Ube3a unsilencer for the potential treatment of Angelman syndrome.

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.

Generation of isogenic models of Angelman syndrome and Prader-Willi syndrome in CRISPR/Cas9-engineered human embryonic stem cells.

Angelman Syndrome (AS) and Prader-Willi Syndrome (PWS), two distinct neurodevelopmental disorders, result from loss of expression from imprinted genes in the chromosome 15q11-13 locus most commonly caused by a megabase-scale deletion on either the maternal or paternal allele, respectively. Each occurs at an approximate incidence of 1/15,000 to 1/30,000 live births and has a range of debilitating phenotypes. Patient-derived induced pluripotent stem cells (iPSCs) have been valuable tools to understand human-relevant gene regulation at this locus and have contributed to the development of therapeutic approaches for AS. Nonetheless, gaps remain in our understanding of how these deletions contribute to dysregulation and phenotypes of AS and PWS. Variability across cell lines due to donor differences, reprogramming methods, and genetic background make it challenging to fill these gaps in knowledge without substantially increasing the number of cell lines used in the analyses. Isogenic cell lines that differ only by the genetic mutation causing the disease can ease this burden without requiring such a large number of cell lines. Here, we describe the development of isogenic human embryonic stem cell (hESC) lines modeling the most common genetic subtypes of AS and PWS. These lines allow for a facile interrogation of allele-specific gene regulation at the chromosome 15q11-q13 locus. Additionally, these lines are an important resource to identify and test targeted therapeutic approaches for patients with AS and PWS.

The role of UBE3A in the autism and epilepsy-related Dup15q syndrome using patient-derived, CRISPR-corrected neurons.

Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurodevelopmental disorder caused by maternal duplications of this region. Autism and epilepsy are key features of Dup15q. UBE3A, which encodes an E3 ubiquitin ligase, is likely a major driver of Dup15q because UBE3A is the only imprinted gene expressed solely from the maternal allele. Nevertheless, the exact role of UBE3A has not been determined. To establish whether UBE3A overexpression is required for Dup15q neuronal deficits, we generated an isogenic control line for a Dup15q patient-derived induced pluripotent stem cell line. Dup15q neurons exhibited hyperexcitability compared with control neurons, and this phenotype was generally prevented by normalizing UBE3A levels using antisense oligonucleotides. Overexpression of UBE3A resulted in a profile similar to that of Dup15q neurons except for synaptic phenotypes. These results indicate that UBE3A overexpression is necessary for most Dup15q cellular phenotypes but also suggest a role for other genes in the duplicated region.

Hyperexcitable Phenotypes in Induced Pluripotent Stem Cell-Derived Neurons From Patients With 15q11-q13 Duplication Syndrome, a Genetic Form of Autism.

BACKGROUND: Chromosome 15q11-q13 duplication syndrome (Dup15q) is a neurogenetic disorder caused by duplications of the maternal copy of this region. In addition to hypotonia, motor deficits, and language impairments, patients with Dup15q commonly meet the criteria for autism spectrum disorder and have a high prevalence of seizures. It is known from mouse models that synaptic impairments are a strong component of Dup15q pathophysiology; however, cellular phenotypes that relate to seizures are less clear. The development of patient-derived induced pluripotent stem cells provides a unique opportunity to study human neurons with the exact genetic disruptions that cause Dup15q. METHODS: Here, we explored electrophysiological phenotypes in induced pluripotent stem cell-derived neurons from 4 patients with Dup15q compared with 6 unaffected control subjects, 1 patient with a 15q11-q13 paternal duplication, and 3 patients with Angelman syndrome. RESULTS: We identified several properties of Dup15q neurons that could contribute to neuronal hyperexcitability and seizure susceptibility. Compared with control neurons, Dup15q neurons had increased excitatory synaptic event frequency and amplitude, increased density of dendritic protrusions, increased action potential firing, and decreased inhibitory synaptic transmission. Dup15q neurons also showed impairments in activity-dependent synaptic plasticity and homeostatic synaptic scaling. Finally, Dup15q neurons showed an increased frequency of spontaneous action potential firing compared with control neurons, in part due to disruption of KCNQ2 potassium channels. CONCLUSIONS: Together, these data point to multiple electrophysiological mechanisms of hyperexcitability that may provide new targets for the treatment of seizures and other phenotypes associated with Dup15q.

A protein regulated by UBE3A PEGs a potential biomarker.

New research from Pandya and colleagues1 identifies PEG10 as a UBE3A-regulated protein that may underlie pathophysiology in Angelman syndrome neurons. PEG10 is a secreted protein, and this work suggests that it may be a potential biomarker for Angelman syndrome therapeutics under development.

Communication-related assessments in an Angelman syndrome mouse model.

INTRODUCTION: Angelman syndrome (AS) is a neurodevelopmental disorder characterized by motor deficits, seizures, some autistic-like behaviors, and severe impairment of speech. A dysfunction of the maternally imprinted UBE3A gene, coupled with a functional yet silenced paternal copy, results in AS. Although studies of transgenic mouse models have revealed a great deal about neural populations and rescue timeframes for specific features of AS, these studies have largely failed to examine intermediate phenotypes that contribute to the profound communicative disabilities associated with AS. METHODS: Here, we use a variety of tasks, including assessments of rapid auditory processing and social communication. Expressive vocalizations were directly assessed and correlated against other core behavioral measures (motor, social, acoustic perception) to model putative influences on communication. RESULTS: AS mice displayed the characteristic phenotypes associated with Angelman syndrome (i.e., social and motor deficits), as well as marginal enhancements in rapid auditory processing ability. Our characterization of adult ultrasonic vocalizations further showed that AS mice produce fewer vocalizations and vocalized for a shorter amount of time when compared to controls. Additionally, a strong correlation between motor indices and ultrasonic vocalization output was shown, suggesting that the motor impairments in AS may contribute heavily to communication impairments. CONCLUSION: In summary, the combination of motor deficits, social impairment, marginal rapid auditory enhancements, and altered ultrasonic vocalizations reported in a mouse model of AS clearly parallel the human symptoms of the disorder. This mouse model offers a novel route to interrogate the underlying genetic, physiologic, and behavioral influences on the under-studied topic of impaired communication in AS.

Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons.

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity. This disorder is caused by the absence of paternally expressed gene products from chromosome 15q11-q13. We previously demonstrated that knocking out ZNF274, a Kruppel-associated box-A-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS induced pluripotent stem cells (iPSCs). However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here, we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared with ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

Identification of Small-Molecule Activators of the Ubiquitin Ligase E6AP/UBE3A and Angelman Syndrome-Derived E6AP/UBE3A Variants.

Genetic aberrations of the UBE3A gene encoding the E3 ubiquitin ligase E6AP underlie the development of Angelman syndrome (AS). Approximately 10% of AS individuals harbor UBE3A genes with point mutations, frequently resulting in the expression of full-length E6AP variants with defective E3 activity. Since E6AP exists in two states, an inactive and an active one, we hypothesized that distinct small molecules can stabilize the active state and that such molecules may rescue the E3 activity of AS-derived E6AP variants. Therefore, we established an assay that allows identifying modulators of E6AP in a high-throughput format. We identified several compounds that not only stimulate wild-type E6AP but also rescue the E3 activity of certain E6AP variants. Moreover, by chemical cross-linking coupled to mass spectrometry we provide evidence that the compounds stabilize an active conformation of E6AP. Thus, these compounds represent potential lead structures for the design of drugs for AS treatment.

Prader-Willi syndrome: reflections on seminal studies and future therapies.

Prader-Willi syndrome (PWS) is caused by the loss of function of the paternally inherited 15q11-q13 locus. This region is governed by genomic imprinting, a phenomenon in which genes are expressed exclusively from one parental allele. The genomic imprinting of the 15q11-q13 locus is established in the germline and is largely controlled by a bipartite imprinting centre. One part, termed the Prader-Willi syndrome imprinting center (PWS-IC), comprises a CpG island that is unmethylated on the paternal allele and methylated on the maternal allele. The second part, termed the Angelman syndrome imprinting centre, is required to silence the PWS_IC in the maternal germline. The loss of the paternal contribution of the imprinted 15q11-q13 locus most frequently occurs owing to a large deletion of the entire imprinted region but can also occur through maternal uniparental disomy or an imprinting defect. While PWS is considered a contiguous gene syndrome based on large-deletion and uniparental disomy patients, the lack of expression of only non-coding RNA transcripts from the SNURF-SNRPN/SNHG14 may be the primary cause of PWS. Patients with small atypical deletions of the paternal SNORD116 cluster alone appear to have most of the PWS related clinical phenotypes. The loss of the maternal contribution of the 15q11-q13 locus causes a separate and distinct condition called Angelman syndrome. Importantly, while much has been learned about the regulation and expression of genes and transcripts deriving from the 15q11-q13 locus, there remains much to be learned about how these genes and transcripts contribute at the molecular level to the clinical traits and developmental aspects of PWS that have been observed.

Abundance and localization of human UBE3A protein isoforms.

Loss of UBE3A expression, a gene regulated by genomic imprinting, causes Angelman syndrome (AS), a rare neurodevelopmental disorder. The UBE3A gene encodes an E3 ubiquitin ligase with three known protein isoforms in humans. Studies in mouse suggest that the human isoforms may have differences in localization and neuronal function. A recent case study reported mild AS phenotypes in individuals lacking one specific isoform. Here we have used CRISPR/Cas9 to generate isogenic human embryonic stem cells (hESCs) that lack the individual protein isoforms. We demonstrate that isoform 1 accounts for the majority of UBE3A protein in hESCs and neurons. We also show that UBE3A predominantly localizes to the cytoplasm in both wild type and isoform-null cells. Finally, we show that neurons lacking isoform 1 display a less severe electrophysiological AS phenotype.

IPSC Models of Chromosome 15Q Imprinting Disorders: From Disease Modeling to Therapeutic Strategies.

The chromosome 15q11-q13 region of the human genome is regulated by genomic imprinting, an epigenetic phenomenon in which genes are expressed exclusively from one parental allele. Several genes within the 15q11-q13 region are expressed exclusively from the paternally inherited chromosome 15. At least one gene UBE3A, shows exclusive expression of the maternal allele, but this allele-specific expression is restricted to neurons. The appropriate regulation of imprinted gene expression across chromosome 15q11-q13 has important implications for human disease. Three different neurodevelopmental disorders result from aberrant expression of imprinted genes in this region: Prader-Willi syndrome (PWS), Angelman syndrome (AS), and 15q duplication syndrome.

A bipartite boundary element restricts UBE3A imprinting to mature neurons.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele of UBE3A, a gene encoding an E3 ubiquitin ligase. UBE3A is only expressed from the maternally inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression of UBE3A is restricted to neurons by expression of UBE3A antisense transcript (UBE3A-ATS) from the paternally inherited allele, which silences the paternal allele of UBE3A in cis However, the mechanism restricting UBE3A-ATS expression and UBE3A imprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression of UBE3A-ATS in humans. Removal of this element led to up-regulation of UBE3A-ATS without repressing paternal UBE3A However, increasing expression of UBE3A-ATS in the absence of the boundary element resulted in full repression of paternal UBE3A, demonstrating that UBE3A imprinting requires both the loss of function from the boundary element as well as the up-regulation of UBE3A-ATS These results suggest that manipulation of the competition between UBE3A-ATS and UBE3A may provide a potential therapeutic approach for AS.

Disrupted neuronal maturation in Angelman syndrome-derived induced pluripotent stem cells.

Angelman syndrome (AS) is a neurogenetic disorder caused by deletion of the maternally inherited UBE3A allele and is characterized by developmental delay, intellectual disability, ataxia, seizures and a happy affect. Here, we explored the underlying pathophysiology using induced pluripotent stem cell-derived neurons from AS patients and unaffected controls. AS-derived neurons showed impaired maturation of resting membrane potential and action potential firing, decreased synaptic activity and reduced synaptic plasticity. These patient-specific differences were mimicked by knocking out UBE3A using CRISPR/Cas9 or by knocking down UBE3A using antisense oligonucleotides. Importantly, these phenotypes could be rescued by pharmacologically unsilencing paternal UBE3A expression. Moreover, selective effects of UBE3A disruption at late stages of in vitro development suggest that changes in action potential firing and synaptic activity may be secondary to altered resting membrane potential. Our findings provide a cellular phenotype for investigating pathogenic mechanisms underlying AS and identifying novel therapeutic strategies.

RBFOX1 and RBFOX2 are dispensable in iPSCs and iPSC-derived neurons and do not contribute to neural-specific paternal UBE3A silencing.

Angelman Syndrome (AS) is a rare neurodevelopmental disorder caused by loss of function of the maternally inherited copy of UBE3A, an imprinted gene expressed biallelically in most tissues, but expressed exclusively from the maternal allele in neurons. Active transcription of the neuron-specific long non-coding RNA (lncRNA), UBE3A-ATS, has been shown to silence paternal UBE3A. We hypothesized that alternative splicing factors RBFOX2 and RBFOX1 might mediate splicing changes and result in the transcription of UBE3A-ATS in neurons. We found that RBFOX2 and RBFOX1 both bind to UBE3A-ATS transcript in neurons, but are not required for gene expression and/or neuron-specific processing in the SNURF/SNRPN-UBE3A region. However, we found that depletion of RBFOX2 causes a proliferation phenotype in immature neural cultures, suggesting that RBFOX2 is involved in division versus differentiation decisions in iPSC-derived neural progenitors. Absence of RBFOX2 also altered the expression of some genes that are important for glutamatergic neocortical development and Wnt-Frizzled signalling in mature neuronal cultures. Our data show that while RBFOX1 and RBFOX2 do not mediate neuron-specific processing of UBE3A-ATS, these proteins play important roles in developing neurons and are not completely functionally redundant.

Modeling Genomic Imprinting Disorders Using Induced Pluripotent Stem Cells.

Induced pluripotent stem cell (iPSC) technology has allowed for the invaluable modeling of many genetic disorders including disorders associated with genomic imprinting. Genomic imprinting involves differential DNA and histone methylation and results in allele-specific gene expression. Most of the epigenetic marks in somatic cells are erased and reestablished during the process of reprogramming into iPSCs. Therefore, in generating models of disorders associated with genomic imprinting, it is important to verify that the imprinting status and allele-specific gene expression patterns of the parental somatic cells are maintained in their derivative iPSCs. Here, we describe three techniques: DNA methylation analysis, allele-specific PCR, and RNA FISH, which we use to analyze genomic imprinting in iPSC models of neurogenetic disorders involving copy number variations of the chromosome 15q11-q13 region.

Epigenetic regulation of UBE3A and roles in human neurodevelopmental disorders.

The E3 ubiquitin ligase UBE3A, also known as E6-AP, has a multitude of ascribed functions and targets relevant to human health and disease. Epigenetic regulation of the UBE3A gene by parentally imprinted noncoding transcription within human chromosome 15q11.2-q13.3 is responsible for the maternal-specific effects of 15q11.2-q13.3 deletion or duplication disorders. Here, we review the evidence for diverse and emerging roles for UBE3A in the proteasome, synapse and nucleus in regulating protein stability and transcription as well as the current mechanistic understanding of UBE3A imprinting in neurons. Angelman and Dup15q syndromes as well as experimental models of these neurodevelopmental disorders are highlighted as improving understanding of UBE3A and its complex regulation for improving therapeutic strategies.

Prader-Willi, Angelman, and 15q11-q13 Duplication Syndromes.

Three distinct neurodevelopmental disorders arise primarily from deletions or duplications that occur at the 15q11-q13 locus: Prader-Willi syndrome, Angelman syndrome, and 15q11-q13 duplication syndrome. Each of these disorders results from the loss of function or overexpression of at least 1 imprinted gene. This article discusses the clinical background, genetic cause, diagnostic strategy, and management of each of these 3 disorders.

Gene expression analysis of human induced pluripotent stem cell-derived neurons carrying copy number variants of chromosome 15q11-q13.1.

BACKGROUND: Duplications of the chromosome 15q11-q13.1 region are associated with an estimated 1 to 3% of all autism cases, making this copy number variation (CNV) one of the most frequent chromosome abnormalities associated with autism spectrum disorder (ASD). Several genes located within the 15q11-q13.1 duplication region including ubiquitin protein ligase E3A (UBE3A), the gene disrupted in Angelman syndrome (AS), are involved in neural function and may play important roles in the neurobehavioral phenotypes associated with chromosome 15q11-q13.1 duplication (Dup15q) syndrome. METHODS: We have generated induced pluripotent stem cell (iPSC) lines from five different individuals containing CNVs of 15q11-q13.1. The iPSC lines were differentiated into mature, functional neurons. Gene expression across the 15q11-q13.1 locus was compared among the five iPSC lines and corresponding iPSC-derived neurons using quantitative reverse transcription PCR (qRT-PCR). Genome-wide gene expression was compared between neurons derived from three iPSC lines using mRNA-Seq. RESULTS: Analysis of 15q11-q13.1 gene expression in neurons derived from Dup15q iPSCs reveals that gene copy number does not consistently predict expression levels in cells with interstitial duplications of 15q11-q13.1. mRNA-Seq experiments show that there is substantial overlap in the genes differentially expressed between 15q11-q13.1 deletion and duplication neurons, Finally, we demonstrate that UBE3A transcripts can be pharmacologically rescued to normal levels in iPSC-derived neurons with a 15q11-q13.1 duplication. CONCLUSIONS: Chromatin structure may influence gene expression across the 15q11-q13.1 region in neurons. Genome-wide analyses suggest that common neuronal pathways may be disrupted in both the Angelman and Dup15q syndromes. These data demonstrate that our disease-specific stem cell models provide a new tool to decipher the underlying cellular and genetic disease mechanisms of ASD and may also offer a pathway to novel therapeutic intervention in Dup15q syndrome.

Imprinted expression of UBE3A in non-neuronal cells from a Prader-Willi syndrome patient with an atypical deletion.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are two neurodevelopmental disorders most often caused by deletions of the same region of paternally inherited and maternally inherited human chromosome 15q, respectively. AS is a single gene disorder, caused by the loss of function of the ubiquitin ligase E3A (UBE3A) gene, while PWS is still considered a contiguous gene disorder. Rare individuals with PWS who carry atypical microdeletions on chromosome 15q have narrowed the critical region for this disorder to a 108 kb region that includes the SNORD116 snoRNA cluster and the Imprinted in Prader-Willi (IPW) non-coding RNA. Here we report the derivation of induced pluripotent stem cells (iPSCs) from a PWS patient with an atypical microdeletion that spans the PWS critical region. We show that these iPSCs express brain-specific portions of the transcripts driven by the PWS imprinting center, including the UBE3A antisense transcript (UBE3A-ATS). Furthermore, UBE3A expression is imprinted in most of these iPSCs. These data suggest that UBE3A imprinting in neurons only requires UBE3A-ATS expression, and no other neuron-specific factors. These data also suggest that a boundary element lying within the PWS critical region prevents UBE3A-ATS expression in non-neural tissues.