FOXG1-Dependent Dysregulation of GABA/Glutamate Neuron Differentiation in Autism Spectrum Disorders

Abstract

Autism spectrum disorder (ASD) is a disorder of brain development. Most cases lack a clear etiology or genetic basis, and the difficulty of re-enacting human brain development has precluded understanding of ASD pathophysiology. Here we use three-dimensional neural cultures (organoids) derived from induced pluripotent stem cells (iPSCs) to investigate neurodevelopmental alterations in individuals with severe idiopathic ASD. While no known underlying genomic mutation could be identified, transcriptome and gene network analyses revealed upregulation of genes involved in cell proliferation, neuronal differentiation, and synaptic assembly. ASD-derived organoids exhibit an accelerated cell cycle and overproduction of GABAergic inhibitory neurons. Using RNA interference, we show that overexpression of the transcription factor FOXG1 is responsible for the overproduction of GABAergic neurons. Altered expression of gene network modules and FOXG1 are positively correlated with symptom severity. Our data suggest that a shift toward GABAergic neuron fate caused by FOXG1 is a developmental precursor of ASD.

Figure 1. ASD organoids display normal neuronal differentiation potential

A, Both control-derived and ASD-derived organoids express markers for proliferating neural progenitors (SOX2), and the neuronal markers TUBB3. The organoids have apico-basal polarity with N-CADHERIN⁺ apical end feet of radial glial cells and pH3⁺ cells undergoing mitosis at the apical side of the neuroepithelium. B, Stereological quantification of SOX2⁺ proliferating radial glia progenitors and TUBB3⁺ neuronal cells at TD31. Scale bars, 10 μm or 20 μm as indicated. C, Action potentials elicited by depolarizing current injections in iPSC-derived neurons from a patient and the corresponding parental control. The dashed lines indicate a membrane potential of 0 mV. D, Examples of spontaneous excitatory postsynaptic currents (EPSCs) recorded at a holding potential of −70 mV in a neuron from a parental control that was maintained in vitro for 52 days. E, average EPSC obtained from 30 spontaneous events. The decay of the current was fitted with a single exponential that gave a time constant of 1.85 ms. F, Histogram of the amplitude of spontaneous inward EPSCs recorded from the same neuron (604 events). The overall frequency of events detected in this neuron was 0.81 per second. G–I, Transcriptome correlation between organoids at terminal differentiation day 0 (d0) (rosette stage), day 11 and 31 (d11 and d31) (n= 45 samples, see Table S1) and postmorterm human brain samples from the BRAINSPAN project (n=524 samples). The X-axis shows the postconceptional age in weeks or the brain region of the BrainSpan postmorterm brain samples. The Y-axis is the number of times each iPSC-derived neuron sample was classified. Classification was based on the maximum correlation coefficient and on the 95% confidence interval of the maximum correlation (see Supplementary Methods). AMY, amygdala; CBC, cerebellar cortex; DFC, dorsolateral frontal cortex; HIP, hippocampus; ITC, inferolateral temporal cortex; MFC, medial prefrontal cortex; OFC, orbital frontal cortex; STC, superior temporal cortex; STR, striatum. In I, X-axis shows BRAINSPAN agglomerated brain regions (i.e. more brain regions were merged into a single ‘larger’ region): NCX (i.e. FC, PC, TC, OC), neocortex; HIP, hippocampus; AMY, amygdala; VF (i.e. VF, MGE, LGE, CGE, STR), ventral forebrain; URL (i.e. CBC, CB, URL), upper rhombic lip. See also Figure S3.

Figure 2. WGCNA network in neuronal cells from ASD patients and unaffected family controls and correlation with clinical phenotypes

A, Relationship between the modules’ eigengenes with diagnosis and time in culture. TD11: 11 days of terminal differentiation; TD31: 31 days of terminal differentiation; F: fathers; P: probands. B, Module-to-module relationship by hierarchical clustering. C, Top 200 hub genes networks for three representative modules. Circles: genes; diamonds: genes overlapping with genes in the SFARI database classified as associated to ASD; red: genes overexpressed at either TD11 or TD31; grey: no changes in gene expression; larger font: genes differentially expressed at both TD11 and TD31; D, Pearson’s correlation between modules’ eigengenes with log transformed HC Z-scores and ADOS severity scores at TD11 and TD31; *nominal p-value <0.1; E, F, Pearson’s correlation between HC Z-scores and modules’ eigengenes or FOXG1 expression levels for the unaffected fathers (E) and the affected probands (F) at TD31. See also Figure S4.

Figure 3. Decreased cell cycle length and increased neuronal/synaptic differentiation in ASD probands

A, B, Cell cycle time determined by the formula Tc= Ts/(%BrdU/Ki67), where Tc= cell cycle time and Ts= S phase time. A, Representative image of double immunostaining for BrdU (red) and Ki67 (green) of undifferentiated iPSCs from a control individual and (B) early neuronal progenitors in monolayer cultures at TD11. *p < 0.05, ANOVA with family as covariant. To prepare monolayer cultures for neuronal progenitors the neural rosettes were dissociated into single cells and cultured in adhesion as monolayers until TD11. C, D, Representative images (C) and stereological quantification (D) of the proportion of proliferating Ki67⁺ neuronal progenitor cells in both control and ASD-derived organoids at TD31. E-J, Increased neuronal maturation and synaptic formation in ASD-derived neurons (TD 31). Representative images of controls and probands organoids at TD 31 labeled with MAP2 and SynI (E), or MAP2 and VGAT (H), and relative quantification of MAP2 density (F), number of SynI⁺ puncta (G), number of VGAT⁺ puncta (I), and number of VGLUT1⁺ puncta (J). The data in (F, G, I, J) are presented as means ± s.e.m; **p < 0.01, ***p< 0.001; t test analysis. Scale bars, 10 μm (A), 10–20 μm (B, as indicated), 10 μm (C), 5 μm (E, H).

Figure 4. ASD organoids show imbalance between glutamatergic and GABAergic neuron fate

Representative images of control-derived and ASD proband-derived organoids: A-F, Immunostaining and respective stereological quantification of SOX1⁺ and PAX6+ proliferating radial glia progenitors, cortical excitatory TBR2⁺ intermediate progenitors, and more mature TBR1⁺ and CTIP2⁺ excitatory neurons at TD31. G-J, Immunostaining and quantification of GABAergic inhibitory progenitor cells (DLX1-2⁺) and mature GABAergic interneurons (GAD1⁺) at TD11 and TD31. K, Box plots showing percentages of inhibitory (GAD1⁺) and excitatory (TBR1⁺, CITIP2⁺) neurons in ASD-derived and control-derived organoids at TD31. Total cells are estimated by counting DAPI⁺ nuclei. C=Controls, P=Probands. **p < 0.01, ***p < 0.001, t test analysis. Scale bars, 10 μm (A, C, E, G, K), 20 μm (I, J). See also Figure S4 and Figure S5.

Figure 5. Sodium currents in iPSC-derived neurons from ASD patients and controls show different voltage dependence of steady-state inactivation

A, B, Inward sodium currents evoked by depolarizing test jumps to −20 mV from pre-test potentials of −90 mV to −25 mV (A) or −45 mV (B) in steps of 5 mV. C, Peak inward current amplitudes as a function of pre-test potential from the data in A and B. The results were normalized to Imax values obtained for each neuron from fits to the raw data (as in D and E). D, E, Steady-state inactivation data obtained from 10 control neurons (D) and seven patient neurons (E). Data for individual cells are shown with different symbols and colors (normalized data in C are shown here with the same symbols: control, open black square; patient, filled black circle). For some neurons, the results were better fitted as the sum of two components (arrowheads point to E_h1/2 values for each component). F, Bar graph depicting the percentage of control and patient neurons (n = 10 and 7) with E_h1/2 values that fell within the indicated ranges. When two components were present, the fractional amplitudes of each were used in the calculation of mean percentages for each group. Cells that gave E_h1/2 values ≤ −65 mV gave half-activation voltages that ranged from −39.1 to −55.8 mV, a phenotype most consistent with the Na_v1.1 brain sodium channel isoform (Catterall et al., 2005).

Figure 6. FOXG1 knockdown in ASD-derived organoids is able to restore the balance between GABA/glutamate neuron fate

A, Relative expression levels of FOXG1 by qPCR among non-virally transduced undifferentiated iPSCs from proband #9 (i07-P#9), TD11 organoids from the proband (07-P#9), his father (lines 07-F#1), and proband’s organoids harboring a non-targeting shRNA-control (shRNA-C) or three different shRNAs targeting FOXG1 (shRNA-1, shRNA-2, shRNA-3). B–F, Double immunostaining for FOXG1 and PAX6 in TD11 non-transduced proband’s organoids (B), or transduced with shRNA-C (C), shRNA-1 (D), shRNA-2 (E), shRNA-3 (F). G–J, qPCR for DLX1 (G), DLX2 (H), GAD1 (I), and PAX6 (J) in TD11 organoids from shRNA-C and shRNA-1/2/3. K, DLX1-2 and GAD1 double immunostaining in organoids derived from the father or the proband transduced with shRNA-C or shRNA-3 at TD11 and TD 30. L, M, Stereological quantification of immunocytochemical (ICC) staining for GABAergic (DLX1-2, GAD1) and glutamatergic markers (PAX6, TBR1) at TD11 (L) and TD 31 (M). (Sample names: 07= family name from which iPSCs were derived; F=Father; P=Proband; #=iPS clone number). Data in (G–J, L, M) are presented as means ± s.e.m; * p < 0.05, ** p < 0.01, *** p < 0.001, t test analysis. Scale bars, 10 μm.

Figure 7. Increased proportion of proliferating GABAergic neuronal progenitors and mature GABAergic interneurons at TD31 in ASD-derived organoids

Representative images (A, D) and stereological quantification of BrdU⁺/DLX1-2⁺ proliferating cells (B, C, E, F) and BrdU⁺/GAD1⁺ neurons (G, H) in TD11 and TD31 organoids derived from 07-F#2 (father), 07-P#9 shRNA-C, and 07-P#9 shRNA-3 (proband’s iPSC lines transduced with shRNA-C or with shRNA-3). The selectively increased proportion of DLX 1-2⁺/BrdU⁺ double positive cells in patient-derived organoids at both TD11 (B, C) and TD31 (E, F), was restored to a physiological level after FOXG1-knockdown at both time points. The increased proportion of proliferating DLX1-2 GABAergic progenitors (A and D, upper panel) resulted in an overproduction of more mature GABAergic GAD1+/BrdU+ double positive interneurons (D, bottom panel), overproduction that was restored to physiological levels after FOXG1-knockdown (G,H). Data in (B, C, E, F, G, H) are presented as means ± s.e.m; * p < 0.05, ** p< 0.01, *** p < 0.001, t test analysis. Scale bars, 10 μm. See also Figures S6, S7.