Molecular and behavioral consequences of Ube3a gene overdosage in mice
Chromosome 15q11.2–q13.1 duplication syndrome (Dup15q syndrome) is a severe neurodevelopmental disorder characterized by intellectual disability, impaired motor coordination, and autism spectrum disorder. Chromosomal multiplication of the UBE3A gene is presumed to be the primary driver of Dup15q pathophysiology, given that UBE3A exhibits maternal monoallelic expression in neurons and that maternal duplications typically yield far more severe neurodevelopmental outcomes than paternal duplications. However, studies into the pathogenic effects of UBE3A overexpression in mice have yielded conflicting results. Here, we investigated the neurodevelopmental impact of Ube3a gene overdosage using bacterial artificial chromosome–based transgenic mouse models (Ube3aOE) that recapitulate the increases in Ube3a copy number most often observed in Dup15q. In contrast to previously published Ube3a overexpression models, Ube3aOE mice were indistinguishable from wild-type controls on a number of molecular and behavioral measures, despite suffering increased mortality when challenged with seizures, a phenotype reminiscent of sudden unexpected death in epilepsy. Collectively, our data support a model wherein pathogenic synergy between UBE3A and other overexpressed 15q11.2–q13.1 genes is required for full penetrance of Dup15q syndrome phenotypes.
Human chromosome 15q11.2–q13.1 is exceptionally vulnerable to structural abnormalities that result in neurological disorders (1–5). Clusters of repetitive sequence, which originated in part from duplications of the GOLGA8 gene, bring about the existence of 5 distinct breakpoint sites (BP1–BP5) spanning this region (6–11). These breakpoints increase the risk of homologous recombination during meiosis, resulting in either deletions or duplications of 15q11.2–q13.1 (4, 6, 11, 12).
Genomic imprinting underlies the monoallelic, parent-of-origin–specific expression of different 15q11.2–q13.1 genes. Consequently, paternal and maternal 15q11.2–q13.1 deletions produce distinct pathophysiologies, which in turn result in Prader-Willi and Angelman syndromes, respectively (13, 14). Maternal duplications of this same region are deemed to be causative of a neuropsychiatric disorder called Dup15q syndrome (11, 15). Dup15q syndrome is clinically defined by moderate to profound intellectual disability, impaired motor coordination, and autism spectrum disorder (ASD) (16–18). Some patients may present with interstitial duplications, a condition referred to as a 15q11.2–q13.1 trisomy. However, in the majority of the duplication events, BP1–BP3 recombination results in an isodicentric triplication of the 15q11.2–q13.1 region [idic(15)], giving rise to a supernumerary chromosome 15 or a 15q11.2–q13.1 tetrasomy (11, 19). Dup15q syndrome pathological severity increases proportionally with the number of 15q11.2–q13.1 copies, meaning that idic(15) individuals, in general, have more severe symptomology than individuals with interstitial duplications (16, 19).
Efforts to elucidate the pathophysiological contributions of specific genes to Dup15q syndrome have focused on UBE3A (20–22). Of all the genes in the 15q11.2–q13.1 region, UBE3A alone exhibits cell type–specific, maternal monoallelic expression. The paternal UBE3A allele is silenced in mature neurons, leaving maternal UBE3A as the sole source of UBE3A protein in these cells (23–25). Thus, neuronal UBE3A overdosage is unique to maternally inherited 15q11.2–q13.1 duplications, which yield far more severe neurodevelopmental phenotypes as compared with those of paternal origin (11, 15, 26). Additionally, maternal inheritance of a circumscribed UBE3A gene duplication has been linked to developmental delay and neuropsychiatric phenotypes in multiple members of a single family; family members with paternal inheritance of the same mutation were unaffected (27). Such findings have focused the lens on UBE3A gene duplications as being a major driver of disease pathology in Dup15q syndrome.
UBE3A encodes a HECT E3 ubiquitin ligase involved in ubiquitin-mediated protein turnover (28, 29). It is commonly believed that the ability of UBE3A to control the abundance of its protein substrates is imperative to prevent disease (30, 31). UBE3A is also strongly implicated in transcriptional coactivation (32–35), another function that may be critical to maintaining cellular homeostasis. Although UBE3A deficiency indisputably leads to Angelman syndrome (14), a causal connection between UBE3A overexpression and Dup15q syndrome phenotypes has proved elusive. Not only is clinical evidence of UBE3A microduplication sparse (27), but also studies of the consequences of UBE3A overexpression in mouse models are contradictory. In 2009, Nakatani and colleagues reported on mice harboring a duplication of chromosome 7, the syntenic 15q11.2–q13.1 region in mice. Surprisingly, it was mice with paternal duplication that showed ASD-like phenotypes in this study. Mice with maternal duplication showed no notable behavioral abnormalities, challenging expectations based on maternal inheritance of Dup15q syndrome (36). In later research, various groups homed in on UBE3A overexpression alone. These efforts yielded novel transgenic mice that were shown to display phenotypes reminiscent of Dup15q syndrome pathology (20–22). However, design features inherent to these models — overly excessive Ube3a copy number (21, 22), homozygous inheritance of transgenic alleles (20, 22), restricted UBE3A isoform representation (21), and the incorporation of function-altering protein tags (20–22) — have confounded interpretations of their pathophysiological relevance to Dup15q syndrome.
In this study we describe transgenic mice for modeling UBE3A overdosage as it would most likely occur in Dup15q syndrome. Our model prioritizes disease-relevant excess of Ube3a gene copies, the full representation of enzymatically competent UBE3A isoforms, and the faithful recapitulation of endogenous UBE3A expression patterns in the brain. By rigorously testing these mice for changes in gene expression, synaptic physiology, and performance in UBE3A-sensitive and Dup15q-relevant behavioral assays, we revisit UBE3A’s contribution to Dup15q syndrome pathophysiology from a position of improved construct validity.