Potassium channel dysfunction in human neuronal models of Angelman syndrome

Abstract

Disruptions in the ubiquitin protein ligase E3A (UBE3A) gene cause Angelman syndrome (AS). Whereas AS model mice have associated synaptic dysfunction and altered plasticity with abnormal behavior, whether similar or other mechanisms contribute to network hyperactivity and epilepsy susceptibility in AS patients remains unclear. Using human neurons and brain organoids, we demonstrate that UBE3A suppresses neuronal hyperexcitability via ubiquitin-mediated degradation of calcium- and voltage-dependent big potassium (BK) channels. We provide evidence that augmented BK channel activity manifests as increased intrinsic excitability in individual neurons and subsequent network synchronization. BK antagonists normalized neuronal excitability in both human and mouse neurons and ameliorated seizure susceptibility in an AS mouse model. Our findings suggest that BK channelopathy underlies epilepsy in AS and support the use of human cells to model human developmental diseases.

Fig. 1.. Altered functional properties of UBE3A-deficient human neurons.

(A to D) Generation of human induced neurons from WT and UBE3A KO hESCs. (A) Schematic illustrating the CRISPR-Cas9-mediated gene editing approach used to knock out UBE3A in hESCs (top) and immunoblot showing the absence of UBE3A protein in UBE3A KO hESCs (bottom). (B) Schematic illustrating the protocol used to generate the human neurons used in this study (top) and representative morphological images of day-35 WT and KO neurons immunostained for MAP2 (blue) and synapsin (magenta) (bottom). Scale bar, 20 μm. (C) Sholl analysis of WT and KO induced neurons at day 35. (D) Representative images and quantification of synaptic puncta densities, calculated from neurons immunostained for synapsin. Scale bar, 1 μm. (E to I) Altered excitability in KO neurons (H9 derived). (E) Frequency-current (F-I) curves showing spike frequency versus current injections in WT and KO induced neurons. (F and G) Representative traces and quantification of maximal spike frequencies by current injection in induced WT and KO neurons. (H and I) Representative traces and quantification of spike fAHP amplitudes of induced WT and KO neurons. (J to N) Induced neurons derived from AS iPSCs showed reproduced excitability and fAHP changes. (J) Immunostaining for UBE3A in AS neurons, with and without ectopic expression of UBE3A. Scale bar, 10 μm. (K) F-I curves showing spike frequencies versus current injection in induced neurons derived from AS iPSCs. (L) Quantification of maximal spike frequencies in the current injections. (M and N) Representative traces and amplitude quantification of spike fAHP. Data represent means ± SEM. The two-tailed unpaired Student’s t test was used for all analyses. The numerals in all bars indicate the number of analyzed neurons. **P < 0.01; *P < 0.05; N.S., not significant.

Fig. 2.. UBE3A deletions increase BK channel function in human neurons.

(A) Representative traces and quantification of BK currents isolated from WT and KO neurons treated with paxilline (5 μM). (B) Diagrams illustrating the detection of BK channels using a functionalized probe with AFM. (C) Representative heatmaps of specific BK probe binding events. Force-distance curves (10 data points by 10 data points) were obtained over 1-μm² areas. Colors indicate the measured force of specific binding events. (D) Unbinding force distribution and BK channel density on the surface of WT and KO neurons. (E to H) Pharmacological rescue by the BK antagonist paxilline. (E and F) Representative traces and quantification of fAHP with and without paxilline (5 μM). DMSO, dimethyl sulfoxide. (G and H) F-I curves showing spike frequencies versus current injections and related quantification in induced neurons. Data represent means ± SEM. In all bars, the values indicate the number of analyzed neurons. The two-tailed unpaired Student’s t test was used. *P < 0.05; N.S., not significant.

Fig. 3.. Altered functional properties of neurons and enhanced network activity in UBE3A-deficient organoids.

(A) Bright-field images of organoids and immunostaining of cortical layer markers in WT and KO organoids at day 120. Scale bars, 5 mm (bright-field images) or 50 μm (immunostaining). (B to D) Altered electrophysiological properties in neurons from KO organoids, as illustrated with representative traces (B), F-I curves evoked by current injection (C), and quantifications (D), with and without paxilline (10 μM). a.u., arbitrary units. (E to K) Two-photon (2P) live calcium imaging of WT and KO organoids. (E) Calcium transient traces extracted from individual neurons of WT and KO organoids. (F) Cumulative distribution of interevent intervals (IEI) in time bins for calcium transients recorded in WT and KO organoids. The inserted bar graph shows the quantification. n = 131 and 236 neurons from N = 12 and 17 organoids, respectively, for WT and KO organoids. (G) Quantification of the amplitudes of calcium transients recorded in WT and KO organoids. (H) Quantification of the frequencies and amplitudes of calcium transients before and after paxilline treatment (10 μM). N = 10 and 11 organoids for WT and KO, respectively. (I and J) Representative traces of calcium transients in individual neurons (left) and correlation heatmaps (right) obtained from WT (I) and KO (J) organoids. SI, synchronization index. (K) Summary of the synchronization index recorded in WT and KO organoids upon paxilline treatment (10 μM). N = 10 and 11 organoids for WT and KO, respectively. Data represent means ± SEM. Numerals in bars indicate the number of analyzed neurons. The two-tailed paired Student’s t test was used for analysis of data in (H) and (K); the two-tailed unpaired Student’s t test was used for analysis of data in all other panels. **P < 0.01; ***P < 0.001.

Fig. 4.. BK modulation ameliorates seizure susceptibility in a mouse model of AS.

(A) Latency to myoclonic seizure induced by flurothyl in WT and KO (Ube3a^m−/p+) mice. WT without paxilline, N = 23; KO without paxilline, N = 17; WT with paxilline, N = 16; KO with paxilline, N = 12. (B) Epilepsy grade induced by picrotoxin in WT and KO mice. WT without paxilline, N = 16; KO without paxilline, N = 18; WT with paxilline, N = 16; KO with paxilline, N = 16. (C) Samples of spectrogram and averaged delta power data for LFP from BIC of WT and KO mice with sound stimuli. Delta rhythmicity below the dashed line was averaged for analysis. (D) Summary of power spectral density (PSD) of LFP recorded from BIC of WT and KO mice with sound stimuli. N = 8 mice from two batches. Normalization was performed to remove the baseline difference due to batch effect. A two-way analysis of variance (ANOVA) with the Bonferroni post hoc test was used for frequencies of 1 to 4 Hz to analyze differences between WT and KO mice. (E) Mean delta (1 to 4 Hz) power of WT and KO mice LFPs. N = 8 mice from two batches. (F) Sample spectrogram and averaged delta power of LFPs in BIC of WT and KO mice with paxilline treatment (0.35 mg/kg; four times, at 1-hour intervals). (G) Summary of PSD of LFPs in BIC recorded in WT and KO mice with paxilline treatment. Two-way ANOVA with the Bonferroni post hoc test was used for frequencies of 1 to 4 Hz to analyze differences between WT and KO mice. (H) Mean delta power (1 to 4 Hz) of WT and KO mice LFPs with paxilline treatment. N = 8 mice from two batches. Numerals in bars indicate the number of animals analyzed. Data represent means ± SEM. The two-tailed unpaired Student’s t test was used to analyze data for all panels except for (D) and (G). *P < 0.05; **P < 0.01; N.S., not significant.