Figure 2.

Short-term read-out of neurite outgrowth, growth cone formation and axonal swellings in iPSC-derived cortical neurons. (A) Timeline of the neurite outgrowth and growth cone assay. Cortical cultures were triturated to single cells and replated at low density on day 57. (B) Cells were fixed and stained for TUBB3 after 24 hours on day 58. Scale bar: 100 µm. (C,D) Quantification of the neurite outgrowth after 24 hours. SPG4 neurons exhibit a significant reduction in neurite length after 24 hours compared to controls. Data points depicted in (C) represent mean values of n = 18 (control) and n = 28 (HSP) biological replicates comprising ≥100 cells each. (D) Cumulative bar graph based on C; Mann-Whitney-U test; ****p < 0.0001; error bars show SD. (E) Immunocytochemistry for TUBB3 and actin on day 58. Scale bar: 50 µm. Insets show magnifications of typical growth cones. Scale bar: 10 µm. (F,G) SPG4 neurons exhibit a significant increase in growth cone area after 24 hours. Data points depicted in (F) represent mean values of n = 15 (control) and n = 25 (HSP) biological replicates comprising ≥100 cells each. (G) Cumulative bar graph based on (F); Mann-Whitney-U test; ****p < 0.0001; error bars show SD. (H) Timeline of the axonal swelling assay: neurons were stimulated with 3 nM taxol on day 44 and cultivated until day 50. (I) Cortical neurons were fixed and stained for the dendrite marker MAP2 and the axon marker TAU1 on day 50. SPG4 neurons exhibit prominent TAU1 positive axonal swellings. Scale bar: 100 µm. (J) Quantification of TAU1 positive axonal swellings. The number of TAU1 positive axonal swellings in patient neurons was significantly higher (mean of 1.537/mm) compared to that of control neurons (mean of 0.073/mm). Data are based on ≥100 cells per biological replicate (C-61f: n = 7, C-62m: n = 7, C-31f: n = 4, HSP-22f: n = 10, HSP-23m: n = 7, HSP-24m: n = 9); unpaired t-test; ****p < 0.0001; error bars: SD. Figure adapted from corresponding PhD thesis⁷².