Lysosomal Trafficking and Axon Degeneration

BODIPY–pepstatin A (Thermo Scientific, P12271) is a probe used to detect active cathepsin D distribution and trafficking in lysosomes in vitro [14 (link)]. On DIV 11, co-cultured cortical neurons and astrocytes transduced with the mScarlet-LC3 viral vector were loaded with BODIPY–pepstatin A (1 μM) for 1 h in both soma and axonal compartments of the microfluidic chamber prior to real-time imaging. Afterwards, the medium at both sides was replaced to remove the fluorescent probe. In order to distinguish individual axons and vesicle fusion rather than spatial overlap, 3-min videos were acquired on the axon side before and at different time points after axotomy. For colocalization analysis between active cathepsin D, as indicated by BODIPY-pepstatin A with the EGFP fluorophore and mScarlet-LC3, the overlapped vesicles labeled with yellow signals were counted manually in individual axons and separately divided by the number of active cathepsin D and LC3 vesicles at each time point.