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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here a protocol to investigate the early effects of amyloid-β (Aβ) in the brain is presented. This shows that Aβ induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of Aβ on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Abstract

Amyloid-β (Aβ) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce Aβ levels in the brains of AD patients, these do not improve memory functions. Since Aβ aggregates in the brain before the appearance of memory impairments, targeting Aβ may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to Aβ deposition should be blocked before AD development. Aβ induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of Aβ toxicity, the source of Aβ toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by Aβ in axonal growth cones of cultured neurons. This protocol revealed that Aβ induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents Aβ toxicity. This protocol will be useful in studying the early effects of Aβ and may lead to more efficient and preventative AD treatment.

Introduction

Amyloid-β (Aβ) deposits are found in the brain of patients with Alzheimer's disease (AD) and are considered a critical cause of AD1 that disrupt neuronal networks, leading to memory impairments2,3,4. Many clinical drug candidates have been shown to effectively prevent amyloid-β (Aβ) production or remove Aβ deposits. However, none have succeeded in improving memory function in AD patients5. Aβ is already deposited in the brain prior to the onset of memory impairments6; therefo....

Protocol

All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama and were approved by the Committee for Animal Care and Use of Laboratory Animals at the Sugitani Campus of the University of Toyama (A2014INM-1, A2017INM-1).

1. Collapse Assay

  1. Poly-D-lysine coating
    1. Coat 8-well culture slides with 400 μL of 5 μg/mL poly-D-lysine (PDL) in phosphate-buffered saline (PBS) an.......

Representative Results

In this protocol, Aβ1-42 was incubated at 37 °C for 7 days before use, because incubation of Aβ1-42 was needed for producing toxic forms27,28,30,35. After this incubation, aggregated forms of Aβ were observed (Figure 1A). It has been reported that similar incubation of Aβ1-42 produced the fibril form of A .......

Discussion

The protocol described in this study enabled the observation of early phenomena in axonal growth cones after Aβ1-42 treatment. Aβ1-42 induced endocytosis in axonal growth cones within 20 min, and growth cone collapse was observed within 1 h of treatment. This endocytosis was probably mediated by clathrin. By using this protocol, the inhibition of clathrin-mediated endocytosis was confirmed to prevent Aβ1-42-induced growth cone collapse and axonal degeneration in cultured neurons27. .......

Acknowledgements

This work was partially supported by research grants from JSPS (KAKENHI 18K07389), Japan, Takeda Science Foundation, Japan, and Kobayashi Pharmaceutical Co., Ltd., Japan.

....

Materials

NameCompanyCatalog NumberComments
ddY miceSLC
Eight-well culture slideFalcon354108
poly D lysineWako168-19041
Culture medium, Neurobasal mediumGibco21103-049
house serumGibco26050-088
glucoseWako049-31165
L-glutamineWako074-00522
0.05% trypsinGibco25300-054
DNase IWorthingtonDP
soybean trypsin inhibitorGibco17075-029
Filter with 70 µm mesh size, cell strainerFalcon352350
B-27 supplementGibco17504-044
CO2 incubatorAstecSCA-165DS
Amyloid β1-42Sigma-AldrichA9810
paraformaldehydeWako162-16065
sucrose Wako196-00015
Aqueous mounting medium, Aqua-Poly/Mountpolysciences18606-20
Inverted microscope ACarl ZeissAxio Observer Z1 Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1
Objective Plan-Apochromat 20xCarl Zeiss420650-9901
Objective Plan-Apochromat 63xCarl Zeiss440762-9904
Objective, CFI Plan Apo Lambda 40XNikon
anti-MAP2 IgGAbcamab32454
anti-tau-1 IgGChemiconMAB3420
anti-amyloid β antibodyIBL10379clone 11A1
normal goat serumWako143-06561
bovine serum albuminWako010-25783
t-octylphenoxypolyethoxyethanolWako169-21105
goat anti-mouse IgG conjugated with AlexaFluor 594InvitrogenA11032
goat anti-rabbit IgG conjugated with AlexaFluor 488InvitrogenA11029
hot plateNISSINNHP-M30N
cover glassFisher Scientific12-545-85
35 mm dishIWAKI1000-035
Silicone RTVShin-EtsuKE42T
hand punchRoper WhitneyNo. XX
Fluorescence membrane probe, FM1-43FXInvitrogenF35355
Ca2+- and Mg2+-free Hanks' balanced salt solutionGibco14175-095
Transfection solution, Nucleofector solutionLonzaVPG-1001
Electroporator, Nucleofector IAmaxa
Inverted microscope BKeyenceBZ-X710
Image software, ImageJNIHhttps://imagej.nih.gov/ij/

References

  1. Selkoe, D. J., Hardy, J. The amyloid hypothesis of Alzheimer's disease at 25 years. EMBO Molecular Medicine. 8 (6), 595-608 (2016).
  2. Dickson, T. C., Vickers, J. C. The morphological phenoty....

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