Topoisomerase inhibitors unsilence the dormant allele of Ube3a in neurons

Abstract

Angelman syndrome is a severe neurodevelopmental disorder caused by deletion or mutation of the maternal allele of the ubiquitin protein ligase E3A (UBE3A). In neurons, the paternal allele of UBE3A is intact but epigenetically silenced, raising the possibility that Angelman syndrome could be treated by activating this silenced allele to restore functional UBE3A protein. Using an unbiased, high-content screen in primary cortical neurons from mice, we identify twelve topoisomerase I inhibitors and four topoisomerase II inhibitors that unsilence the paternal Ube3a allele. These drugs included topotecan, irinotecan, etoposide and dexrazoxane (ICRF-187). At nanomolar concentrations, topotecan upregulated catalytically active UBE3A in neurons from maternal Ube3a-null mice. Topotecan concomitantly downregulated expression of the Ube3a antisense transcript that overlaps the paternal copy of Ube3a. These results indicate that topotecan unsilences Ube3a in cis by reducing transcription of an imprinted antisense RNA. When administered in vivo, topotecan unsilenced the paternal Ube3a allele in several regions of the nervous system, including neurons in the hippocampus, neocortex, striatum, cerebellum and spinal cord. Paternal expression of Ube3a remained elevated in a subset of spinal cord neurons for at least 12 weeks after cessation of topotecan treatment, indicating that transient topoisomerase inhibition can have enduring effects on gene expression. Although potential off-target effects remain to be investigated, our findings suggest a therapeutic strategy for reactivating the functional but dormant allele of Ube3a in patients with Angelman syndrome.

Figure 1. A small-molecule screen identifies a topoisomerase inhibitor that unsilences the paternal allele of Ube3a in neurons

a, High-content screen flowchart. E15.5 cortical neurons with a paternally inherited Ube3a-YFP allele were cultured in 384-well plates and treated with small molecules from DIV7–DIV10. Active compounds that unsilence the paternal Ube3a-YFP allele were detected with antibody-enhanced fluorescence and high-content imaging. b, High-content imaging of DIV7 neurons that inherited Ube3a-YFP maternally (m^YFP/p⁺) or paternally (m⁺/p^YFP). Nuclei were stained with DAPI. Scale bar = 50 µm. c, Mean ± s.e.m. levels of UBE3A-YFP fluorescence in neurons cultured from maternal Ube3a-YFP (m^YFP/p⁺) or wild-type (m⁺/p⁺) mice, normalized to levels in age-matched neurons cultured from paternal Ube3a-YFP mice (m⁺/p^YFP). Two-way ANOVA revealed main effects of genotype, duration, and a genotype-duration interaction (P<0.001); Bonferroni post hoc test examined comparisons between maternal and paternal Ube3a-YFP mice from DIV4 to DIV10, *P<0.001; n=2–6 culture wells/day. d, Pie chart depicting categories of the 2,306 screened compounds and graph summarizing presumptive UBE3A-YFP in arbitrary fluorescence units (A.F.U.) after small molecule treatments. Small molecules that were subsequently found to be autofluorescent (Supplementary Fig. 2) are depicted in gray. The initial screen identified one active compound, irinotecan (red). e, High magnification view of wells treated with vehicle (0.2% DMSO) or 10 µM irinotecan for 72 hr. Neuron density and health is similar in vehicle- and irinotecan-treated cells as evidenced by counterstaining with the nuclear marker DAPI. Scale bar = 100 µm. f,g Western blots of cultured cortical neurons probed with GFP or UBE3A antibodies, respectively, ± irinotecan (10 µM for 72h). f, *P<0.05; two-tailed t-test, n=3/group. g, *P<0.001, one-way ANOVA with Bonferroni post hoc, n=7–8/group. All data are presented as means ± s.e.m.

Figure 2. Topotecan unsilences the paternal allele of Ube3a and the unsilenced protein is catalytically active

a, Dose-response curves for unsilencing paternal Ube3a-YFP. Inactive = lactam E ring-camptothecin. n=4/data point. b, UBE3A-YFP levels in neurons from Ube3a^m+/pYFP mice increase with duration of topotecan (300 nM) or irinotecan (1 µM) treatment. *P<0.05, oneway ANOVA with Bonferroni post hoc tests relative to day zero, n=4–8/group. A.F.U.=arbitrary fluorescence units. c, Western blots and quantification of UBE3A and the loading control actin. *P<0.05, one-way ANOVA with Bonferroni post hoc tests, n=4/group. d, Quantification of unbound TOP1 and representative western blots. β-tubulin was used as a loading control. One-way ANOVA with Bonferroni post hoc tests, *P<0.05; n=3/group. e, Western blot from vehicle- and topotecan-treated neurons from wild-type (m⁺/p⁺) and maternal Ube3a-deficient (m^–/p⁺) mice. f, Western blots examining UBE3A ubiquitin-thioester formation following immunoprecipitation with an anti-UBE3A antibody and in vitro ubiquitination in the presence or absence of the ubiquitin conjugating enzyme (E2), UBCH7. All data are presented as means ± s.e.m. g, Schematic demonstrating location of 4 primer sets used to probe mRNA expression shown in h. h, Normalized mRNA levels in cultured Ube3a^m–/p+ neurons following vehicle or 300 nM topotecan. Expression is given as a ratio of expression in drug treated cells to vehicle treated cells, normalized to the housekeeping gene RPL22. *P<0.05 compared to 0 hr, Kruskal-Wallis one-way ANOVA followed by post hoc tests, n=4–5 cultures/data point. i, Schematic summarizing methylation status of the Snrpn promoter region on the maternal and paternal chromosome following treatment with vehicle or 300 nM topotecan (see complete primer 1 data set in Supplementary Fig.12). Average methylation status is indicated using a grayscale.

Figure 3. Topotecan enduringly unsilences the paternal allele of Ube3a in vivo

a, Unilateral delivery of topotecan (i.c.v.) using a mini-osmotic pump into the lateral ventricle of Ube3a^m+/pYFP mice in vivo. Two weeks of topotecan infusion (3.74 µg/h) unsilenced the paternal Ube3a-YFP allele in the hippocampus of the infused hemisphere near the site of drug delivery, while only modestly unsilencing Ube3a-YFP in the contralateral (non-infused) hemisphere. Scale bar = 500 µm. Pharmacokinetic analyses measuring topotecan levels in the infused and non-infused hemisphere immediately (t=0) or five hours (t=5) after cessation of drug delivery. *P<0.05, one-way ANOVA with Bonferroni post hoc test, n=5–9/group. b, Representative sections and c, quantification of optical intensity of UBE3A-YFP in hippocampal regions (CA1, CA2/3, and dentate gyrus=DG) of the topotecan-infused hemisphere or the hemisphere of vehicle-treated mice. *P<0.05, Mann-Whitney rank sum test, n=5/group. d, Representative sections and e, quantification of paternal UBE3A-YFP in the striatum following i.c.v. infusion of topotecan. *P<0.05, two-tailed t test, n=4/group. f, Schematic depicting schedule for i.t. delivery of topotecan (50 nmol/day for 10 of 14 days) and endpoints (arrows) immediately, 4 weeks, and 12 weeks after cessation of drug. g,h Topotecan (i.t.) increased the number of UBE3A-YFP-positive spinal neurons compared to vehicle, and the unsilencing of Ube3a-YFP was maintained for at least 12 weeks. *P<0.001, one-way ANOVA with Bonferroni post hoc test, n=7–8/group.