Neuronal excitatory-to-inhibitory balance is altered in cerebral organoid models of genetic neurological diseases

Abstract

The neuro-physiological properties of individuals with genetic pre-disposition to neurological disorders are largely unknown. Here we aimed to explore these properties using cerebral organoids (COs) derived from fibroblasts of individuals with confirmed genetic mutations including PRNP^E200K, trisomy 21 (T21), and LRRK2^G2019S, which are associated with Creutzfeldt Jakob disease, Down Syndrome, and Parkinson's disease. We utilized no known disease/healthy COs (HC) as normal function controls. At 3-4 and 6-10 months post-differentiation, COs with mutations showed no evidence of disease-related pathology. Electrophysiology assessment showed that all COs exhibited mature neuronal firing at 6-10 months old. At this age, we observed significant changes in the electrophysiology of the COs with disease-associated mutations (dCOs) as compared with the HC, including reduced neuronal network communication, slowing neuronal oscillations, and increased coupling of delta and theta phases to the amplitudes of gamma oscillations. Such changes were linked with the detection of hypersynchronous events like spike-and-wave discharges. These dysfunctions were associated with altered production and release of neurotransmitters, compromised activity of excitatory ionotropic receptors including receptors of kainate, AMPA, and NMDA, and changed levels and function of excitatory glutamatergic synapses and inhibitory GABAergic synapses. Neuronal properties that modulate GABAergic inhibition including the activity of Na-K-Cl cotransport 1 (NKCC1) in Cl^- homeostasis and the levels of synaptic and extra-synaptic localization of GABA receptors (GABARs) were altered in the T21 COs only. The neurosteroid allopregnanolone, a positive modulator of GABARs, was downregulated in all the dCOs. Treatment with this neurosteroid significantly improved the neuronal communication in the dCOs, possibly through improving the GABAergic inhibition. Overall, without the manifestation of any disease-related pathology, the genetic mutations PRNP^E200K, T21, and LRRK2^G2019S significantly altered the neuronal network communication in dCOs by disrupting the excitatory-to-inhibitory balance.

Keywords: Neural oscillation; Neurodegenerative diseases; Neuronal network communication.

Fig. 1

Neuronal firing and network communication at 3–4 and 6–10 months old. a Top panel: a picture of cerebral organoids on Multi-electrode arrays (MEA) with the black dots indicating the electrodes; Middle panel: a representative diagram of local field potential detected by each electrode shown in the top panel); Bottom panel: a diagram displaying how the raw data were processed. b Representative raster blots displaying neuronal firings or spikes (dots), bursts (clusters of dots), and network communication (depicted by electrodes with overlapping bursts) in the healthy controls (HC) and the organoids with genetic defects (PRNP^E200K1, PRNP^E200K2, T21, and LRRK2^G2019S). c Spike rate (n = 18 to 20 at 3–4 months; n = 36 to 44 at 6–10 months). d Burst rate (n = 14 to 19 at 3 months; n = 50 at 6–10 months). e Periodicity of network firing (n = 18 to 20 at 3–4 months; n = 62 to 67 at 6–10 months). f Percentage of connected electrodes based on spike correlations (left panel; n = 8 to 11 at 3–4 months; n = 30 to 46 at 6–10 months) and overlapping bursts (right panel; n = 17 to 29 at 6–10 months). g Inter-spike interval coefficient of variation (CV; n = 18 to 20 at 3–4 months; n = 20 to 23 at 6–10 months). h Representative graphs of the connectivity between electrodes (nodes) based on the spike correlation with the node colour and size representing the burst rate and spike rate. c–f The parameters of neuronal spiking in the organoids with mutations were compared to the age-matched HC by One-way ANOVA on ranks with Dunnett’s correction for multiple comparisons. g Paired Student’s t-test on ranks was used to analyse the age-dependent change in the inter-spike interval CV. Each point on the graphs represents an individual organoid. If not otherwise indicated CO colour code are as follows; HC (blue), PRNP^E200K−1 (red), PRNP^E200K−2 (yellow), T21 (purple) and LRRK2^G2019S (grey). Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 2

Genetic defects altered the neuronal oscillations. a–d Relative oscillatory power of delta, theta, low gamma, and upper gamma oscillations in 6–10 month- old HC (n = 30) and dCOs (PRNP^E200K1, PRNP^E200K2, T21, and LRRK2^G2019S; n = 29). e Representative connectivity graphs of electrodes based on the inter-electrode correlation of delta, lower gamma, and upper gamma oscillatory power. f–h The weight (based on Pearson’s correlation coefficient) of the connectivity in e (n = 9 to 25). i Representative traces of local field potential (LFP) generated by each organoid line. The right panel is a magnification of the left panel. The bottom panel is a raster plot showing the spikes detected in the corresponding LFP. j The peak amplitudes of LFP. k–n The modulation index of the coupling of delta and theta phases with the amplitudes of the lower gamma oscillations (k, l; n = 30) and amplitudes of upper gamma oscillations (m, n; n = 30). a–d f–h, j–n Measurements were compared to the HC by One-way ANOVA on ranks with Dunnett’s correction for multiple comparisons. Each point on the graphs represents an individual organoid. Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

Genetic defects altered the expression levels and functions of synaptic markers that are essential for synapse formation. a A heatmap displaying the Z-scores of the total protein levels of essential synaptic proteins in 6–10-month-old HC and dCOs (PRNP^E200K1, PRNP^E200K2, T21, and LRRK2^G2019S). See Additional file 13 for the representative images and quantifications. b A heatmap displaying the Z-scores of the active levels of essential synaptic proteins in HC and genetic defect organoids. See Additional file 14 for the representative images and quantifications. a, b Protein levels in the mutants were compared with the HC by One-way ANOVA with Dunnett’s correction multiple comparisons. c A heatmap displaying the Z-scores of the Delta Ct from the qRT-PCR analysis of various neurotransmitter receptors of HC, PRNP^E200K1 and T21 organoids. The mRNA levels in the organoids with genetic defects were compared to the HC by Two-way ANOVA with Dunnett’s correction for multiple comparisons. See Additional file 15 for additional data. d Representative confocal immunofluorescence images displaying the colocalizations of SYN1(red), MAP2(green), and NR1 (magenta). e Representative immunofluorescence images showing the colocalizations of VGLUT1 (red), PSD95 (green), and GABA(A)Rs (magenta). f–j The degree of colocalization between SYN1 and MAP2 (f), VGLUT1 and PSD95 (g), NR1 and SYN1 (h), VGLUT1 and GABA(A)Rs (i), and PSD95 and GABA(A)Rs (j). f–j n = 6 in HC and n = 3 in the genetic defect organoids. The average Pearson’s correlation coefficient or degree of colocalization was compared between organoid lines by One-way ANOVA with Dunnett’s correction for multiple comparisons. a–c The heatmap key is on the right panel of (a). d scale bar represents 50 μm. Each point on the graphs represents an individual organoid. Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

The genetic defects altered the production and release of neurotransmitters. a Heatmaps displaying the Z-scores of the levels of neurotransmitters, extracted from organoids and media, in HC and dCOs (PRNP^E200K1, PRNP^E200K2, T21, LRRK2^G2019S). The heatmap key is on the right panel. b–e Some of the raw data summarized in a including serine (b), aspartate (c), dopamine (d), and GABA (e). b–e consisted of n = 12 for the HC and n = 4 for the other organoid lines. Levels of neurotransmitters in the organoids with genetic defects were compared to the HC by One-way ANOVA with Dunnett’s correction for multiple comparisons. Each point on the graphs represents an individual organoid. Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

The genetic defects altered the agonist-dependent activity of the major ionotropic receptors. a, c, e, g Burst rate recorded in 6–10-month-old HC and dCOs (PRNP^E200K1, PRNP^E200K2, T21, and LRRK2^G2019S) before and after exposure to increasing concentrations of kainate (a; n = 4), AMPA (c; n = 4), NMDA (e; with 5 µM of glycine; n = 4), and GABA (g; n = 4). b, d, f, h Intracellular levels of calcium before and after exposure to increasing concentrations of kainate (b; n = 4-), AMPA (d; n = 4–7), NMDA (f; with 5 µM of glycine; n = 4–7), and GABA (h; n = 4). i–k Burst rate before and after treatments with 30 μM NBQX (i; n = 4), 100 μM AP5 and 10 µM maleate solution (j; n = 4), and 100 µm Bicuculine and 10 μM CGP55845 hydrochloride (k; n = 4). l Burst rate before and after stimulating the organoids with 500 μM NMDA/5 µM glycine in the presence of GABAR (GB) receptors blockers, 100 µM Bicuculine and 10 μM CGP55845 hydrochloride (n = 7 for the HC; n = 4 for other organoid lines). The dose- response (mean burst rate or intracellular calcium) to each treatment was compared between organoids by Repeated Measures Two-way ANOVA with Dunnett’s correction for multiple comparisons. i–l We used paired Student’s t-test to compare the average burst rate before and after treatments. Each point on the graphs represents an individual organoid. If not otherwise indicated CO colour code are as follows; HC (blue), PRNP^E200K−1 (red), PRNP^E200K−2 (yellow), T21 (purple) and LRRK2^G2019S (grey). Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. a–h Asterisk colour signifies which organoid line with the statistically significant results

Fig. 6

T21 impaired NKCC1 activity and increased the activity of extra-synaptic GABARs. a, b Protein levels of NKCC1 and NKCC2 in 6–10-month-old HC (n = 6) and dCOs (PRNP^E200K1 with n = 3, PRNP^E200K2with n = 5, T21 with n = 4, and LRRK2^G2019S with n = 4). Representative images are in Additional file 17. c Burst rate in response to a treatment with 10 µM NKCC1 blocker bumetanide (n = 4 per an organoid line). d Representative images of surface GABARs (green) before and after treatments with GABAR blockers (100 µM Bicuculine and 10 μM CGP55845 hydrochloride) or 500 μM ZnCl₂ in HC and T21 organoids. Scale bar is 100 µm. e Quantifications of the levels of surface GABARs before and after treatments with either GABARs blockers or ZnCl₂ (n = 4). f Burst rate before (n = 24 for the HC and n = 16 for the other organoid lines) and after exposure to 500 μM ZnCl2 (n = 6 for the HC and n = 4 for the other organoid lines). c–f Average burst rates and levels of surface GABARs before and after the treatments with Bumetanide or ZnCl2 were compared by paired Student’s t-test. Each point on the graphs represents an individual organoid. If not otherwise indicated CO colour code are as follows; HC (blue), PRNP^E200K−1 (red), PRNP^E200K−2 (yellow), T21 (purple) and LRRK2^G2019S (grey). Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

The genetic defects altered neuronal communication associated with neurosteroid Allopregnanolone. a, b Representative images and quantifications of neurosteroid allopregnanolone detected in 6–10-month-old HC (n = 9) and dCOs (PRNP^E200K1, PRNP^E200K2, T21, LRRK2^G2019S; n = 4). Scale bar is 100 µm. c Burst rate before and after treatments with 1 µM pregnanolone (n = 10 for the HC; n = 8 for the other organoid lines). d Relative intracellular calcium levels measured before and after treatments with either 1 µM pregnanolone or 5 μM nimodipine (n = 4 per an organoid line). e Burst rate before and after the treatments with 5 µM nimodipine (n = 4 per an organoid line). f, g The modulation index of the coupling between the delta/theta phases and the amplitudes of the upper gamma oscillations after treatments with pregnanolone (f; n = 4) and nimodipine (g; n = 4). b Allopregnanolone levels were compared between groups by One-way ANOVA with Dunnett’s correction for multiple comparisons. c, e Effect of the treatments within an organoid line was analysed by Paired Student’s t test. d Calcium levels after treatments were normalized to the pre-treatment levels and the effect of the treatments on the calcium flux was determined by One-sample t-test based on a hypothetical value of 1. f, g Measurements were analysed by Two-way ANOVA with Dunnett’s correction for multiple comparisons. Each point on the graphs represents an individual organoid. If not otherwise indicated CO colour code are as follows; HC (blue), PRNP^E200K−1 (red), PRNP^E200K−2 (yellow), T21 (purple) and LRRK2^G2019S (grey). Bars and error denote mean and SEM. * p < 0.05, **p < 0.01, ***p < 0.001