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Arthur L Beaudet - One of the best experts on this subject based on the ideXlab platform.

  • abstract ia28 towards a therapy for angelman syndrome by targeting a long noncoding rna to active UBE3A
    Cancer Research, 2016
    Co-Authors: Linyan Meng, Arthur L Beaudet, Amanda J Ward, Frank C Bennett, Frank Rigo
    Abstract:

    Angelman syndrome (AS) is a single gene disorder characterized by intellectual disability, developmental delay, behavioral uniqueness, speech impairment, seizures, and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine UBE3A-ATS reduction by either transcription termination or topoisomerase I inhibition increased paternal UBE3A expression. Despite a clear understanding of the disease-causing event in AS and the potential to harness the intact paternal allele to correct disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for AS by reducing UBE3A-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of UBE3A-ATS and sustained unsilencing of paternal UBE3A in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an AS mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, for the first time we developed a sequence-specific and clinically feasible method to activate expression of the paternal UBE3A allele. Citation Format: Linyan Meng, Amanda J. Ward, C. Frank Bennett, Arthur Beaudet, Frank Rigo. Towards a therapy for Angelman syndrome by targeting a long noncoding RNA to active UBE3A. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr IA28.

  • towards a therapy for angelman syndrome by targeting a long non coding rna
    Nature, 2015
    Co-Authors: Linyan Meng, Arthur L Beaudet, Amanda J Ward, Seung Chun, Frank C Bennett, Frank Rigo
    Abstract:

    Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia. It is caused by maternal deficiency of the imprinted gene UBE3A, encoding an E3 ubiquitin ligase. All patients carry at least one copy of paternal UBE3A, which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript (UBE3A-ATS). Murine UBE3A-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal UBE3A expression. Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing UBE3A-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of UBE3A-ATS and sustained unsilencing of paternal UBE3A in neurons in vitro and in vivo. Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal UBE3A allele.

  • truncation of UBE3A ats unsilences paternal UBE3A and ameliorates behavioral defects in the angelman syndrome mouse model
    PLOS Genetics, 2013
    Co-Authors: Linyan Meng, Richard E Person, Wei Huang, Ping Jun Zhu, Mauro Costamattioli, Arthur L Beaudet
    Abstract:

    Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by maternal deficiency of the imprinted gene UBE3A. Individuals with AS suffer from intellectual disability, speech impairment, and motor dysfunction. Currently there is no cure for the disease. Here, we evaluated the phenotypic effect of activating the silenced paternal allele of UBE3A by depleting its antisense RNA UBE3A-ATS in mice. Premature termination of UBE3A-ATS by poly(A) cassette insertion activates expression of UBE3A from the paternal chromosome, and ameliorates many disease-related symptoms in the AS mouse model, including motor coordination defects, cognitive deficit, and impaired long-term potentiation. Studies on the imprinting mechanism of UBE3A revealed a pattern of biallelic transcription initiation with suppressed elongation of paternal UBE3A, implicating transcriptional collision between sense and antisense polymerases. These studies demonstrate the feasibility and utility of unsilencing the paternal copy of UBE3A via targeting UBE3A-ATS as a treatment for Angelman syndrome.

  • UBE3A ats is an atypical rna polymerase ii transcript that represses the paternal expression of UBE3A
    Human Molecular Genetics, 2012
    Co-Authors: Linyan Meng, Richard E Person, Arthur L Beaudet
    Abstract:

    The Angelman syndrome gene, UBE3A, is subject to genomic imprinting controlled by mechanisms that are only partially understood. Its antisense transcript, UBE3A-ATS, is also imprinted and hypothesized to suppress UBE3A in cis. In this research, we showed that the mouse antisense ortholog, UBE3A-ATS, was transcribed by RNA polymerase (RNAP) II. However, unlike typical protein-coding transcripts, UBE3A-ATS was not poly-adenylated and was localized exclusively in the nucleus. It was relatively unstable with a half-life of 4 h, shorter than most protein-coding RNAs tested. To understand the role of UBE3A-ATS in vivo, a mouse model with a 0.9-kb genomic deletion over the paternal Snrpn major promoter was studied. The mice showed partial activation of paternal UBE3A, with decreased expression of UBE3A-ATS but not any imprinting defects in the Prader–Willi syndrome/Angelman syndrome region. A novel cell culture model was also generated with a transcriptional termination cassette inserted downstream of UBE3A on the paternal chromosome to reduce UBE3A-ATS transcription. In neuronally differentiated embryonic stem (ES) cells, paternal UBE3A was found to be expressed at a high level, comparable with that of the maternal allele. To further characterize the antisense RNA, a strand-specific microarray was performed. UBE3A-ATS was detectable across the entire locus of UBE3A and extended beyond the transcriptional start site of UBE3A. In summary, we conclude that UBE3A-ATS is an atypical RNAPII transcript that represses UBE3A on the paternal chromosome. These results suggest that the repression of human UBE3A-ATS may activate the expression of UBE3A from the paternal chromosome, providing a potential therapeutic strategy for patients with Angelman syndrome.

  • imprinted expression of the murine angelman syndrome gene UBE3A in hippocampal and purkinje neurons
    Nature Genetics, 1997
    Co-Authors: Urs Albrecht, Arthur L Beaudet, James S Sutcliffe, Bruce M Cattanach, C V Beechey, Dawna L Armstrong, Gregor Eichele
    Abstract:

    Angelman syndrome (AS) is a human genetic disorder characterized by mental retardation, seizures, inappropriate laughter, abnormal gait, tremor and ataxia1–3. There is strong genetic evidence that the disorder is associated with a maternally expressed, imprinted gene mapping to chromosome 15q11–13. Affected patients demonstrate varied molecular abnormalities including large maternal deletions, uniparental paternal disomy (UPD), imprinting mutations4 and loss of function mutations of E6–associated-protein (E6-AP) ubiquitin–protein ligase (UBE3A)5,6. All of these abnormalities are associated with loss of maternal expression of UBE3A. Although mutations in UBE3A cause AS, indicating that maternal-specific expression of UBE3A is essential for a normal phenotype, evidence for maternal-specific expression of UBE3A has been lacking7,8. Using mice with partial paternal UPD encompassing UBE3A to differentiate maternal and paternal expression, we found by in situ hybridization that expression of UBE3A in Purkinje cells, hippocampal neurons and mitral cells of the olfactory bulb in UPD mice was markedly reduced compared to non-UPD littermates. In contrast, expression of UBE3A in other regions of the brain was only moderately or not at all reduced in UPD mice. The major phenotypic features of AS correlate with the loss of maternal-specific expression of UBE3A in hippocampus and cerebellum as revealed in the mouse model.

Mark J Zylka - One of the best experts on this subject based on the ideXlab platform.

  • cas9 gene therapy for angelman syndrome traps UBE3A ats long non coding rna
    Nature, 2020
    Co-Authors: Justin M Wolter, Hanqian Mao, Giulia Fragola, Jeremy M Simon, James L Krantz, Hannah O Bazick, Baris Oztemiz, Jason L Stein, Mark J Zylka
    Abstract:

    Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal UBE3A in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3' region of UBE3A-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring UBE3A is predicted to be greatest1,2. This early treatment unsilenced paternal UBE3A throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of UBE3A-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder.

  • cas9 gene therapy for angelman syndrome traps UBE3A ats long non coding rna
    Nature, 2020
    Co-Authors: Justin M Wolter, Hanqian Mao, Giulia Fragola, Jeremy M Simon, James L Krantz, Hannah O Bazick, Baris Oztemiz, Jason L Stein, Mark J Zylka
    Abstract:

    Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by a mutation or deletion of the maternally inherited UBE3A allele. In neurons, the paternally inherited UBE3A allele is silenced in cis by a long non-coding RNA called UBE3A-ATS. Here, as part of a systematic screen, we found that Cas9 can be used to activate ('unsilence') paternal UBE3A in cultured mouse and human neurons when targeted to Snord115 genes, which are small nucleolar RNAs that are clustered in the 3′ region of UBE3A-ATS. A short Cas9 variant and guide RNA that target about 75 Snord115 genes were packaged into an adeno-associated virus and administered to a mouse model of AS during the embryonic and early postnatal stages, when the therapeutic benefit of restoring UBE3A is predicted to be greatest1,2. This early treatment unsilenced paternal UBE3A throughout the brain for at least 17 months and rescued anatomical and behavioural phenotypes in AS mice. Genomic integration of the adeno-associated virus vector into Cas9 target sites caused premature termination of UBE3A-ATS at the vector-derived polyA cassette, or when integrated in the reverse orientation, by transcriptional collision with the vector-derived Cas9 transcript. Our study shows that targeted genomic integration of a gene therapy vector can restore the function of paternally inherited UBE3A throughout life, providing a path towards a disease-modifying treatment for a syndromic neurodevelopmental disorder. Genomic integration of an adeno-associated virus vector in a mouse model of Angelman syndrome unsilences paternal UBE3A and rescues anatomical and behavioural phenotypes, suggesting a pathway towards the treatment of this neurodevelopmental disorder.

  • the autism linked UBE3A t485a mutant e3 ubiquitin ligase activates the wnt β catenin pathway by inhibiting the proteasome
    Journal of Biological Chemistry, 2017
    Co-Authors: Smita R Paranjape, Justin M Wolter, Giulia Fragola, Matthew P Walker, Rajarshi Choudhury, Michael J Emanuele, Michael B Major, Mark J Zylka
    Abstract:

    UBE3A is a HECT domain E3 ubiquitin ligase whose dysfunction is linked to autism, Angelman syndrome, and cancer. Recently, we characterized a de novo autism-linked UBE3A mutant (UBE3AT485A) that disrupts phosphorylation control of UBE3A activity. Through quantitative proteomics and reporter assays, we found that the UBE3AT485A protein ubiquitinates multiple proteasome subunits, reduces proteasome subunit abundance and activity, stabilizes nuclear β-catenin, and stimulates canonical Wnt signaling more effectively than wild-type UBE3A. We also found that UBE3AT485A activates Wnt signaling to a greater extent in cells with low levels of ongoing Wnt signaling, suggesting that cells with low basal Wnt activity are particularly vulnerable to UBE3AT485A mutation. Ligase-dead UBE3A did not stimulate Wnt pathway activation. Overexpression of several proteasome subunits reversed the effect of UBE3AT485A on Wnt signaling. We also observed that subunits that interact with UBE3A and affect Wnt signaling are located along one side of the 19S regulatory particle, indicating a previously unrecognized spatial organization to the proteasome. Altogether, our findings indicate that UBE3A regulates Wnt signaling in a cell context-dependent manner and that an autism-linked mutation exacerbates these signaling effects. Our study has broad implications for human disorders associated with UBE3A gain or loss of function and suggests that dysfunctional UBE3A might affect additional proteins and pathways that are sensitive to proteasome activity.

  • gabaergic neuron specific loss of UBE3A causes angelman syndrome like eeg abnormalities and enhances seizure susceptibility
    Neuron, 2016
    Co-Authors: Matthew C. Judson, Michael S. Sidorov, Michael L Wallace, Alain C Burette, Geeske M Van Woerden, Ian F G King, Ji Eun E Han, Mark J Zylka, Ype Elgersma, Richard J Weinberg
    Abstract:

    Summary Loss of maternal UBE3A causes Angelman syndrome (AS), a neurodevelopmental disorder associated with severe epilepsy. We previously implicated GABAergic deficits onto layer (L) 2/3 pyramidal neurons in the pathogenesis of neocortical hyperexcitability, and perhaps epilepsy, in AS model mice. Here we investigate consequences of selective UBE3A loss from either GABAergic or glutamatergic neurons, focusing on the development of hyperexcitability within L2/3 neocortex and in broader circuit and behavioral contexts. We find that GABAergic UBE3A loss causes AS-like increases in neocortical EEG delta power, enhances seizure susceptibility, and leads to presynaptic accumulation of clathrin-coated vesicles (CCVs)—all without decreasing GABAergic inhibition onto L2/3 pyramidal neurons. Conversely, glutamatergic UBE3A loss fails to yield EEG abnormalities, seizures, or associated CCV phenotypes, despite impairing tonic inhibition onto L2/3 pyramidal neurons. These results substantiate GABAergic UBE3A loss as the principal cause of circuit hyperexcitability in AS mice, lending insight into ictogenic mechanisms in AS.

  • maternal UBE3A loss disrupts sleep homeostasis but leaves circadian rhythmicity largely intact
    The Journal of Neuroscience, 2015
    Co-Authors: J C Ehlen, Mark J Zylka, Richard J Weinberg, Kelly A Jones, Lennisha Pinckney, Cloe L Gray, Susan Burette, Jennifer A Evans, Allison J Brager, Ketema Paul
    Abstract:

    Individuals with Angelman syndrome (AS) suffer sleep disturbances that severely impair quality of life. Whether these disturbances arise from sleep or circadian clock dysfunction is currently unknown. Here, we explored the mechanistic basis for these sleep disorders in a mouse model of Angelman syndrome (UBE3Am−/p+ mice). Genetic deletion of the maternal UBE3A allele practically eliminates UBE3A protein from the brain of UBE3Am−/p+ mice, because the paternal allele is epigenetically silenced in most neurons. However, we found that UBE3A protein was present in many neurons of the suprachiasmatic nucleus—the site of the mammalian circadian clock—indicating that UBE3A can be expressed from both parental alleles in this brain region in adult mice. We found that while UBE3Am−/p+ mice maintained relatively normal circadian rhythms of behavior and light-resetting, these mice exhibited consolidated locomotor activity and skipped the timed rest period (siesta) present in wild-type (UBE3Am+/p+) mice. Electroencephalographic analysis revealed that alterations in sleep regulation were responsible for these overt changes in activity. Specifically, UBE3Am−/p+ mice have a markedly reduced capacity to accumulate sleep pressure, both during their active period and in response to forced sleep deprivation. Thus, our data indicate that the siesta is governed by sleep pressure, and that UBE3A is an important regulator of sleep homeostasis. These preclinical findings suggest that therapeutic interventions that target mechanisms of sleep homeostasis may improve sleep quality in individuals with AS. SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by loss of expression of the maternal copy of the UBE3A gene. Individuals with AS have severe sleep dysfunction that affects their cognition and presents challenges to their caregivers. Unfortunately, current treatment strategies have limited efficacy due to a poor understanding of the mechanisms underlying sleep disruptions in AS. Here we demonstrate that abnormal sleep patterns arise from a deficit in accumulation of sleep drive, uncovering the UBE3A gene as a novel genetic regulator of sleep homeostasis. Our findings encourage a re-evaluation of current treatment strategies for sleep dysfunction in AS, and suggest that interventions that promote increased sleep drive may alleviate sleep disturbances in individuals with AS.

Joseph Wagstaff - One of the best experts on this subject based on the ideXlab platform.

  • sleep disturbances in UBE3A maternal deficient mice modeling angelman syndrome
    Neurobiology of Disease, 2005
    Co-Authors: Damien Colas, Joseph Wagstaff, Denise Salvert, Patrice Fort, Nicole Sarda
    Abstract:

    Abstract Background: Angelman syndrome (AS) is a severe neurodevelopmental disorder with electroencephalographic (EEG) abnormalities and sleep disturbances. It results from lack of the functional maternal allele of UBE3A, which encodes a ubiquitin–protein ligase. Different mechanisms of UBE3A inactivation correlate with clinical phenotypes of varying severity; the majority of cases of AS are due to a de novo maternal deletion of the 15q11–q13 region. Methods: UBE3A maternal-deficient mice ( UBE3A m−/p+) were generated in a C57Bl/6J background. This study compares cortical EEG and architecture of the sleep–waking cycle in adult UBE3A m−/p+ mice compared with those of age-matched WT (m+/p+) mice, under baseline conditions or after 4-h sleep deprivation (SD). Results: UBE3A m−/p+ mice exhibited: reduced slow-wave sleep (SWS) amount with increase waking (W) at the dark/light transitions; increased SWS and W episode numbers; and deterioration of paradoxical sleep (PS) over 24 h [amount: −44%; episode duration: −46%; episode number: −40%; theta peak frequency (TPF) acceleration: 7.6 Hz vs. 7.0 Hz in WT mice]. Characteristic paroxysmal EEG discharges are observed during W and SWS associated with synchronous muscle bursting activity during hypoactive W. During the recovery period following SD, UBE3A m−/p+ mice exhibited no rebound either in slow-wave activity (+89% in WT) or in δ-power spectra but a slight rebound in PS amount (+20%). Conclusions: These data validate the mouse model produced by null mutation of the maternal UBE3A gene and provide useful results to investigate and better understand the molecular basis of sleep disturbances in AS patients.

  • snurf snrpn and UBE3A transcript levels in patients with angelman syndrome
    Human Genetics, 2004
    Co-Authors: Maren Runte, Joseph Wagstaff, Peter M Kroisel, Gabriele Gillessenkaesbach, Raymonda Varon, Denise Horn, Monika Y Cohen, Bernhard Horsthemke, Karin Buiting
    Abstract:

    The imprinted domain on human chromosome 15 consists of two oppositely imprinted gene clusters, which are under the control of an imprinting center (IC). The paternally expressed SNURF-SNRPN gene hosts several snoRNA genes and overlaps the UBE3A gene, which is encoded on the opposite strand, expressed — at least in brain cells — from the maternal chromosome only, and affected in patients with Angelman syndrome (AS). In contrast to SNURF-SNRPN, imprinted expression of UBE3A is not regulated by a 5′ differentially methylated region. Here we report that splice forms of the SNURF-SNRPN transcript overlapping UBE3A in an antisense orientation are present in brain but barely detectable in blood. In contrast, splice forms that do not overlap with UBE3A are of similar abundance in brain and blood. The tissue distribution of the splice forms parallels that of the snoRNAs encoded in the respective parts of the SNURF-SNRPN transcript. Using a quantitative PCR assay, we have found that the ratio of SNURF-SNRPN/UBE3A transcript levels is increased in blood cells of AS patients with an imprinting defect, but not in AS patients with a UBE3A mutation or an unknown defect. Our findings are compatible with the assumption that imprinted UBE3A expression is regulated through the SNURF-SNRPN sense-UBE3A antisense transcript.

  • neurons but not glial cells show reciprocal imprinting of sense and antisense transcripts of UBE3A
    Human Molecular Genetics, 2003
    Co-Authors: Kentaro Yamasaki, Joseph Wagstaff, Keiichiro Joh, Tohru Ohta, Hideaki Masuzaki, Tadayuki Ishimaru, Tsunehiro Mukai, Norio Niikawa, Michio Ogawa, Tatsuya Kishino
    Abstract:

    The human UBE3A gene shows brain-specific partial imprinting, and lack of a maternally inherited allele causes Angelman syndrome (AS), which is characterized by neurobehavioral anomalies. In several AS model mice, imprinted UBE3A expression is detected predominantly in the hippocampus, cerebellar Purkinje cells and the olfactory bulb. Therefore, imprinting of mouse UBE3A is thought to be region-specific with different levels of silencing of the paternal UBE3A allele in different brain regions. To determine cell types of imprinted UBE3A expression, we analyzed its imprinting status in embryonic brain cells by using primary cortical cell cultures. RT-PCR and immunofluorescence were performed to determine the allelic expression of the gene. The UBE3A gene encodes two RNA transcripts in the brain, sense and antisense. The sense transcript was expressed maternally in neurons but biallelically in glial cells in the embryonic brain, whereas the antisense transcript was expressed only in neurons and only from the paternal allele. Our data present evidence of brain cell type-specific imprinting, i.e. neuron-specific imprinting of UBE3A in primary brain cell cultures. Reciprocal imprinting of sense and antisense transcripts present only in neurons suggests that the neuron-specific imprinting mechanism is related to the lineage determination of neural stem cells.

  • neurobehavioral and electroencephalographic abnormalities in UBE3A maternal deficient mice
    Neurobiology of Disease, 2002
    Co-Authors: Kiyonori Miura, Tatsuya Kishino, Hayley A Webber, Pieter Dikkes, Gregory L Holmes, Joseph Wagstaff
    Abstract:

    Angelman syndrome (AS), characterized by motor dysfunction, mental retardation, and seizures, is caused by several genetic etiologies involving chromosome 15q11–q13, including mutations of the UBE3A gene. UBE3A encodes UBE3A/E6-AP, a ubiquitin-protein ligase, and shows brain-specific imprinting, with brain expression predominantly from the maternal allele. Lack of a functional maternal allele of UBE3A causes AS. In order to understand the causal relationship between maternal UBE3A mutations and AS, we have constructed a mouse model with targeted inactivation of UBE3A. The inactive allele contains a lacZ reporter gene for analysis of brain-specific imprinting. Maternal, but not paternal, transmission of the targeted allele leads to β-galactosidase activity in hippocampal and cerebellar neurons. Maternal inheritance of the UBE3A mutant allele also causes impaired performance in tests of motor function and spatial learning, as well as abnormal hippocampal EEG recordings. As predicted from the dependence of UBE3A-mediated ubiquitination of p53 on HPV E6 protein, our maternal-deficient mice show normal brain p53 levels.

  • Genomic organization of the UBE3A/E6-AP gene and related pseudogenes.
    Genomics, 1998
    Co-Authors: Tatsuya Kishino, Joseph Wagstaff
    Abstract:

    The UBE3A gene encodes the E6-AP ubiquitin-protein ligase and has recently been shown to be mutated in Angelman syndrome patients who lack 15q11-q13 deletions or chromosome 15 paternal uniparental disomy. Previous UBE3A cDNA analysis has shown a coding region of approximately 2.6 kb and a 3'-untranslated region (UTR) of 2 kb of 3'-UTR. We have established the genomic organization of UBE3A and the sequence of intron-exon borders. We have also mapped two highly homologous processed pseudogenes, UBE3AP1 and UBE3AP2, to chromosomes 2 and 21, respectively, and determined their genomic organization. These results will form the basis for studies of mutation and imprinting of UBE3A.

Benjamin D. Philpot - One of the best experts on this subject based on the ideXlab platform.

  • delayed loss of UBE3A reduces the expression of angelman syndrome associated phenotypes
    Molecular Autism, 2019
    Co-Authors: Monica Sonzogni, Benjamin D. Philpot, Matthew C. Judson, Geeske M Van Woerden, Sara Silvasantos, Johanna Hakonen, Mireia Bernabe Kleijn, Ype Elgersma
    Abstract:

    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.

  • subcellular organization of UBE3A in human cerebral cortex
    Molecular Autism, 2018
    Co-Authors: Alain C Burette, Benjamin D. Philpot, Matthew C. Judson, Edward F Chang, William W Seeley, Richard J Weinberg
    Abstract:

    Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A’s localization is integral to its function. We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals. We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals. By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder.

  • characterization and structure activity relationships of indenoisoquinoline derived topoisomerase i inhibitors in unsilencing the dormant UBE3A gene associated with angelman syndrome
    Molecular Autism, 2018
    Co-Authors: Hyeong Min Lee, Ellen P Clark, Bram M Kuijer, Mark Cushman, Yves Pommier, Benjamin D. Philpot
    Abstract:

    Angelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A (UBE3A), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of UBE3A in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal UBE3A in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics. We tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of UBE3A in mouse neurons. Primary cortical neurons were isolated from embryonic day 14.5 (E14.5) mice with a UBE3A-YFP fluorescent tag on the paternal allele (UBE3Am+/pYFP mice) or mice that lack the maternal UBE3A allele and hence model AS (UBE3Am−/p+ mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay. All 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal UBE3A. Several compounds exhibited favorable paternal UBE3A unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated Emax (maximum response of unsilencing paternal UBE3A) and EC50 (half maximal effective concentration). We provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal UBE3A unsilencers. All 13 tested compounds were effective at unsilencing paternal UBE3A, although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of UBE3A unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed.

  • Characterization and structure-activity relationships of indenoisoquinoline-derived topoisomerase I inhibitors in unsilencing the dormant UBE3A gene associated with Angelman syndrome
    BMC, 2018
    Co-Authors: Hyeong Min Lee, Ellen P Clark, Bram M Kuijer, Mark Cushman, Yves Pommier, Benjamin D. Philpot
    Abstract:

    Abstract Background Angelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A (UBE3A), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of UBE3A in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal UBE3A in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics. Methods We tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of UBE3A in mouse neurons. Primary cortical neurons were isolated from embryonic day 14.5 (E14.5) mice with a UBE3A-YFP fluorescent tag on the paternal allele (UBE3A m+/pYFP mice) or mice that lack the maternal UBE3A allele and hence model AS (UBE3A m−/p+ mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay. Results All 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal UBE3A. Several compounds exhibited favorable paternal UBE3A unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated Emax (maximum response of unsilencing paternal UBE3A) and EC50 (half maximal effective concentration). Conclusions We provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal UBE3A unsilencers. All 13 tested compounds were effective at unsilencing paternal UBE3A, although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of UBE3A unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed

  • Persistent neuronal UBE3A expression in the suprachiasmatic nucleus of Angelman syndrome model mice
    Scientific Reports, 2016
    Co-Authors: Kelly A Jones, Jason P. Debruyne, Benjamin D. Philpot
    Abstract:

    Mutations or deletions of the maternal allele of the UBE3A gene cause Angelman syndrome (AS), a severe neurodevelopmental disorder. The paternal UBE3A/UBE3A allele becomes epigenetically silenced in most neurons during postnatal development in humans and mice; hence, loss of the maternal allele largely eliminates neuronal expression of UBE3A protein. However, recent studies suggest that paternal UBE3A may escape silencing in certain neuron populations, allowing for persistent expression of paternal UBE3A protein. Here we extend evidence in AS model mice ( UBE3A ^ m –/ p +) of paternal UBE3A expression within the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Paternal UBE3A-positive cells in the SCN show partial colocalization with the neuropeptide arginine vasopressin (AVP) and clock proteins (PER2 and BMAL1), supporting that paternal UBE3A expression in the SCN is often of neuronal origin. Paternal UBE3A also partially colocalizes with a marker of neural progenitors, SOX2, implying that relaxed or incomplete imprinting of paternal UBE3A reflects an overall immature molecular phenotype. Our findings highlight the complexity of UBE3A imprinting in the brain and illuminate a subpopulation of SCN neurons as a focal point for future studies aimed at understanding the mechanisms of UBE3A imprinting.

Stormy J Chamberlain - One of the best experts on this subject based on the ideXlab platform.

  • a bipartite boundary element restricts UBE3A imprinting to mature neurons
    Proceedings of the National Academy of Sciences of the United States of America, 2019
    Co-Authors: Jack S Hsiao, Noelle D Germain, Andrea Wilderman, Christopher Stoddard, Luke A Wojenski, Geno J Villafano, Leighton J Core, Justin Cotney, Stormy J Chamberlain
    Abstract:

    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.

  • a bipartite boundary element restricts UBE3A imprinting to mature neurons
    bioRxiv, 2018
    Co-Authors: Jack S Hsiao, Noelle D Germain, Andrea Wilderman, Christopher Stoddard, Luke A Wojenski, Geno J Villafano, Leighton J Core, Justin Cotney, Stormy J Chamberlain
    Abstract:

    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 upregulation of UBE3A-ATS without repressing paternal UBE3A. However, increasing expression of UBE3AATS 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 upregulation of UBE3A-ATS. These results suggest that manipulation of the competition between UBE3A-ATS and UBE3A may provide a potential therapeutic approach for AS.

  • epigenetic regulation of UBE3A and roles in human neurodevelopmental disorders
    Epigenomics, 2015
    Co-Authors: Janine M Lasalle, Lawrence T. Reiter, Stormy J Chamberlain
    Abstract:

    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.

  • imprinted expression of UBE3A in non neuronal cells from a prader willi syndrome patient with an atypical deletion
    Human Molecular Genetics, 2014
    Co-Authors: Kristen Martinstaylor, Marc Lalande, Jack S Hsiao, Heather Glattdeeley, Pinfang Chen, Adam J De Smith, Alexandra I F Blakemore, Stormy J Chamberlain
    Abstract:

    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.

  • induced pluripotent stem cell models of the genomic imprinting disorders angelman and prader willi syndromes
    Proceedings of the National Academy of Sciences of the United States of America, 2010
    Co-Authors: Stormy J Chamberlain, Pinfang Chen, Khong Y Ng, Fany Bourgoisrocha, Fouad Lemtirichlieh, Eric S Levine, Marc Lalande
    Abstract:

    Angelman syndrome (AS) and Prader–Willi syndrome (PWS) are neurodevelopmental disorders of genomic imprinting. AS results from loss of function of the ubiquitin protein ligase E3A (UBE3A) gene, whereas the genetic defect in PWS is unknown. Although induced pluripotent stem cells (iPSCs) provide invaluable models of human disease, nuclear reprogramming could limit the usefulness of iPSCs from patients who have AS and PWS should the genomic imprint marks be disturbed by the epigenetic reprogramming process. Our iPSCs derived from patients with AS and PWS show no evidence of DNA methylation imprint erasure at the cis-acting PSW imprinting center. Importantly, we find that, as in normal brain, imprinting of UBE3A is established during neuronal differentiation of AS iPSCs, with the paternal UBE3A allele repressed concomitant with up-regulation of the UBE3A antisense transcript. These iPSC models of genomic imprinting disorders will facilitate investigation of the AS and PWS disease processes and allow study of the developmental timing and mechanism of UBE3A repression in human neurons.