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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.
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.
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....
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
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 .......
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. .......
This work was partially supported by research grants from JSPS (KAKENHI 18K07389), Japan, Takeda Science Foundation, Japan, and Kobayashi Pharmaceutical Co., Ltd., Japan.
....Name | Company | Catalog Number | Comments |
ddY mice | SLC | ||
Eight-well culture slide | Falcon | 354108 | |
poly D lysine | Wako | 168-19041 | |
Culture medium, Neurobasal medium | Gibco | 21103-049 | |
house serum | Gibco | 26050-088 | |
glucose | Wako | 049-31165 | |
L-glutamine | Wako | 074-00522 | |
0.05% trypsin | Gibco | 25300-054 | |
DNase I | Worthington | DP | |
soybean trypsin inhibitor | Gibco | 17075-029 | |
Filter with 70 µm mesh size, cell strainer | Falcon | 352350 | |
B-27 supplement | Gibco | 17504-044 | |
CO2 incubator | Astec | SCA-165DS | |
Amyloid β1-42 | Sigma-Aldrich | A9810 | |
paraformaldehyde | Wako | 162-16065 | |
sucrose | Wako | 196-00015 | |
Aqueous mounting medium, Aqua-Poly/Mount | polysciences | 18606-20 | |
Inverted microscope A | Carl Zeiss | Axio Observer Z1 | Connected with AxioCam MRm, Heating Unit XL S, CO2 Module S1, and TempModule S1 |
Objective Plan-Apochromat 20x | Carl Zeiss | 420650-9901 | |
Objective Plan-Apochromat 63x | Carl Zeiss | 440762-9904 | |
Objective, CFI Plan Apo Lambda 40X | Nikon | ||
anti-MAP2 IgG | Abcam | ab32454 | |
anti-tau-1 IgG | Chemicon | MAB3420 | |
anti-amyloid β antibody | IBL | 10379 | clone 11A1 |
normal goat serum | Wako | 143-06561 | |
bovine serum albumin | Wako | 010-25783 | |
t-octylphenoxypolyethoxyethanol | Wako | 169-21105 | |
goat anti-mouse IgG conjugated with AlexaFluor 594 | Invitrogen | A11032 | |
goat anti-rabbit IgG conjugated with AlexaFluor 488 | Invitrogen | A11029 | |
hot plate | NISSIN | NHP-M30N | |
cover glass | Fisher Scientific | 12-545-85 | |
35 mm dish | IWAKI | 1000-035 | |
Silicone RTV | Shin-Etsu | KE42T | |
hand punch | Roper Whitney | No. XX | |
Fluorescence membrane probe, FM1-43FX | Invitrogen | F35355 | |
Ca2+- and Mg2+-free Hanks' balanced salt solution | Gibco | 14175-095 | |
Transfection solution, Nucleofector solution | Lonza | VPG-1001 | |
Electroporator, Nucleofector I | Amaxa | ||
Inverted microscope B | Keyence | BZ-X710 | |
Image software, ImageJ | NIH | https://imagej.nih.gov/ij/ |
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