A preclinical pig model of Angelman syndrome mirrors the early developmental trajectory of the human condition

Abstract

Angelman syndrome is a neurodevelopmental disorder characterized by severe motor and cognitive deficits. It is caused by the loss of the maternally inherited allele of the imprinted ubiquitin-protein ligase E3A (UBE3A) gene. Rodent models of Angelman syndrome do not fully recapitulate all the symptoms associated with the condition and are limited as a preclinical model for therapeutic development. Here, we show that pigs (Sus scrofa) with a maternally inherited deletion of UBE3A (UBE3A^-/+) have altered postnatal behaviors, impaired vocalizations, reduced brain growth, motor incoordination, and ataxia. Neonatal UBE3A^-/+ pigs exhibited several symptoms observed in infants with Angelman syndrome, including hypotonia, suckling deficits, and failure to thrive. Collectively, these findings are consistent with the pathophysiology and developmental trajectory observed in individuals with Angelman syndrome. We anticipate that this pig model will advance our understanding of the pathophysiology of Angelman syndrome and be used as a preclinical large animal model for therapeutic development.

Keywords: CRISPR/Cas9; Sus scrofa; UBE3A; angelman syndrome; pig.

Fig. 1.

Generation of a pig model of Angelman syndrome. (A) Cactus alignment (Zoonomia project) between humans and the orthologous regions in cynomolgus macaques, rats, mice, pigs, and armadillos. (B) Percent identity of UBE3A DNA, mRNA, and protein sequences, relative to humans. (C) Phylogenetic tree illustrating the high substitution rate of Ube3a in rats and mice relative to other mammalian species. (D) Schematic illustrating the CRISPR-Cas9 target sites (red boxes) in exon 1 and exon 13 in the pig UBE3A gene. (E) Schematic of a Sanger sequencing chromatogram confirming the CRISPR/Cas9 mediated 97 kb deletion of UBE3A. (F) Image of founder male pigs generated by somatic cell nuclear transfer carrying a compound heterozygous deletion of UBE3A (UBE3A^97kb/1bp). (G) Schematic of pedigree showing the breeding strategy to produce pigs with a maternal (UBE3A^-/+) or paternal (UBE3A^+/-) derived deletion of UBE3A.

Fig. 2.

The pig UBE3A gene is imprinted in the central nervous system. (A) RT-PCR quantification of UBE3A RNA expression in WT, UBE3A^-/+, and UBE3A^+/- tissues, normalized to WT. Data are presented as mean ± SEM. (B) Western blot quantification of UBE3A protein expression in WT, UBE3A^-/+, and UBE3A^+/- tissues, normalized to total protein and WT. Data are presented as mean ± SEM. (C) Immunohistochemical analysis of UBE3A protein expression in the parietal cortex of WT and UBE3A^-/+ pigs; (Scale bar, 0.05 mm.) Abbreviations: wild-type (WT), maternal UBE3A deletion (UBE3A^-/+), and paternal UBE3A deletion (UBE3A^+/-).

Fig. 3.

Impaired neonatal, juvenile, and adolescent development in UBE3A^-/+ pigs. (A) Muscle tone scores (1 to 5 scale) of WT and UBE3A^-/+ pigs and representative images showing size and muscle tone differences between UBE3A^-/+ and WT pigs. A score of 1 indicates low muscle tone with prominent shoulders, spine, and hips, and a score of five indicates high muscle tone with no visible rib or hip bones. Data distribution is presented as a boxplot, Chi-square ordinal logistic test. (B) Vocalization scores during the modified restraint test performed on WT and UBE3A^-/+ pigs (1: no vocalization; 2: grunting; 3: squealing). Data distribution is presented as a boxplot, Ordinal Logistic Fit, FDR correction. (C) Resistance scores from the modified restraint test performed on WT and UBE3A^-/+ pigs (1: no movement; 2: leg kicking; 3: whole-body thrashing). Data distribution is presented as a boxplot, Ordinal Logistic Fit, FDR correction. (D) Percent of neonatal (19-d old) WT and UBE3A^-/+ pigs successfully navigating the incline test. (E) Latency time for adolescent (71-d old) WT and UBE3A^-/+ pigs to transition from supine to standing in the righting reflex test. Data are presented as mean ± SEM, Mann–Whitney test. (F) Kaplan–Meier survival analysis of WT and UBE3A^-/+ pigs, Gehan-Breslow-Wilcoxon test. Abbreviations: wild-type (WT) and maternal UBE3A deletion (UBE3A^-/+). *P < 0.05, **P < 0.01, and ***P < 0.0001.

Fig. 4.

UBE3A^-/+ pigs vocalize less and have an abnormal vocalization structure. (A) Quantification of total vocalizations made by the WT and UBE3A^-/+ pigs during development. (B) Quantification of low-frequency grunts (0 to 8,000 Hz) made by the WT and UBE3A^-/+ pigs. (C) Quantification of medium-frequency grunts (8,001 to 12,500 Hz) made by the WT and UBE3A^-/+ pigs. (D) Quantification of high-frequency squeals (12,501 to 22,000 Hz) made by the WT and UBE3A^-/+ pigs. (E and F) Representative spectrograms of 15-d-old WT and UBE3A^-/+ pigs illustrate fewer vocalizations and abnormal vocalization structure in the UBE3A^-/+ pigs. Spectrograms display intensity and frequency over 10 s. Data are presented as mean ± SEM, Student’s t, all pairwise comparison. Abbreviations: wild-type (WT) and maternal UBE3A deletion (UBE3A^-/+). *P < 0.05, **P < 0.01, and ***P < 0.0001.

Fig. 5.

Juvenile UBE3A^-/+ pigs have an ataxic gait. (A and B) Gait map measurements of the front and hind step lengths and (C and D) front and hind stride lengths of juvenile (33 to 44 d old) WT and UBE3A^-/+ pigs. Data are presented as mean ± SEM, Student’s t, all pairwise comparison. Abbreviations: wild-type (WT) and maternal UBE3A deletion (UBE3A^-/+). *P < 0.05 and **P < 0.01.

Fig. 6.

Neonatal and juvenile UBE3A^-/+ pigs are hypoactive. (A and B) Activity (distance traveled and percent time moving) of WT and UBE3A^-/+ pigs in an open-field arena. Data are presented as mean ± SEM, Wilcoxon two-sample exact test. (C) Representative heat maps showing the typical and atypical exploratory behavior of 5-d-old WT and UBE3A^-/+ pigs, respectively, in the open-field arena. Abbreviations: wild-type (WT) and maternal UBE3A deletion (UBE3A^-/+). *P < 0.05, **P < 0.01, and ***P < 0.0001.

Fig. 7.

UBE3A^-/+ pigs have reduced body weight and brain volume. (A) Longitudinal analysis of body weight during neonatal, juvenile, and adolescent development. Data are presented as mean ± SEM, all pairwise comparisons—Tukey HSD. (B) Gross brain weights of the WT and UBE3A^-/+ at different age ranges during postnatal development, including the brain weights of the UBE3A^+/- pigs at 116 to 136 d old. Data are presented as mean ± SEM, ANOVA Tukey–Kramer HSD. (C and D) Postmortem MRI analysis of whole brain and cerebellum volumes of adolescent (96 to 115 d old) WT and UBE3A^-/+ pigs. Data are presented as mean ± SEM, Student’s t, all pairwise comparison. Abbreviations: wild-type (WT), maternal UBE3A deletion (UBE3A^-/+), and paternal UBE3A deletion (UBE3A^+/-); **P < 0.01 and ***P < 0.0001; ns, not significant.