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; therefore, decreasing Aβ levels in the brains of patients exhibiting memory impairments may be ineffective. Aβ deposition is present in preclinical AD patients; however, these patients rarely present with neuronal degeneration and memory deficits⁶. There is a time lag between Aβ deposition and memory impairments. Therefore, a critical strategy for the prevention of AD is blocking Aβ toxicity signaling during the early stages of AD, prior to the development of memory deficits. Aβ deposition induces axon degeneration^{7,8,9,10,11,12,13}, which may lead to a disruption of neural networks and permanent impairment of memory function. Many studies have investigated the mechanisms of Aβ toxicity; for example, the degenerated axons of AD mice brains have been shown to have increased autophagy¹⁴. Calcineurin activation has been reported as a possible mechanism of Aβ-induced axonal degeneration¹⁵; however, the direct trigger of axonal degeneration remains unknown.

This study focuses on the collapse of axonal endings called growth cones. The collapse of axonal growth cones can be caused by axonal growth repellents, such as semaphorin-3A and ephrin-A5^{16,17,18,19,20}. Collapse-like dystrophic axonal endings have been observed in the brains of AD patients^21,22. Additionally, a failure of growth cone functioning can provoke axonal degeneration²³. However, it is unknown whether Aβ induces growth cone collapse. Therefore, this study presents a novel protocol to observe the early effects of Aβ in cultured neurons and investigate Aβ-induced growth cone collapse.

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) and incubate them at 37 °C overnight.
   2. Remove the PDL solution and wash the wells 3 times with distilled water.
2. Neuron culture²⁴
   1. Mince freshly isolated cerebral cortices from embryonic day 14 (E14) ddY mice with microscissors in neuron culture medium containing 12% horse serum, 0.6% glucose, and 2 mM L-glutamine (medium A). Do not add antibiotics.
      NOTE: In this protocol, the ddY mouse is used. This is an outbred strain commonly used in Japan. This neuron culture protocol can be also applied for rat cortical neurons^7,25.
   2. Centrifuge the tissues at 87 x g for 3 min.
   3. Remove the supernatant. Then to the pellet, add 2 mL of 0.05% trypsin and incubate for 15 min at 37°C. Mix by tapping every 5 min.
   4. Add 4 mL of medium A and mix by tapping.
   5. Centrifuge the tissues at 178 x g for 3 min.
   6. Remove the supernatant, and incubate the tissues with 600 U/mL DNase I and 0.3 mg/mL soybean trypsin inhibitor dissolved in PBS for 15 min at 37 °C. Mix by tapping every 5 min.
   7. After incubation, add 4 mL of medium A and mix by tapping.
   8. Centrifuge the tissues at 178 x g for 3 min.
   9. After removing the supernatant, add 4 mL of medium A and triturate the tissues with a polished Pasteur pipette.
   10. Filter the triturated tissues with a 70 µm pore-size mesh. After filtration, calculate the density of cells with a hemocytometer.
   11. Culture the cells in the 8-well culture slide at 0.8 x 10⁴ cells/well with medium A and maintain them in a CO₂ incubator with a humidified atmosphere of 10% CO₂ at 37 °C.
   12. After 4 h of culturing, replace the culture medium to one containing 2% supplement for neuronal culture, 0.6% glucose, and 2 mM L-glutamine (medium B).
       NOTE: The purity of neurons was approximately 75%, as described previously²⁶.
3. Collapse assay²⁷
   1. Dissolve commercially obtained full-length amyloid β1-42 (Aβ1-42) in distilled water at a concentration of 0.5 mM and incubate at 37 °C for 7 days. After the incubation, store the aggregated Aβ1-42 solution in a -30 °C freezer until use.
      NOTE: This incubation is necessary for aggregation and toxicity of Aβ^27,28,29,30.
   2. After 4 days of neuronal culture, treat the wells with 100 μL of new medium B, containing 0.5 μM aggregated Aβ1-42 or vehicle solution (distilled water) for 1 h.
      NOTE: Effects of Aβ1-42 were dose-dependently increased from 0.1 to 5 μM, and peaked at 0.5 μM as described previously²⁷. Similar results can be observed when by Aβ1-42 treatment for 1 h after 3 days of neuronal culture³¹.
   3. Remove the culture medium and immediately fix the neurons with 4% paraformaldehyde containing 4% sucrose in PBS for 1 h at 37 °C on a hot plate.
   4. After fixation, wash the neurons 3 times with PBS and mount them with an aqueous mounting medium. Dry the mounting medium at 4 °C for 2–4 days.
   5. Capture the entire area (7.8 x 9 mm²) of each well with a 20X dry objective lens on an inverted microscope.
   6. Classify the longest neurites of each neuron in stage 3 or 4 as axons, as previously described^32,33.
   7. Classify growth cones according to the following criteria: 1) axonal growth cones lacking lamellipodia or 2) possessing fewer than three filopodia are considered collapsed growth cones, as described previously¹⁷.
      NOTE: Healthy growth cones are scored as 0 point; collapsed growth cones are scored as 1 point. Mean collapse scores are calculated for each treatment.

2. Amyloid β Immunostaining

1. Culture mouse cortical neurons for 3 days, as described in step 1.2.
2. Treat with aggregated Aβ1-42 (5 μM) or vehicle for 4 h at 37 °C in a CO₂ incubator.
3. Without removing the medium, add an equal volume of 4% paraformaldehyde containing 4% sucrose in PBS to each well, and maintain the culture at 37 °C on a hot plate for 5 min.
4. Replace the solution with 400 μL of 4% paraformaldehyde containing 4% sucrose in PBS, and maintain at 37 °C on the hot plate for 1 h. This fixation protocol was modified from a previous report³⁴.
5. Wash the neurons 3 times with PBS.
6. Block with 5% normal goat serum in PBS.
7. Incubate the neurons with mouse anti-amyloid β immunoglobulin G (IgG) (1:50) and 1% bovine serum albumin in PBS at 4 °C overnight.
8. Wash the neurons 3 times with PBS.
9. Incubate the neurons with a fluorescence-conjugated secondary antibody (1:400) and 1% bovine serum albumin in PBS at room temperature for 2 h.
10. Wash the neurons 3 times with PBS and mount them with an aqueous mounting medium.
11. Capture fluorescence images and bright field images with oblique illumination by using a 40X dry objective lens on inverted microscope B.

3. Axonal Immunostaining²⁷

1. Wash the neurons 3 times with PBS after cultured neuron fixation, as described in step 1.3.3.
2. Incubate the neurons with mouse anti-tau-1 IgG (1:500), rabbit anti-microtubule associated protein 2 (MAP2) IgG (1:500) in 5% normal goat serum, and 0.3% t-octylphenoxypoly-ethoxyethanol in PBS at 4 °C overnight.
3. Wash the neurons 3 times with PBS.
4. Incubate the neurons with fluorescence-conjugated secondary antibodies (1:400) and 0.3% t-octylphenoxypolyethoxyethanol in PBS at room temperature for 2 h.
5. Wash the neurons 3 times with PBS, and mount using an aqueous mounting medium.
6. Capture fluorescence and differential interference contrast (DIC) images by using a 20× dry objective lens on inverted microscope A.

4. Live Cell Imaging²⁷

1. Coat glass-based dishes with 500 μL of PDL (5 μg/mL), as described in step 1.1.1.
   NOTE: In this protocol, homemade glass-based dishes were used. Commercially available glass-based dishes can also be used for live imaging. Homemade glass-based dishes were prepared as follows: 1) make a hole approximately 1.4 mm in diameter in the center of a 35-mm dish with a hand punch, and 2) attach a glass coverslip (diameter of 22 mm) to the back of the dish with silicone.
2. Wash the plates with distilled water, as described in step 1.1.2, and culture the cortical neurons in the glass-based dish at 3 x 10⁴ cells/dish with medium A, as described in step 1.2.
3. After 4 days of cell culture, replace the medium with 2 mL of new medium B, and transfer the dish to inverted microscope A. Maintain the culture in a humidified atmosphere of 10% CO₂ at 37 °C.

5. Endocytosis Experiment

1. Culture the mouse cortical neurons as described in step 1.2.
2. Four days later, replace the medium with 100 μL of new medium B containing 20 μM fluorescence membrane probe for 1 min.
3. Add 1 μL of 0.05 mM aggregated Aβ1-42 (final 0.5 μM) or vehicle (distilled water) solution and mix by pipetting. Incubate for 20 min.
4. Remove the medium and wash the wells twice with medium B that has been pre-warmed to 37 °C.
5. Fix, wash, and mount the neurons as described in steps 1.3.3 and 1.3.4.
6. Capture fluorescent and DIC images with a 63X oil objective lens on inverted microscope A.
7. Quantify the density of the fluorescence membrane probe-positive area in each healthy growth cone by using an image software.

6. Gene Transfection

1. Prepare cortical neurons as described in step 1.2. After completing steps 1.2.1 to 1.2.10, centrifuge the neurons at 178 x g for 3 min.
2. Remove the supernatant, add 4 mL of Ca²⁺-free and Mg²⁺-free Hanks' balanced salt solution (CMF-HBSS), and mix by pipetting.
3. Centrifuge the cells at 178 x g for 3 min.
4. Remove the supernatant, add 4 mL of CMF-HBSS, and mix by pipetting. Next, calculate the cell density, as described in step 1.2.10.
5. Transfer 5 x 10⁶ cells to a 1.5 mL tube and centrifuge at 1,677 x g for 1 min.
6. Remove the supernatant, add 100 μL of transfection solution with supplement and 3 μg of DNA plasmid encoding EGFP or EGFP-AP180 C-terminus, and mix by pipetting.
7. Transfer the above solution (step 6.6) to a certified cuvette and transfect with an electroporator, according to the manufacturer's protocol.
8. Immediately after transfection, add 500 μL of medium A into the cuvette and transfer the solution to a 1.5-mL tube with a certified pipette. Next, calculate the cell density, as described in step 1.2.10.
9. Culture the cells in an 8-well culture slide at 0.8 x 10⁴ cells/well, as described in steps 1.2.11 and 1.2.12.
10. After 4 days of cell culture, perform a collapse assay as described in step 1.3.

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 forms^27,28,30,35. 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 Aβ³⁶....

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

List of materials used in this article

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/