Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice

AbstractIn humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in > 1–2% of patients with autism spectrum disorders—a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 can be directly ubiquitylated by UBE3A. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

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Deleting a UBE3A substrate rescues impaired hippocampal physiology and learning in Angelman syndrome mice

10.1101/625418 ◽

2019 ◽

Author(s):

Gabrielle L. Sell ◽

Wendy Xin ◽

Emily K. Cook ◽

Mark A. Zbinden ◽

Thomas B. Schaffer ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Angelman Syndrome ◽

Developmental Disorders ◽

Motor Coordination ◽

Synapse Formation ◽

Neurodevelopmental Disorder ◽

Autism Spectrum ◽

Loss Of Function ◽

Altered Expression ◽

Exciting Potential

ABSTRACTIn humans, loss-of-function mutations in the UBE3A gene lead to the neurodevelopmental disorder Angelman syndrome (AS). AS patients have severe impairments in speech, learning and memory, and motor coordination, for which there is currently no treatment. In addition, UBE3A is duplicated in >1-2% of patients with autism spectrum disorders – a further indication of the significant role it plays in brain development. Altered expression of UBE3A, an E3 ubiquitin ligase, is hypothesized to lead to impaired levels of its target proteins, but identifying the contribution of individual UBE3A targets to UBE3A-dependent deficits remains of critical importance. Ephexin5 is a putative UBE3A substrate that has restricted expression early in development, regulates synapse formation during hippocampal development, and is abnormally elevated in AS mice, modeled by maternally-derived Ube3a gene deletion. Here, we report that Ephexin5 is a direct substrate of UBE3A ubiquitin ligase activity. Furthermore, removing Ephexin5 from AS mice specifically rescued hippocampus-dependent behaviors, CA1 physiology, and deficits in dendritic spine number. Our findings identify Ephexin5 as a key driver of hippocampal dysfunction and related behavioral deficits in AS mouse models. These results demonstrate the exciting potential of targeting Ephexin5, and possibly other UBE3A substrates, to improve symptoms of AS and other UBE3A-related developmental disorders.

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Seizure-like activity in a juvenile Angelman syndrome mouse model is attenuated by reducing Arc expression

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1504809112 ◽

2015 ◽

Vol 112 (16) ◽

pp. 5129-5134 ◽

Cited By ~ 45

Author(s):

Caleigh Mandel-Brehm ◽

John Salogiannis ◽

Sameer C. Dhamne ◽

Alexander Rotenberg ◽

Michael E. Greenberg

Keyword(s):

Angelman Syndrome ◽

Motor Coordination ◽

Neural Circuit ◽

Brain Activity ◽

Neurodevelopmental Disorder ◽

Ultrasonic Vocalizations ◽

Therapeutic Interventions ◽

Motor Deficits ◽

Loss Of Function ◽

Circuit Defects

Angelman syndrome (AS) is a neurodevelopmental disorder arising from loss-of-function mutations in the maternally inherited copy of the UBE3A gene, and is characterized by an absence of speech, excessive laughter, cognitive delay, motor deficits, and seizures. Despite the fact that the symptoms of AS occur in early childhood, behavioral characterization of AS mouse models has focused primarily on adult phenotypes. In this report we describe juvenile behaviors in AS mice that are strain-independent and clinically relevant. We find that young AS mice, compared with their wild-type littermates, produce an increased number of ultrasonic vocalizations. In addition, young AS mice have defects in motor coordination, as well as abnormal brain activity that results in an enhanced seizure-like response to an audiogenic challenge. The enhanced seizure-like activity, but not the increased ultrasonic vocalizations or motor deficits, is rescued in juvenile AS mice by genetically reducing the expression level of the activity-regulated cytoskeleton-associated protein, Arc. These findings suggest that therapeutic interventions that reduce the level of Arc expression have the potential to reverse the seizures associated with AS. In addition, the identification of aberrant behaviors in young AS mice may provide clues regarding the neural circuit defects that occur in AS and ultimately allow new approaches for treating this disorder.

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Intranasal oxytocin administration ameliorates social behavioral deficits in a POGZWT/Q1038R mouse model of autism spectrum disorder

Molecular Brain ◽

10.1186/s13041-021-00769-8 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Kohei Kitagawa ◽

Kensuke Matsumura ◽

Masayuki Baba ◽

Momoka Kondo ◽

Tomoya Takemoto ◽

...

Keyword(s):

Autism Spectrum Disorder ◽

Social Behavior ◽

Mouse Model ◽

Mouse Models ◽

De Novo ◽

Neurodevelopmental Disorder ◽

Autism Spectrum ◽

Spectrum Disorder ◽

De Novo Mutation ◽

Behavioral Deficits

AbstractAutism spectrum disorder (ASD) is a highly prevalent neurodevelopmental disorder characterized by core symptoms of impaired social behavior and communication. Recent studies have suggested that the oxytocin system, which regulates social behavior in mammals, is potentially involved in ASD. Mouse models of ASD provide a useful system for understanding the associations between an impaired oxytocin system and social behavior deficits. However, limited studies have shown the involvement of the oxytocin system in the behavioral phenotypes in mouse models of ASD. We have previously demonstrated that a mouse model that carries the ASD patient-derived de novo mutation in the pogo transposable element derived with zinc finger domain (POGZWT/Q1038R mice), showed ASD-like social behavioral deficits. Here, we have explored whether oxytocin (OXT) administration improves impaired social behavior in POGZWT/Q1038R mice and found that intranasal oxytocin administration effectively restored the impaired social behavior in POGZWT/Q1038R mice. We also found that the expression level of the oxytocin receptor gene (OXTR) was low in POGZWT/Q1038R mice. However, we did not detect significant changes in the number of OXT-expressing neurons between the paraventricular nucleus of POGZWT/Q1038R mice and that of WT mice. A chromatin immunoprecipitation assay revealed that POGZ binds to the promoter region of OXTR and is involved in the transcriptional regulation of OXTR. In summary, our study demonstrate that the pathogenic mutation in the POGZ, a high-confidence ASD gene, impairs the oxytocin system and social behavior in mice, providing insights into the development of oxytocin-based therapeutics for ASD.

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Integrating de novo and inherited variants in over 42,607 autism cases identifies mutations in new moderate risk genes

10.1101/2021.10.08.21264256 ◽

2021 ◽

Author(s):

Xueya Zhou ◽

Pamela Feliciano ◽

Tianyun Wang ◽

Irina Astrovskaya ◽

Chang Shu ◽

...

Keyword(s):

Genetic Risk ◽

De Novo ◽

Meta Analysis ◽

Neurodevelopmental Disorder ◽

Autism Spectrum ◽

Loss Of Function ◽

Moderate Risk ◽

Full Spectrum ◽

Risk Genes ◽

Biological Parents

AbstractDespite the known heritable nature of autism spectrum disorder (ASD), studies have primarily identified risk genes with de novo variants (DNVs). To capture the full spectrum of ASD genetic risk, we performed a two-stage analysis of rare de novo and inherited coding variants in 42,607 ASD cases, including 35,130 new cases recruited online by SPARK. In the first stage, we analyzed 19,843 cases with one or both biological parents and found that known ASD or neurodevelopmental disorder (NDD) risk genes explain nearly 70% of the genetic burden conferred by DNVs. In contrast, less than 20% of genetic risk conferred by rare inherited loss-of-function (LoF) variants are explained by known ASD/NDD genes. We selected 404 genes based on the first stage of analysis and performed a meta-analysis with an additional 22,764 cases and 236,000 population controls. We identified 60 genes with exome-wide significance (p < 2.5e-6), including five new risk genes (NAV3, ITSN1, MARK2, SCAF1, and HNRNPUL2). The association of NAV3 with ASD risk is entirely driven by rare inherited LoFs variants, with an average relative risk of 4, consistent with moderate effect. ASD individuals with LoF variants in the four moderate risk genes (NAV3, ITSN1, SCAF1, and HNRNPUL2, n = 95) have less cognitive impairment compared to 129 ASD individuals with LoF variants in well-established, highly penetrant ASD risk genes (CHD8, SCN2A, ADNP, FOXP1, SHANK3) (59% vs. 88%, p= 1.9e-06). These findings will guide future gene discovery efforts and suggest that much larger numbers of ASD cases and controls are needed to identify additional genes that confer moderate risk of ASD through rare, inherited variants.

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UBE3A-mediated PTPA ubiquitination and degradation regulate PP2A activity and dendritic spine morphology

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1820131116 ◽

2019 ◽

Vol 116 (25) ◽

pp. 12500-12505 ◽

Cited By ~ 7

Author(s):

Jie Wang ◽

Sen-Sen Lou ◽

Tingting Wang ◽

Rong-Jie Wu ◽

Guangying Li ◽

...

Keyword(s):

Ubiquitin Ligase ◽

Dendritic Spine ◽

Isotope Labeling ◽

Neurodevelopmental Disorder ◽

Critical Role ◽

Motor Impairment ◽

Autism Spectrum ◽

Loss Of Function ◽

Pp2a Activity

Deficiency in the E3 ubiquitin ligase UBE3A leads to the neurodevelopmental disorder Angelman syndrome (AS), while additional dosage of UBE3A is linked to autism spectrum disorder. The mechanisms underlying the downstream effects of UBE3A gain or loss of function in these neurodevelopmental disorders are still not well understood, and effective treatments are lacking. Here, using stable-isotope labeling of amino acids in mammals and ubiquitination assays, we identify PTPA, an activator of protein phosphatase 2A (PP2A), as a bona fide ubiquitin ligase substrate of UBE3A. Maternal loss of Ube3a (Ube3am−/p+) increased PTPA level, promoted PP2A holoenzyme assembly, and elevated PP2A activity, while maternal 15q11–13 duplication containing Ube3a down-regulated PTPA level and lowered PP2A activity. Reducing PTPA level in vivo restored the defects in dendritic spine maturation in Ube3am−/p+ mice. Moreover, pharmacological inhibition of PP2A activity with the small molecule LB-100 alleviated both reduction in excitatory synaptic transmission and motor impairment in Ube3am−/p+ mice. Together, our results implicate a critical role of UBE3A-PTPA-PP2A signaling in the pathogenesis of UBE3A-related disorders and suggest that PP2A-based drugs could be potential therapeutic candidates for treatment of UBE3A-related disorders.

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BIOLOGICAL SCIENCES: Genetics

10.1101/404061 ◽

2018 ◽

Author(s):

Jack S. Hsiao ◽

Noelle D. Germain ◽

Andrea Wilderman ◽

Christopher Stoddard ◽

Luke A. Wojenski ◽

...

Keyword(s):

Boundary Element ◽

Angelman Syndrome ◽

Antisense Transcript ◽

Neurodevelopmental Disorder ◽

Boundary Function ◽

Paternal Allele ◽

Loss Of Function ◽

Maternal Allele ◽

Specific Expression ◽

Human Neurons

ABSTRACTAngelman 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 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 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.SIGNIFICANCE STATEMENTAngelman syndrome is a neurodevelopmental disorder caused by loss of function from the maternal allele of UBE3A, an imprinted gene. The paternal allele of UBE3A is silenced by a long, non-coding antisense transcript in mature neurons. We have identified a boundary element that stops the transcription of the antisense transcript in human pluripotent stem cells, and thus restricts UBE3A imprinted expression to neurons. We further determined that UBE3A imprinting requires both the loss of the boundary function and sufficient expression of the antisense transcript to silence paternal UBE3A. These findings provide essential details about the mechanisms of UBE3A imprinting that may suggest additional therapeutic approaches for Angelman syndrome.

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Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis

Nature Communications ◽

10.1038/s41467-021-27156-0 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Kellan P. Weston ◽

Xiaoyi Gao ◽

Jinghan Zhao ◽

Kwang-Soo Kim ◽

Susan E. Maloney ◽

...

Keyword(s):

Angelman Syndrome ◽

Large Scale ◽

Phenotypic Diversity ◽

Function Analysis ◽

Genetic Disorders ◽

Neurodevelopmental Disorder ◽

Loss Of Function ◽

Monogenic Disorders ◽

Functional Variant ◽

Variant Analysis

AbstractThe mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

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Insulin-like growth factor-2 does not improve behavioral deficits in mouse and rat models of Angelman Syndrome

10.1101/2021.08.13.456299 ◽

2021 ◽

Author(s):

Elizabeth Berg ◽

Stela Petkova ◽

Heather A Born ◽

Anna Adhikari ◽

Anne E Anderson ◽

...

Keyword(s):

Growth Factor ◽

Angelman Syndrome ◽

Motor Behavior ◽

Neurodevelopmental Disorder ◽

Model Systems ◽

Seizure Threshold ◽

Insulin Like Growth Factor ◽

Behavioral Deficits ◽

Rat Models

Background: Angelman Syndrome (AS) is a rare neurodevelopmental disorder for which there is currently no cure or effective therapeutic. Since the genetic cause of AS is known to be dysfunctional expression of the maternal allele of ubiquitin protein ligase E3A (UBE3A), several genetic animal models of AS have been developed. Both the Ube3a maternal deletion mouse and rat models of AS reliably demonstrate behavioral phenotypes of relevance to AS and therefore offer suitable in vivo systems in which to test potential therapeutics. One promising candidate treatment is insulin-like growth factor-2 (IGF-2), which has recently been shown to ameliorate behavioral deficits in the mouse model of AS and improve cognitive abilities across model systems. Methods: We used both the Ube3a maternal deletion mouse and rat models of AS to evaluate the ability of IGF-2 to improve electrophysiological and behavioral outcomes. Results: Acute systemic administration of IGF-2 had an effect on electrophysiological activity in the brain and on a metric of motor ability, however the effects were not enduring or extensive. Additional metrics of motor behavior, learning, ambulation, and coordination were unaffected and IGF-2 did not improve social communication, seizure threshold, or cognition. Limitations: The generalizability of these results to humans is difficult to predict and it remains possible that dosing schemes (i.e., chronic or subchronic dosing), routes, and/or post-treatment intervals other than that used herein may show more efficacy. Conclusions: Despite a few observed effects of IGF-2, our results taken together indicate that IGF-2 treatment does not profoundly improve behavioral deficits in mice or rat models of AS. These findings shed cautionary light on the potential utility of acute systemic IGF-2 administration in the treatment of AS.

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Biophysical classification of a CACNA1D de novo mutation as a high-risk mutation for a severe neurodevelopmental disorder

Molecular Autism ◽

10.1186/s13229-019-0310-4 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 10

Author(s):

Nadja T. Hofer ◽

Petronel Tuluc ◽

Nadine J. Ortner ◽

Yuliia V. Nikonishyna ◽

Monica L. Fernándes-Quintero ◽

...

Keyword(s):

High Risk ◽

Neurodevelopmental Disorders ◽

Developmental Disorder ◽

De Novo ◽

Disease Risk ◽

Neurodevelopmental Disorder ◽

Autism Spectrum ◽

De Novo Mutation ◽

Loss Of Function ◽

Gain Of Function

Abstract Background There is increasing evidence that de novo CACNA1D missense mutations inducing increased Cav1.3 L-type Ca2+-channel-function confer a high risk for neurodevelopmental disorders (autism spectrum disorder with and without neurological and endocrine symptoms). Electrophysiological studies demonstrating the presence or absence of typical gain-of-function gating changes could therefore serve as a tool to distinguish likely disease-causing from non-pathogenic de novo CACNA1D variants in affected individuals. We tested this hypothesis for mutation S652L, which has previously been reported in twins with a severe neurodevelopmental disorder in the Deciphering Developmental Disorder Study, but has not been classified as a novel disease mutation. Methods For functional characterization, wild-type and mutant Cav1.3 channel complexes were expressed in tsA-201 cells and tested for typical gain-of-function gating changes using the whole-cell patch-clamp technique. Results Mutation S652L significantly shifted the voltage-dependence of activation and steady-state inactivation to more negative potentials (~ 13–17 mV) and increased window currents at subthreshold voltages. Moreover, it slowed tail currents and increased Ca2+-levels during action potential-like stimulations, characteristic for gain-of-function changes. To provide evidence that only gain-of-function variants confer high disease risk, we also studied missense variant S652W reported in apparently healthy individuals. S652W shifted activation and inactivation to more positive voltages, compatible with a loss-of-function phenotype. Mutation S652L increased the sensitivity of Cav1.3 for inhibition by the dihydropyridine L-type Ca2+-channel blocker isradipine by 3–4-fold. Conclusions and limitations Our data provide evidence that gain-of-function CACNA1D mutations, such as S652L, but not loss-of-function mutations, such as S652W, cause high risk for neurodevelopmental disorders including autism. This adds CACNA1D to the list of novel disease genes identified in the Deciphering Developmental Disorder Study. Although our study does not provide insight into the cellular mechanisms of pathological Cav1.3 signaling in neurons, we provide a unifying mechanism of gain-of-function CACNA1D mutations as a predictor for disease risk, which may allow the establishment of a more reliable diagnosis of affected individuals. Moreover, the increased sensitivity of S652L to isradipine encourages a therapeutic trial in the two affected individuals. This can address the important question to which extent symptoms are responsive to therapy with Ca2+-channel blockers.

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Cul3 regulates cytoskeleton protein homeostasis and cell migration during a critical window of brain development

Nature Communications ◽

10.1038/s41467-021-23123-x ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Jasmin Morandell ◽

Lena A. Schwarz ◽

Bernadette Basilico ◽

Saren Tasciyan ◽

Georgi Dimchev ◽

...

Keyword(s):

Cell Migration ◽

Transcriptional Activation ◽

Neuronal Migration ◽

Motor Coordination ◽

De Novo ◽

Autism Spectrum ◽

Loss Of Function ◽

Critical Window ◽

Cytoskeleton Organization

AbstractDe novo loss of function mutations in the ubiquitin ligase-encoding gene Cullin3 (CUL3) lead to autism spectrum disorder (ASD). In mouse, constitutive Cul3 haploinsufficiency leads to motor coordination deficits as well as ASD-relevant social and cognitive impairments. However, induction of Cul3 haploinsufficiency later in life does not lead to ASD-relevant behaviors, pointing to an important role of Cul3 during a critical developmental window. Here we show that Cul3 is essential to regulate neuronal migration and, therefore, constitutive Cul3 heterozygous mutant mice display cortical lamination abnormalities. At the molecular level, we found that Cul3 controls neuronal migration by tightly regulating the amount of Plastin3 (Pls3), a previously unrecognized player of neural migration. Furthermore, we found that Pls3 cell-autonomously regulates cell migration by regulating actin cytoskeleton organization, and its levels are inversely proportional to neural migration speed. Finally, we provide evidence that cellular phenotypes associated with autism-linked gene haploinsufficiency can be rescued by transcriptional activation of the intact allele in vitro, offering a proof of concept for a potential therapeutic approach for ASDs.

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