In Vitro Assessment of Aggregated Amyloid-β on Neuronal Growth Cone Collapse

Abstract

Source: Kuboyama, T. Visualizing Axonal Growth Cone Collapse and Early Amyloid β Effects in Cultured Mouse Neurons. J. Vis. Exp. (2018)

This video demonstrates an in vitro assessment of neuronal growth cone collapse by amyloid-β (Aβ) aggregates. Treating the neurons with Aβ aggregates causes cytoskeletal destabilization, leading to the collapse of growth cones at the tip of the axons. The treated cells are compared with control cells to assess the extent of growth cone collapse.

Protocol

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 culture
   1. Mince freshly isolated cerebral cortices from embryonic day 14 (E14) ddY (Deutschland, Denken, and Yoken) 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.
   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 deoxyribonuclease I (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 previously.
3. Collapse assay
   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.
   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 previously. Similar results can be observed when by Aβ1-42 treatment for 1 h after 3 days of neuronal culture.
   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.
   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 previously.
      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.

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, AquaPoly/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