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.

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.

Recovery of Angelman syndrome rat deficits with UBE3A protein supplementation.

We recently generated a novel Angelman syndrome (AS) rat model with a complete Ube3a gene deletion, that recapitulates the loss of UBE3A protein and shows cognitive and EEG deficits. We also recently published the identification of extracellular UBE3A protein within the brain using microdialysis. Here we explored the effects of supplementation of exogenous UBE3A protein to hippocampal slices and intrahippocampal injection of AS rats. We report that the AS rat model demonstrates deficits in hippocampal long-term potentiation (LTP) which can be recovered with the application of exogenous UBE3A protein. Furthermore, injection of recombinant UBE3A protein into the hippocampus of the AS rat can rescue the associative learning and memory deficits seen in the fear conditioning task. These data suggest that extracellular UBE3A protein may play a role in synaptic function, LTP induction and hippocampal-dependent memory formation.

Excessive Laughter-like Vocalizations, Microcephaly, and Translational Outcomes in the Ube3a Deletion Rat Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder characterized by intellectual disabilities, motor and balance deficits, impaired communication, and a happy, excitable demeanor with frequent laughter. We sought to elucidate a preclinical outcome measure in male and female rats that addressed communication abnormalities of AS and other neurodevelopmental disorders in which communication is atypical and/or lack of speech is a core feature. We discovered, and herein report for the first time, excessive laughter-like 50 kHz ultrasonic emissions in the Ube3amat-/pat+ rat model of AS, which suggests an excitable, playful demeanor and elevated positive affect, similar to the demeanor of individuals with AS. Also in line with the AS phenotype, Ube3amat-/pat+ rats demonstrated aberrant social interactions with a novel partner, distinctive gait abnormalities, impaired cognition, an underlying LTP deficit, and profound reductions in brain volume. These unique, robust phenotypes provide advantages compared with currently available mouse models and will be highly valuable as outcome measures in the evaluation of therapies for AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurogenetic disorder for which there is no cure, despite decades of research using mouse models. This study used a recently developed rat model of AS to delineate disease-relevant outcome measures to facilitate therapeutic development. We found the rat to be a strong model of AS, offering several advantages over mouse models by exhibiting numerous AS-relevant phenotypes, including overabundant laughter-like vocalizations, reduced hippocampal LTP, and volumetric anomalies across the brain. These findings are unconfounded by detrimental motor abilities and background strain, issues plaguing mouse models. This rat model represents an important advancement in the field of AS, and the outcome metrics reported herein will be central to the therapeutic pipeline.

Imprinting effects of UBE3A loss on synaptic gene networks and Wnt signaling pathways.

Ubiquitin E3 ligase 3A (UBE3A) encodes an E3 ubiquitin ligase whose loss from the maternal allele causes the neurodevelopmental disorder Angelman syndrome (AS). Previous studies of UBE3A function have not examined full Ube3a deletion in mouse, the complexity of imprinted gene networks in brain nor the molecular basis of systems-level cognitive dysfunctions in AS. We therefore utilized a systems biology approach to elucidate how UBE3A loss impacts the early postnatal brain in a novel CRISPR/Cas9-engineered rat Angelman model of a complete Ube3a deletion. Strand-specific transcriptome analysis of offspring from maternally or paternally inherited Ube3a deletions revealed the expected parental expression patterns of Ube3a sense and antisense transcripts by postnatal day 2 (P2) in hypothalamus and day 9 (P9) in cortex, compared to wild-type littermates. The dependency of genome-wide effects on parent-of-origin, Ube3a genotype and time (P2 and P9) was investigated through transcriptome (RNA sequencing of cortex and hypothalamus) and methylome (whole-genome bisulfite sequencing of hypothalamus). Weighted gene co-expression and co-methylation network analyses identified co-regulated networks in maternally inherited Ube3a deletion offspring enriched in postnatal developmental processes including Wnt signaling, synaptic regulation, neuronal and glial functions, epigenetic regulation, ubiquitin, circadian entrainment and splicing. Furthermore, we showed that loss of the paternal Ube3a antisense transcript resulted in both unique and overlapping dysregulated gene pathways with maternal loss, predominantly at the level of differential methylation. Together, these results provide a holistic examination of the molecular impacts of UBE3A loss in brain, supporting the existence of interactive epigenetic networks between maternal and paternal transcripts at the Ube3a locus.

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

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.

Administration of CoQ10 analogue ameliorates dysfunction of the mitochondrial respiratory chain in a mouse model of Angelman syndrome.

Genetic defects in the UBE3A gene, which encodes for the imprinted E6-AP ubiquitin E3 ligase (UBE3A), is responsible for the occurrence of Angelman syndrome (AS), a neurodegenerative disorder which arises in 1 out of every 12,000-20,000 births. Classical symptoms of AS include delayed development, impaired speech, and epileptic seizures with characteristic electroencephalography (EEG) readings. We have previously reported impaired mitochondrial structure and reduced complex III in the hippocampus and cerebellum in the Ube3a(m-/p+) mice. CoQ10 supplementation restores the electron flow to the mitochondrial respiratory chain (MRC) to ultimately increase mitochondrial antioxidant capacity. A number of recent studies with CoQ10 analogues seem promising in providing therapeutic benefit to patients with a variety of disorders. CoQ10 therapy has been reported to be safe and relatively well-tolerated at doses as high as 3000mg/day in patients with disorders of CoQ10 biosynthesis and MRC disorders. Herein, we report administration of idebenone, a potent CoQ10 analogue, to the Ube3a(m-/p+) mouse model corrects motor coordination and anxiety levels, and also improves the expression of complexes III and IV in hippocampus CA1 and CA2 neurons and cerebellum in these Ube3a(m-/p+) mice. However, treatment with idebenone illustrated no beneficial effects in the reduction of oxidative stress. To our knowledge, this is the first study to suggest an improvement in mitochondrial respiratory chain dysfunction via bioenergetics modulation with a CoQ10 analogue. These findings may further elucidate possible cellular and molecular mechanism(s) and ultimately a clinical therapeutic approach/benefit for patients with Angelman syndrome.

Regulatory elements associated with paternally-expressed genes in the imprinted murine Angelman/Prader-Willi syndrome domain.

The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain - such as MKRN3 and NDN - are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRF's) and YY1 specifically on the paternal allele. NRF's and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRF's and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRF's also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRF's and YY1.

Human disorders caused by the disruption of the regulation of excitatory neurotransmission.

The nicotinic acetylcholine receptors (nAChRs) are members of the large family of ligand-gated ion channels, and are constituted by the assembly of five subunits arranged pseudosymmetrically around the central axis that forms a cation-selective ion pore. They are widely distributed in both the nervous system and non-neuronal tissues, and can be activated by endogenous agonists such as acetylcholine or exogenous ligands such as nicotine. Mutations in neuronal nAChRs are found in a rare form of familial nocturnal frontal lobe epilepsy (ADNFLE), while mutations in the neuromuscular subtype of the nAChR are responsible for either congenital myasthenia syndromes (adult subtype of neuromuscular nAChR) or a form of arthrogryposis multiplex congenita type Escobar (fetal subtype of neuromuscular nAChR).

Pharmacologic evidence for abnormal thalamocortical functioning in GABA receptor beta3 subunit-deficient mice, a model of Angelman syndrome.

PURPOSE: gamma-Aminobutyric acid receptor (GABA(A)r) subunit beta3-deficient mice model Angelman syndrome by displaying impaired learning, abnormal EEG with interictal spikes and slowing, myoclonus, and convulsions. The beta3-subunit deficiency causes a failure of intrathalamic reticular nucleus inhibition, leading to abnormally synchronized thalamocortical oscillations. We postulated that this pathophysiology underlies the abnormal cortical EEG and triggers interictal spikes and seizures, but extrathalamic regions also contribute to interictal spikes and seizures, so that the EEG slowing should reveal an absence-like response profile, whereas spikes and seizures have dual responsiveness to absence and partial-seizure drugs. METHODS: Recording electrodes were implanted over the parietal cortices of wild-type, heterozygotes, and homozygous null mice. In each experiment, EEG was recorded for 45 min, either drug or vehicle administered, and EEG recorded for another 3 h. Each EEG was scored for slow-wave activity, interictal spikes, and seizures by a reader blinded to treatments. RESULTS: Interictal spiking and percentage of time in EEG slowing in heterozygotes were increased by the proabsence drug baclofen (GABA(B)-receptor agonist), whereas CGP 35348 (GABA(B)-receptor antagonist) had the opposite effect. The antiabsence drug ethosuximide markedly suppressed EEG slowing and interictal spiking in heterozygote and null mice. Broad-spectrum clonazepam and valproate were more effective on interictal spiking than on EEG slowing, and fosphenytoin suppressed only interictal spiking. CONCLUSIONS: The results suggest that this model of Angelman syndrome, although not expressing typical absence seizures, is characterized by hypersynchronous thalamocortical oscillations that possess absence-like pharmacologic responsiveness and promote EEG slowing, interictal spikes, and convulsive seizures.