Cortical neurons obtained from patient-derived iPSCs with GNAO1 p.G203R variant show altered differentiation and functional properties

Abstract

Pathogenic variants in the GNAO1 gene, encoding the alpha subunit of an inhibitory heterotrimeric guanine nucleotide-binding protein (Go) highly expressed in the mammalian brain, have been linked to encephalopathy characterized by different combinations of neurological symptoms, including developmental delay, hypotonia, epilepsy and hyperkinetic movement disorder with life-threatening paroxysmal exacerbations. Currently, there are only symptomatic treatments, and little is known about the pathophysiology of GNAO1-related disorders. Here, we report the characterization of a new in vitro model system based on patient-derived induced pluripotent stem cells (hiPSCs) carrying the recurrent p.G203R amino acid substitution in Gαo, and a CRISPR-Cas9-genetically corrected isogenic control line. RNA-Seq analysis highlighted aberrant cell fate commitment in neuronal progenitor cells carrying the p.G203R pathogenic variant. Upon differentiation into cortical neurons, patients' cells showed reduced expression of early neural genes and increased expression of astrocyte markers, as well as premature and defective differentiation processes leading to aberrant formation of neuronal rosettes. Of note, comparable defects in gene expression and in the morphology of neural rosettes were observed in hiPSCs from an unrelated individual harboring the same GNAO1 variant. Functional characterization showed lower basal intracellular free calcium concentration ([Ca²⁺]_i), reduced frequency of spontaneous activity, and a smaller response to several neurotransmitters in 40- and 50-days differentiated p.G203R neurons compared to control cells. These findings suggest that the GNAO1 pathogenic variant causes a neurodevelopmental phenotype characterized by aberrant differentiation of both neuronal and glial populations leading to a significant alteration of neuronal communication and signal transduction.

Keywords: Encephalopathy; GNAO1; Induced pluripotent stem cell; Movement disorder; neural rosette; wnt.

Fig. 1

RNA-Seq analysis of neural progenitors A. Outline of the origin of the hiPSC lines used in this study. B. Heatmap showing the expression in GNAO1^+/G203R and GNAO1^+/G203G NPCs at day 7 and 11 of the leading edge genes identified via GSEA for the “cell fate commitment” Gene Ontology Biological Process category. Genes that were identified as leading edge ones only at day 7, only at day 11, or at both time points were split into three groups (Only_D7, Only D11, Common_D7-D11, respectively). Genes belonging to the “neuron fate commitment” or “neuron fate specification” categories were colored in purple. C. Heatmap showing the expression in GNAO1^+/G203R and GNAO1^+/G203G NPCs at day 7 and 11 of selected neuron progenitor- and reactive astrocyte-specific genes. The adjusted p-values displayed on the right pertain to the DGE analyses performed between GNAO1^+/G203R and GNAO1^+/G203G NPCs at day 7 (FDR D7) and day 11 (FDR D11). The expression values reported in both heatmaps correspond to row-scaled (Z-score), rlog-transformed count data.

Fig. 2

Cortical neuron differentiation and analysis at day 25. A. Schematic representation of the protocol used for cortical neuron differentiation of human iPSCs in conventional 2D cultures. NPC: neural progenitor cell; SB: SB431542 (TGFβ type I receptor/ALK5 inhibitor); LDN: LDN193189 (BMP inhibitor); DAPT: Notch inhibitor; BDNF: Brain Derived Neurotrophic Factor; GDNF: Glial Derived Neurotrophic Factor; AA: ascorbic acid; cAMP: cyclic AMP. B. Phase contrast images of differentiating cells at day 25. C. Real-time qRT-PCR analysis of the expression of the indicated markers in differentiating cells. The graphs show the average and standard deviation (Student's t-test; paired; two tails; **p < 0.01, ***p < 0.001).

Fig. 3

Immunostaining analysis at day 25 of differentiation A-B. Immunostaining analysis with the indicated primary antibodies and DAPI to label nuclei. PAX6 and NESTIN are neural progenitor markers; TBR2 is a neuronal precursor marker; TUJ1 and MAP2 are neuronal markers.

Fig. 4

Analysis of neuronal rosettes morphology in the independent GNAO1^+/G203R#2 line A. Phase contrast images of GNAO1^+/G203R#2 cells at the neural rosette stage, corresponding to day 30 of differentiation. Note that the in the protocol used for differentiation of GNAO1^+/G203R#2 cells (as described in the Methods section) has a different timing from the one depicted in Fig. 2A. B-D. Immunostaining was performed using the specified primary antibodies along with DAPI for nuclear labeling. TBR1 is a marker of early-born neurons (B); and TUJ1 (B) and MAP2 (C) are markers for neurons; NESTIN serves as a marker for neural progenitors (C); β-catenin as a marker of apical polarization in neural rosettes (D). Number of individual cultures for experiments was 3.

Fig. 5

Marker expression analysis at day 40 of differentiation A. Real-time qRT-PCR analysis of the expression of the indicated markers in differentiating cells. The graphs show the average and standard deviation (Student's t-test; paired; two tails; *p < 0.05, ***p < 0.001, n.s. nonsignificant). B. Immunostaining analysis with the indicated primary antibodies and DAPI to label nuclei.

Fig. 6

Marker expression analysis at day 50 and 70 of differentiation A-B. Real-time qRT-PCR analysis of the expression of the indicated markers in differentiating cells at the indicated time points. The graphs show the average and standard deviation (Student's t-test; paired; two tails; *p < 0.05, ***p < 0.001, n.s. nonsignificant). C. Immunostaining analysis with the indicated primary antibodies and DAPI to label nuclei.

Fig. 7

Neurons carrying the p.G203R GNAO1 substitution exhibit lower values of basal [Ca²⁺]_i and a reduced spontaneous Ca²⁺ activity. A. representative spontaneous Ca²⁺ transients recorded in control cells. B. up, [Ca²⁺]_i basal values measured in individual cells. Please note the reduced basal [Ca²⁺]_i for p.G203R mutant neurons. Number of examined cells: 160, 159, 229 and 230, for p.G203G DIV40, p.G203R DIV40, p.G203G DIV50 and p.G203R DIV50, respectively. Bottom, percentage of neurons exhibiting spontaneous Ca²⁺ transients, same cells as upper panel. *, p < 0.001.

Fig. 8

The p.G203R GNAO1 substitution reduces the response of differentiated neurons to several neurotransmitters in terms of [Ca²⁺]_i elevations. A. left, percentage of neurons exhibiting Ca²⁺ transients induced by the application of glutamate (1 mM, 3 s); inset, representative Ca²⁺ transients elicited by glutamate administration (1 mM, 3 s), in a control (black) and GNAO1 p.G203R (red) iPSC-derived neuronal cells. Number of examined cells: 160, 159, 229 and 230, for p.G203G DIV40, p.G203R DIV40, p.G203G DIV50 and p.G203R DIV50, respectively. Right, amplitude of the [Ca²⁺]_i increase induced by glutamate application in the responding cells. B. left, percentage of neurons exhibiting Ca²⁺ transients induced by the application of GABA (100 μM, 3 s). Same cells as Fig. 7A. Right, amplitude of the [Ca²⁺]_i increase induced by GABA application in the responding cells. C. left, percentage of neurons exhibiting Ca²⁺ transients induced by the application of baclofen (100 μM, 3 s). Same cells as Fig. 7A. Right, amplitude of the [Ca²⁺]_i increase induced by baclofen application in the responding cells. D. left, percentage of neurons exhibiting Ca²⁺ transients induced by the application of glycine (30 μM, 3 s). Same cells as Fig. 7A. Right, amplitude of the [Ca²⁺]_i increase induced by glycine application in the responding cells. E. left, percentage of neurons exhibiting Ca²⁺ transients induced by the application of ACh (100 μM, 3 s). Same cells as Fig. 7A. Right, amplitude of the [Ca²⁺]_i increase induced by ACh application in the responding cells. F. left, percentage of neurons exhibiting Ca²⁺ transients induced by the application of ATP (100 μM, 3 s). Same cells as Fig. 7A. Right, amplitude of the [Ca²⁺]_i increase induced by ATP application in the responding cells. *, p < 0.001; a, p = 0.008; b, p = 0.022; c, p = 0.006, d, p = 0.007.