Fig. 7: Rhosin treatment rescued functional deficits of the 16p11.2 deletion DA neuron networks in vitro. | Nature Communications

Fig. 7: Rhosin treatment rescued functional deficits of the 16p11.2 deletion DA neuron networks in vitro.

From: 16p11.2 deletion is associated with hyperactivation of human iPSC-derived dopaminergic neuron networks and is rescued by RHOA inhibition in vitro

Fig. 7

a Schematic presentation of the network functional analyses and Rhosin (1 μM) or vehicle treatment on the LD-MEA. Cells were plated to LD-MEA after sorting and recorded up to 28 days. b KCTD13 expression in the DA neurons with 16p11.2 CNVs. Reduced KCTD13 expression was detected in the 16pdel neurons compared to control cells (p = 0.0117). c RHOA expression in the DA neurons with 16p11.2 CNVs. Increased RHOA expression was detected in the 16pdel neurons compared to control cells (p = 0.0323). Data are presented as mean ± SEM, and statistical analyses were done with unpaired t test with Welch’s correction and with one-tailed p values, *p < 0.05 and **p < 0.01 (b, c). The number of biologically independent samples are n = 8 control, n = 8 16pdup, and n = 8 16pdel (b, c). d Weighted mean firing rate (Hz) of the active electrodes in DA neuron networks were analyzed during 28 days of recording. Higher weighted mean firing rates were detected in 16pdel neurons compared to control neurons at days 24 and 28 (p = 0.0015 day 24, p = 0.0004 day 28), and compared to 16dup cells at days 24 and 28 (p = 0.0003 d24, p = 0.0002 day 28). The number of biologically independent samples is n = 16 per each genotype. e After 24 days of plating the DA neurons on the LD-MEA, the weighted mean firing rate (Hz) of 16pdel cells was higher compared to control cells (p = 0.0021) or 16pdup cells (p = 0.0012), and the long-term Rhosin treatment reduced the weighted mean firing rate in the 16pdel neurons significantly (p = 0.0032). f The number of spikes were recorded during development of networks up to 28 days. Rhosin or vehicle treatments were started after 5 days of plating of the cells on the LD-MEA. 16pdel cells had higher spike rate compared to control neurons at day 17 (p = 0.0038), day 22 (p = 0.0117), day 24 (p < 0.0001), and day 28 (p < 0.0001), and compared to 16dup cells at day 17 (p < 0.0001), day 22 (p < 0.0001), day 24 (p < 0.0001), and day 28 (p < 0.0001). Rhosin treatment reduced the spike rate in the 16pdel neurons significantly at day 17 (p = 0.0073), day 22 (p = 0.0032), day 24 (p = 0.0061), and day 28 (p = 0.0032). Data presented as mean ± SEM (d–f). The number of biologically independent samples are n = 16 for control + veh, 16pdup + veh, 16pdel + veh, and n = 7 control + Rhosin, n = 6 16pdup + Rhosin, n = 6 16pdel + Rhosin (e, f). Two-way ANOVA followed by Tukey’s multiple comparisons test, and adjusted p values, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 in (d, f), and unpaired t test with Welch’s correction and one-tailed p values (e). g After 24 days of plating the DA neurons, the number of bursts was higher in the 16pdel group compared to control (p = 0.0035) or 16pdup cells (p = 0.025), and the long-term Rhosin treatment reduced the number of bursts significantly in the 16pdel neurons (p = 0.0247). Data are presented as mean ± SEM, unpaired t test with Welch’s correction and one-tailed p values, p values presented *p < 0.05 and **p < 0.01. The number of biologically independent samples are n = 16 for control + veh, 16pdup + veh, 16pdel + veh, and n = 7 control + Rhosin, n = 6 16pdup + Rhosin, and n = 6 16pdel + Rhosin (g). The burst duration was not significantly different between different groups. The number of samples per group are control + veh n = 9, 16pdup + veh n = 5, 16pdel + veh n = 12, control + Rhosin n = 3, 16pdup + Rhosin n = 2, 16pdel + Rhosin n = 3 (g). h, i Representative raster plots of DA neuron network firing and bursting patterns after 24 days of recording on LD-MEA, cells were treated with vehicle or Rhosin. Source data are provided as a Source Data file.

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