UBE3A reinstatement as a disease-modifying therapy for Angelman syndrome.

Half a century ago, Harry Angelman reported three patients with overlapping clinical features, now well known as Angelman syndrome. Angelman syndrome is caused by mutations affecting the maternally inherited UBE3A gene, which encodes an E3-ubiquitin ligase that is critical for typical postnatal brain development. Emerging evidence indicates that UBE3A plays a particularly important role in the nucleus. However, the critical substrates that are controlled by UBE3A remain elusive, which hinders the search for effective treatments. Moreover, given the multitude of signalling mechanisms that are derailed, it is unlikely that targeting a single pathway is going to be very effective. Therefore, expectations are very high for approaches that aim to restore UBE3A protein levels. A particular promising strategy is an antisense oligonucleotide approach, which activates the silenced paternal UBE3A gene. When successful, such treatments potentially offer a disease-modifying therapy for Angelman syndrome and several other neurodevelopmental disorders. What this paper adds Loss of UBE3A affects multiple signalling pathways in the brain. Emerging evidence suggests that UBE3A plays a critical role in the cell nucleus. Trials using antisense oligonucleotides to restore UBE3A levels are continuing.

Assessing the requirements of prenatal UBE3A expression for rescue of behavioral phenotypes in a mouse model for Angelman syndrome.

BACKGROUND: Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of functional ubiquitin protein ligase E3A (UBE3A). In neurons, UBE3A expression is tightly regulated by a mechanism of imprinting which suppresses the expression of the paternal UBE3A allele. Promising treatment strategies for AS are directed at activating paternal UBE3A gene expression. However, for such strategies to be successful, it is important to know when such a treatment should start, and how much UBE3A expression is needed for normal embryonic brain development. METHODS: Using a conditional mouse model of AS, we further delineated the critical period for UBE3A expression during early brain development. Ube3a gene expression was induced around the second week of gestation and mouse phenotypes were assessed using a behavioral test battery. To investigate the requirements of embryonic UBE3A expression, we made use of mice in which the paternal Ube3a allele was deleted. RESULTS: We observed a full behavioral rescue of the AS mouse model phenotypes when Ube3a gene reactivation was induced around the start of the last week of mouse embryonic development. We found that full silencing of the paternal Ube3a allele was not completed till the first week after birth but that deletion of the paternal Ube3a allele had no significant effect on the assessed phenotypes. LIMITATIONS: Direct translation to human is limited, as we do not precisely know how human and mouse brain development aligns over gestational time. Moreover, many of the assessed phenotypes have limited translational value, as the underlying brain regions involved in these tasks are largely unknown. CONCLUSIONS: Our findings provide further important insights in the requirement of UBE3A expression during brain development. We found that loss of up to 50% of UBE3A protein during prenatal mouse brain development does not significantly impact the assessed mouse behavioral phenotypes. Together with previous findings, our results indicate that the most critical function for mouse UBE3A lies in the early postnatal period between birth and P21.

Conserved UBE3A subcellular distribution between human and mice is facilitated by non-homologous isoforms.

The human UBE3A gene, which is essential for normal neurodevelopment, encodes three Ubiquitin E3 ligase A (UBE3A) protein isoforms. However, the subcellular localization and relative abundance of these human UBE3A isoforms are unknown. We found, as previously reported in mice, that UBE3A is predominantly nuclear in human neurons. However, this conserved subcellular distribution is achieved by strikingly distinct cis-acting mechanisms. A single amino-acid deletion in the N-terminus of human hUBE3A-Iso3, which is homologous to cytosolic mouse mUBE3A-Iso2, results in its translocation to the nucleus. This singe amino-acid deletion is shared with apes and Old World monkeys and was preceded by the appearance of the cytosolic hUBE3A-Iso2 isoform. This hUBE3A-Iso2 isoform arose after the lineage of New World monkeys and Old World monkeys separated from the Tarsiers (Tarsiidae). Due to the loss of a single nucleotide in a non-coding exon, this exon became in frame with the remainder of the UBE3A protein. RNA-seq analysis of human brain samples showed that the human UBE3A isoforms arise by alternative splicing. Consistent with the predominant nuclear enrichment of UBE3A in human neurons, the two nuclear-localized isoforms, hUBE3A-Iso1 and -Iso3, are the most abundantly expressed isoforms of UBE3A, while hUBE3A-Iso2 maintains a small pool of cytosolic UBE3A. Our findings provide new insight into UBE3A localization and evolution and may have important implications for gene therapy approaches in Angelman syndrome.

Loss of nuclear UBE3A causes electrophysiological and behavioral deficits in mice and is associated with Angelman syndrome.

Mutations affecting the gene encoding the ubiquitin ligase UBE3A cause Angelman syndrome. Although most studies focus on the synaptic function of UBE3A, we show that UBE3A is highly enriched in the nucleus of mouse and human neurons. We found that the two major isoforms of UBE3A exhibit highly distinct nuclear versus cytoplasmic subcellular localization. Both isoforms undergo nuclear import through direct binding to PSMD4 (also known as S5A or RPN10), but the amino terminus of the cytoplasmic isoform prevents nuclear retention. Mice lacking the nuclear UBE3A isoform recapitulate the behavioral and electrophysiological phenotypes of Ube3am-/p+ mice, whereas mice harboring a targeted deletion of the cytosolic isoform are unaffected. Finally, we identified Angelman syndrome-associated UBE3A missense mutations that interfere with either nuclear targeting or nuclear retention of UBE3A. Taken together, our findings elucidate the mechanisms underlying the subcellular localization of UBE3A, and indicate that the nuclear UBE3A isoform is the most critical for the pathophysiology of Angelman syndrome.

Delayed loss of UBE3A reduces the expression of Angelman syndrome-associated phenotypes.

Background: Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by mutations affecting UBE3A gene expression. Previous studies in mice revealed distinct critical periods during neurodevelopment in which reactivation of Ube3a gene expression can prevent the onset of behavioral deficits. Whether UBE3A is required for brain function throughout life is unknown. Here, we address the importance of maintaining UBE3A expression after normal brain development. Findings: Using a conditional mouse, we deleted the Ube3a gene at three ages spanning brain maturation. We assessed the consequences of Ube3a gene deletion by testing the mice in behavioral tasks previously shown to produce robust phenotypes in AS model mice. Early embryonic deletion of Ube3a recapitulated all behavioral deficits of AS mice. In contrast, Ube3a gene deletion at 3 or 12 weeks of age did not have a significant effect on most behavioral tasks and did not increase seizure sensitivity. Conclusions: Taken together, these results emphasize that UBE3A critically impacts early brain development, but plays a more limited role in adulthood. Our findings provide important considerations for upcoming clinical trials in which UBE3A gene expression is reactivated and suggest that even transient UBE3A reinstatement during a critical window of early development is likely to prevent most adverse Angelman syndrome phenotypes. However, sustained UBE3A expression into adulthood is probably needed for optimal clinical benefit.

A behavioral test battery for mouse models of Angelman syndrome: a powerful tool for testing drugs and novel Ube3a mutants.

Background: Angelman syndrome (AS) is a neurodevelopmental disorder caused by mutations affecting UBE3A function. AS is characterized by intellectual disability, impaired motor coordination, epilepsy, and behavioral abnormalities including autism spectrum disorder features. The development of treatments for AS heavily relies on the ability to test the efficacy of drugs in mouse models that show reliable, and preferably clinically relevant, phenotypes. We previously described a number of behavioral paradigms that assess phenotypes in the domains of motor performance, repetitive behavior, anxiety, and seizure susceptibility. Here, we set out to evaluate the robustness of these phenotypes when tested in a standardized test battery. We then used this behavioral test battery to assess the efficacy of minocycline and levodopa, which were recently tested in clinical trials of AS. Methods: We combined data of eight independent experiments involving 111 Ube3a mice and 120 wild-type littermate control mice. Using a meta-analysis, we determined the statistical power of the subtests and the effect of putative confounding factors, such as the effect of sex and of animal weight on rotarod performance. We further assessed the robustness of these phenotypes by comparing Ube3a mutants in different genetic backgrounds and by comparing the behavioral phenotypes of independently derived Ube3a-mutant lines. In addition, we investigated if the test battery allowed re-testing the same animals, which would allow a within-subject testing design. Results: We find that the test battery is robust across different Ube3a-mutant lines, but confirm and extend earlier studies that several phenotypes are very sensitive to genetic background. We further found that the audiogenic seizure susceptibility phenotype is fully reversible upon pharmacological treatment and highly suitable for dose-finding studies. In agreement with the clinical trial results, we found that minocycline and levodopa treatment of Ube3a mice did not show any sign of improved performance in our test battery. Conclusions: Our study provides a useful tool for preclinical drug testing to identify treatments for Angelman syndrome. Since the phenotypes are observed in several independently derived Ube3a lines, the test battery can also be employed to investigate the effect of specific Ube3a mutations on these phenotypes.