This study investigates the effects of aggregated amyloid-�� (A��) on the structural integrity of growth cones in mouse embryonic neurons. The findings demonstrate that A�� induces collapse of growth cones, highlighting its toxic effects on neuronal development.
Begin with a multi-well culture slide containing neurons from a mouse embryo in a medium.
The neurons exhibit growth cones at the tips of their growing axons. These structures include thin projections called filopodia and sheet-like extensions called lamellopodia, which consist of actin filaments and microtubules, components of the cytoskeleton.
Treat the test wells with aggregated amyloid-β, or Aβ, protein and the control wells with a vehicle solution.
In the test wells, aggregated Aβ binds to membrane receptors, initiating downstream signaling that phosphorylates cytoskeleton-regulating proteins.
The phosphorylated proteins disrupt actin filaments and microtubules, destabilizing the growth cone's structural integrity and causing its collapse.
Incubate with a fixative to preserve the cellular structure, then wash the cells and mount them for imaging.
Using a microscope, compare the cellular structures in the test and control wells.
An increased occurrence of growth cone collapse in Aβ-treated neurons indicates the toxic effect of Aβ aggregates.
Begin this assay at least a week in advance by opening a fresh vial of commercially-obtained, full-length A-beta-42 and dissolving the contents in sterile distilled water to a concentration of 0.5 millimolar. Incubate at 37 degrees Celsius for seven days to induce aggregation and toxicity. Prepare aliquots of aggregated A-beta-42, and store at minus 80 degrees Celsius until use.
On day four of the neuronal culture, treat the wells with 100 microliters of 0.5 micromolar aggregated A-beta-42 or vehicle solution in medium B for one hour as previously demonstrated. After the hour has elapsed, remove the culture medium, and immediately fix the neurons with 4% paraformaldehyde containing 4% sucrose in PBS for one hour at 37 degrees Celsius on a hot plate.
Fixation is mostly important as maintaining the shape of growth cones is critical for this protocol.
After fixation, wash the neurons three times with PBS. Then, after removing the chamber, mount the neurons with an aqueous mounting medium. Dry the mounting medium at 4 degrees Celsius for two to four days. Capture the entire area of each well with a 20x dry objective lens on an inverted microscope.
Capturing and analyzing the entire area in each well is important for avoiding subjectivity.
Classify the longest neurites of each neuron in stage 3 or 4 as axons. Axonal growth cones lacking lamellipodia or possessing fewer than three filopodia are considered collapsed growth cones.
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