Sleep EEG signatures in mouse models of 15q11.2-13.1 duplication (Dup15q) syndrome.

BACKGROUND: Sleep disturbances are a prevalent and complex comorbidity in neurodevelopmental disorders (NDDs). Dup15q syndrome (duplications of 15q11.2-13.1) is a genetic disorder highly penetrant for NDDs such as autism and intellectual disability and it is frequently accompanied by significant disruptions in sleep patterns. The 15q critical region harbors genes crucial for brain development, notably UBE3A and a cluster of gamma-aminobutyric acid type A receptor (GABAAR) genes. We previously described an electrophysiological biomarker of the syndrome, marked by heightened beta oscillations (12-30 Hz) in individuals with Dup15q syndrome, akin to electroencephalogram (EEG) alterations induced by allosteric modulation of GABAARs. Those with Dup15q syndrome exhibited increased beta oscillations during the awake resting state and during sleep, and they showed profoundly abnormal NREM sleep. This study aims to assess the translational validity of these EEG signatures and to delve into their neurobiological underpinnings by quantifying sleep physiology in chromosome-engineered mice with maternal (matDp/ + mice) or paternal (patDp/ + mice) inheritance of the full 15q11.2-13.1-equivalent duplication, and mice with duplication of just the UBE3A gene (Ube3a overexpression mice; Ube3a OE mice) and comparing the sleep metrics with their respective wildtype (WT) littermate controls. METHODS: We collected 48-h EEG/EMG recordings from 35 (23 male, 12 female) 12-24-week-old matDp/ + , patDp/ + , Ube3a OE mice, and their WT littermate controls. We quantified baseline sleep, sleep fragmentation, spectral power dynamics during sleep states, and recovery following sleep deprivation. Within each group, distinctions between Dup15q mutant mice and WT littermate controls were evaluated using analysis of variance (ANOVA) and student's t-test. The impact of genotype and time was discerned through repeated measures ANOVA, and significance was established at p < 0.05. RESULTS: Our study revealed that across brain states, matDp/ + mice mirrored the elevated beta oscillation phenotype observed in clinical EEGs from individuals with Dup15q syndrome. Time to sleep onset after light onset was significantly reduced in matDp/ + and Ube3a OE mice. However, NREM sleep between Dup15q mutant and WT littermate mice remained unaltered, suggesting a divergence from the clinical presentation in humans. Additionally, while increased beta oscillations persisted in matDp/ + mice after 6-h of sleep deprivation, recovery NREM sleep remained unaltered in all groups, thus suggesting that these mice exhibit resilience in the fundamental processes governing sleep-wake regulation. CONCLUSIONS: Quantification of mechanistic and translatable EEG biomarkers is essential for advancing our understanding of NDDs and their underlying pathophysiology. Our study of sleep physiology in the Dup15q mice underscores that the beta EEG biomarker has strong translational validity, thus opening the door for pre-clinical studies of putative drug targets, using the biomarker as a translational measure of drug-target engagement. The unaltered NREM sleep may be due to inherent differences in neurobiology between mice and humans. These nuanced distinctions highlight the complexity of sleep disruptions in Dup15q syndrome and emphasize the need for a comprehensive understanding that encompasses both shared and distinct features between murine models and clinical populations.

Interlinked destinies: How ubiquitin-proteasome and autophagy systems underpin neurocognitive outcomes.

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.

NEAT1 Promotes Valproic Acid-Induced Autism Spectrum Disorder by Recruiting YY1 to Regulate UBE3A Transcription.

Evidence suggests that long non-coding RNAs (lncRNAs) play a significant role in autism. Herein, we explored the functional role and possible molecular mechanisms of NEAT1 in valproic acid (VPA)-induced autism spectrum disorder (ASD). A VPA-induced ASD rat model was constructed, and a series of behavioral tests were performed to examine motor coordination and learning-memory abilities. qRT-PCR and western blot assays were used to evaluate target gene expression levels. Loss-and-gain-of-function assays were conducted to explore the functional role of NEAT1 in ASD development. Furthermore, a combination of mechanistic experiments and bioinformatic tools was used to assess the relationship and regulatory role of the NEAT1-YY1-UBE3A axis in ASD cellular processes. Results showed that VPA exposure induced autism-like developmental delays and behavioral abnormalities in the VPA-induced ASD rat model. We found that NEAT1 was elevated in rat hippocampal tissues after VPA exposure. NEAT1 promoted VPA-induced autism-like behaviors and mitigated apoptosis, oxidative stress, and inflammation in VPA-induced ASD rats. Notably, NEAT1 knockdown improved autism-related behaviors and ameliorated hippocampal neuronal damage. Mechanistically, it was observed that NEAT1 recruited the transcription factor YY1 to regulate UBE3A expression. Additionally, in vitro experiments further confirmed that NEAT1 knockdown mitigated hippocampal neuronal damage, oxidative stress, and inflammation through the YY1/UBE3A axis. In conclusion, our study demonstrates that NEAT1 is highly expressed in ASD, and its inhibition prominently suppresses hippocampal neuronal injury and oxidative stress through the YY1/UBE3A axis, thereby alleviating ASD development. This provides a new direction for ASD-targeted therapy.

Regional and cellular organization of the autism-associated protein UBE3A/E6AP and its antisense transcript in the brain of the developing rhesus monkey.

Angelman syndrome (AS) is a neurogenetic disorder caused by mutations or deletions in the maternally-inherited UBE3A allele, leading to a loss of UBE3A protein expression in neurons. The paternally-inherited UBE3A allele is epigenetically silenced in neurons during development by a noncoding transcript (UBE3A-ATS). The absence of neuronal UBE3A results in severe neurological symptoms, including speech and language impairments, intellectual disability, and seizures. While no cure exists, therapies aiming to restore UBE3A function-either by gene addition or by targeting UBE3A-ATS-are under development. Progress in developing these treatments relies heavily on inferences drawn from mouse studies about the function of UBE3A in the human brain. To aid translational efforts and to gain an understanding of UBE3A and UBE3A-ATS biology with greater relevance to human neurodevelopmental contexts, we investigated UBE3A and UBE3A-ATS expression in the developing brain of the rhesus macaque, a species that exhibits complex social behaviors, resembling aspects of human behavior to a greater degree than mice. Combining immunohistochemistry and in situ hybridization, we mapped UBE3A and UBE3A-ATS regional and cellular expression in normal prenatal, neonatal, and adolescent rhesus macaque brains. We show that key hallmarks of UBE3A biology, well-known in rodents, are also present in macaques, and suggest paternal UBE3A silencing in neurons-but not glial cells-in the macaque brain, with onset between gestational day 48 and 100. These findings support proposals that early-life, perhaps even prenatal, intervention is optimal for overcoming the maternal allele loss of UBE3A linked to AS.

Cobalamins Function as Allosteric Activators of an Angelman Syndrome-Associated UBE3A/E6AP Variant.

Genetic aberrations of the maternal UBE3A allele, which encodes the E3 ubiquitin ligase E6AP, are the cause of Angelman syndrome (AS), an imprinting disorder. In most cases, the maternal UBE3A allele is not expressed. Yet, approximately 10 percent of AS individuals harbor distinct point mutations in the maternal allele resulting in the expression of full-length E6AP variants that frequently display compromised ligase activity. In a high-throughput screen, we identified cyanocobalamin, a vitamin B12-derivative, and several alloxazine derivatives as activators of the AS-linked E6AP-F583S variant. Furthermore, we show by cross-linking coupled to mass spectrometry that cobalamins affect the structural dynamics of E6AP-F583S and apply limited proteolysis coupled to mass spectrometry to obtain information about the regions of E6AP that are involved in, or are affected by binding cobalamins and alloxazine derivatives. Our data suggest that dietary supplementation with vitamin B12 can be beneficial for AS individuals.

Impact of Deletion on Angelman Syndrome Phenotype Variability: Phenotype-Genotype Correlation in 97 Patients with Motor Developmental Delay.

Angelman syndrome (AS) is a rare neurodevelopmental disorder due to genetic defects involving chromosome 15, known by intellectual disability, cognitive and behavioral disorders, ataxia, delayed motor development, and seizures. This study highlights the clinical spectrum and molecular research to establish the genotype-phenotype correlation in the pediatric Moroccan population. Methylation-specific-polymerase chain reaction (MS-PCR) is a primordial technique not only to identify the genetic mechanism of AS but also to characterize the different molecular classes induced in the appearance of the clinical symptoms. Patients with positive methylation profile were additionally studied by fluorescent in situ hybridization. Sequencing analysis of the UBE3A gene was performed for patients with negative MS-PCR. We used Fisher's test to assess differences in the distribution of features frequencies among the deletional and the nondeletional group. Statistical analysis was performed using R project. We identified from 97 patients diagnosed with AS, 14 (2.06%) had a classical AS phenotype, while 70 (84.5%) patients displayed a subset of consistent and frequent criteria. Development delay was shown severe in 63% and moderate in 37%. Nineteen out of 97 of them had MS-PCR positive in which 17 (89.47%) had 15q11-q13 deletion. Deletion patients presented a higher incidence of epileptic seizures ( p = 0.04), ataxia ( p = 0.0008), and abnormal electroencephalogram (EEG) profile ( p = 0.003). We further found out a frameshift deletion located at exon 9 of the UBE3A gene discovered in a 5 years old patient. We report in this study the genotype-phenotype correlation using different molecular testing. Correlation analysis did not reveal any statistical differences in phenotypic dissimilarity between deletion and nondeletion groups for most clinical features, except the correlation was highly significant in the abnormal EEG. According to our findings, we recommend offering MS-PCR analysis to all patients with severe intellectual disability, developmental delay, speech impairment, happy demeanor, and hypopigmentation.

Functional analysis of the ube3a response in Japanese flounder (Paralichthys olivaceus) to CSBV infection.

Ube3a is a member of the E3 ubiquitin ligase HECTc family, and its role has been established in neurodevelopmental disorders. However, studies on its role in Japanese flounder are scarce. Thus, in this study, the ube3a of Japanese flounder was cloned, and its role in conferring resistance against Chinook salmon bafnivirus (CSBV) was analyzed. Japanese flounder ube3a encoded a protein containing 834 amino acids. Interestingly, its homology with the Atlantic halibut was determined to be 94%. In addition, there were differential expressions of ube3a in different tissues of Japanese flounder, with the highest expression level observed in the fin, followed by the gills and skin (P ≤ 0.05). Subcellular localization analysis revealed that Ube3a is a cytoplasmic protein. We established an in vitro CSBV infection model using Japanese flounder gill cell line (FG). After ube3a overexpression, the viral load was significantly lower than that of the control group (P ≤ 0.05). Contrastingly, after incubation of FG cells with an E3 ubiquitin ligase inhibitor, the viral load was significantly higher than in the control group (P ≤ 0.01). Then, the expression levels of nf-κb, traf3, and tnf-α after incubation with an E3 ubiquitin ligase inhibitor were examined. The results demonstrated that ube3a may exerted a significant antiviral effect in Japanese flounder via the ubiquitination pathway.

The role of glia in the dysregulation of neuronal spinogenesis in Ube3a-dependent ASD.

Overexpression of the Ube3a gene and the resulting increase in Ube3a protein are linked to autism spectrum disorder (ASD). However, the cellular and molecular processes underlying Ube3a-dependent ASD remain unclear. Using both male and female mice, we find that neurons in the somatosensory cortex of the Ube3a 2× Tg ASD mouse model display reduced dendritic spine density and increased immature filopodia density. Importantly, the increased gene dosage of Ube3a in astrocytes alone is sufficient to confer alterations in neurons as immature dendritic protrusions, as observed in primary hippocampal neuron cultures. We show that Ube3a overexpression in astrocytes leads to a loss of astrocyte-derived spinogenic protein, thrombospondin-2 (TSP2), due to a suppression of TSP2 gene transcription. By neonatal intraventricular injection of astrocyte-specific virus, we demonstrate that Ube3a overexpression in astrocytes in vivo results in a reduction in dendritic spine maturation in prelimbic cortical neurons, accompanied with autistic-like behaviors in mice. These findings reveal an astrocytic dominance in initiating ASD pathobiology at the neuronal and behavior levels. SIGNIFICANCE STATEMENT: Increased gene dosage of Ube3a is tied to autism spectrum disorders (ASDs), yet cellular and molecular alterations underlying autistic phenotypes remain unclear. We show that Ube3a overexpression leads to impaired dendritic spine maturation, resulting in reduced spine density and increased filopodia density. We find that dysregulation of spine development is not neuron autonomous, rather, it is mediated by an astrocytic mechanism. Increased gene dosage of Ube3a in astrocytes leads to reduced production of the spinogenic glycoprotein thrombospondin-2 (TSP2), leading to abnormalities in spines. Astrocyte-specific Ube3a overexpression in the brain in vivo confers dysregulated spine maturation concomitant with autistic-like behaviors in mice. These findings indicate the importance of astrocytes in aberrant neurodevelopment and brain function in Ube3a-depdendent ASD.

Azadiradione up-regulates the expression of parvalbumin and BDNF via Ube3a.

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.

Stem cell models of Angelman syndrome.

Angelman syndrome (AS) is an imprinted neurodevelopmental disorder that lacks a cure, characterized by developmental delay, intellectual impairment, seizures, ataxia, and paroxysmal laughter. The condition arises due to the loss of the maternally inherited copy of the UBE3A gene in neurons. The paternally inherited UBE3A allele is unable to compensate because it is silenced by the expression of an antisense transcript (UBE3A-ATS) on the paternal chromosome. UBE3A, encoding enigmatic E3 ubiquitin ligase variants, regulates target proteins by either modifying their properties/functions or leading them to degradation through the proteasome. Over time, animal models, particularly the Ube3a mat-/pat+ Knock-Out (KO) mice, have significantly contributed to our understanding of the molecular mechanisms underlying AS. However, a shift toward human pluripotent stem cell models (PSCs), such as human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), has gained momentum. These stem cell models accurately capture human genetic and cellular characteristics, offering an alternative or a complement to animal experimentation. Human stem cells possess the remarkable ability to recapitulate neurogenesis and generate "brain-in-a-dish" models, making them valuable tools for studying neurodevelopmental disorders like AS. In this review, we provide an overview of the current state-of-the-art human stem cell models of AS and explore their potential to become the preclinical models of choice for drug screening and development, thus propelling AS therapeutic advancements and improving the lives of affected individuals.

Light activates Ube3a, an Angelman syndrome-associated gene, by mediating the chromatin structures during postnatal development of mouse retina.

Angelman syndrome, a severe neurodevelopmental disorder, is primarily caused by mutations or deletions of maternally inherited ubiquitin protein ligase E3A (UBE3A). Activation of the silenced paternal copy of UBE3A can occur with pharmacological perturbation; however, an environmental approach has not been examined. Here, we found Ube3a is highly expressed in embryonic and early neonatal mouse retina and is maternally-, but not paternally-, expressed in ganglion cells, amacrine cells, and horizontal cells. Moreover, we analyzed UBE3A expression in the retina and visual cortex of postnatal day 28 mice (P28) following exposure to light emissions from white compact-fluorescent bulbs or blue light-emitting diodes from postnatal day 0 (P0) to 28 (P28), encompassing a crucial phase of visual system development. We found higher levels of Ube3a RNA and protein in the retina, but not visual cortex compared with tissues from P28 mice exposure to typical lighting (controls). Levels of both paternal- and maternal-UBE3A protein in mouse retina were higher than controls in P28 mice exposed to white or blue light. Moreover, levels of open and repressive chromatin structures, indicated by histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3), respectively, were increased in the Ube3a promoter from mouse retina exposed to white or blue light. Our findings strongly suggest that extended exposure to white or blue light constitutes a substantial environmental factor that can effectively promote UBE3A expression within the central nervous system.

Ubiquitin-protein ligase E3A (UBE3A) mediation of viral infection and human diseases.

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.

Enabling endpoint development for interventional clinical trials in individuals with Angelman syndrome: a prospective, longitudinal, observational clinical study (FREESIAS).

BACKGROUND: Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by the absence of a functional UBE3A gene, which causes developmental, behavioral, and medical challenges. While currently untreatable, comprehensive data could help identify appropriate endpoints assessing meaningful improvements in clinical trials. Herein are reported the results from the FREESIAS study assessing the feasibility and utility of in-clinic and at-home measures of key AS symptoms. METHODS: Fifty-five individuals with AS (aged < 5 years: n = 16, 5-12 years: n = 27, ≥ 18 years: n = 12; deletion genotype: n = 40, nondeletion genotype: n = 15) and 20 typically developing children (aged 1-12 years) were enrolled across six USA sites. Several clinical outcome assessments and digital health technologies were tested, together with overnight 19-lead electroencephalography (EEG) and additional polysomnography (PSG) sensors. Participants were assessed at baseline (Clinic Visit 1), 12 months later (Clinic Visit 2), and during intermittent home visits. RESULTS: The participants achieved high completion rates for the clinical outcome assessments (adherence: 89-100% [Clinic Visit 1]; 76-91% [Clinic Visit 2]) and varied feasibility of and adherence to digital health technologies. The coronavirus disease 2019 (COVID-19) pandemic impacted participants' uptake of and/or adherence to some measures. It also potentially impacted the at-home PSG/EEG recordings, which were otherwise feasible. Participants achieved Bayley-III results comparable to the available natural history data, showing similar scores between individuals aged ≥ 18 and 5-12 years. Also, participants without a deletion generally scored higher on most clinical outcome assessments than participants with a deletion. Furthermore, the observed AS EEG phenotype of excess delta-band power was consistent with prior reports. CONCLUSIONS: Although feasible clinical outcome assessments and digital health technologies are reported herein, further improved assessments of meaningful AS change are needed. Despite the COVID-19 pandemic, remote assessments facilitated high adherence levels and the results suggested that at-home PSG/EEG might be a feasible alternative to the in-clinic EEG assessments. Taken altogether, the combination of in-clinic/at-home clinical outcome assessments, digital health technologies, and PSG/EEG may improve protocol adherence, reduce patient burden, and optimize study outcomes in AS and other rare disease populations.

Autism-linked UBE3A gain-of-function mutation causes interneuron and behavioral phenotypes when inherited maternally or paternally in mice.

The E3 ubiquitin ligase Ube3a is biallelically expressed in neural progenitors and glial cells, suggesting that UBE3A gain-of-function mutations might cause neurodevelopmental disorders irrespective of parent of origin. Here, we engineered a mouse line that harbors an autism-linked UBE3AT485A (T503A in mouse) gain-of-function mutation and evaluated phenotypes in animals that inherited the mutant allele paternally, maternally, or from both parents. We find that paternally and maternally expressed UBE3AT503A results in elevated UBE3A activity in neural progenitors and glial cells. Expression of UBE3AT503A from the maternal allele, but not the paternal one, leads to a persistent elevation of UBE3A activity in neurons. Mutant mice display behavioral phenotypes that differ by parent of origin. Expression of UBE3AT503A, irrespective of its parent of origin, promotes transient embryonic expansion of Zcchc12 lineage interneurons. Phenotypes of Ube3aT503A mice are distinct from Angelman syndrome model mice. Our study has clinical implications for a growing number of disease-linked UBE3A gain-of-function mutations.

A Case Study of Early Diagnosed Angelman Syndrome: Recognizing Atypical Clinical Presentations.

Angelman syndrome (AS) is a rare pediatric neurological condition in which patients most commonly present with inappropriate laughter, microcephaly, speech difficulties, seizures, and movement disorders. AS can be diagnosed clinically and confirmed with genetic testing. In this case report, the patient presented with 9.3% weight loss at two days of age. Although there were multiple attempts at lactational counseling and nutritional guidance, the patient was admitted to the hospital due to failure to thrive. Due to continued global developmental delay and upper and lower extremities hypotonia by the age of nine months, the patient was referred to a neurologist. Brain MRI was negative, and genetic testing revealed 15q11.2q13.1 deletion, which is consistent with AS. Through different therapies and intervention, the patient showed slow improvements in symptoms. This case illustrates the importance of early recognition of nonspecific clinical manifestations of AS. The general management for all AS patients includes physical therapy, speech therapy, mobility support devices, education, and behavioral therapy as they progress through life. Establishing an early diagnosis has potential long-term benefits of improved quality of life and outcomes for patients via early interventions such as physical therapy starting at the age of six months to improve gross motor function. When infants present with nonspecific clinical presentations such as failure to thrive and hypotonia, clinicians should maintain a lower threshold for suspecting genetic conditions, which will facilitate early diagnosis of AS.

MiR-218-5p promotes trophoblast infiltration and inhibits endoplasmic reticulum/oxidative stress by reducing UBE3A-mediated degradation of SATB1.

This research evaluated the effects of miR-218-5p on trophoblast infiltration and endoplasmic reticulum/oxidative stress during preeclampsia (PE). The expression of miR-218-5p and special AT-rich sequence binding protein 1 (SATB1) in placental tissues from 25 patients with PE and 25 normal pregnant subjects was determined using qRT-PCR and western blotting. Cell invasion and cell migration were detected by performing Transwell assays and scratch assays, respectively. MMP-2/9, TIMP1/2, HIF-1α, p-eIF2α, and ATF4 expression in cells was assessed through western blotting. Intracellular reactive oxygen species were detected using 2,7-dichlorodihydrofluorescein diacetate, and intracellular malondialdehyde and superoxide dismutase activities were determined with kits. Dual-luciferase and RNA pull-down assays were performed to verify the interaction between miR-218-5p and UBE3A. Co-immunoprecipitation and western blotting were used to detect the ubiquitination levels of SATB1. A rat model of PE was established, and an miR-218-5p agomir was injected into rat placental tissues. The pathological characteristics of placental tissues were detected via HE staining, and MMP-2/9, TIMP1/2, p-eIF2α, and ATF4 expression in rat placental tissues was determined through western blotting. MiR-218-5p and SATB1 were expressed at low levels, while UBE3A was highly expressed in the placental tissues of patients with PE. The transfection of an miR-218-5p mimic, UBE3A shRNA, or an SATB1 overexpression vector into HTR-8/SVneo cells promoted trophoblast infiltration and inhibited endoplasmic reticulum/oxidative stress. It was determined that UBE3A is a target of miR-218-5p; UBE3A induces ubiquitin-mediated degradation of SATB1. In PE model rats, miR-218-5p alleviated pathological features, promoted trophoblast infiltration, and inhibited endoplasmic reticulum/oxidative stress. MiR-218-5p targeted and negatively regulated UBE3A expression to inhibit ubiquitin-mediated SATB1 degradation, promote trophoblast infiltration, and inhibit endoplasmic reticulum/oxidative stress.

Coexistence of junctional epidermolysis bullosa, autosomal recessive deafness type 57, and Angelman syndrome: A case report.

Key Clinical Message: The presence of more than one genetic/genomic disorder is not uncommon. It is therefore essential to continuously consider new signs and symptoms over time. Administration of gene therapy could be extremely difficult in particular situations. Abstract: A 9-month-old boy presented to our department for evaluation of developmental delay. We found that he was affected by intermediate junctional epidermolysis bullosa (COL17A1, c.3766 + 1G > A, homozygous), Angelman syndrome (5,5 Mb deletion of 15q11.2-q13.1), and autosomal recessive deafness type 57 (PDZD7, c.883C > T, homozygous).

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

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

Transcriptomic analysis provides insight into the mechanism of IKKß-mediated suppression of HPV18E6-induced cellular abnormalities.

High-risk human papillomaviruses (HPVs) 16 and 18 are responsible for more than 70% of cervical cancers and majority of other HPV-associated cancers world-wide. Current treatments for these cancers have limited efficacy, which in turn has resulted in disease recurrence and poor survival rates in advanced disease stages. Hence, there is a significant need for development of novel molecularly-targeted therapeutics. This can only be achieved through improved understanding of disease mechanism. Recently, we developed a Drosophila model of HPV18E6 plus human E3 ubiquitin ligase (hUBE3A) and demonstrated that the E6-induced cellular abnormalities are conserved between humans and flies. Subsequently, we demonstrated that reduced level and activity of IKKß, a regulator of NF-κB, suppresses the cellular abnormalities induced by E6 oncoprotein and that the interaction of IKKß and E6 is conserved in human cells. In this study, we performed transcriptomic analysis to identify differentially expressed genes that play a role in IKKß-mediated suppression of E6-induced defects. Transcriptome analysis identified 215 genes whose expression was altered due to reduced levels of IKKß. Of these 215 genes, 151 genes showed annotations. These analyses were followed by functional genetic interaction screen using RNAi, overexpression, and mutant fly strains for identified genes. The screen identified several genes including genes involved in Hippo and Toll pathways as well as junctional complexes whose downregulation or upregulation resulted in alterations of E6-induced defects. Subsequently, RT-PCR analysis was performed for validation of altered gene expression level for a few representative genes. Our results indicate an involvement for Hippo and Toll pathways in IKKß-mediated suppression of E6 + hUBE3A-induced cellular abnormalities. Therefore, this study enhances our understanding of the mechanisms underlying HPV-induced cancer and can potentially lead to identification of novel drug targets for cancers associated with HPV.

E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure.

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates in vitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1 UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2 UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.