RESUMEN
The protein homeostasis, or proteostasis, is maintained through the coupling of two pivotal systems: the ubiquitin-proteasome and autophagy. Cumulative evidence has suggested E3 ubiquitin ligases specifically play a central role in this coupling, ensuring the regulation of synaptic and cognitive functions. Defects in these ligases have been identified as hallmarks in a range of neurodevelopmental and neurodegenerative disorders. Recent literature has spotlighted the E3 ubiquitin ligase, UBE3A, as a key player in this domain. Dysregulation or loss of UBE3A function has been linked to disrupted proteostasis, leading to synaptic and cognitive anomalies. Notably, such defects are prominently observed in conditions like Angelman syndrome, a neurodevelopmental disorder characterized by severe cognitive impairments. The emerging understanding of UBE3A's role in bridging the ubiquitin-proteasome and autophagy systems offers a promising therapeutic avenue. Targeting the defective pathways caused by UBE3A loss could pave the way for innovative treatments, potentially ameliorating the cognitive deficits observed in neurological disorders like Angelman syndrome. As the scientific community delves deeper into the molecular intricacies of E3 ubiquitin ligases, there is burgeoning hope for devising effective interventions for associated neurological conditions.
Asunto(s)
Autofagia , Complejo de la Endopetidasa Proteasomal , Ubiquitina-Proteína Ligasas , Ubiquitina , Humanos , Autofagia/fisiología , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Animales , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patología , Cognición/fisiologíaRESUMEN
Angelman syndrome (AS) is a rare genetic disorder due to lack of UBE3A function on chromosome 15q11.2q13 caused by a deletion, uniparental paternal disomy (UPD), imprinting center disorder (ICD), or pathological variant of the UBE3A gene. AS is characterized by developmental delay, epilepsy, and lack of speech. Although fractures are observed frequently in our clinical practice, there are few studies on bone health in AS. The aim of this study is to investigate bone health in children with AS. In this prospective cohort study, we describe bone health in 91 children with AS visiting the ENCORE Expertise Center for AS between April 2010 and December 2021. Bone health was assessed with the bone health index (BHI) in standard deviation score (SDS) measured by digital radiogrammetry of the left hand using BoneXpert software. Risk factors analyzed were age, sex, genetic subtype, epilepsy, anti-seizure medication use, mobility, body mass index (BMI), and onset of puberty. Children with AS had a mean BHI of -1.77 SDS (SD 1.4). A significantly lower BHI was found in children with a deletion (-2.24 SDS) versus non-deletion (-1.02 SDS). Other factors associated with reduced BHI-SDS were inability to walk and late onset of puberty. Children with a history of one or more fractures (22%) had a significantly lower BHI than children without fractures (-2.60 vs -1.56 SDS). Longitudinal analysis showed a significant decrease in BHI-SDS with age in all genetic subtypes. Conclusions: Children with AS have a reduced bone health. Risk factors are deletion genotype, no independent walking, and late onset of puberty. Bone health decreased significantly with age. What is Known: ⢠Children with neurological disorders often have a low bone health and higher risk of fractures. ⢠Little is known about bone health in children with Angelman syndrome (AS). What is New: ⢠Children with AS showed a reduced bone health and this was significantly associated with having a deletion, not being able to walk independently, and late onset of puberty. ⢠Longitudinal analysis showed a significant decrease in bone health as children got older.
Asunto(s)
Síndrome de Angelman , Epilepsia , Niño , Humanos , Síndrome de Angelman/complicaciones , Síndrome de Angelman/genética , Síndrome de Angelman/patología , Densidad Ósea , Estudios Prospectivos , Genotipo , Ácido Láctico , Cromosomas Humanos Par 15/genéticaRESUMEN
Azadiradione is a small bioactive limonoid found in the seed of Azadirachta Indica, an Indian medicinal plant commonly known as Neem. Recently, it has been shown to ameliorate the disease pathology in fly and mouse model of Huntington's disease by restoring impaired proteostasis. Here we report that the azadiradione could be involved in modulating the synaptic function through increased expression of Ube3a, a dual function protein having ubiquitin ligase and co-activator functions and associated with Angelman syndrome and autism. Treatment of azadiradione to HT22 hippocampal cell line and in adult mice induced the expression of Ube3a as well as two important synaptic function and plasticity regulating proteins, parvalbumin and brain-derived neurotropic factor (BDNF). Interestingly, another synaptic plasticity modulating protein Arc (activity-regulated cytoskeletal associated protein) was down-regulated by azadiradione. Partial knockdown of Ube3a in HT22 cell abrogated azadiradione induced expression of parvalbumin and BDNF. Ube3a-maternal deficient mice also exhibited significantly decreased expression of parvalbumin and BDNF in their brain and treatment of azadiradione in these animals did not rescue the altered expression of either parvalbumin or BDNF. These results indicate that azadiradione-induced expression of parvalbumin and BDNF in the brain is mediated through Ube3a and suggest that azadiradione could be implicated in restoring synaptic dysfunction in many neuropsychiatric/neurodegenerative disorders.
Asunto(s)
Síndrome de Angelman , Limoninas , Ratones , Animales , Limoninas/farmacología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Parvalbúminas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Encéfalo/metabolismo , Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Síndrome de Angelman/patología , Modelos Animales de EnfermedadRESUMEN
The Ubiquitin-protein ligase E3A, UBE3A, also known as E6-associated protein (E6-AP), is known to play an essential role in regulating the degradation of various proteins by transferring Ub from E2 Ub conjugating enzymes to the substrate proteins. Several studies indicate that UBE3A regulates the stabilities of key viral proteins in the virus-infected cells and, thereby, the infected virus-mediated diseases, even if it were reported that UBE3A participates in non-viral-related human diseases. Furthermore, mutations such as deletions and duplications in the maternally inherited gene in the brain cause human neurodevelopmental disorders such as Angelman syndrome (AS) and autism. It is also known that UBE3A functions as a transcriptional coactivator for the expression of steroid hormone receptors. These reports establish that UBE3A is distinguished by its multitudinous functions that are paramount to viral pathology and human diseases. This review is focused on molecular mechanisms for such intensive participation of UBE3A in disease formation and virus regulation.
Asunto(s)
Síndrome de Angelman , Virosis , Humanos , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Mutación , Encéfalo/patología , Síndrome de Angelman/genética , Síndrome de Angelman/metabolismo , Síndrome de Angelman/patología , Virosis/genéticaRESUMEN
Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder caused by the maternally inherited loss of function of the UBE3A gene. AS is characterized by a developmental delay, lack of speech, motor dysfunction, epilepsy, autistic features, happy demeanor, and intellectual disability. While the cellular roles of UBE3A are not fully understood, studies suggest that the lack of UBE3A function is associated with elevated levels of reactive oxygen species (ROS). Despite the accumulating evidence emphasizing the importance of ROS during early brain development and its involvement in different neurodevelopmental disorders, up to date, the levels of ROS in AS neural precursor cells (NPCs) and the consequences on AS embryonic neural development have not been elucidated. In this study we show multifaceted mitochondrial aberration in AS brain-derived embryonic NPCs, which exhibit elevated mitochondrial membrane potential (ΔΨm), lower levels of endogenous reduced glutathione, excessive mitochondrial ROS (mROS) levels, and increased apoptosis compared to wild-type (WT) littermates. In addition, we report that glutathione replenishment by glutathione-reduced ethyl ester (GSH-EE) corrects the excessive mROS levels and attenuates the enhanced apoptosis in AS NPCs. Studying the glutathione redox imbalance and mitochondrial abnormalities in embryonic AS NPCs provides an essential insight into the involvement of UBE3A in early neural development, information that can serve as a powerful avenue towards a broader view of AS pathogenesis. Moreover, since mitochondrial dysfunction and elevated ROS levels were associated with other neurodevelopmental disorders, the findings herein suggest some potential shared underlying mechanisms for these disorders as well.
Asunto(s)
Síndrome de Angelman , Células-Madre Neurales , Animales , Síndrome de Angelman/genética , Síndrome de Angelman/patología , Especies Reactivas de Oxígeno , Células-Madre Neurales/patología , Neuronas/patología , Glutatión , Ubiquitina-Proteína Ligasas/genética , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Angelman syndrome (AS) is a genetic disorder that affects neurodevelopment. The investigation of changes in the brain white matter network, which would contribute to a better understanding of the pathogenesis of AS brain, was lacking. PURPOSE: To investigate both local and global alterations of white matter in patients with AS. STUDY TYPE: Prospective. SUBJECTS: A total of 29 AS patients (6.6 ± 1.4 years, 15 [52%] females) and 19 age-matched healthy controls (HC) (7.0 ± 1.5 years, 10 [53%] females). FIELD STRENGTH/SEQUENCE: A 3-T, three-dimensional (3D) T1-weighted imaging by using gradient-echo-based sequence, single shell diffusion tensor imaging by using spin-echo-based echo-planar imaging. ASSESSMENT: Network metrics including global efficiency (Eg ), local efficiency (Eloc ), small world coefficient (Swc), rich-club coefficient (Φ), and nodal degree (ND) were estimated from diffusion MR (dMR) data. Connections among highly connected (hub) regions and less connected (peripheral) regions were also assessed. Correlation between the topological parameters and age for each group was also calculated to assess the development of the brain. STATISTICAL TESTS: Linear regression model, permutation test. P values estimated from the regression model for each brain region were adjusted by false discovery rate (FDR) correction. RESULTS: AS patients showed significantly lower Eg and higher swc compared to HC. Φn significantly increased at higher k-levels in AS patients. In addition, the connections among hub regions and peripheral regions were significantly interrupted in AS patients. DATA CONCLUSION: The AS brain showed diminished connectivity, reflected by reduced network efficiency compared to HC. Compared to densely connected regions, less connected regions were more vulnerable in AS. EVIDENCE LEVEL: 2 TECHNICAL EFFICACY: Stage 3.
Asunto(s)
Síndrome de Angelman , Sustancia Blanca , Femenino , Humanos , Preescolar , Niño , Masculino , Imagen de Difusión Tensora/métodos , Síndrome de Angelman/patología , Estudios Prospectivos , Encéfalo/patologíaRESUMEN
Angelman syndrome (AS) is one of the common genetic disorders that could emerge either from a 15q11-q13 deletion or paternal uniparental disomy (UPD) or imprinting or UBE3A mutations. AS comes with various behavioral and phenotypic variability, but the acquisition of subjects for experiment and automating the landmarking process to characterize facial morphology for Angelman syndrome variation investigation are common challenges. By automatically detecting and annotating subject faces, we collected 83 landmarks and 10 anthropometric linear distances were measured from 17 selected anatomical landmarks to account for shape variability. Statistical analyses were performed on the extracted data to investigate facial variation in each age group. There is a correspondence in the results achieved by relative warp (RW) of the principal component (PC) and the thin-plate spline (TPS) interpolation. The group is highly discriminated and the pattern of shape variability is higher in children than other groups when judged by the anthropometric measurement and principal component.
Asunto(s)
Síndrome de Angelman/patología , Cara/anomalías , Adolescente , Adulto , Envejecimiento , Síndrome de Angelman/genética , Antropometría , Niño , Cara/patología , Humanos , Fenotipo , Adulto JovenRESUMEN
Angelman-like syndromes are a group of neurodevelopmental disorders that entail clinical presentation similar to Angelman Syndrome (AS). In our previous study, we showed that calcium signaling is disrupted in AS, and we identified calcium-target and calcium-regulating gene signatures that are able to differentiate between AS and their controls in different models. In the herein study, we evaluated these sets of calcium-target and calcium-regulating genes as signatures of AS-like and non-AS-like syndromes. We collected a number of RNA-seq datasets of various AS-like and non-AS-like syndromes and performed Principle Component Analysis (PCA) separately on the two sets of signature genes to visualize the distribution of samples on the PC1-PC2 plane. In addition to the evaluation of calcium signature genes, we performed differential gene expression analyses to identify calcium-related genes dysregulated in each of the studied syndromes. These analyses showed that the calcium-target and calcium-regulating signatures differentiate well between AS-like syndromes and their controls. However, in spite of the fact that many of the non-AS-like syndromes have multiple differentially expressed calcium-related genes, the calcium signatures were not efficient classifiers for non-AS-like neurodevelopmental disorders. These results show that features based on clinical presentation are reflected in signatures derived from bioinformatics analyses and suggest the use of bioinformatics as a tool for classification.
Asunto(s)
Síndrome de Angelman/genética , Señalización del Calcio/genética , Biología Computacional , Síndrome de Angelman/patología , Calcio/metabolismo , Deleción Cromosómica , Regulación de la Expresión Génica/genética , Humanos , Análisis de Componente Principal , RNA-SeqRESUMEN
BACKGROUND: There are few cohort studies describing the adaptive functioning profile for Pitt-Hopkins syndrome (PTHS). In this study we examine the adaptive functioning profile for PTHS and compare it to Angelman syndrome (AS). METHOD: Caregivers of 14 individuals with PTHS, 33 with deletion AS and 23 with non-deletion AS, completed the Vineland Adaptive Behavior Scales-II. RESULTS: The profile of adaptive functioning in PTHS was characterised by strengths in socialisation, followed by motor skills, communication then daily living skills. The PTHS group scored significantly lower than the non-deletion AS group on all domains except socialisation and significantly lower than the deletion AS group, for motor skills only. CONCLUSIONS: An uneven adaptive behavior profile for individuals with PTHS mirrors that of AS, with implications for assessment and intervention.
Asunto(s)
Adaptación Fisiológica , Hiperventilación/patología , Discapacidad Intelectual/patología , Fenotipo , Actividades Cotidianas , Adolescente , Adulto , Síndrome de Angelman/patología , Niño , Facies , Humanos , Destreza Motora , Conducta SocialRESUMEN
Angelman syndrome is a neurodevelopmental disorder characterized by intellectual disability (ID), a distinctive gait pattern, abnormal behaviors, severe impairment in language development, and characteristic facial features. Most cases are caused by the absence of a maternal contribution to the imprinted region on chromosome 15q11-q13. Here, we present the first reported case of a 3-year-old boy with an atypical phenotype of Angelman syndrome due to uniparental isodisomy with two recessive homozygous pathogenic variants: in HERC2 and AP3B2. Known phenotypes related to HERC2 and AP3B2 include ID and early infantile epileptic encephalopathy, respectively. The patient had severe global developmental delay and profound ID and showed a happy demeanor, stereotypic laughter, and hand-flapping movements, but also irritability. Craniofacial dysmorphic features, including brachycephaly, strabismus, wide ala nasi, short philtrum, wide open mouth, and slight hypopigmentation were seen. Progressive microcephaly was noted. Magnetic resonance imaging of the brain showed delayed myelination and cerebral atrophy. Trio whole exome sequencing and CGH-SNP array analysis revealed paternal uniparental isodisomy of chromosome 15 and two coexisting recessive diseases resulting from homozygous HERC2 and AP3B2 pathogenic variants. The pathogenic variant in HERC2 was inherited from his heterozygous-carrier father, and the variant in AP3B2 was de novo. We suppose that these unusual features were the combination of the effect of three concomitant disorders.
Asunto(s)
Complejo 3 de Proteína Adaptadora/genética , Subunidades beta de Complejo de Proteína Adaptadora/genética , Síndrome de Angelman/genética , Discapacidad Intelectual/genética , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/diagnóstico , Síndrome de Angelman/patología , Preescolar , Cromosomas Humanos Par 15/genética , Predisposición Genética a la Enfermedad , Homocigoto , Humanos , Discapacidad Intelectual/patología , Masculino , Fenotipo , Disomía Uniparental/genética , Secuenciación del ExomaRESUMEN
Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by impaired communication skills, ataxia, motor and balance deficits, intellectual disabilities, and seizures. The genetic cause of AS is the neuronal loss of UBE3A expression in the brain. A novel approach, described here, is a stem cell gene therapy which uses lentivector-transduced hematopoietic stem and progenitor cells to deliver functional UBE3A to affected cells. We have demonstrated both the prevention and reversal of AS phenotypes upon transplantation and engraftment of human CD34+ cells transduced with a Ube3a lentivector in a novel immunodeficient Ube3amat-/pat+ IL2rg-/y mouse model of AS. A significant improvement in motor and cognitive behavioral assays as well as normalized delta power measured by electroencephalogram was observed in neonates and adults transplanted with the gene modified cells. Human hematopoietic profiles observed in the lymphoid organs by detection of human immune cells were normal. Expression of UBE3A was detected in the brains of the adult treatment group following immunohistochemical staining illustrating engraftment of the gene-modified cells expressing UBE3A in the brain. As demonstrated with our data, this stem cell gene therapy approach offers a promising treatment strategy for AS, not requiring a critical treatment window.
Asunto(s)
Síndrome de Angelman/terapia , Terapia Genética , Discapacidad Intelectual/terapia , Convulsiones/terapia , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patología , Animales , Antígenos CD34/genética , Ataxia/genética , Ataxia/patología , Encéfalo/metabolismo , Encéfalo/patología , Disfunción Cognitiva/genética , Disfunción Cognitiva/terapia , Modelos Animales de Enfermedad , Electroencefalografía , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Trasplante de Células Madre Hematopoyéticas , Humanos , Discapacidad Intelectual/genética , Interleucina-2/genética , Lentivirus/genética , Ratones , Trastornos de la Destreza Motora/genética , Trastornos de la Destreza Motora/patología , Trastornos de la Destreza Motora/terapia , Convulsiones/genéticaRESUMEN
UBE3A is an E3 ubiquitin ligase encoded by the neurally imprinted UBE3A gene. The abundance and subcellular distribution of UBE3A has been the topic of many previous studies as its dosage and localization has been linked to neurodevelopmental disorders including Autism, Dup15q syndrome, and Angelman syndrome. While commercially available antibodies have been widely employed to determine UBE3A localization, an extensive analysis and comparison of the performance of different UBE3A antibodies has not been conducted. Here we evaluated the specificities of seven commercial UBE3A antibodies in two of the major experimental models used in UBE3A research, mouse and human pluripotent stem cell-derived neural cells and tissues. We tested these antibodies in their two most common assays, immunofluorescence and western blot. In addition, we also assessed the ability of these antibodies to capture dynamic spatiotemporal changes of UBE3A by utilizing human cerebral organoid models. Our results reveal that among the seven antibodies tested, three antibodies demonstrated substantial nonspecific immunoreactivity. While four antibodies show specific localization patterns in both mouse brain sections and human cerebral organoids, these antibodies varied significantly in background signals and staining patterns in undifferentiated human pluripotent stem cells.
Asunto(s)
Anticuerpos/genética , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/genética , Síndrome de Angelman/patología , Animales , Anticuerpos/inmunología , Trastorno Autístico/genética , Trastorno Autístico/patología , Cerebelo/citología , Modelos Animales de Enfermedad , Impresión Genómica , Humanos , Ratones , Células-Madre Neurales/metabolismo , Neuronas/patología , Organoides/citología , Células Madre Pluripotentes , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by deletion (~75%) or mutation (~10%) of the ubiquitin E3 ligase A (UBE3A) gene, which encodes a HECT type E3 ubiquitin protein ligase. Although the critical substrates of UBE3A are unknown, previous studies have suggested a critical role of nuclear UBE3A in AS pathophysiology. Here, we investigated to what extent UBE3A missense mutations disrupt UBE3A subcellular localization as well as catalytic activity, stability and protein folding. Our functional screen of 31 UBE3A missense mutants revealed that UBE3A mislocalization is the predominant cause of UBE3A dysfunction, accounting for 55% of the UBE3A mutations tested. The second major cause (29%) is a loss of E3-ubiquitin ligase activity, as assessed in an Escherichia coli in vivo ubiquitination assay. Mutations affecting catalytic activity are found not only in the catalytic HECT domain, but also in the N-terminal half of UBE3A, suggesting an important contribution of this N-terminal region to its catalytic potential. Together, our results show that loss of nuclear UBE3A E3 ligase activity is the predominant cause of UBE3A-linked AS. Moreover, our functional analysis screen allows rapid assessment of the pathogenicity of novel UBE3A missense variants which will be of particular importance when treatments for AS become available.
Asunto(s)
Síndrome de Angelman/patología , Núcleo Celular/metabolismo , Mutación Missense , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación , Síndrome de Angelman/genética , Animales , Escherichia coli/metabolismo , Células HEK293 , Humanos , Ratones , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/químicaRESUMEN
Angelman syndrome (AS) is a neurogenetic disorder involving ataxia and motor dysfunction, resulting from the absence of the maternally inherited functional Ube3a protein in neurons. Since adenosine A2A receptor (A2AR) blockade relieves synaptic and motor impairments in Parkinson's or Machado-Joseph's diseases, we now tested if A2AR blockade was also effective in attenuating motor deficits in an AS (Ube3am-/p+) mouse model and if this involved correction of synaptic alterations in striatum and cerebellum. Chronic administration of the A2AR antagonist SCH58261 (0.1 mg/kg/day, ip) promoted motor learning of AS mice in the accelerating-rotarod task and rescued the grip strength impairment of AS animals. These motor impairments were accompanied by synaptic alterations in cerebellum and striatum typified by upregulation of synaptophysin and vesicular GABA transporters (vGAT) in the cerebellum of AS mice along with a downregulation of vGAT, vesicular glutamate transporter 1 (vGLUT1) and the dopamine active transporter in AS striatum. Notably, A2AR blockade prevented the synaptic alterations found in AS mice cerebellum as well as the downregulation of striatal vGAT and vGLUT1. This provides the first indications that A2AR blockade may counteract the characteristic motor impairments and synaptic changes of AS, although more studies are needed to unravel the underlying mechanisms.
Asunto(s)
Antagonistas del Receptor de Adenosina A2/farmacología , Síndrome de Angelman/patología , Síndrome de Angelman/fisiopatología , Cerebelo/fisiopatología , Cuerpo Estriado/fisiopatología , Actividad Motora , Receptor de Adenosina A2A/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Fosfoproteína 32 Regulada por Dopamina y AMPc/metabolismo , Fuerza de la Mano , Ratones Endogámicos C57BL , Actividad Motora/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neurotransmisores/metabolismo , Fosforilación/efectos de los fármacos , Pirimidinas/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Triazoles/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Angelman syndrome is a complex neurodevelopmental disorder characterized by delayed development, intellectual disability, speech impairment, and ataxia. It results from the loss of UBE3A protein, an E3 ubiquitin ligase, in neurons of the brain. Despite the dynamic spatiotemporal expression of UBE3A observed in rodents and the potential clinical importance of when and where it is expressed, its expression pattern in humans remains unknown. This reflects a common challenge of studying human neurodevelopment: prenatal periods are hard to access experimentally. In this work, human cerebral organoids reveal a change from weak to strong UBE3A in neuronal nuclei within 3 weeks of culture. Angelman syndrome human induced pluripotent stem cell-derived organoids also exhibit early silencing of paternal UBE3A, with topoisomerase inhibitors partially rescuing UBE3A levels and calcium transient phenotypes. This work establishes human cerebral organoids as an important model for studying UBE3A and motivates their broader use in understanding complex neurodevelopmental disorders.
Asunto(s)
Cerebro/metabolismo , Organoides/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Síndrome de Angelman/patología , Línea Celular , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Impresión Genómica/efectos de los fármacos , Humanos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Organoides/efectos de los fármacos , Factores de Tiempo , Inhibidores de Topoisomerasa/farmacologíaRESUMEN
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.
Asunto(s)
Síndrome de Angelman/genética , Neuronas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Empalme Alternativo/genética , Síndrome de Angelman/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Impresión Genómica/genética , Humanos , Ratones , Neuronas/patología , Isoformas de Proteínas/genéticaRESUMEN
Prader-Willi syndrome (PWS) is a prototypic genetic condition related to imprinting. Causative mechanisms include paternal 15q11-q13 deletion, maternal chromosome 15 uniparental disomy (UPD15), Prader-Willi Syndrome/Angelman Syndrome (PWS/AS) critical region imprinting defects, and complex chromosomal rearrangements. Maternal UPD15-related PWS poses risks of concomitant autosomal recessive (AR) disorders when the mother carries a pathogenic variant in one of the genes on chromosome 15 associated with autosomal recessive inherited disease. Co-occurrence of autosomal recessive conditions in the setting of UPD leads to increased complexity of the clinical phenotype, and may delay the diagnosis of PWS. We report a patient with PWS and associated congenital ichthyosis due to maternal UPD15, and a homozygous novel pathogenic variant in ceramide synthase 3 (CERS3). We also review the literature of associated disorders reported in the setting of maternal UPD15-related PWS and provide a summary of the previously described CERS3 variants. This represents the second case of autosomal recessive congenital ichthyosis (ARCI) in the setting of PWS and UPD15. There needs to be a high index of suspicion of this genetic mechanism when there is unexpected phenotype or evolution of the clinical course in a patient with PWS.
Asunto(s)
Síndrome de Angelman/genética , Ictiosis/genética , Síndrome de Prader-Willi/genética , Esfingosina N-Aciltransferasa/genética , Adolescente , Adulto , Síndrome de Angelman/patología , Niño , Preescolar , Cromosomas Humanos Par 15/genética , Anomalías Congénitas/diagnóstico , Anomalías Congénitas/genética , Anomalías Congénitas/patología , Femenino , Genes Recesivos/genética , Impresión Genómica/genética , Humanos , Ictiosis/complicaciones , Ictiosis/patología , Hibridación Fluorescente in Situ , Lactante , Recién Nacido , Herencia Materna/genética , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/patología , Disomía Uniparental/diagnóstico , Disomía Uniparental/genética , Disomía Uniparental/patología , Adulto JovenRESUMEN
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.
Asunto(s)
Síndrome de Angelman/genética , Predisposición Genética a la Enfermedad , Ubiquitina-Proteína Ligasas/genética , Síndrome de Angelman/patología , Animales , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Fenómenos Electrofisiológicos/genética , Impresión Genómica/genética , Células Madre Embrionarias Humanas/metabolismo , Células Madre Embrionarias Humanas/patología , Humanos , Ratones , Neuronas/metabolismo , Neuronas/patología , Isoformas de Proteínas/genéticaRESUMEN
Angelman syndrome (AS) is a genetic neurodevelopmental disorder caused by loss or deficient expression of UBE3A on the maternally inherited allele. In 10-15% of individuals with a clinical diagnosis of AS, a molecular diagnosis cannot be established with conventional testing. We describe a 13-year-old male with an atypical presentation of AS, who was found to have a novel, maternally inherited, intronic variant in UBE3A (c.3-12T>A) using genome sequencing (GS). Targeted sequencing of RNA isolated from blood confirmed the creation of a new acceptor splice site. These GS results ended a six-year diagnostic odyssey and revealed a 50% recurrence risk for the unaffected parents. This case illustrates a previously unreported splicing variant causing AS. Intronic variants identifiable by GS may account for a proportion of individuals who are suspected of having well-known genetic disorders despite negative prior genetic testing.
Asunto(s)
Síndrome de Angelman/genética , Predisposición Genética a la Enfermedad , Intrones/genética , Ubiquitina-Proteína Ligasas/genética , Adolescente , Alelos , Síndrome de Angelman/patología , Niño , Mapeo Cromosómico , Variación Genética/genética , Humanos , Masculino , Mutación/genética , Sitios de Empalme de ARN/genética , Secuenciación Completa del Genoma/métodosRESUMEN
Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by speech impairment, intellectual disability, ataxia, and epilepsy. AS is caused by mutations in the maternal copy of UBE3A located on chromosome 15q11-13. UBE3A codes for E6AP (E6 Associated Protein), a prominent member of the HECT (Homologous to E6AP C-Terminus) E3 ubiquitin ligase family. E6AP catalyzes the posttranslational attachment of ubiquitin via its HECT domain onto various intracellular target proteins to regulate DNA repair and cell cycle progression. The HECT domain consists of an N-lobe, required for E2~ubiquitin recruitment, while the C-lobe contains the conserved catalytic cysteine required for ubiquitin transfer. Previous genetic studies of AS patients have identified point mutations in UBE3A that result in amino acid substitutions or premature termination during translation. An AS transversion mutation (codon change from ATA to AAA) within the region of the gene that codes for the catalytic HECT domain of E6AP has been annotated (I827K), but the molecular basis for this loss of function substitution remained elusive. Here, we demonstrate that the I827K substitution destabilizes the 3D fold causing protein aggregation of the C-terminal lobe of E6AP using a combination of spectropolarimetry and nuclear magnetic resonance (NMR) spectroscopy. Our fluorescent ubiquitin activity assays with E6AP-I827K show decreased ubiquitin thiolester formation and ubiquitin discharge. Using 3D models in combination with our biochemical and biophysical results, we rationalize why the I827K disrupts E6AP-dependent ubiquitylation. This work provides new insight into the E6AP mechanism and how its malfunction can be linked to the AS phenotype.