Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 43 Figure 3. Extra developmental stage and gradual action potential maturation during axon specification A. Schematic illustration and timing of neurodevelopmental stages 2a, 2b, 3a and 3b in human iPSC- derived NSCs/neurons. B. Representative images of stage 2a, 2b, 3a, and 3b hiPSC-derived neurons. Cells were subjected to FUGW-GFP lentivirus and immunostained for AnkG and Trim46. Arrowheads mark Trim46 and AnkG accumulations. Scale bar: 40 µm overview, 5 µm zooms. C. Quantifications of percentage neurons in stage 2a, 2b, 3a or 3b. N=2, n=50-55 neurons. D. Quantifications total Trim46 length in neurites of stage 2b, 3a and 3b human iPSC-derived neurons. N=2, n=20 cells. E. Quantifications of distance soma to start of the Trim46 signal in neurites of stage 2b, 3a and 3b human iPSC-derived neurons. N=2, n=20 cells. F. Phase plots of a representative AP recorded of a human iPSC-derived neuron at 10 days and 14 days. G. Scatter plot of AP amplitude versus AP half-width grouped by days after plating (N=4; 7 days: n=7 cells, 10-11 days: n=15 cells, 13-14 days: n=36 cells). H. AP amplitude recorded in human iPSC-derived neurons of 10-11 days (n=15 cells) and 13-14 days (n=36 cells). I. AP half-width recorded in human iPSC-derived neurons of 10-11 days (n=15 cells) and 13-14 days (n=36 cells). Used tests: Chi-square test with Bonferroni post-hoc correction ( C ), One-way ANOVA with Bonferroni post-hoc correction ( D , E ), Student’s t-test ( H ), Mann-Whitney U test ( I ), *** p<0.001, ** p<0.01, * p<0.05, ns p≥0.05; error bars are ± SEM. form relatively long, noncontinuous structures in the distal axon before accumulating at the proximal axon to form the AIS structure. Action potential maturation coinciding with onset of axon development We next investigated whether the different organization of AIS components in stage 3a and 3b neurons is accompanied by differences in electrical properties. Local clustering of voltage-gated ion channels at AIS structures, as observed in stage 3b neurons, are important to facilitate mature APs (Kole et al. 2008). Hence, we hypothesized that the noncontinuous appearance of NaV channels at distal axons in the newly identified stage 3a neurons affect AP firing. To address this, we performed electrophysiological experiments of neurons from day 7 to 14 to capture the developmental transition from stage 3a to stage 3b neurons. Our recordings showed progressive maturation of physiological properties during this developmental time window (Fig 3F,G). Neurons recorded on day 13-14 fired APs with a larger amplitude and shorter half-width compared to 10-11 days old neurons (Fig 3H,I). No differences in AP amplitude and half-width were found between neurons of 7 and 10-11