Fig. 1: ATXN2-ALS motor neurons show early signs of motor neuron disease. | Nature Communications

Fig. 1: ATXN2-ALS motor neurons show early signs of motor neuron disease.

From: ATAXIN-2 intermediate-length polyglutamine expansions elicit ALS-associated metabolic and immune phenotypes

Fig. 1

A, B Immunocytochemistry for TUJ1, CHAT, and ISL1 on human iPSC-derived hMN cultures. Scale bar is 200 µm. C Current-clamp recording of CTL hMNs following current injections at DIV12. D t-SNE map of scRNAseq experiments on hMN cultures showing individual cells and cluster numbers assigned by RaceID2, where 60% of the cells correspond to motor neurons. n = 384 cells divided in 4 libraries from 1 control (CTL_2) and 1 ATXN2-ALS (ALS_G) line (n = 1 differentiation). E, F Representative scanning electron microscopy images of DIV12 control (CTL) and ATXN2-ALS hMN cultures. CTL MNs contain smooth neurite thickenings (white arrowhead) whereas ATXN-ALS cultures show ruffled and damaged structures (black arrowhead). Scale bar is 2 µm. F Quantification of damaged neurites as in E. n = 149 neurites from 3 CTL lines and 308 neurites from 4 ATXN2-ALS lines (two-tailed upaired t test; *P = 0.02). Data show individual lines and mean ± SD. G Immunocytochemistry for PABP in DIV9 hMNs after 60 min of sodium arsenite (ARS) treatment. Scale bar is 5 µm. H Quantification of number of stress granules (SGs) per neuron. n = 100 cells per condition from 3 CTL and 4 ATXN2-ALS lines, 3 experimental replicates/line (Kruskal–Wallis test, CTL 60’ vs ATXN2-ALS 60’ *P = 0.0169; ALS-ATXN2 60’ vs ALS-ATXN2 180’ *P = 0.0135). Violin plot of individual cells. I Quantification of SG area. 3 CTL and 4 ATXN2-ALS lines, 3 experimental replicates/line (2-way ANOVA and Tukey’s multiple comparison, CTL 60’ vs CTL180’ *** P < 0.0001; CTL 180’ vs ATXN2-ALS 180’ **P = 0.0062). Bar plots show mean ± SEM (See Supplementary Data 1 for N, SEM, and all P values). J Distribution of SG area in DIV9 hMNs at 60 minutes after ARS and 120 min of recovery (180’). K Immunocytochemistry for ATXN2 in DIV12 hMN cultures. Phalloidin stains F-actin. Scale bar is 10 µm. L, M Current-clamp recording of hMN cultures from 2 CTL and 2 ATXN2-ALS lines. L At DIV12, ATXN2-ALS hMNs are hypoexcitable (n = 30 neurons; P = 0.049, P = 0.005, and P = 0.0001 for 30, 40, and 50 nA, respectively; 2-way ANOVA and Sidak’s multiple comparisons test) and M at DIV24 hyperexcitable as compared to CTL (n = 46 neurons; *P = 0.01 at 30 nA, ***P < 0.0001 at 40 and ***P = 0.0006 at 50 nA; 2-way ANOVA and Sidak’s multiple comparisons test). Data are mean ± SD. Source data are provided as a Source Data file for F, H, I, J, L, M.

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