Lysosomal abnormalities in human spastin-HSP patients. (A) Skin fibroblasts from control subjects (top) or three spastin-HSP patients (SPG4; bottom) were fixed and visualized by confocal immunofluorescence microscopy (IF) for LAMP1. In each image, the inset panels show higher-magnification views of the boxed region. The diameter of the largest lysosome was measured in 100 fibroblast cells from each subject, and the mean percentage of cells with largest lysosome >2 µm was plotted in the corresponding histogram (n = 3 experimental repeats). Histogram shows mean ± SEM. (B) EM of fibroblasts from spastin-HSP patient and control (bottom, magnified views of boxed areas). (C) Cells from two human cortical neuron lines (SPG4-111 and 112) and a control line (Ctrl-231) were fixed and labeled for βIII-tubulin (as a marker of neuronal differentiation) and LAMP1. The diameter of the largest lysosome was measured in βIII-tubulin–positive cells, and the mean ± SEM percentage of neurons with largest lysosome >2 µm was plotted in the corresponding histogram (n = 3/line, 100 cells/repeat). The two SPG4 neuronal lines were differentiated from separate NPC cultures derived from patient SPG4-1 iPSCs. (D) Cells from two human cortical neuron lines (SPG4-111 and 112) were labeled for βIII-tubulin and LAMP1. The images show a typical axonal swelling. The number of lysosomes in swellings (box 1) and adjacent axonal segments (box 2) was counted and normalized for segment length, and the mean ± SEM number of LAMP1-positive vesicles/µm of axonal length are presented in the corresponding histogram (n = 20 swellings). (E) EM of lysosomal structures in patient SPG4-1 iPSC-derived cortical neurons. Bars: (EM) 500 nm; (IF main panels) 10 µm; (IF magnified insets) 5 µm. P-values generated by two-tailed Student’s t test.