 2017). The following endocytosis assay has been optimized to assess the amyloid precursor protein (APP) endocytosis and degradation by live murine cortical primary neurons (Ubelmann et al., 2017).

Keywords: APP (APP)

Endocytosis (内吞作用)

Degradation (降解)

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). The endocytosis of APP has been analysed in pulse-chase kinetic experiments in bulk by classical biotinylation of surface proteins (Sannerud et al., 2011; Xiao et al., 2012; Sullivan et al., 2014), in single cells by specific labelling of surface APP using antibodies against N-terminal extracellular domain of APP (Yamazaki et al., 1995; Xiao et al., 2012). The majority of these studies used non-neuronal cells (Yamazaki et al., 1996; Lee et al., 2008; Sullivan et al., 2014), and neuronal-like cell lines (Xiao et al., 2012), few used primary neurons (Yamazaki et al., 1995; Sullivan et al., 2014). Primary neurons differentiate like in vivo axons and dendrites, with their specialized presynaptic terminals and post-synaptic compartments. However, careful measurements and distinction between these neuronal compartments are lacking in these reports. We developed a method of analysing APP endocytosis in the different neuronal compartments, the soma or cell body, dendrites and axons that we describe in this bio-protocol. Our protocol details the procedure for following and measuring APP endocytosis in polarized neurons using classical immunofluorescence and semi-quantitative cell biology analysis methods.

We believe our method will allow the field to move forward by reliably measuring semi-quantitatively the compartmentalized endocytosis of APP specific to polarized neurons.

Materials and Reagents

24-well dishes (SARSTEDT, catalog number: 83.1836 ) for mammalian cell culture
Circular glass coverslips, 13 mm (VWR, Marienfeld, catalog number: 630-1597 )
Note: Autoclaved, pre-washed with 40% ethanol/60% HCl for 1 h at RT and washed 4 times, 15 min each, with Milli-Q water at RT; coated overnight with 200 µl 0.1% (w/v) poly-D-lysine at 37 °C in a 5% CO₂ and 20% O₂ humidified incubator and washed 3 x with sterile Milli-Q water.
Superfrost glass slides (MENZEL GERHARD, catalog number: 2586E )
Plastic Pasteur pipette (SARSTEDT, catalog number: 86.1171 )
Parafilm (Fisher Scientific, catalog number: 11782644)
Manufacturer: Bemis, Parafilm, catalog number: PM999 .
Wild-type females and males mouse embryos (Balbc, embryonic day 16; Charles River)
APP-RFP plasmid (Szodorai et al., 2009) (S. Kins, University of Kaiserslautern)
0.1% (w/v) poly-D-lysine (Sigma-Aldrich, catalog number: P1149 )
Plating medium:
1. DMEM, high glucose, pyruvate (Thermo Fisher Scientific, Gibco^TM, catalog number: 11995065 )
2. 10% fetal bovine serum (FBS), qualified, heat inactivated, US origin (Thermo Fisher Scientific, Gibco^TM, catalog number: 16140071 )
3. 1% penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, Gibco^TM, catalog number: 15140122 )
Neurobasal medium:
1. Neurobasal medium (Thermo Fisher Scientific, Gibco^TM, catalog number: 21103049 )
2. 2% B-27^® supplement, custom (50x) (Thermo Fisher Scientific, Gibco^TM, catalog number: 0080085SA )
3. 0.1% GlutaMAX^TM supplement (Thermo Fisher Scientific, Gibco^TM, catalog number: 35050038 )
Trypsin (2.5%), no phenol red (Thermo Fisher Scientific, Gibco^TM, catalog number: 15090046 )
Lipofectamine 2000 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 11668019 )
Opti-MEM (Thermo Fisher Scientific, catalog number: 31985062 )
Murine anti-APP N-terminal monoclonal (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 )
Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
Sucrose (NZYTech, catalog number: MB18601 )
Saponin (Sigma-Aldrich, catalog number: 47036 )
Donkey anti-mouse Alexa 488 (Thermo Fisher Scientific, catalog number: A-21202 )
Coverslip-Slide Mounting solution (FluoroMount-G) (SouthernBiotech, catalog number: 0100-01 )
DAPI (Sigma-Aldrich, catalog number: D9542 )
HBSS (GE Healthcare, Hyclone^TM, catalog number: SH30031.03 )
50% glucose in sterile water (NZYTech, catalog number: MB16801 )
Bovine serum albumin fraction V (BSA) (NZYTech, catalog number: MB04602 )

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

Procedure

English

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Ubelmann, F., Burrinha, T. and Guimas Almeida, C. (2017). A Novel Protocol to Quantitatively Measure the Endocytic Trafficking of Amyloid Precursor Protein (APP) in Polarized Primary Neurons with Sub-cellular Resolution. Bio-protocol 7(23): e2629. DOI: 10.21769/BioProtoc.2629.