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Angelman syndrome (AS) is a severe neurodevelopmental disorder (NDD) caused by loss of functional ubiquitin protein ligase E3A (UBE3A). Previous studies showed that UBE3A plays an important role in the first postnatal weeks of mouse brain development, but its precise role is unknown. Since impaired striatal maturation has been implicated in several mouse models for NDDs, we studied the importance of UBE3A in striatal maturation. We used inducible Ube3a mouse models to investigate the maturation of medium spiny neurons (MSNs) from dorsomedial striatum. MSNs of mutant mice matured properly till postnatal day 15 (P15) but remained hyperexcitable with fewer excitatory synaptic events at later ages, indicative of stalled striatal maturation in Ube3a mice. Reinstatement of UBE3A expression at P21 fully restored MSN excitability but only partially restored synaptic transmission and the operant conditioning behavioral phenotype. Gene reinstatement at P70 failed to rescue both electrophysiological and behavioral phenotypes. In contrast, deletion of Ube3a after normal brain development did not result in these electrophysiological and behavioral phenotypes. This study emphasizes the role of UBE3A in striatal maturation and the importance of early postnatal reinstatement of UBE3A expression to obtain a full rescue of behavioral phenotypes associated with striatal function in AS.

Angelman Syndrome (AS) is a rare genetic disorder caused by lack of maternal UBE3A protein due to a deletion of the chromosome 15q11.2-q13 region, uniparental paternal disomy, imprinting center defect, or pathogenic variant in the UBE3A gene. Characteristics are developmental delay, epilepsy, behavioral, and sleep problems. There is some evidence for hyperphagia, shorter stature, and higher BMI compared to neurotypical children, but longitudinal studies on growth are lacking. In this study, we analyzed prospectively collected data of 145 children with AS, who visited the ENCORE Expertise Center between 2010 and 2021, with a total of 853 visits. Children showed an elevated mean score of 25 on the Dykens Hyperphagia questionnaire (range 11–55) without genotype association. Higher scores were significantly associated with higher body mass index (BMI) standard deviation scores (SDS) (p = 0.004). Mean height was −1.2 SDS (SD 1.3), mean BMI-SDS was 0.6 (SD 1.7); 43% had a BMI-SDS > 1 and 20% had a BMI-SDS > 2. Higher BMI-SDS was significantly associated with non-deletion genotype (p = 0.037) and walking independently (p = 0.023). Height SDS decreased significantly with age (p < 0.001) and BMI-SDS increased significantly with age (p < 0.001. Onset of puberty was normal. In conclusion, children with AS showed moderate hyperphagia, lower height SDS, and higher BMI-SDS compared to norm data, with increasing deviation from the norm with age. It is uncertain how loss of maternal UBE3A function may influence growth. Attention to diet, exercise, and hyperphagia from an early age is recommended to prevent obesity and associated health problems.

Chromosome 15q11.2–q13.1 duplication syndrome (Dup15q syndrome) is a severe neurodevelopmental disorder characterized by intellectual disability, impaired motor coordination, and autism spectrum disorder. Chromosomal multiplication of the UBE3A gene is presumed to be the primary driver of Dup15q pathophysiology, given that UBE3A exhibits maternal monoallelic expression in neurons and that maternal duplications typically yield far more severe neurodevelopmental outcomes than paternal duplications. However, studies into the pathogenic effects of UBE3A overexpression in mice have yielded conflicting results. Here, we investigated the neurodevelopmental impact of Ube3a gene overdosage using bacterial artificial chromosome–based transgenic mouse models (Ube3aOE) that recapitulate the increases in Ube3a copy number most often observed in Dup15q. In contrast to previously published Ube3a overexpression models, Ube3aOE mice were indistinguishable from wild-type controls on a number of molecular and behavioral measures, despite suffering increased mortality when challenged with seizures, a phenotype reminiscent of sudden unexpected death in epilepsy. Collectively, our data support a model wherein pathogenic synergy between UBE3A and other overexpressed 15q11.2–q13.1 genes is required for full penetrance of Dup15q syndrome phenotypes.

Abstract Background RASopathies are a group of disorders that result from mutations in genes coding for proteins involved in regulating the Ras-MAPK signaling pathway, and have an increased incidence of autism spectrum disorder (ASD). Legius syndrome is a rare RASopathy caused by loss-of-function mutations in the SPRED1 gene. The patient phenotype is similar to, but milder than, Neurofibromatosis type 1—another RASopathy caused by loss-of-function mutations in the NF1 gene. RASopathies exhibit increased activation of Ras-MAPK signaling and commonly manifest with cognitive impairments and ASD. Here, we investigated if a Spred1-/- mouse model for Legius syndrome recapitulates ASD-like symptoms, and whether targeting the Ras-MAPK pathway has therapeutic potential in this RASopathy mouse model. Methods We investigated social and communicative behaviors in Spred1-/- mice and probed therapeutic mechanisms underlying the observed behavioral phenotypes by pharmacological targeting of the Ras-MAPK pathway with the MEK inhibitor PD325901. Results Spred1-/- mice have robust increases in social dominance in the automated tube test and reduced adult ultrasonic vocalizations during social communication. Neonatal ultrasonic vocalization was also altered, with significant differences in spectral properties. Spred1-/- mice also exhibit impaired nesting behavior. Acute MEK inhibitor treatment in adulthood with PD325901 reversed the enhanced social dominance in Spred1-/- mice to normal levels, and improved nesting behavior in adult Spred1-/- mice. Limitations This study used an acute treatment protocol to administer the drug. It is not known what the effects of longer-term treatment would be on behavior. Further studies titrating the lowest dose of this drug that is required to alter Spred1-/- social behavior are still required. Finally, our findings are in a homozygous mouse model, whereas patients carry heterozygous mutations. These factors should be considered before any translational conclusions are drawn. Conclusions These results demonstrate for the first time that social behavior phenotypes in a mouse model for RASopathies (Spred1-/-) can be acutely reversed. This highlights a key role for Ras-MAPK dysregulation in mediating social behavior phenotypes in mouse models for ASD, suggesting that proper regulation of Ras-MAPK signaling is important for social behavior.

ObjectiveThe etiology of autism spectrum disorder (ASD) remains unclear, due to genetic heterogeneity and heterogeneity in symptoms across individuals. This study compares ASD symptomatology between monogenetic syndromes with a high ASD prevalence, in order to reveal syndrome specific vulnerabilities and to clarify how genetic variations affect ASD symptom presentation.MethodsWe assessed ASD symptom severity in children and young adults (aged 0-28 years) with Fragile X Syndrome (FXS, n = 60), Angelman Syndrome (AS, n = 91), Neurofibromatosis Type 1 (NF1, n = 279) and Tuberous Sclerosis Complex (TSC, n = 110), using the Autism Diagnostic Observation Schedule and Social Responsiveness Scale. Assessments were part of routine clinical care at the ENCORE expertise center in Rotterdam, the Netherlands. First, we compared the syndrome groups on the ASD classification prevalence and ASD severity scores. Then, we compared individuals in our syndrome groups with an ASD classification to a non-syndromic ASD group (nsASD, n = 335), on both ASD severity scores and ASD symptom profiles. Severity scores were compared using MANCOVAs with IQ and gender as covariates.ResultsOverall, ASD severity scores were highest for the FXS group and lowest for the NF1 group. Compared to nsASD, individuals with an ASD classification in our syndrome groups showed less problems on the instruments' social domains. We found a relative strength in the AS group on the social cognition, communication and motivation domains and a relative challenge in creativity; a relative strength of the NF1 group on the restricted interests and repetitive behavior scale; and a relative challenge in the FXS and TSC groups on the restricted interests and repetitive behavior domain.ConclusionThe syndrome-specific strengths and challenges we found provide a frame of reference to evaluate an individual's symptoms relative to the larger syndromic population and to guide treatment decisions. Our findings support the need for personalized care and a dimensional, symptom-based diagnostic approach, in contrast to a dichotomous ASD diagnosis used as a prerequisite for access to healthcare services. Similarities in ASD symptom profiles between AS and FXS, and between NF1 and TSC may reflect similarities in their neurobiology. Deep phenotyping studies are required to link neurobiological markers to ASD symptomatology.

Abstract Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by brain-specific loss of UBE3A, an E3 ubiquitin protein ligase. A substantial number of possible ubiquitination targets of UBE3A have been identified, although evidence of being direct UBE3A substrates is often lacking. Here we identified the synaptic protein Rabphilin-3a (RPH3A), an effector of the RAB3A small GTPase involved in axonal vesicle priming and docking, as a ubiquitination target of UBE3A. We found that the UBE3A and RAB3A binding sites on RPH3A partially overlap, and that RAB3A binding to RPH3A interferes with UBE3A binding. We confirmed previous observations that RPH3A levels are critically dependent on RAB3A binding but, rather surprisingly, we found that the reduced RPH3A levels in the absence of RAB3A are not mediated by UBE3A. Indeed, while we found that RPH3A is ubiquitinated in a UBE3A-dependent manner in mouse brain, UBE3A mono-ubiquitinates RPH3A and does not facilitate RPH3A degradation. Moreover, we found that an AS-linked UBE3A missense mutation in the UBE3A region that interacts with RPH3A, abrogates the interaction with RPH3A. In conclusion, our results identify RPH3A as a novel target of UBE3A and suggest that UBE3A-dependent ubiquitination of RPH3A serves a non-degradative function.

BackgroundA low test positivity rate is key to keeping the COVID-19 pandemic under control. Throughout the pandemic, several migrant groups in Norway have seen higher rates of confirmed COVID-19 and related hospitalizations, while test positivity has remained high in the same groups. The Norwegian government has used several platforms for communication, and targeted social media advertisements have in particular been an important part of the communication strategy to reach these groups. ObjectiveIn this study, we aimed to investigate whether such a targeted Facebook campaign increased the rate of COVID-19 tests performed in certain migrant groups. MethodsWe randomly assigned 386 Norwegian municipalities and city districts to intervention or control groups. Individuals born in Eritrea, Iraq, Pakistan, Poland, Russia, Somalia, Syria, and Turkey residing in intervention areas were targeted with a social media campaign aiming at increasing the COVID-19 test rate. The campaign message was in a simple language and conveyed in the users’ main language or in English. ResultsDuring the 2-week follow-up period, the predicted probability of having a COVID-19 test taken was 4.82% (95% CI 4.47%-5.18%) in the control group, and 5.58% (95% CI 5.20%-5.99%) in the intervention group (P=.004). ConclusionsOur targeted social media intervention led to a modest increase in test rates among certain migrant groups in Norway. Trial RegistrationClinicalTrials.gov NCT04866589; https://clinicaltrials.gov/ct2/show/NCT04866589

Teaching early Christianity in Nigeria and elsewhere in Africa since 2002, has convinced this author how important it is for African Christians to know of the deep roots of Christianity in Africa, and recognize the important early African theologians, Tertullian, Origen, Athanasius and Augustine, just to name a few. This argument has a significant precedent among 19th century African Christians encouraged by the unbroken presence of Christianity from antiquity in Ethiopia. In the US, Thomas Oden promoted the study of pre-Islamic Christian Africa through the Centre for Early African Christianity, and publications like the series, Ancient Christian Commentary on Scripture. This Centre has also encouraged universities in Africa to get involved in deciphering archeological materials and documents from North African sites as evidence for Christianity from its earliest days; the study of such documents has recently been established at the University of Jos (Plateau State, Nigeria). These initiatives are doubly significant because Christianity is growing phenomenally throughout Africa and is often accused of being “mile wide and an inch deep”.

The RASopathies are a group of disorders due to variations of genes associated with the Ras/MAPK pathway. Some of the RASopathies include neurofibromatosis type 1 (NF1), Noonan syndrome, Noonan syndrome with multiple lentigines, cardiofaciocutaneous (CFC) syndrome, Costello syndrome, Legius syndrome, and capillary malformation-arteriovenous malformation (CM-AVM) syndrome. In combination, the RASopathies are a frequent group of genetic disorders. This report summarizes the proceedings of the 4th International Symposium on Genetic Disorders of the Ras/MAPK pathway and highlights gaps in the field. © 2016 Wiley Periodicals, Inc.

Cognitive impairments are a major clinical feature of the common neurogenetic disease neurofibromatosis type 1 (NF1). Previous studies have demonstrated that increased neuronal inhibition underlies the learning deficits in NF1, however, the molecular mechanism underlying this cell-type specificity has remained unknown. Here, we identify an interneuron-specific attenuation of hyperpolarization-activated cyclic nucleotide-gated (HCN) current as the cause for increased inhibition in Nf1 mutants. Mechanistically, we demonstrate that HCN1 is a novel NF1-interacting protein for which loss of NF1 results in a concomitant increase of interneuron excitability. Furthermore, the HCN channel agonist lamotrigine rescued the electrophysiological and cognitive deficits in two independent Nf1 mouse models, thereby establishing the importance of HCN channel dysfunction in NF1. Together, our results provide detailed mechanistic insights into the pathophysiology of NF1-associated cognitive defects, and identify a novel target for clinical drug development.

Abstract Background Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by the loss of functional ubiquitin protein ligase E3A (UBE3A). In neurons, UBE3A expression is tightly regulated by a mechanism of imprinting which suppresses the expression of the paternal UBE3A allele. Promising treatment strategies for AS are directed at activating paternal UBE3A gene expression. However, for such strategies to be successful, it is important to know when such a treatment should start, and how much UBE3A expression is needed for normal embryonic brain development. Methods Using a conditional mouse model of AS, we further delineated the critical period for UBE3A expression during early brain development. Ube3a gene expression was induced around the second week of gestation and mouse phenotypes were assessed using a behavioral test battery. To investigate the requirements of embryonic UBE3A expression, we made use of mice in which the paternal Ube3a allele was deleted. Results We observed a full behavioral rescue of the AS mouse model phenotypes when Ube3a gene reactivation was induced around the start of the last week of mouse embryonic development. We found that full silencing of the paternal Ube3a allele was not completed till the first week after birth but that deletion of the paternal Ube3a allele had no significant effect on the assessed phenotypes. Limitations Direct translation to human is limited, as we do not precisely know how human and mouse brain development aligns over gestational time. Moreover, many of the assessed phenotypes have limited translational value, as the underlying brain regions involved in these tasks are largely unknown. Conclusions Our findings provide further important insights in the requirement of UBE3A expression during brain development. We found that loss of up to 50% of UBE3A protein during prenatal mouse brain development does not significantly impact the assessed mouse behavioral phenotypes. Together with previous findings, our results indicate that the most critical function for mouse UBE3A lies in the early postnatal period between birth and P21.

BackgroundMajor depressive disorder (MDD) is a severe psychiatric disorder that is associated with various cognitive impairments, including learning and memory deficits. As synaptic plasticity is considered an important mechanism underlying learning and memory, deficits in cortical plasticity might play a role in the pathophysiology of patients with MDD. We used Transcranial Magnetic Stimulation (TMS) to assess inhibitory neurotransmission and cortical plasticity in the motor cortex of MDD patients and controls.MethodsWe measured the cortical silent period (CSP) and short interval cortical inhibition (SICI), as well as intermittent theta-burst stimulation (iTBS), in 9 drug-free MDD inpatients and 18 controls.ResultsThe overall response to the CSP, SICI, and iTBS paradigms was not significantly different between the patient and control groups. iTBS induction resulted in significant potentiation after 20 mins in the control group (t(17) = −2.8, p = 0.01), whereas no potentiation was observed in patients.ConclusionsPotentiation of MEP amplitudes was not observed within the MDD group. No evidence was found for medium-to-large effect size differences in CSP and SICI measures in severely depressed drug-free patients, suggesting that reduced cortical inhibition is unlikely to be a robust correlate of the pathophysiological mechanism in MDD. However, these findings should be interpreted with caution due to the high inter-subject variability and the small sample size.SignificanceThese findings advance our understanding of neurophysiological functioning in drug-free severely depressed inpatients.

Healing of burn injury is a complex process that often leads to the development of functional and aesthetic complications. To study skin regeneration in more detail, organotypic skin models, such as full skin equivalents (FSEs) generated from dermal matrices, can be used. Here, FSEs were generated using de-epidermalized dermis (DED) and collagen matrices MatriDerm® and Mucomaix®. Our aim was to validate the MatriDerm- and Mucomaix-based FSEs for the use as in vitro models of wound healing. Therefore, we first characterized the FSEs in terms of skin development and cell proliferation. Proper dermal and epidermal morphogenesis was established in all FSEs and was comparable to ex vivo human skin models. Extension of culture time improved the organization of the epidermal layers and the basement membrane in MatriDerm-based FSE but resulted in rapid degradation of the Mucomaix-based FSE. After applying a standardized burn injury to the models, re-epithelization occurred in the DED- and MatriDerm-based FSEs at 2 weeks after injury, similar to ex vivo human skin. High levels of pro-inflammatory cytokines were present in the culture media of all models, but no significant differences were observed between models. We anticipate that these animal-free in vitro models can facilitate research on skin regeneration and can be used to test therapeutic interventions in a preclinical setting to improve wound healing.

Abstract Background Although neuronal extracellular sensing is emerging as crucial for brain wiring and therefore plasticity, little is known about these processes in neurodevelopmental disorders. Ubiquitin protein ligase E3A (UBE3A) plays a key role in neurodevelopment. Lack of UBE3A leads to Angelman syndrome (AS), while its increase is among the most prevalent genetic causes of autism (e.g., Dup15q syndrome). By using microstructured substrates that can induce specific directional stimuli in cells, we previously found deficient topographical contact guidance in AS neurons, which was linked to a dysregulated activation of the focal adhesion pathway. Methods Here, we study axon and dendrite contact guidance and neuronal morphological features of wild-type, AS, and UBE3A-overexpressing neurons (Dup15q autism model) on micrograting substrates, with the aim to clarify the role of UBE3A in neuronal guidance. Results We found that loss of axonal contact guidance is specific for AS neurons while UBE3A overexpression does not affect neuronal directional polarization along microgratings. Deficits at the level of axonal branching, growth cone orientation and actin fiber content, focal adhesion (FA) effectors, and actin fiber–binding proteins were observed in AS neurons. We tested different rescue strategies for restoring correct topographical guidance in AS neurons on microgratings, by either UBE3A protein re-expression or by pharmacological treatments acting on cytoskeleton contractility. Nocodazole, a drug that depolymerizes microtubules and increases cell contractility, rescued AS axonal alignment to the gratings by partially restoring focal adhesion pathway activation. Surprisingly, UBE3A re-expression only resulted in partial rescue of the phenotype. Conclusions We identified a specific in vitro deficit in axonal topographical guidance due selectively to the loss of UBE3A, and we further demonstrate that this defective guidance can be rescued to a certain extent by pharmacological or genetic treatment strategies. Overall, cytoskeleton dynamics emerge as important partners in UBE3A-mediated contact guidance responses. These results support the view that UBE3A-related deficits in early neuronal morphogenesis may lead to defective neuronal connectivity and plasticity.

Tuberous sclerosis complex (TSC), caused by heterozygous mutations in TSC1 or TSC2, frequently results in intractable epilepsy. Here, we made use of an inducible Tsc1-knockout mouse model, allowing us to study electrophysiological and molecular changes of Tsc1-induced epileptogenesis over time. We recorded from pyramidal neurons in the hippocampus and somatosensory cortex (L2/L3) and combined this with an analysis of transcriptome changes during epileptogenesis. Deletion of Tsc1 resulted in hippocampus-specific changes in excitability and adaptation, which emerged before seizure onset and progressed over time. All phenotypes were rescued after early treatment with rapamycin, an mTOR inhibitor. Later in epileptogenesis, we observed a hippocampal increase of excitation-to-inhibition ratio. These cellular changes were accompanied by dramatic transcriptional changes, especially after seizure onset. Most of these changes were rescued upon rapamycin treatment. Of the genes encoding ion channels or belonging to the Gene Ontology term action potential, 27 were differentially expressed just before seizure onset, suggesting a potential driving role in epileptogenesis. Our data highlight the complex changes driving epileptogenesis in TSC, including the changed expression of multiple ion channels. Our study emphasizes inhibition of the TSC/mTOR signaling pathway as a promising therapeutic approach to target epilepsy in patients with TSC.

Angelman Syndrome (AS) is a severe neurodevelopmental disorder, caused by the neuronal absence of the ubiquitin protein ligase E3A (UBE3A). UBE3A promotes ubiquitin-mediated protein degradation and functions as a transcriptional coregulator of nuclear hormone receptors, including the glucocorticoid receptor (GR). Previous studies showed anxiety-like behavior and hippocampal-dependent memory disturbances in AS mouse models. Hippocampal GR is an important regulator of the stress response and memory formation, and we therefore investigated whether the absence of UBE3A in AS mice disrupted GR signaling in the hippocampus. We first established a strong cortisol-dependent interaction between the GR ligand binding domain and a UBE3A nuclear receptor box in a high-throughput interaction screen. In vivo, we found that UBE3A-deficient AS mice displayed significantly more variation in circulating corticosterone levels throughout the day compared to wildtypes (WT), with low to undetectable levels of corticosterone at the trough of the circadian cycle. Additionally, we observed an enhanced transcriptomic response in the AS hippocampus following acute corticosterone treatment. Surprisingly, chronic corticosterone treatment showed less contrast between AS and WT mice in the hippocampus and liver transcriptomic responses. This suggests that UBE3A limits the acute stimulation of GR signaling, likely as a member of the GR transcriptional complex. Altogether, these data indicate that AS mice are more sensitive to acute glucocorticoid exposure in the brain compared to WT mice. This suggests that stress responsiveness is altered in AS which could lead to anxiety symptoms.

"The Third International Meeting on Genetic Disorders in the RAS/MAPK Pathway: Towards a Therapeutic Approach" was held at the Renaissance Orlando at SeaWorld Hotel (August 2-4, 2013). Seventy-one physicians and scientists attended the meeting, and parallel meetings were held by patient advocacy groups (CFC International, Costello Syndrome Family Network, NF Network and Noonan Syndrome Foundation). Parent and patient advocates opened the meeting with a panel discussion to set the stage regarding their hopes and expectations for therapeutic advances. In keeping with the theme on therapeutic development, the sessions followed a progression from description of the phenotype and definition of therapeutic endpoints, to definition of genomic changes, to identification of therapeutic targets in the RAS/MAPK pathway, to preclinical drug development and testing, to clinical trials. These proceedings will review the major points of discussion.

Loss of the maternal UBE3A allele causes Angelman syndrome (AS), a debilitating neurodevelopmental disorder. Here, we devised an AS treatment strategy based on reinstating dual-isoform expression of human UBE3A (hUBE3A) in the developing brain. Kozak sequence engineering of our codon-optimized vector (hUBE3Aopt) enabled translation of both short and long hUBE3A protein isoforms at a near-endogenous 3:1 (short/long) ratio, a feature that could help to support optimal therapeutic outcomes. To model widespread brain delivery and early postnatal onset of hUBE3A expression, we packaged the hUBE3Aopt vector into PHP.B capsids and performed intracerebroventricular injections in neonates. This treatment significantly improved motor learning and innate behaviors in AS mice, and it rendered them resilient to epileptogenesis and associated hippocampal neuropathologies induced by seizure kindling. hUBE3A overexpression occurred frequently in the hippocampus but was uncommon in the neocortex and other major brain structures; furthermore, it did not correlate with behavioral performance. Our results demonstrate the feasibility, tolerability, and therapeutic potential for dual-isoform hUBE3A gene transfer in the treatment of AS.

Angelman syndrome (AS) is a severe neurodevelopmental disorder for which only symptomatic treatment with limited benefits is available. AS is caused by mutations affecting the maternally inherited ubiquitin protein ligase E3A (UBE3A) gene. Previous studies showed that the silenced paternal Ube3a gene can be activated by targeting the antisense Ube3a-ATS transcript. We investigated antisense oligonucleotide–induced (ASO-induced) Ube3a-ATS degradation and its ability to induce UBE3A reinstatement and rescue of AS phenotypes in an established Ube3a mouse model. We found that a single intracerebroventricular injection of ASOs at postnatal day 1 (P1) or P21 in AS mice resulted in potent and specific UBE3A reinstatement in the brain, with levels up to 74% of WT levels in the cortex and a full rescue of sensitivity to audiogenic seizures. AS mice treated with ASO at P1 also showed rescue of established AS phenotypes, such as open field and forced swim test behaviors, and significant improvement on the reversed rotarod. Hippocampal plasticity of treated AS mice was comparable to WT but not significantly different from PBS-treated AS mice. No rescue was observed for the marble burying and nest building phenotypes. Our findings highlight the promise of ASO-mediated reactivation of UBE3A as a disease-modifying treatment for AS.