Abnormal lysosomes in spastin-HSP mouse neurons. (A) Primary cortical neurons derived from spastin^wt/wt (WT), spastin^wt/N384K (HET), and spastin^N384K/N384K (KI) animals were processed for immunofluorescence microscopy with the neuronal marker βIII-tubulin (red) and LAMP1 (green). The percentage of cells with largest lysosome >2 µm diameter was plotted for each animal (100 cells/animal). Bars indicate mean ± SEM. (B) EM of primary neuron cell bodies and neurites from animals with spastin^N384K genotypes indicated. Boxed areas are in higher magnification on the right. (C) Confocal immunofluorescence microscopy (IF) of axonal swelling in spastin^N384K/N384K mouse primary cortical neuron, labeled for LAMP1 and the axonal marker tau. Boxed areas shown in higher power in bottom panels. The mean ± SEM number of LAMP1-positive vesicles/µm of axonal length in 20 swellings versus adjacent axonal segments is plotted for three animals. Bars: (EM) 500 nm; (IF main panels) 10 µm; (IF magnified insets) 5 µm. P-values generated by two-tailed Student’s t test.