Feline Lindhout

3 Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons 79 Figure 2. Centriole loss in NSCs perturbs subsequent axonal Trim46 targeting and action potential maturation A . Typical examples of Centrinone-B treated or control human iPSC-derived NSCs immunostained for Pericentrin and Centrin. Inserts represent centriole(s). Scale bar: 5 µm in overview, 2 µm in inserts. B. Quantifications of the percentage of cells with 0, 1 or 2 centrioles per cell after 0 (control), 2 or 5 days Centrinone-B treatment. N=2, n=48-51 cells. C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Inserts represent centrosomes, zooms on the right represent AIS structures. Scale bars: 20 µm in overview, 2 µm in insert, 10 µm in zooms. D. Quantifications of percentage of human iPSC-derived neurons (12-15 days) treated with Centrinone-B containing a Trim46-positive or Trim46-negative process. Neurons are subdivided in populations containing 2 centrioles, or less than 2 centrioles, based on Centrin immunostaining. N=3, n=31-53 cells. E. Quantifications of percentage of human iPSC-derived neurons (12-15 days) treated with Centrinone-B containing an AnkG-positive or AnkG-negative process. Neurons are subdivided in populations containing 2 centrioles, or less than 2 centrioles, based on Centrin immunostaining. N=2, n=27-32 cells. F. Quantifications of percentage human iPSC-derived neurons (12-15 days) treated with Centrinone-B containingAnkG-positive processes that areTrim46-positive or Trim46-negative. Neurons are subdivided in populations containing 2 centrioles, or less than 2 centrioles, based on Centrin immunostaining. N=2, n=27 cells. G. Top: Schematic illustration of the experimental electrophysiology setup. To determine action potential (AP) frequency, somatic current injections from -10 pA to 50 pA (steps of 5 pA, 400 ms) were applied. Bottom: Representative example of evoked AP firing in a Centrinone-B treated human iPSC-derived neuron, response to hyperpolarizing and first two depolarizing current steps, recorded at day 13. H. Neuronal excitability was recorded in 54/61 control cells and 53/54 Centrinone-B treated cells. Percentage of cells firing zero, one or multiple APs in control (N=4; no AP: n=2, single AP: n=32, multiple APs: n=22) versus Centrinone-B treated cultures (N=3; no AP: n=9, single AP: n=34, multiple APs: n=10). I. Scatter plots of AP amplitude versus AP half-width grouped by days after plating for Centrinone-B treated (10-11 days: n=14 cells, 13-14 days: n=28 cells) and control (7 days: n=7 cells, 10-11 days: n=15 cells, 13-14 days: n=36 cells) human iPSC-derived neurons. J. Phase plots of a representative AP of a human iPSC-derived neuron treated with Centrinone-B and a control neuron of 13 and 14 days, respectively. K . AP half-width recorded in Centrinone-B treated (n=43 cells) and control human iPSC-derived neurons (n=59 cells). L. AP threshold, amplitude and after-hyperpolarization recorded in Centrinone-B treated (n=43 cells) and control human iPSC-derived neuron cultures (n=59 cells). M. Left top: Schematic representation of the voltage ramp protocol used to determine maximum sodium current; membrane potential was changed from -100 mV to 200 mV in 400 ms. Left bottom: Representative example of maximum sodium peak recorded of a control neuron at day 13. Right: Maximum sodium peak in Centrinone-B treated (n=52 cells) and control human iPSC-derived neurons (n=48 cells). Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction ( B , D , E , F ); Mann Whitney test ( K , L : after-hyperpolarization, M ), Student’s t test ( L : AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05