搜索

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   

评审
匿名评审
下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

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

  1. 24-well dishes (SARSTEDT, catalog number: 83.1836 ) for mammalian cell culture
  2. 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.
  3. Superfrost glass slides (MENZEL GERHARD, catalog number: 2586E )
  4. Plastic Pasteur pipette (SARSTEDT, catalog number: 86.1171 )
  5. Parafilm (Fisher Scientific, catalog number: 11782644)
    Manufacturer: Bemis, Parafilm, catalog number: PM999 .
  6. Wild-type females and males mouse embryos (Balbc, embryonic day 16; Charles River)
  7. APP-RFP plasmid (Szodorai et al., 2009) (S. Kins, University of Kaiserslautern)
  8. 0.1% (w/v) poly-D-lysine (Sigma-Aldrich, catalog number: P1149 )
  9. Plating medium:
    1. DMEM, high glucose, pyruvate (Thermo Fisher Scientific, GibcoTM, catalog number: 11995065 )
    2. 10% fetal bovine serum (FBS), qualified, heat inactivated, US origin (Thermo Fisher Scientific, GibcoTM, catalog number: 16140071 )
    3. 1% penicillin-streptomycin (10,000 U/ml) (Thermo Fisher Scientific, GibcoTM, catalog number: 15140122 )
  10. Neurobasal medium:
    1. Neurobasal medium (Thermo Fisher Scientific, GibcoTM, catalog number: 21103049 )
    2. 2% B-27® supplement, custom (50x) (Thermo Fisher Scientific, GibcoTM, catalog number: 0080085SA )
    3. 0.1% GlutaMAXTM supplement (Thermo Fisher Scientific, GibcoTM, catalog number: 35050038 )
  11. Trypsin (2.5%), no phenol red (Thermo Fisher Scientific, GibcoTM, catalog number: 15090046 )
  12. Lipofectamine 2000 (Thermo Fisher Scientific, InvitrogenTM, catalog number: 11668019 )
  13. Opti-MEM (Thermo Fisher Scientific, catalog number: 31985062 )
  14. Murine anti-APP N-terminal monoclonal (22C11) (Merck, catalog number: MAB348 )
  15. HEPES (1 M) (Thermo Fisher Scientific, GibcoTM, catalog number: 15630080 )
  16. Phosphate buffer saline (PBS) (Thermo Fisher Scientific, GibcoTM, catalog number: 10010031 )
  17. Paraformaldehyde (Sigma-Aldrich, catalog number: P6148 )
  18. Sucrose (NZYTech, catalog number: MB18601 )
  19. Saponin (Sigma-Aldrich, catalog number: 47036 )
  20. Donkey anti-mouse Alexa 488 (Thermo Fisher Scientific, catalog number: A-21202 )
  21. Coverslip-Slide Mounting solution (FluoroMount-G) (SouthernBiotech, catalog number: 0100-01 )
  22. DAPI (Sigma-Aldrich, catalog number: D9542 )
  23. HBSS (GE Healthcare, HycloneTM, catalog number: SH30031.03 )
  24. 50% glucose in sterile water (NZYTech, catalog number: MB16801 )
  25. Bovine serum albumin fraction V (BSA) (NZYTech, catalog number: MB04602 )

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 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 endocytosis assay is performed on neurons cultivated for 9 days in vitro (9 DIV).

  1. Cell culture and transfection (Day 1, Day 6, Day 8)
    1. Day 1: Prepare primary cortical neurons from cortices and hippocampi from wild-type females and males mouse embryos (E16) as previously described (Almeida et al., 2005 and 2006; Ubelmann et al., 2017). Briefly, dissociated neurons are plated on 24-well plates containing poly-D-lysine coated glass coverslips (5-10 x 104 cells/cm2) in plating medium (DIV 0). After 3 h or overnight, replace the plating medium with Neurobasal medium allowing only neurons to grow and differentiate until use. (Almeida et al., 2005 and 2006; Ubelmann et al., 2017)
    2. Day 8: For expression of APP-RFP cDNA, 8 DIV primary cortical neurons are transiently transfected with Lipofectamine 2000 according to manufacturer’s protocol, using 0.5 µg cDNA: 0.5 µl Lipofectamine mix in 25 µl Opti-MEM per well of 24-well plate with 250 µl fresh antibiotics-free Neurobasal medium and incubated overnight until assaying APP endocytosis .
      Note: Expect on average 10 transfected healthy neurons at 8 DIV at stage 5 of differentiation (Dotti et al., 1988) and the neurites should not show bead-like structures that indicate degeneration. This efficiency can be achieved using freshly prepared cDNA with a regular midi-prep kit (we use a local NZYTECH brand).
    3. (Optional) Day 6: For knockdown analysis, primary neurons at 6 days in vitro (6 DIV) were transfected with siRNA using Lipofectamine RNAiMax according to manufacturer’s protocol, after substituting culture media with fresh antibiotics-free Neurobasal medium.

  2. APP endocytosis assay of 9 DIV primary neurons expressing APP-RFP (Figure 1) by the following steps (Day 9)


    Figure 1. Schematic of APP endocytosis monitored using anti-APP antibody incubation for 10 min in axon, dendrite and cell body. EE: Early endosome; Lys: Lysosome.

    1. Remove cell culture media with a plastic Pasteur pipette, add 400 µl B27-free Neurobasal medium for 30 min at 37 °C in cell culture incubator. 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 complete Neurobasal 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. Place the coverslips, carefully and as fast as possible, over the droplets with cells facing the antibody solution, cover the reaction in a container to avoid evaporation and incubate at 37 °C for 10 min in a cell culture incubator. This step allows for anti-APP antibody binding to cell surface APP and subsequent endocytosis.
      (Optional)
      1. To monitor endocytosed APP lysosomal degradation, further incubate cells for 60 min upon washing by dipping coverslips once for 4 s in 500 µl pre-warmed PBS.
      2. To detect APP at the plasma membrane, incubate cells with anti-APP antibody for only 4 min at 37 °C in a cell culture incubator. This optional step allows labelling anti-APP antibody bound to cell surface APP without significant detection of endocytosis.
    5. Wash each coverslip by dipping once in 500 µl pre-warmed PBS for 4 s.
    6. Fix cells by placing coverslips back in a 24-well plate and add 500 µl 4% paraformaldehyde/4% sucrose for 20 min at room temperature (RT). Replace fixative solution with 500 µl PBS, and after 3 washes proceed for detection of anti-APP.

  3. Anti-APP antibody detection (Day 9)
    1. For detection of endocytosed APP bound to anti-APP antibody, permeabilize fixed cells with 500 µl 0.1% saponin/PBS (permeabilization buffer) for 60 min at RT. Remove the permeabilization buffer and wash each coverslip with PBS.
      Optional: For detection of anti-APP bound to APP at the cell surface (4 min), no permeabilization is required. Do not include saponin in steps C2 and C3.
    2. Block non-specific binding of the antibody to serum proteins with 500 µl of blocking buffer (3% FBS/0.1% saponin/PBS) for 60 min at RT.
    3. 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.
      Optional: Include DAPI in the antibody solution (1:10,000 of a stock of 1 mg/ml) to counterstain cell nuclei.
    4. Cover the reaction and incubate it for 60 min at RT, in the dark.
    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 at RT on microscope slides and let dry overnight in the dark to preserve the fluorescence signal.
      Note: Pipette Fluoromount G quickly to prevent it from drying but gently to avoid bubbles.

  4. Image acquisition (Day 10)
    Image acquisition is 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). See Figure 2 for a representative image.
    1. Neuronal cell body, portions of dendrites and axons must be in focus. Not well-developed neurons or neurons expressing low or high levels of APP-RFP should be excluded. 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 (ideally use a camera with 16-bit binary range) without saturating any of the pixels.
    2. Acquire 10-20 neurons per condition to have sufficient data for statistical analysis. To image the whole neuron, it may be necessary to acquire multiple fields.


      Figure 2. Representative image of APP endocytosis by primary neurons expressing APP-RFP incubated for 10 min with anti-APP (22C11) detected with anti-mouse Alexa 488. Dd: dendrite; Ax: axon; scale bars = 10 µm (adapted from Ubelmann et al., 2017).

  5. Image analysis & APP endocytosis quantification in cell body, dendrite and axon (Figure 3) (Day 10-11)
    Note: Quantification of endocytosed fluorescent signal acquired with ImageJ/Fiji.
    1. Identify the cell body, a dendrite and the axon based on neuronal morphology and on RFP signal and roughly outline composite selections (non-contiguous ROIs) corresponding to cell body, two to five ~20 µm segments in dendrites and one in the axon (usually thinner, longer and with branches at 90° angle) using ‘polygon selection’ while pressing ‘shift’ (see Figure 3). Axons and dendrites can alternatively be counterstained with Ankyrin-G (Santa Cruz Biotechnology) or MAP2 (Sigma-Aldrich).


      Figure 3. Image analysis of APP endocytosis: Step 1

    2. Duplicate image (shift + D) containing composite selections and remove unselected pixels by using ‘Clear outside’ (see Figure 4).


      Figure 4. Image analysis of APP endocytosis: Step 2

    3. To refine the ROI to the cell boundary: auto ‘Threshold’ the APP-RFP signal in the cell body; create a new ROI by clicking on the thresholded neurite with the ‘Magic Wand’; add ROI to ‘ROI manager’ (shift + t) (see Figure 5).


      Figure 5. Image analysis of APP endocytosis: Step 3

    4. To refine the ROI to the dendrites and axons: adjust the auto ‘Threshold’ to the APP-RFP signal in the dendrites and in the axon if necessary; create new ROIs by clicking on the thresholded neurites with the ‘Magic Wand’; add ROIs to ‘ROI manager’ (shift + t) (see Figure 6).


      Figure 6. Image analysis of APP endocytosis: Step 4

    5. Transfer all regions to endocytosed Alexa488-anti-APP channel, draw a background ROI in a cell-free region using the tool ‘polygon selection’ and add it to the ‘ROI manager’. In the ROI manager, press ‘measure’ to obtain the mean Alexa488-anti-APP fluorescence intensities per ROI (background, cell body, dendrites and axon). Select, copy and export measurements to Microsoft Excel (see Figure 7).


      Figure 7. Image analysis of APP endocytosis: Step 5

    6. Transfer all regions back to APP-RFP channel, and in the ROI manager, press ‘measure’ to obtain the mean APP-RFP fluorescence intensities per ROI (background, cell body, dendrites and axon). Select, copy and export measurements to Microsoft Excel (see Figure 8). 


      Figure 8. Image analysis of APP endocytosis: Step 6

Data analysis

Note: Data analysis is done using Microsoft Excel.

  1. Subtract the background mean fluorescence intensity from all measurements, per single neuron.
  2. Normalize the Alexa488-anti-APP fluorescence intensities (from the 3 neuronal compartments) by the APP-RFP fluorescence intensity in the cell body to control for the different expression level of APP-RFP, per single neuron.
  3. Different conditions can be compared using a classical t-test provided the data follow a normal distribution.
  4. The 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.
  5. Statistical significance for nonparametric data was tested by Mann-Whitney test.

Notes

  1. Variability is often due to the culture of primary neurons; the level of differentiation should be kept constant between independent experiments.
  2. In our hands, APP endocytosis is robust with the average results very reproducible. However, it is expected variability between different neurons in the same experiment.
  3. For optimal APP transfection and thus experimental conditions, good quality DNA plasmid and healthy and well developed primary neurons are paramount.

Acknowledgments

We thank for the gift of APP-RFP plasmids from Dr. S. Kins (University of Kaiserslautern). 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 is acknowledged. CGA is Investigator FCT (IF/00998/2012, FCT). FU was 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. Almeida, C. G., Takahashi, R. H. and Gouras, G. K. (2006). β-amyloid accumulation impairs multivesicular body sorting by inhibiting the ubiquitin-proteasome system. J Neurosci 26(16): 4277-4288.
  2. Almeida, C. G., Tampellini, D., Takahashi, R. H., Greengard, P., Lin, M. T., Snyder, E. M. and Gouras, G. K. (2005). β-amyloid accumulation in APP mutant neurons reduces PSD-95 and GluR1 in synapses. Neurobiol Dis 20(2): 187-198.
  3. 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-beta in vivo. Neuron 58(1): 42-51.
  4. Dotti, C. G., Sullivan, C. A. and Banker, G. A. (1988). The establishment of polarity by hippocampal neurons in culture. J Neurosci 8: 1454-1468.
  5. Edgar, J. R., Willen, K., Gouras, G. K. and Futter, C. E. (2015). ESCRTs regulate amyloid precursor protein sorting in multivesicular bodies and intracellular amyloid-beta accumulation. J Cell Sci 128(14): 2520-2528.
  6. 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.
  7. Haass, C., Koo, E. H., Mellon, A., Hung, A. Y. and Selkoe, D. J. (1992). Targeting of cell-surface beta-amyloid precursor protein to lysosomes: alternative processing into amyloid-bearing fragments. Nature 357(6378): 500-503.
  8. 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.
  9. Lee, J., Retamal, C., Cuitino, L., Caruano-Yzermans, A., Shin, J. E., van Kerkhof, P., Marzolo, M. P. and Bu, G. (2008). Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes. J Biol Chem 283(17): 11501-11508.
  10. Morel, E., Chamoun, Z., Lasiecka, Z. M., Chan, R. B., Williamson, R. L., Vetanovetz, C., Dall'Armi, C., Simoes, S., Point Du Jour, K. S., McCabe, B. D., Small, S. A. and Di Paolo, G. (2013). Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system. Nat Commun 4: 2250.
  11. Mucke, L. and Selkoe, D. J. (2012). Neurotoxicity of amyloid β-protein: synaptic and network dysfunction. Cold Spring Harb Perspect Med 2(7): a006338.
  12. Rajendran, L. and Annaert, W. (2012). Membrane trafficking pathways in Alzheimer's disease. Traffic 13(6): 759-770.
  13. 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-secretase by membrane targeting. Science 320(5875): 520-523.
  14. Sannerud, R., Declerck, I., Peric, A., Raemaekers, T., Menendez, G., Zhou, L., Veerle, B., Coen, K., Munck, S., De Strooper, B., Schiavo, G. and Annaert, W. (2011). ADP ribosylation factor 6 (ARF6) controls amyloid precursor protein (APP) processing by mediating the endosomal sorting of BACE1. Proc Natl Acad Sci U S A 108(34): E559-568.
  15. Sullivan, S. E., Dillon, G. M., Sullivan, J. M. and Ho, A. (2014). Mint proteins are required for synaptic activity-dependent amyloid precursor protein (APP) trafficking and amyloid β generation. J Biol Chem 289(22): 15374-15383.
  16. 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.
  17. 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.
  18. Xiao, Q., Gil, S. C., Yan, P., Wang, Y., Han, S., Gonzales, E., Perez, R., Cirrito, J. R. and Lee, J. M. (2012). Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis. J Biol Chem 287(25): 21279-21289.
  19. 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.
  20. Yamazaki, T., Selkoe, D. J. and Koo, E. H. (1995). Trafficking of cell surface beta-amyloid precursor protein: retrograde and transcytotic transport in cultured neurons. J Cell Biol 129(2): 431-442.

简介

阿尔茨海默病确定的主要触发因素是β-淀粉样蛋白(Aβ)(Mucke和Selkoe,2012)。 淀粉样蛋白前体蛋白(APP)内吞作用是在早期内体中产生Aβ所必需的(Rajendran和Annaert,2012)。 内涵体上的APP保留取决于其对溶酶体中的降解的分选(Haass等人,1992; Morel等人,2013; Edgar等人, 2015年; Ubelmann等人,2017年)。 已经优化了以下内吞作用测定法以评估活的小鼠皮层原代神经元的淀粉状蛋白前体蛋白(APP)内吞作用和降解(Ubelmann等人,2017)。

【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)。已经通过表面蛋白的经典生物素化在脉冲追踪动力学实验中分析了APP的内吞作用(Sannerud等人,2011; Xiao等人,2012; Sullivan等人,2014),在单细胞中通过使用针对APP的N-末端胞外结构域的抗体(Yamazaki等人,1995; Xiao ,2012)。这些研究中的大多数使用非神经元细胞(Yamazaki等人,1996; Lee等人,2008; Sullivan等人, ,2014)和神经元样细胞系(Xiao等人,2012),很少使用原代神经元(Yamazaki等人,1995; Sullivan等人,2014)。原代神经元像体内轴突和树突一样分化,具有特殊的突触前末梢和突触后隔室。然而,在这些报道中缺乏对这些神经元区室的仔细测量和区分。我们开发了一种方法来分析不同的神经元室,体细胞体,树突和轴突,我们在这个生物协议中描述的内吞。我们的协议详细说明了使用经典免疫荧光和半定量细胞生物学分析方法来跟踪和测量极化神经元中的APP胞吞作用的程序。

我们相信我们的方法将通过可靠地测量半定量测量对于极化神经元特异性的APP的分隔内吞作用来允许该领域向前迈进。

关键字:APP, 内吞作用, 降解, 阿尔茨海默病, 免疫荧光

材料和试剂

  1. 用于哺乳动物细胞培养的24孔培养皿(SARSTEDT,目录编号:83.1836)
  2. 圆形玻璃盖玻片,13毫米(VWR,Marienfeld,目录号:630-1597)
    注意:高压灭菌,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;用200μl0.1%(w / v)聚-D-赖氨酸在37℃,5%CO 2和20%加湿的培养箱中,用无菌的Milli-Q水洗涤3次。
  3. 超级玻璃滑梯(MENZEL GERHARD,产品目录号:2586E)
  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. 野生型雌性和雄性小鼠胚胎(Balbc,胚胎第16天; Charles River)
  7. APP-RFP质粒(Szodorai等人,2009)(S.Kins,凯泽斯劳滕大学)
  8. 0.1%(w / v)聚-D-赖氨酸(Sigma-Aldrich,目录号:P1149)
  9. 电镀介质:
    1. DMEM,高葡萄糖,丙酮酸(Thermo Fisher Scientific,Gibco TM,目录号:11995065)
    2. 10%胎牛血清(FBS),合格的,热灭活的,美国来源(Thermo Fisher Scientific,Gibco TM,产品目录号:16140071)
    3. 1%青霉素 - 链霉素(10,000U / ml)(Thermo Fisher Scientific,Gibco TM,目录号:15140122)
  10. 神经基础培养基:
    1. 神经基础培养基(Thermo Fisher Scientific,Gibco TM,目录号:21103049)
    2. 2%B-27补充剂,定制(50x)(Thermo Fisher Scientific,Gibco TM,产品目录号:0080085SA)
    3. 0.1%GlutaMAX TM补充物(Thermo Fisher Scientific,Gibco TM,目录号:35050038)
  11. 胰蛋白酶(2.5%),无酚红(Thermo Fisher Scientific,Gibco TM,产品目录号:15090046)
  12. Lipofectamine 2000(Thermo Fisher Scientific,Invitrogen TM,目录号:11668019)
  13. Opti-MEM(Thermo Fisher Scientific,目录号:31985062)
  14. 鼠抗APP N-末端单克隆(22C11)(Merck,目录号:MAB348)
  15. HEPES(1M)(Thermo Fisher Scientific,Gibco TM,目录号:15630080)
  16. 磷酸盐缓冲盐水(PBS)(Thermo Fisher Scientific,Gibco TM,目录号:10010031)
  17. 多聚甲醛(Sigma-Aldrich,目录号:P6148)
  18. 蔗糖(NZYTech,目录号:MB18601)
  19. 皂苷(Sigma-Aldrich,目录号:47036)
  20. 驴抗小鼠Alexa 488(Thermo Fisher Scientific,目录号:A-21202)
  21. 盖玻片安装解决方案(FluoroMount-G)(SouthernBiotech,目录号:0100-01)
  22. DAPI(Sigma-Aldrich,目录号:D9542)
  23. HBSS(GE Healthcare,Hyclone TM,产品目录号:SH30031.03)
  24. 无菌水中含50%葡萄糖(NZYTech,目录号:MB16801)
  25. 牛血清白蛋白V(BSA)(NZYTech,目录号:MB04602)

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:在体外培养9天的神经元上进行APP胞吞作用试验(9DIV)。

  1. 细胞培养和转染(第1天,第6天,第8天)
    1. 第1天:如先前所述(Almeida等人,2005和2006; Ubelmann等人),从野生型雌性和雄性小鼠胚胎(E16)制备来自皮层和海马的原代皮层神经元,2017)。简而言之,将解离的神经元接种在含有聚D-赖氨酸包被的玻璃盖玻片(5-10×10 4细胞/ cm 2)的24孔平板上, DIV 0)。 3小时或过夜后,用Neurobasal培养基更换电镀培养基,只允许神经元生长和分化,直到使用。 (Almeida等人,2005和2006; Ubelmann等人,2017),
    2. 第8天:为了表达APP-RFP cDNA,根据制造商的方案,用Lipofectamine 2000瞬时转染8个DIV初级皮质神经元,使用0.5μgcDNA:0.5μlLipofectamine混合物在25μlOpti-MEM中,每孔24-孔板250微升新鲜无抗生素Neurobasal培养基,孵育过夜,直到测定APP内吞。
      注意:在分化的第5阶段,平均期望10个转染的健康神经元在8个DIV处(Dotti等,1988),并且神经突不应显示指示退化的珠状结构。使用新制备的cDNA和常规的midi-prep试剂盒(我们使用当地的NZYTECH品牌)可以达到这一效率。
    3. (可选)第6天:对于敲低分析,在用新鲜无抗生素的Neurobasal替代培养基后,使用Lipofectamine RNAiMax,使用Lipofectamine RNAiMax,用siRNA转染6天体外原代神经元(6DIV)中等。

  2. 通过以下步骤(第9天)表达9个表达APP-RFP的DIV初级神经元的APP胞吞作用测定(图1)


    图1.使用抗APP抗体在轴突,树突和细胞体中温育10分钟监测的APP胞吞作用的示意图。EE:早期内体; Lys:溶酶体。

    1. 用塑料巴斯德吸管移出细胞培养基,加入400μLB27免费Neurobasal培养基30分钟在37°C在细胞培养孵化器。所有的移液都应该慢慢地靠在井壁上,而不要接触细胞。
    2. 将25μl抗APP抗体(22C11;0.25μg/μl原液浓度)稀释到25μl完全Neurobasal培养基中,每个盖玻片含10mM HEPES(0.25μl储备液,1M,pH7.2-7.5)。
    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. 将盖玻片小心并尽可能快地覆盖在面对抗体溶液的细胞的液滴上,盖上容器中的反应物以避免蒸发,并在37℃下在细胞培养箱中孵育10分钟。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)
      1. 为了监测胞吞的APP溶酶体降解,通过在500μl预热的PBS中一次浸渍盖玻片一次4s进一步孵育细胞60分钟。
      2. 为了在质膜上检测APP,在37℃下在细胞培养箱中用抗APP抗体孵育细胞仅4分钟。这个可选的步骤允许标记抗APP抗体绑定到细胞表面的APP没有显着检测内吞作用。

    5. 在500μl预热的PBS中浸渍4次,每次盖上盖玻片
    6. 通过将盖玻片放回到24孔板中并在室温(RT)下加入500μl4%多聚甲醛/ 4%蔗糖20分钟来固定细胞。用500μlPBS代替固定液,洗3次后进行抗APP检测。

  3. 抗APP抗体检测(第9天)
    1. 为了检测与抗APP抗体结合的内吞的APP,在室温下用500μl0.1%皂苷/ PBS(透化缓冲液)透化固定的细胞60分钟。去除透化缓冲液,并用PBS清洗每个盖玻片。
      可选:为了在细胞表面检测与APP结合的抗APP(4分钟),不需要透化。 不要在步骤C2和C3中加入皂苷。
    2. 用500μl封闭缓冲液(3%FBS / 0.1%皂角苷/ PBS)在室温封闭抗体与血清蛋白的非特异性结合60分钟。
    3. 将每个盖玻片(细胞朝下)放在50μL3%FBS / 0.1%皂素/ PBS稀释的驴抗小鼠Alexa 488(1:250)液滴。
      可选:将DAPI包含在抗体溶液(1:10,000的1毫克/毫升的股票)中以使细胞核复染。
    4. 覆盖反应,并在室温下孵育60分钟,在黑暗中。
    5. 将盖玻片放回到24孔板上,在室温下用PBS清洗3次。
    6. 在显微镜载玻片上放置盖有细胞的细胞,将细胞面朝下放在25μlFluoromount G液滴上,并在黑暗中干燥过夜以保持荧光信号。
      注意:快速移取Fluoromount G以防止其干燥,但要轻轻地避免气泡。

  4. 图像采集(第10天)
    图像采集是使用epifluorescence显微镜(如蔡司Z2)与60x NA 1.4油浸物镜和CCD相机(见设备的细节使用显微镜)。有关代表性图像,请参见图2。
    1. 神经元细胞体,部分树突和轴突必须集中。应该排除表达低或高水平的APP-RFP的发育不良的神经元或神经元。曝光时间应根据具有最明亮的预期信号的样本来确定,以便尽可能多地使用相机的动态范围(理想情况下使用具有16位二进制范围的相机),而不使任何像素饱和。
    2. 每个条件获得10-20个神经元,以获得足够的数据进行统计分析。为了对整个神经元进行成像,可能需要获取多个场。


      图2.表达APP-RFP的原代神经元的APP内吞作用的代表性图像,其中用抗小鼠Alexa 488检测的抗APP(22C11)温育10分钟.Dd:树突;斧:轴突;比例尺=10μm(由Ubelmann等人改编,2017)。

  5. 图像分析与放大APP胞吞作用在细胞体,树突和轴突中定量(图3)(第10-11天)
    注意:使用ImageJ / Fiji获得的内吞荧光信号的定量
    1. 根据神经元形态学和RFP信号确定细胞体,树突和轴突,并粗略地勾画与细胞体对应的复合体选择(非邻接的ROI),树突中2至5μm〜20μm区段和轴突中的一个(通常更薄,更长,并在90°角有分支)使用“多边形选择”,同时按下“移位”(见图3)。轴突和树突也可以用Ankyrin-G(Santa Cruz Biotechnology)或MAP2(Sigma-Aldrich)复染。


      图3. APP内吞作用的图像分析:第1步

    2. 复制包含复合选项的图像(shift + D),并使用“清除外部”来移除未选中的像素(见图4)。


      图4. APP胞吞作用的图像分析:第2步

    3. 将ROI细化到细胞边界:自动“阈值”细胞体中的APP-RFP信号;通过用“魔杖”点击阈值的神经突,创造一个新的投资回报。将ROI添加到“ROI manager”(shift + t)(参见图5)。


      图5. APP内吞作用的图像分析:第3步

    4. 为了提高树突和轴突的ROI:如果必要的话,调整树突和轴突中的APP-RFP信号的自动“阈值”通过用“魔杖”点击阈值的神经突,创造新的投资回报。将ROI添加到“ROI manager”(shift + t)(参见图6)。


      图6. APP内吞作用的图像分析:第4步

    5. 将所有区域转移至内吞的Alexa488抗APP通道,使用工具“多边形选择”在无细胞区域绘制背景ROI,并将其添加到“ROI管理器”。在ROI管理器中,按“测量”以获得每个ROI(背景,细胞体,树突和轴突)的平均Alexa488-抗APP荧光强度。选择,复制和导出测量到Microsoft Excel(见图7)。


      图7. APP内吞作用的图像分析:第5步

    6. 将所有区域转移回APP-RFP通道,并在ROI管理器中按“测量”以获得每ROI(背景,细胞体,树突和轴突)的平均APP-RFP荧光强度。选择,复制和导出测量结果到Microsoft Excel(见图8)。


      图8. APP内吞作用的图像分析:第6步

数据分析

注意:数据分析是使用Microsoft Excel完成的。


  1. 每个单个神经元的所有测量值减去背景平均荧光强度
  2. 通过细胞体中的APP-RFP荧光强度来标准化Alexa488-抗APP荧光强度(来自3个神经元区室)以控制每个单个神经元的APP-RFP的不同表达水平。
  3. 如果数据遵循正态分布,则可以使用经典的 t 测试来比较不同的条件。
  4. 根据以前的研究,在每个独立实验中样品大小为每个条件约20个细胞。对至少三个独立实验的统计学显着性是通过双尾Student的吨 -test和用于多重比较单向ANOVA与Tukey检验使用关于正常的数据(达戈斯蒂诺皮尔森总括正态性检验)来确定GraphPad棱镜6.
  5. 非参数数据的统计显着性通过Mann-Whitney检验来检验。

笔记

  1. 可变性通常是由于原代神经元的培养;
    在独立实验中,分化程度应保持不变
  2. 在我们的手中,APP内吞是稳健的,平均结果非常可重复。但是,在同一个实验中,不同神经元之间的差异是可以预料的。
  3. 对于最佳的APP转染和实验条件,高质量的DNA质粒和健康和发达的原代神经元是至关重要的。

致谢

我们感谢S.Kins博士(凯泽斯劳滕大学)的APP-RFP质粒。这项工作得到了CEDOC和玛丽•居里综合补助金(334366 TrafficInAD FP7-PEOPLE-2012-CIG; Marie Curie Actions,EC)的支持。 iNOVA4Health-UID / Multi / 04462/2013是一个由FundaçãoparaCiênciae Teologologia(FCT)/EducaçãoeCiênciaMinistériodaEducaçãoeCiência资助的计划,由FEDER根据PT2020合作协议共同资助。 CGA是Investigator FCT(IF / 00998/2012,FCT)。 FU获得FRM博士后奖学金(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. Almeida,C.G。,Takahashi,R.H。和Gouras,G.K。(2006)。 β-淀粉样蛋白蓄积通过抑制泛素 - 蛋白酶体系统损害多泡体分选。 em> J Neurosci 26(16):4277-4288。
  2. Almeida,C.G.,Tampellini,D.,Takahashi,R.H.,Greengard,P.,Lin,M.T.,Snyder,E.M。和Gouras,G.K。(2005)。 APP突变型神经元中β-淀粉样蛋白的积累减少了突触中的PSD-95和GluR1。 Neurobiol Dis 20(2):187-198。
  3. 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蛋白需要内吞作用。 神经元 58(1):42-51。
  4. Dotti,C.G.,Sullivan,C.A。和Banker,G.A。(1988)。 建立海马神经元在文化中的极性 J Neurosci em> 8:1454-1468。
  5. Edgar,J. R.,Willen,K.,Gouras,G. K.和Futter,C. E.(2015)。 ESCRTs调节多泡体中的淀粉样前体蛋白分选和细胞内淀粉状蛋白-β积累。 J Cell Sci 128(14):2520-2528。
  6. 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。“生物化学杂志”278(33):31261-31268。
  7. Haass,C.,Koo,E.H。,Mellon,A.,Hung,A.Y。和Selkoe,D.J。(1992)。 细胞表面β-淀粉样蛋白前体蛋白靶向溶酶体:替代加工成淀粉样蛋白片段。 Nature 357(6378):500-503。
  8. 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。
  9. Lee,J.,Retamal,C.,Cuitino,L.,Caruano-Yzermans,A.,Shin,J.E.,van Kerkhof,P.,Marzolo,M.P.and Bu,G.(2008)。 衔接蛋白分选nexin 17调节淀粉样蛋白前体蛋白的运输和在早期内体中的加工
    J Biol Chem 283(17):11501-11508
  10. Morel,E.,Chamoun,Z.,Lasiecka,ZM,Chan,RB,Williamson,RL,Vetanovetz,C.,Dall'Armi,C.,Simoes,S.,Point Du Jour,KS,McCabe,BD,Small ,SA和Di Paolo,G。(2013)。 磷脂酰肌醇-3-磷酸通过内体系统调节淀粉样蛋白前体蛋白的分选和加工 Nat Commun 4:2250.
  11. 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。
  12. Rajendran,L.和Annaert,W。(2012)。 阿尔茨海默病中的膜贩运途径 Traffic 13( 6):759-770。
  13. 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-分泌酶 科学 320(5875):520-523。
  14. Sannerud,R.,Declerck,I.,Peric,A.,Raemaekers,T.,Menendez,G.,Zhou,L.,Veerle,B.,Coen,K.,Munck,S.,De Strooper,B. ,Schiavo,G。和Annaert,W。(2011)。 (ADF)核糖基化因子6(ARF6)通过调节淀粉样前体蛋白(APP)的内体分选来控制淀粉样前体蛋白(APP) BACE1。美国国家科学院院刊108(34):E559-568。
  15. Sullivan,S.E。,Dillon,G.M.,Sullivan,J.M。和Ho,A。(2014)。 薄荷蛋白是突触活动依赖性淀粉样前体蛋白(APP)运输和β淀粉样蛋白产生所必需的。 J生物化学杂志289(22):15374-15383。
  16. 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。
  17. 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。
  18. Xiao,Q.,Gil,S.C.,Yan,P.,Wang,Y.,Han,S.,Gonzales,E.,Perez,R.,Cirrito,J.R。和Lee,J.M。(2012)。 磷脂酰肌醇网格蛋白聚集淋巴样髓样白血病(PICALM)在细胞内淀粉样前体蛋白(APP)加工中的作用和淀粉状蛋白斑发病机理。“生物化学杂志”287(25):21279-21289。
  19. Yamazaki,T.,Koo,E.H。和Selkoe,D.J。(1996)。 贩卖细胞表面β淀粉样蛋白前体。 II。通过免疫定位检测的内吞作用,再循环和溶酶体靶向。 J Cell Sci 109(Pt 5):999-1008。
  20. Yamazaki,T.,Selkoe,D.J。和Koo,E.H。(1995)。 贩卖细胞表面β-淀粉样前体蛋白:在培养的神经元中逆行和转胞吞转运
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。