AntimiR targeting of microRNA-134 reduces seizures in a mouse model of Angelman syndrome

Abstract

Angelman syndrome (AS) is a severe neurodevelopmental disorder featuring ataxia, cognitive impairment, and drug-resistant epilepsy. AS is caused by mutations or deletion of the maternal copy of the paternally imprinted UBE3A gene, with current precision therapy approaches focusing on re-expression of UBE3A. Certain phenotypes, however, are difficult to rescue beyond early development. Notably, a cluster of microRNA binding sites was reported in the untranslated Ube3a1 transcript, including for miR-134, suggesting that AS may be associated with microRNA dysregulation. Here, we report levels of miR-134 and key targets are normal in the hippocampus of mice carrying a maternal deletion of Ube3a (Ube3a ^m-/p+ ). Nevertheless, intracerebroventricular injection of an antimiR oligonucleotide inhibitor of miR-134 (Ant-134) reduced audiogenic seizure severity over multiple trials in 21- and 42-day-old AS mice. Interestingly, Ant-134 also improved distance traveled and center crossings of AS mice in the open-field test. Finally, we show that silencing miR-134 can upregulate targets of miR-134 in neurons differentiated from Angelman patient-derived induced pluripotent stem cells. These findings indicate that silencing miR-134 and possibly other microRNAs could be useful to treat clinically relevant phenotypes with a later developmental window in AS.

Keywords: Angelman syndrome; Behavior; Cerebellum; Hippocampus; MT: Oligonucleotides: Therapies and Applications; epilepsy; miR-134; microRNAs; seizures.

Graphical abstract

Figure 1

Ube3a^m−/p+ mice model multiple AS phenotypes (A) Lack of Ube3a (E6-AP) confirmed by western blotting lysates from P21 F1 Ube3a^(m−/p+) (AS) mice compared with wild-type (WT) littermate controls. n = 4/genotype; ∗∗∗p < 0.001. (B) Total time (s) spent exploring the open-field arena over a 10 min trial in P21 mice; WT, n = 15; AS, n = 19; ∗p = 0.022. (C) Representative track plot from each genotype in the open field. (D) Latency to fall from the rotarod during four test phases; WT, n = 12; AS, n = 18; ∗∗∗p = 0.0008. (E) Number of marbles buried over a 30 min trial; WT, n = 5; AS, n = 7; ∗p = 0.045. (F) Representative image of cages 30 min after marbles trial. (G) Total power of baseline EEG over 4–6 h recordings in F1 P21 mice; WT, n = 9; AS, n = 8; p = 0.95. (H) Spectral analysis of individual band frequencies over baseline recordings. WT, n = 9; AS, n = 8; delta (1–4 Hz), p = 0.5; theta (4–8 Hz), ∗p = 0.015; alpha (8–12 Hz), p = 0.81; beta (12–30 Hz); ∗p = 0.011. (I) Total power of baseline EEG recording over 4–6 h in F1 P42 mice; WT, n = 13; AS, n = 15; ∗∗∗p < 0.0001. (J) Spectral analysis of individual band frequencies over baseline recordings, n = 11/genotype. Delta (1–4 Hz), p = 0.6; theta (4–8 Hz), ∗∗p = 0.003; alpha (8–12 Hz), p = 0.7; beta (12–30 Hz), p = 0.14. Data are expressed as SEM. A Shapiro-Wilk test was used to test for normality. Open symbols represent female mice and closed symbols represent male mice. ns, non-significant.

Figure 2

miR-134 levels and effects of Ant-134 in the open field in F1 Ube3a^m−/p+ mice (A) Expression of miR-134 in the cerebellum of P21 mice; WT, n = 9; AS, n = 7; ∗∗p = 0.008. (B) Limk1 in the cerebellum of P21 mice; WT, n = 9; AS, n = 8; p = 0.048. (C) miR-134 in the hippocampus of P21 mice; WT, n = 8; AS, n = 10; p = 0.069. (D) Ago-bound miR-134 in the hippocampus of P21 mice; n = 10/genotype, p = 0.209. (E) Mature miR-134 sequence and alignment of Ant-134 and scramble (Scr). (F) miR-134 levels in the hippocampus 24 h after ICV injection of Ant-134 or Scr (0.1 nmol); Scr, n = 4; Ant-134, n = 5; ∗p = 0.0174. (G) miR-134 levels in the cerebellum of F1 mice 24 h after Ant-134/Scr treatment (0.1 nmol); Scr, n = 5; AS, n = 4; ∗∗p = 0.006. (H) Representative track plots of F1 Ube3a^m−/p+ mice 24 h after treatment at P21 with either Scr or Ant-134 (0.1 nmol). (I) Exploration time (s) during open field; Scr, n = 10; Ant-134, n = 8; ∗∗∗p = 0.0002. (J) Number of entries into inner zone of open field; Scr, n = 9; Ant-134, n = 7; ∗p = 0.01. (K) Distance traveled (cm) in open-field arena in P21 mice; Scr, n = 9; Ant-134, n = 7; p = 0.09. (L) Latency to fall (s) on accelerating rotarod; Scr, n = 10; Ant-134, n = 9; p = 0.81. (M) Number of marbles buried during 30 min marble-burying test; Scr, n = 10; Ant-134, n = 9; p = 0.75. A t test was used for analysis between Scr- and Ant-134-treated mice. Data are expressed as SEM except in F (median with interquartile range). Open symbols represent female mice and closed symbols represent male mice. Multiple corrections were performed on molecular data and α was adjusted to 0.01. ns, non-significant.

Figure 3

Silencing miR-134 reduces hippocampal excitability in N4 Ube3a^m−/p+ mice (A and B) miR-134 levels in N4 generation Ube3a^m−/p+ P21 mice treated with Scr or Ant-134 with (A) 0.1 nmol or (B) 0.5 nmol; n = 4 or 5 group. Brains were analyzed 24 h later using qRT-PCR. ∗p = 0.042 and ∗∗∗p = 0.0011. (C) Dcx transcript levels 24 h following Scr or Ant-134 (0.5 nmol); ∗p = 0.014. (D) Limk1 transcript levels 24 h following Scr/Ant-134 (0.5 nmol). (E) Creb1 transcript levels 24 h following Scr/Ant-134 (0.5 nmol); p = 0.90. (F) Brain slice recordings show Ant-134 caused a reduction in hippocampal CA1 population synaptic potential; n = 4 mice per group; 2 or 3 slices per mouse; two-way repeated-measures ANOVA, treatment term ∗p = 0.038. (G) Ant-134 had no effect on hippocampal CA1 population spiking; n = 4 mice per group; 2 or 3 slices per mouse; two-way repeated-measures ANOVA, treatment term p = 0.15. (H) Representative raw traces showing population synaptic potential and spiking in hippocampal CA1 in Ant-134 and Scr-treated Ube3a^m−/p+ mice. Data are expressed as SEM. Multiple corrections were performed on molecular data, and α was adjusted to 0.016. ns, non-significant.

Figure 4

Ant-134 reduces audiogenic seizures in N4 Ube3a^m−/p+ mice (A) Schematic of antimiR testing in the audiogenic seizure model. P21 Ube3a^m−/p+ mice received an ICV injection of Scr/Ant-134 (0.5 nmol), and seizures were induced 24 h later. Seizures were repeated at P24 and P26. (B) Tabular representation of the type of seizure each mouse had over three seizures. (C) Percentage of maximum seizure score per treatment group at P22, P24, and P26. There was a significant reduction in seizure severity in Ant-134 treated mice at P26 in comparison with P22; Scr, n = 11; Ant-134, n = 11; ∗p < 0.05. (D) Kaplan-Meier curve of survival rates following Scr/Ant-134. Note, 55% survival in Scr group in comparison with 81% survival in Ant-134 group (ns). (E) Representative western blots show levels of Dcx 24 h after final seizure in the cortex and Creb1 levels in the hippocampus of Scr/Ant-134-treated mice. (F) Dcx in the cortex; n = 4/genotype, ∗p = 0.024. (G) Creb1 levels in the hippocampus; n = 4/genotype, ∗p = 0.026. Data are expressed as SEM. (H) Schematic illustration of experimental design of antimiR testing in P42 AS mice. (I) Tabular representation of the type of seizure each mouse had over three seizures. (J) Percentage of maximum Racine score per treatment group at P22, P24, and P26; Scr, n = 4; Ant-134, n = 5; ∗p = 0.04. Mortality was compared between treatments using incidence rate ratios and a Poisson model, with number of trials as the exposure variable. Seizure severity was modeled using ordinal logistic regression with robust variance estimation used to adjust for clustering of data with mice. An interaction term was used to test for a change in effectiveness of treatment as a function of trial number.

Figure 5

Ant-134 reduces miR-134 levels and upregulated targets in AS patient-derived neurons (A and B) Recorded calcium transients in differentiated neurons (6 weeks) from an AS patient showing typical spontaneous activity consistent with functioning network. (C and D) Detection of Ant-134 (fluorescein amidite [FAM]-labeled) 24 h after transfection in neurons expressing PSD-95 and NeuN. (E) Potent knockdown of miR-134 by Ant-134 (300 nM) after 24 h treatment in 6 week-differentiated AS patient neurons. (F–I) Transcript levels of miR-134 targets in Ant-134-treated AS neurons. Note increased levels some (DCX, CREB1) but not all (LIMK1, Serpine1). n = 9 Ant-134, n = 9 Scr; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001. Data are expressed as SEM. Multiple corrections were performed on molecular data, and α was adjusted to 0.01. ns, non-significant.