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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   

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本实验方案简略版
EMBO Reports
Jan 2017

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

  1. 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.
  2. Superfrost glass slides (MENZEL GERHARD, catalog number: 2586E )
  3. 24-well dishes (SARSTEDT, catalog number: 83.1836 ) for mammalian cell culture
  4. Plastic Pasteur pipette (SARSTEDT, catalog number: 86.1171 )
  5. Parafilm (Fisher Scientific, catalog number: 11782644)
    Manufacturer: Bemis, Parafilm, catalog number: PM999 .
  6. Neuroblastoma Neuro2a cells (ATCC, catalog number: CCL-131 )
  7. APP-RFP plasmid (Szodorai et al., 2009) (S. Kins, University of Kaiserslautern)
  8. Culture medium: Dulbecco’s modified Eagle medium (DMEM)-GlutaMAX (Thermo Fisher Scientific, GibcoTM, 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, GibcoTM, catalog number: 15140122 )
  9. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  10. Opti-MEM (Thermo Fisher Scientific, GibcoTM, catalog number: 31985062 )
  11. Lipofectamine RNAiMax (Thermo Fisher Scientific, InvitrogenTM, catalog number: 13778150 )
  12. Murine Anti-APP N-terminal monoclonal antibody (22C11) (Merck, catalog number: MAB348 )
  13. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  14. Phosphate buffer saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
  15. Donkey anti-mouse Alexa 488 (Thermo Fisher Scientific, catalog number: A-21202 )
  16. Coverslip-slide Mounting solution (FluoroMount-G) (Southern Biotech, catalog number: 0100-01 )
  17. Saponin (Sigma-Aldrich, catalog number: 47036 )
  18. Sodium chloride (NaCl) (NZYTech, catalog number: MB15901 )
  19. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  20. Acetic acid (Merck, catalog number: 1.00063.2511 )
  21. Acid-stripping buffer (see Recipes)
  22. Fixative solution (see Recipes)

Equipment

  1. CO2 incubator for primary cell culture (BINDER, model: CB 160 )
  2. Counting chamber (Belden, Hirschmann, catalog number: 8100103 )
  3. 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

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

Procedure

Note: The APP recycling assay is performed on Neuro2a cells grown on glass coverslips.

  1. Cell culture and transfection (Day 1, Day 2)
    1. Place one washed coverslip in the bottom of each well of a 24-well plate.
    2. Plate Neuro2a cells (30,000 cells per well of 24-well plate; adjust the cell density so that Neuro2a cells do not reach confluence at the day of the recycling assay) in culture medium (see Reagents) at 37 °C in a 5% CO2 and 20% O2 humidified incubator (Day 1). We recommend using two coverslips per condition.
      Note: Most cells will grow adherent to the glass coverslip.
    3. For expression of APP-RFP cDNA, 60-70% confluent Neuro2a cells are transiently transfected with 0.5 µg cDNA: 0.5 µl Lipofectamine 2000 mix in 25 µl Opti-MEM per well of 24-well plate with 250 µl of antibiotics-free complete medium. (Day 2)
    4. Replace transfection medium 2 h post-transfection with complete culture medium at 37 °C to avoid the excessive expression of APP-RFP (Day 2).
    5. (Optional) siRNA transfection for knockdown analysis with Lipofectamine RNAiMax (Thermo Fisher Scientific) according to manufacturer’s protocol 72 h prior to the recycling assay (Day 2).

  2. APP recycling assay in Neuro2a cells expressing APP-RFP by the following steps (Figure 1) (Day 3)


    Figure 1. Schematic of protocols to monitor APP endocytosis, APP recycling and non-recycling APP. A. Pulse with anti-APP antibody for 10 min; B. Upon 10 min pulse, non-endocytosed APP at the surface is removed with acid-stripping buffer, and recycled APP at the surface is detected in non-permeabilized cells after a 20 min chase; C. Upon the 20 min chase, the non-recycled pool of APP is detected upon acid-stripping and permeabilization.

    1. Remove cell culture media with a plastic Pasteur pipette, add 500 µl serum-free culture medium at 37 °C and incubate cells for 30 min at 37 °C in cell culture incubator.
      Note: All pipetting should be done slowly against the wall of the well without touching the cells.
    2. Dilute 0.25 µl of anti-APP antibody (22C11; 0.25 µg/µl stock concentration) into 25 µl culture medium with 10 mM HEPES (0.25 µl of stock solution at 1 M pH 7.2-7.5) per coverslip.
    3. Place one 25 µl droplet of diluted anti-APP antibody per glass coverslip onto Parafilm stretched on a 24 wells plate lid.
    4. Carefully place the coverslips over the droplets with cells facing the antibody solution and incubate them at 37 °C for 10 min in cell culture incubator. Cover the reaction to avoid evaporation. This step allows for anti-APP antibody binding to cell surface APP and subsequent endocytosis.
      (Optional) For a surface labeling control coverslip, carefully transfer the coverslips over the droplets with cells facing the antibody solution and incubate them at 37 °C for 4 min in cell culture incubator. Wash each coverslip by dipping in PBS at 37 °C for 4 s. Place coverslips back in a 24-well plate and add 250 µl of 4% PFA fixative solution for 10 min at room temperature (RT). After washing 3 times with PBS (RT) proceed for detection of anti-APP. This optional step allows for anti-APP antibody binding to cell surface APP.
    5. Wash each coverslip by dipping in PBS at 37 °C for 4 s.
      (Optional) For controlling the pool of endocytosed APP (see Figure 1A) proceed for fixation by placing the control coverslip(s) back in the 24-well plate and add 250 µl of 4% PFA fixative solution for 10 min at room temperature (RT). After washing 3 times with PBS (RT) proceed for detection of anti-APP.
    6. For assaying the pool of recycled APP bound to anti-APP (see Figure 1B) is necessary to:
      1. Remove of surface anti-APP antibody bound to non-endocytosed APP by dipping the corresponding coverslips into an acid-stripping buffer (see Recipes) for 5 s and careful washing by dipping the coverslips in PBS at 37 °C for 4 s.
      2. Place the coverslips back in a 24-well plate containing pre-warmed culture medium (37 °C) for a 20 min incubation at 37 °C in a CO2 incubator.
      3. Wash cells carefully by dipping the coverslips for 4 s in PBS at RT before fixation in 4% PFA for 20 min. Wash 3 times with PBS and proceed for detection of anti-APP.
    7. For assaying the pool of non-recycled APP bound to anti-APP (see Figure 1C), cells are carefully washed for 4 s in PBS at RT, followed by removal of recycled anti-APP antibody by dipping coverslips into an acid-stripping buffer for 5 s before fixation in 4% PFA for 20 min at RT. After washing 3 times with PBS proceed for detection of anti-APP.
    Note: Cells are fixed for 20 min instead of 10 min because Neuro2a cells, after the extensive manipulation required to detect the recycled APP, tend to detach from the coverslips.

  3. Anti-APP antibody detection (Day 3).
    1. For detection of the recycled APP bound to anti-APP at the plasma membrane (after 20 min chase, see Figure 1B):
      Note: Permeabilization is not necessary since the recycled anti-APP is extracellular.
      1. Place Parafilm stretched on a 24 wells plate lid.
      2. Place each coverslip (cells facing down) onto a 50 µl droplet of diluted donkey anti-mouse Alexa 488 (1:250) in 3% FBS/PBS.
      3. Cover the reaction and incubate it for 60 min at RT, in the dark.
      4. Place coverslips back on a 24-well plate and wash 3 times with PBS at RT.
      5. Mount coverslips with cells facing down onto a 25 µl droplet of Fluoromount G on microscope slides and let dry overnight in the dark to preserve the fluorescence signal.
    2. For detecting the pool of non-recycled APP bound to anti-APP (an optional endocytosis control, see Figure 1C)
      1. Place coverslips on the 24-well plate and add 250 µl of 0.1% saponin/PBS to permeabilize fixed cells for 20 min at RT.
      2. Wash once with PBS at RT.
      3. Place Parafilm stretched on a 24 wells plate lid.
      4. Place each coverslip (cells facing down) onto a 50 µl droplet of diluted donkey anti-mouse Alexa 488 (1:250) in 3% FBS/0.1% saponin/PBS.
      5. Place coverslips back on a 24-well plate and wash 3 times with PBS at RT.
      6. Mount coverslips with cells facing down onto a 25 µl droplet of Fluoromount G on microscope slides and let dry overnight in the dark to preserve the fluorescence signal.
      Optional: Include DAPI (1:10,000 of a stock of 1 mg/ml) in the fluorescently-labeled antibody solution to counterstain cell nuclei.

  4. Image acquisition (Day 4)
    1. Image acquisition can be done using epifluorescence microscopy (such as ZEISS Z2) with a 60x NA 1.4 oil immersion objective and a CCD camera (see Equipment for details on microscope used). Exposure times should be determined based on the sample with the brightest expected signal, to use as much of the dynamic range of the camera and a 16-bit binary range as possible, without saturating any of the pixels.
    2. Acquire 10-20 fields per condition to have sufficient data for statistical analysis. Select fields with isolated cells to facilitate analysis. Focusing on the dorsal plasma membrane may facilitate the visualization of APP recycling, see Figure 2 for representative images.


      Figure 2. Representative images of APP surface labeling (4 min), endocytosed APP (10 min) and recycled APP (+ 20 min chase) by Neuro2a cells expressing APP-RFP with anti-APP (22C11) detected with anti-mouse Alexa 488 (adapted from Ubelmann et al., 2017). Scale bar = 10 μm.

  5. Image analysis & APP recycling quantification (Figure 3) (Day 4)
    Note: Quantification of fluorescent signal of endocytosed APP per single cell acquired with ImageJ/Fiji.


    Figure 3. Image analysis of APP recycling

    1. Select cells with median expression of APP-RFP for image analysis in order to uniformize the quantity of APP present on the surface, available for endocytosis and subsequent recycling. Repeat the procedure for 20-50 cells with good morphology and median APP-RFP expression, about 10 fields per condition.
    2. Outline each cell based on anti-APP or APP-RFP signal, which becomes visible upon increasing the brightness using B&C tool (without altering intensity levels), using the tool ‘polygon selection’. Add it to ‘ROI manager’ (shift + t).
    3. Draw a background ROI using the tool ‘polygon selection’ in the original image and add it to the ‘ROI manager’.
    4. Measure mean intensities of APP-RFP and background ROIs using ‘measure’ (click M) of ROI manager. Fluorescence intensities of the two regions (background and cell) are measured for APP-RFP and anti-APP using the ‘ROI manager’.
    5. Transfer all regions to recycled Alexa488-anti-APP channel, and use ‘measure’ to obtain the mean fluorescence intensities per ROI and repeat this procedure for each ROI.
    6. Export measurements to Microsoft Excel.

Data analysis

Note: Data analysis is done using Microsoft Excel.

  1. Subtract the background mean fluorescence intensity for all measurements, per single cell.
  2. Normalize the Alexa488-anti-APP fluorescence intensities by the APP-RFP fluorescence intensity to control for different expression level of APP-RFP, per single cell.
  3. Sample size is about 20 cells per condition in each independent experiment, based on previous studies. Statistical significance for at least three independent experiments is determined on normal data (D’Agostino-Pearson omnibus normality test) by two-tailed Student’s t-test and for multiple comparisons one-way ANOVA with Tukey’s test using GraphPad Prism 6.
  4. Statistical significance for nonparametric data is tested by Mann-Whitney test.
  5. The numerical analysis and graphic representation can be accessed in Ubelmann et al., 2017.

Notes

  1. In our hands, APP recycling is robust with the average results very reproducible. However, variability among different cells is expected.
  2. Optimal APP transfection depends on necessary optimal experimental conditions, good quality APP-DNA plasmid (recent midi-prep) and healthy early passage Neuro2a cells.

Recipes

  1. Acid-stripping buffer
    0.5 M NaCl
    0.2 M acetic acid
  2. Fixative solution
    4% paraformaldehyde (PFA) in PBS, pH 7

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

  1. Chaufty, J., Sullivan, S. E. and Ho, A. (2012). Intracellular amyloid precursor protein sorting and amyloid-β secretion are regulated by Src-mediated phosphorylation of Mint2. J Neurosci 32(28): 9613-9625.
  2. Cirrito, J. R., Kang, J. E., Lee, J., Stewart, F. R., Verges, D. K., Silverio, L. M., Bu, G., Mennerick, S. and Holtzman, D. M. (2008). Endocytosis is required for synaptic activity-dependent release of amyloid-β in vivo. Neuron 58(1): 42-51.
  3. Grbovic, O. M., Mathews, P. M., Jiang, Y., Schmidt, S. D., Dinakar, R., Summers-Terio, N. B., Ceresa, B. P., Nixon, R. A. and Cataldo, A. M. (2003). Rab5-stimulated up-regulation of the endocytic pathway increases intracellular β-cleaved amyloid precursor protein carboxyl-terminal fragment levels and Aβ production. J Biol Chem 278(33): 31261-31268.
  4. Koo, E. H. and Squazzo, S. L. (1994). Evidence that production and release of amyloid β-protein involves the endocytic pathway. J Biol Chem 269(26): 17386-17389.
  5. Koo, E. H., Squazzo, S. L., Selkoe, D. J. and Koo, C. H. (1996). Trafficking of cell-surface amyloid β-protein precursor. I. Secretion, endocytosis and recycling as detected by labeled monoclonal antibody. J Cell Sci 109 (Pt 5): 991-998.
  6. Mucke, L. and Selkoe, D. J. (2012). Neurotoxicity of amyloid β-protein: synaptic and network dysfunction. Cold Spring Harb Perspect Med 2(7): a006338.
  7. Rajendran, L. and Annaert, W. (2012). Membrane trafficking pathways in Alzheimer's disease. Traffic 13(6): 759-770.
  8. Rajendran, L., Schneider, A., Schlechtingen, G., Weidlich, S., Ries, J., Braxmeier, T., Schwille, P., Schulz, J. B., Schroeder, C., Simons, M., Jennings, G., Knolker, H. J. and Simons, K. (2008). Efficient inhibition of the Alzheimer's disease β-sretase by membrane targeting. Science 320(5875): 520-523.
  9. Szodorai, A., Kuan, Y. H., Hunzelmann, S., Engel, U., Sakane, A., Sasaki, T., Takai, Y., Kirsch, J., Muller, U., Beyreuther, K., Brady, S., Morfini, G. and Kins, S. (2009). APP anterograde transport requires Rab3A GTPase activity for assembly of the transport vesicle. J Neurosci 29(46): 14534-14544.
  10. Ubelmann, F., Burrinha, T., Salavessa, L., Gomes, R., Ferreira, C., Moreno, N. and Guimas Almeida, C. (2017). Bin1 and CD2AP polarise the endocytic generation of beta-amyloid. EMBO Rep 18(1): 102-122.
  11. Yamazaki, T., Koo, E. H. and Selkoe, D. J. (1996). Trafficking of cell-surface amyloid beta-protein precursor. II. Endocytosis, recycling and lysosomal targeting detected by immunolocalization. J Cell Sci 109 (Pt 5): 999-1008.

简介

已确定的阿尔茨海默病的主要诱因是β-淀粉样蛋白(Aβ)(穆克和Selkoe,2012)。淀粉样蛋白前体蛋白(APP)胞吞作用是早期内涵体产生Aβ所必需的(核内体中的APP保留还取决于其回到质膜的再循环(Koo等人,1996; Rajendran和Annaert,2012)。 Ubelmann等人,2017)。已经优化了以下回收测定法以评估活的小鼠神经母细胞瘤细胞系具有Neuro2a细胞(Ubelmann等人,2017)的APP回收。

【背景】Aβ42积聚是阿尔茨海默病的主要触发因素,APP的胞吞作用需要Aβ42代(辜和Squazzo,1994; Grbovic等人,2003; Cirrito等人,2008;拉金德伦等人,2008)内吞作用后,APP可以循环回到质膜上,可能会逃避内体的处理虽然许多研究表征了APP内吞作用,但需要建立调节。 APP回收的机制。因此,强有力的,敏感的和定量的测定是必要的。已经通过免疫荧光和定量地使用表面蛋白的批量生物素化,随后追逐内吞作用和剥离或阻断表面蛋白后的循环追踪,定性地评估APP回收(辜等,1996; Yamazaki,1996; Chaufty等,2012)。
 
我们使用经典的免疫荧光和半定量的细胞生物学方法,开发了用于跟踪和测量具有Neuro2a细胞中的APP回收的方法。我们的测定依赖于用红色荧光蛋白(RFP)标记的APP的瞬时表达,以检测和定量APP的细胞库和使用针对APP胞外域的抗体来选择性检测和定量表面池的运输的APP。

关键字:APP, 回收, 阿尔茨海默病, 免疫荧光

材料和试剂

  1. 圆形玻璃盖玻片,13毫米(VWR,Marienfeld,目录号:630-1597)
    注意:高压灭菌,在室温下用40%乙醇/ 60%HCl预清洗1小时,并在RT下用Milli-Q水洗涤4次,每次15分钟。
  2. 超级玻璃滑梯(MENZEL GERHARD,产品目录号:2586E)
  3. 用于哺乳动物细胞培养的24孔培养皿(SARSTEDT,目录编号:83.1836)
  4. 塑料巴斯德吸管(SARSTEDT,目录号:86.1171)
  5. Parafilm(Fisher Scientific,目录号:11782644)
    制造商:Bemis,Parafilm,目录号:PM999。
  6. 神经母细胞瘤Neuro2a细胞(ATCC,目录号:CCL-131)
  7. APP-RFP质粒(Szodorai等人,2009)(S.Kins,凯泽斯劳滕大学)
  8. 培养基:含有10%胎牛血清(FBS)(Sigma-Aldrich,目录号:F0804)的Dulbecco改良Eagle培养基(DMEM)-GlutaMAX(Thermo Fisher Scientific,Gibco TM,目录号:10566016) ); 100U / ml青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,产品目录号:15140122)
  9. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  10. Opti-MEM(Thermo Fisher Scientific,Gibco TM,目录号:31985062)
  11. Lipofectamine RNAiMax(Thermo Fisher Scientific,Invitrogen TM,目录号:13778150)
  12. 鼠抗APP N-末端单克隆抗体(22C11)(Merck,目录号:MAB348)
  13. HEPES(1M)(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  14. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,产品目录号:10010031)
  15. 驴抗小鼠Alexa 488(Thermo Fisher Scientific,目录号:A-21202)
  16. 盖玻片安装解决方案(FluoroMount-G)(Southern Biotech,目录号:0100-01)
  17. 皂苷(Sigma-Aldrich,目录号:47036)
  18. 氯化钠(NaCl)(NZYTech,目录号:MB15901)
  19. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  20. 醋酸(Merck,目录号:1.00063.2511)
  21. 酸解缓冲液(见食谱)
  22. 固定解决方案(见食谱)

设备

  1. CO 2培养箱用于原代细胞培养(BINDER,型号:CB 160)
  2. 计数室(Belden,Hirschmann,目录号:8100103)
  3. Epifluorescence直立式显微镜Z2(Carl Zeiss,型号:Axio Imager Z2)配备了60x NA-1.4油浸物镜和AxioCam MRm CCD照相机(Carl Zeiss)

软件

  1. ImageJ 2.0 v1.51n软件(可从 http://rsb.info.nih.gov/ij/免费下载
  2. GraphPad Prism 6( https://www.graphpad.com/scientific-software/prism/

程序

注意:APP回收测定是在玻璃盖玻片上生长的Neuro2a细胞上进行的。

  1. 细胞培养和转染(第1天,第2天)

    1. 在一个24孔板的每个孔的底部放置一个洗过的盖玻片
    2. 在37℃下,在5%CO中,平板Neuro2a细胞(24孔板每孔30,000个细胞;调节细胞密度,使得Neuro2a细胞在再循环测定当天不会达到汇合)(见试剂)加湿培养箱(第1天)和20%O 2加湿培养箱。我们建议每个条件使用两个盖玻片。
      注意:大多数细胞会附着在玻璃盖玻片上。
    3. 为了表达APP-RFP cDNA,将60-70%融合的Neuro2a细胞用0.5μgcDNA:每孔25μlOpti-MEM中的0.5μlLipofectamine 2000混合物用24孔板和250μl不含抗生素的完全培养基瞬时转染。 (第二天)
    4. 用完全培养基在37℃下转染2小时后更换转染培养基以避免APP-RFP(第2天)的过度表达。
    5. (可选)在回收测定之前72小时(第2天),根据制造商的方案,用Lipofectamine RNAiMax(赛默飞世尔科技)进行siRNA转染用于敲低分析。

  2. 通过以下步骤(图1)(第3天)表达APP-RFP的Neuro2a细胞中的APP回收测定法(第3天)

    图1.监测APP内吞,APP回收和非回收APP的方案示意图。 :一种。用抗APP抗体脉冲10分钟; B.在10分钟脉冲时,在表面的非内吞的APP用酸洗剥离液除去,在20分钟追踪后,在非透化细胞中检测到表面的再循环的APP; C.经过20分钟追踪,APP的非再循环池在酸解和渗透时被检测到。

    1. 用塑料巴斯德吸管移出细胞培养基,在37°C加入500μL无血清培养基,细胞培养箱孵育细胞30分钟在37°C.
      注意:所有的移液都应该缓慢地靠在井壁上,而不要接触细胞。
    2. 稀释0.25μL的抗APP抗体(22C11; 0.25微克/微升股票浓度)到25μL的培养基与10毫米的HEPES(0.25μL的储备液,1M的pH 7.2-7.5)每盖玻片。
    3. 将每个玻璃盖玻片上的25μl稀释的抗APP抗体滴在24孔板盖上的Parafilm上。
    4. 小心地将盖玻片上的细胞面对抗体溶液,并在37°C培养10分钟细胞培养箱。覆盖反应以避免蒸发。这个步骤允许抗APP抗体结合到细胞表面APP和随后的内吞作用。
      (可选)对于表面标记控制盖玻片,小心地将盖玻片上的细胞面对抗体溶液的细胞转移,并在细胞培养箱中孵育他们在37°C 4分钟。洗涤每个盖玻片浸入PBS中37°C 4秒。将盖玻片放回到24孔板中,并在室温(RT)下加入250μl4%PFA固定溶液10分钟。用PBS(RT)洗涤3次后,进行抗APP的检测。该可选步骤允许抗APP抗体与细胞表面APP结合。
    5. 洗涤每个盖玻片浸入PBS中37°C 4秒。
      (可选)为了控制内吞的APP池(参见图1A),将对照盖玻片重新放回到24孔板中并加入250μl4%PFA固定溶液10分钟以进行固定室温(RT)。用PBS(RT)洗涤3次后,进行抗APP检测。
    6. 为了测定结合抗APP的再循环APP(参见图1B)的池是必要的:
      1. 通过将相应的盖玻片浸入酸性剥离缓冲液(参见食谱)中5秒钟,去除与非内吞APP相结合的表面抗APP抗体,并通过将盖玻片在37℃下在PBS中浸渍4秒小心清洗。
      2. 将盖玻片放回到含有预热的培养基(37℃)的24孔板中,在37℃在CO 2培养箱中孵育20分钟。
      3. 通过在室温下将盖玻片在PBS中浸渍4s仔细清洗细胞,然后在4%PFA中固定20分钟。用PBS清洗3次,继续检测抗APP。
    7. 为了测定结合抗APP的未再循环的APP库(参见图1C),细胞在室温下在PBS中小心地洗涤4秒,接着通过将盖玻片浸入酸洗脱缓冲液中除去再循环的抗APP抗体在4%PFA中固定5分钟,在RT下20分钟。用PBS洗涤3次后进行抗APP的检测。
    注意:细胞被固定20分钟而不是10分钟,因为Neuro2a细胞在检测回收的APP所需的大量操作之后倾向于与盖玻片分离。

  3. 抗APP抗体检测(第3天)。
    1. 为了检测与质膜上的抗APP结合的再循环APP(20分钟追踪后,见图1B):
      注意:因为回收的抗APP是细胞外的,所以通透不是必需的。
      1. 将Parafilm放置在24孔板盖上。
      2. 放置每个盖玻片(细胞面朝下)到50μL稀释驴抗小鼠Alexa 488(1:250)在3%FBS / PBS液滴。
      3. 覆盖反应,并在室温下孵育60分钟,在黑暗中。
      4. 将盖玻片放回到24孔板上,在室温下用PBS洗涤3次。
      5. 将盖玻片上的细胞面朝下放在显微镜载玻片上的25μlFluoromount G液滴上,让其在黑暗中过夜以保存荧光信号。
    2. 为了检测结合抗APP的非再循环APP(任选的内吞对照,参见图1C)
      1. 在24孔板上放置盖玻片,加入250μl的0.1%皂角苷/ PBS在室温下透化固定的细胞20分钟。

      2. 在PBS中用PBS清洗一次
      3. 将Parafilm放置在24孔板盖上。
      4. 将每个盖玻片(细胞朝下)放入50μl的稀释的驴抗小鼠Alexa 488(1:250)在3%FBS / 0.1%皂角苷/ PBS中的液滴。
      5. 将盖玻片放回到24孔板上,在室温下用PBS洗涤3次。
      6. 将盖玻片上的细胞面朝下放在显微镜载玻片上的25μlFluoromount G液滴上,并在黑暗中干燥过夜以保存荧光信号。
      可选:在荧光标记的抗体溶液中包含DAPI(1:10,000的1mg / ml的原液)以使细胞核复染。

  4. 图像采集(第4天)
    1. 图像采集可以使用epifluorescence显微镜(如ZEISS Z2)与一个60x NA 1.4油浸物镜和一个CCD相机(见设备的细节使用显微镜)。曝光时间应根据具有最明亮的预期信号的样本来确定,以尽可能多地使用摄像机的动态范围和尽可能使用16位二进制范围,而不使任何像素饱和。
    2. 每个条件采集10-20个字段,以获得足够的数据进行统计分析。选择具有隔离单元格的字段以方便分析。聚焦于背侧质膜可以促进APP回收的可视化,见图2代表图像。


      图2.表达APP-RFP的Neuro2a细胞的APP表面标记(4分钟),内吞APP(10分钟)和再循环APP(+ 20分钟追踪)的代表性图像,抗-APP(22C11)小鼠Alexa 488(改编自Ubelmann等人,2017)。比例尺= 10微米。

  5. 图像分析与放大APP回收量化(图3)(第4天)
    注意:用ImageJ / Fiji获得的单个细胞内吞的APP的荧光信号的定量


    图3. APP回收的图像分析

    1. 选择具有APP-RFP中值表达的细胞进行图像分析,以便使存在于表面上的APP的量均匀化,可用于内吞作用和随后的再循环。重复20-50细胞的过程,具有良好的形态学和中位APP-RFP表达,每个条件大约10个场。
    2. 根据抗APP或APP-RFP信号勾画出每个细胞,使用工具“多边形选择”,使用B& C工具(不改变强度水平)增加亮度时,该信号变得可见。将其添加到“ROI经理”(shift + t)。
    3. 使用原始图像中的工具“多边形选择”绘制背景投资回报率,并将其添加到“投资回报管理器”。
    4. 使用ROI管理器的“度量”(单击M)度量APP-RFP和背景ROI的平均强度。使用“ROI管理器”测量APP-RFP和抗APP的两个区域(背景和细胞)的荧光强度。
    5. 将所有区域转移至回收的Alexa488-抗APP通道,并使用“测量”获得每个ROI的平均荧光强度,并对每个ROI重复此程序。
    6. 将度量值导出到Microsoft Excel。

数据分析

注意:数据分析是使用Microsoft Excel完成的。


  1. 每个单元格减去所有测量的背景平均荧光强度
  2. 通过APP-RFP荧光强度将Alexa488-抗APP荧光强度标准化以控制每个单细胞的APP-RFP的不同表达水平。
  3. 根据以前的研究,每个独立实验中的样品大小是每个条件约20个细胞。对于至少三个独立实验的统计学显着性是通过双尾学生t检验在正常数据(D'Agostino-Pearson综合正态性检验)上确定的,并且对于使用Tukey检验的多重比较单因素方差分析来确定GraphPad棱镜6.
  4. 非参数数据的统计显着性通过Mann-Whitney检验来检验。

  5. 数值分析和图示可以在Ubelmann等人的“2017年”中找到。

笔记

  1. 在我们的手中,APP回收是稳健的,平均结果是非常可重复的。但是,不同的细胞之间的变化预计。
  2. 最佳的APP转染依赖于必要的最佳实验条件,优质的APP-DNA质粒(最近的midi-prep)和健康的早期传代的Neuro2a细胞。

食谱

  1. 酸解缓冲液
    0.5 M NaCl
    0.2 M乙酸
  2. 固定解决方案
    4%多聚甲醛(PFA)的PBS溶液,pH7

致谢

我们感谢S.Kins博士(凯泽斯劳滕大学)的APP-RFP质粒以及S.Miserey-Lenkei(Institut Curie)的Neuro2a细胞。这项工作得到了CEDOC和居里夫人综合补助金(334366 TrafficInAD FP7-PEOPLE-2012-CIG; Marie Curie Actions,EC)的支持,iNOVA4Health-UID / Multi / 04462/2013是一个由Fundaçãopara a Ciênciae Tecnologia(FCT)/MinêteriodaEducaçãoeCiência,通过国家基金和FEDER根据PT2020合作协议共同出资。 CGA是Investigator FCT(IF / 00998/2012,FCT)。 FU是FRM博士后奖学金获得者(SPE20130326599)和FCT博士后奖学金(SFRH / BPD / 94186/2013)。这个协议是根据我们最近在EMBO报告中发表的(Ubelmann等人,2017)。作者宣称没有利益冲突。

参考

  1. Chaufty,J.,Sullivan,S.E。和Ho,A。(2012)。 细胞内淀粉样前体蛋白分选和淀粉样蛋白β分泌受Src介导的Mint2磷酸化调节。 / J> Neurosci 32(28):9613-9625。
  2. Cirrito,J.R.,Kang,J.E.,Lee,J.,Stewart,F.R.,Verges,D.K.,Silverio,L.M.,Bu,G.,Mennerick,S.and Holtzman,D.M。(2008)。 体内的突触活性依赖性释放淀粉样β蛋白需要胞吞作用。 Neuron 58(1):42-51。
  3. Grbovic,O.M.,Mathews,P.M.,Jiang,Y.,Schmidt,S.D。,Dinakar,R.,Summers-Terio,N.B.,Ceresa,B.P.,Nixon,R.A。和Cataldo,A.M。(2003) Rab5刺激的内吞途径的上调增加细胞内β切割的淀粉样前体蛋白羧基末端片段水平和Aβ产生。 J Biol Che m 278(33):31261-31268。
  4. Koo,E.H。和Squazzo,S.L。(1994)。 证据表明β-淀粉样蛋白的产生和释放涉及内吞途径。 > J Biol Chem 269(26):17386-17389。
  5. Koo,E.H。,Squazzo,S.L.,Selkoe,D.J。和Koo,C.H。(1996)。 贩卖细胞表面淀粉样β蛋白前体。 I.通过标记的单克隆抗体检测到的分泌,内吞和再循环。
  6. Mucke,L。和Selkoe,D.J。(2012)。 淀粉样β蛋白的神经毒性:突触和网络功能障碍 Cold Spring Harb Perspect Med 2(7):a006338。
  7. Rajendran,L.和Annaert,W。(2012)。 阿尔茨海默病中的膜贩运途径 Traffic 13( 6):759-770。
  8. Rajendran,L.,Schneider,A.,Schlechtingen,G.,Weidlich,S.,Ries,J.,Braxmeier,T.,Schwille,P.,Schulz,JB,Schroeder,C.,Simons,M.,Jennings ,G.,Knolker,HJ和Simons,K。(2008)。 通过膜靶向有效抑制阿尔茨海默病β-sretase 科学 320(5875):520-523。
  9. Szodorai,A.,Kuan,YH,Hunzelmann,S.,Engel,U.,Sakane,A.,Sasaki,T.,Takai,Y.,Kirsch,J.,Muller,U.,Beyreuther,K.,Brady ,S.,Morfini,G。和Kins,S。(2009)。 APP顺行运输需要Rab3A GTP酶活性来组装运输囊泡。 J Neurosci 29(46):14534-14544。
  10. Ubelmann,F.,Burrinha,T.,Salavessa,L.,Gomes,R.,Ferreira,C.,Moreno,N.和Guimas Almeida,C.(2017)。 Bin1和CD2AP极化β淀粉样蛋白的内吞性生成 EMBO Rep 18(1):102-122。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
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.
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