Visualizing Axonal Growth Cone Collapse and Early Amyloid β Effects in Cultured Mouse Neurons

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 impairments^2,3,4. 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.......

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. .......

Materials

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/