Centrosome-mediated microtubule remodeling during axon formation in human iPSC-derived neurons (2021)
Figure 2

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 = 5µm in overview, 2µm 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 and prior to neuronal induction. n = 48-51 cells in 2 independent experiments.

C. Typical examples of Centrinone-B treated or control human iPSC-derived neurons (12-15 days) immunostained for AnkG, Trim46, MAP2 and Centrin. Arrowheads mark AIS structures, arrows mark centrosomes. Inserts represent centrosomes, zooms on the right represent AIS structures. Scale bar = 20µm in overview, 2µm in insert, 10µm 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 = 31-53 cells in 3 independent experiments.

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 = 27-32 cells in 2 independent experiments.

F. Quantifications of percentage human iPSC-derived neurons (12-15 days) treated with Centrinone-B containing AnkG-positive processes that are Trim46-positive or Trim46-negative. Neurons are subdivided in populations containing 2 centrioles, or less than 2 centrioles, based on Centrin immunostaining. n = 27 cells in 2 independent experiments.

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 (4 independent experiments; no AP: n=2, single AP: n=32, multiple APs: n=22) versus Centrinone-B treated cultures (3 independent experiments; no AP: n=9, single AP: n=34, multiple APs: n=10).

I. Representative examples of evoked AP firing in Centrinone-B treated human iPSC-derived neurons recorded at day 11. Shown is the response to a single depolarizing current step of a neuron that fires no APs, a neuron that fires a single AP and a neuron that fires multiple APs. The offset current (Ihold) was adjusted to keep the baseline membrane potential at approximately −60 mV (dashed lines).

J. Scatter plots of AP amplitude versus AP half-width grouped by days after plating for Centrinone-B treated (10-11 days: n = 14 cells in 1 independent experiment, 13-14 days: n = 28 cells in 3 independent experiments) and control (7 days: n = 7 cells, 10-11 days: n = 15 cells in 2 independent experiments, 13-14 days: n = 36 cells in 4 independent experiments) human iPSC-derived neurons.

K. 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.

L. AP half-width recorded in Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neurons (n = 59 cells in 4 independent experiments).

M. AP threshold, amplitude and after-hyperpolarization recorded in Centrinone-B treated (n = 43 cells in 3 independent experiments) and control human iPSC-derived neuron cultures (n = 59 cells in 4 independent experiments).

N. 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 in 3 independent experiments) and control human iPSC-derived neurons (n = 48 cells in 3 independent experiments).

Data information: Data represents mean ± SEM. Chi-square-test including post-hoc analysis with Bonferroni correction (B, D, E, F); Mann Whitney test (L, M: after-hyperpolarization, N), Student's t test (M: threshold, AP amplitude). *** p<0.001, ** p<0.01, * p < 0.05, ns p>0.05