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

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 con