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.

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.

A PSD-95 peptidomimetic mitigates neurological deficits in a mouse model of Angelman syndrome.

Angelman Syndrome (AS) is a severe cognitive disorder caused by loss of neuronal expression of the E3 ubiquitin ligase UBE3A. In an AS mouse model, we previously reported a deficit in brain-derived neurotrophic factor (BDNF) signaling, and set out to develop a therapeutic that would restore normal signaling. We demonstrate that CN2097, a peptidomimetic compound that binds postsynaptic density protein-95 (PSD-95), a TrkB associated scaffolding protein, mitigates deficits in PLC-CaMKII and PI3K/mTOR pathways to restore synaptic plasticity and learning. Administration of CN2097 facilitated long-term potentiation (LTP) and corrected paired-pulse ratio. As the BDNF-mTORC1 pathway is critical for inhibition of autophagy, we investigated whether autophagy was disrupted in AS mice. We found aberrantly high autophagic activity attributable to a concomitant decrease in mTORC1 signaling, resulting in decreased levels of synaptic proteins, including Synapsin-1 and Shank3. CN2097 increased mTORC1 activity to normalize autophagy and restore hippocampal synaptic protein levels. Importantly, treatment mitigated cognitive and motor dysfunction. These findings support the use of neurotrophic therapeutics as a valuable approach for treating AS pathology.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Lack of UBE3A-Mediated Regulation of Synaptic SK2 Channels Contributes to Learning and Memory Impairment in the Female Mouse Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by severe developmental delay, motor impairment, language and cognition deficits, and often with increased seizure activity. AS is caused by deficiency of UBE3A, which is both an E3 ligase and a cofactor for transcriptional regulation. We previously showed that the small conductance potassium channel protein SK2 is a UBE3A substrate, and that increased synaptic SK2 levels contribute to impairments in synaptic plasticity and fear-conditioning memory, as inhibition of SK2 channels significantly improved both synaptic plasticity and fear memory in male AS mice. In the present study, we investigated UBE3a-mediated regulation of synaptic plasticity and fear-conditioning in female AS mice. Results from both western blot and immunofluorescence analyses showed that synaptic SK2 levels were significantly increased in hippocampus of female AS mice, as compared to wild-type (WT) littermates. Like in male AS mice, long-term potentiation (LTP) was significantly reduced while long-term depression (LTD) was enhanced at hippocampal CA3-CA1 synapses of female AS mice, as compared to female WT mice. Both alterations were significantly reduced by treatment with the SK2 inhibitor, apamin. The shunting effect of SK2 channels on NMDA receptor was significantly larger in female AS mice as compared to female WT mice. Female AS mice also showed impairment in both contextual and tone memory recall, and this impairment was significantly reduced by apamin treatment. Our results indicate that like male AS mice, female AS mice showed significant impairment in both synaptic plasticity and fear-conditioning memory due to increased levels of synaptic SK2 channels. Any therapeutic strategy to reduce SK2-mediated inhibition of NMDAR should be beneficial to both male and female patients.

A Scalable, Cell-based Method for the Functional Assessment of Ube3a Variants.

The increased use of sequencing in medicine has identified millions of coding variants in the human genome. Many of these variants occur in genes associated with neurodevelopmental disorders, but the functional significance of the vast majority of variants remains unknown. The present protocol describes the study of variants for Ube3a, a gene that encodes an E3 ubiquitin ligase linked to both autism and Angelman syndrome. Duplication or triplication of Ube3a is strongly linked to autism, whereas its deletion causes Angelman syndrome. Thus, understanding the valence of changes in UBE3A protein activity is important for clinical outcomes. Here, a rapid, cell-based method that pairs Ube3a variants with a Wnt pathway reporter is described. This simple assay is scalable and can be used to determine the valence and magnitude of activity changes in any Ube3a variant. Moreover, the facility of this method allows the generation of a wealth of structure-function information, which can be used to gain deep insights into the enzymatic mechanisms of UBE3A.

An Association Study of DNA Methylation and Gene Expression in Angelman Syndrome: A Bioinformatics Approach.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of function of the E3-ligase UBE3A. Despite multiple studies, AS pathophysiology is still obscure and has mostly been explored in rodent models of the disease. In recent years, a growing body of studies has utilized omics datasets in the attempt to focus research regarding the pathophysiology of AS. Here, for the first time, we utilized a multi-omics approach at the epigenomic level and the transcriptome level, for human-derived neurons. Using publicly available datasets for DNA methylation and gene expression, we found genome regions in proximity to gene promoters and intersecting with gene-body regions that were differentially methylated and differentially expressed in AS. We found that overall, the genome in AS postmortem brain tissue was hypo-methylated compared to healthy controls. We also found more upregulated genes than downregulated genes in AS. Many of these dysregulated genes in neurons obtained from AS patients are known to be critical for neuronal development and synaptic functioning. Taken together, our results suggest a list of dysregulated genes that may be involved in AS development and its pathological features. Moreover, these genes might also have a role in neurodevelopmental disorders similar to AS.

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.

A cross-species spatiotemporal proteomic analysis identifies UBE3A-dependent signaling pathways and targets.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of neuronal E3 ligase UBE3A. Restoring UBE3A levels is a potential disease-modifying therapy for AS and has recently entered clinical trials. There is paucity of data regarding the molecular changes downstream of UBE3A hampering elucidation of disease therapeutics and biomarkers. Notably, UBE3A plays an important role in the nucleus but its targets have yet to be elucidated. Using proteomics, we assessed changes during postnatal cortical development in an AS mouse model. Pathway analysis revealed dysregulation of proteasomal and tRNA synthetase pathways at all postnatal brain developmental stages, while synaptic proteins were altered in adults. We confirmed pathway alterations in an adult AS rat model across multiple brain regions and highlighted region-specific differences. UBE3A reinstatement in AS model mice resulted in near complete and partial rescue of the proteome alterations in adolescence and adults, respectively, supporting the notion that restoration of UBE3A expression provides a promising therapeutic option. We show that the nuclear enriched transketolase (TKT), one of the most abundantly altered proteins, is a novel direct UBE3A substrate and is elevated in the neuronal nucleus of rat brains and human iPSC-derived neurons. Taken together, our study provides a comprehensive map of UBE3A-driven proteome remodeling in AS across development and species, and corroborates an early UBE3A reinstatement as a viable therapeutic option. To support future disease and biomarker research, we present an accessible large-scale multi-species proteomic resource for the AS community ( https://www.angelman-proteome-project.org/ ).

The Hippocampal Response to Acute Corticosterone Elevation Is Altered in a Mouse Model for Angelman Syndrome.

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.

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

In 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.

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.

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

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 mouse 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.

Secreted retrovirus-like GAG-domain-containing protein PEG10 is regulated by UBE3A and is involved in Angelman syndrome pathophysiology.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by the loss of maternal UBE3A, a ubiquitin protein ligase E3A. Here, we study neurons derived from patients with AS and neurotypical individuals, and reciprocally modulate UBE3A using antisense oligonucleotides. Unbiased proteomics reveal proteins that are regulated by UBE3A in a disease-specific manner, including PEG10, a retrotransposon-derived GAG protein. PEG10 protein increase, but not RNA, is dependent on UBE3A and proteasome function. PEG10 binds to both RNA and ataxia-associated proteins (ATXN2 and ATXN10), localizes to stress granules, and is secreted in extracellular vesicles, modulating vesicle content. Rescue of AS patient-derived neurons by UBE3A reinstatement or PEG10 reduction reveals similarity in transcriptome changes. Overexpression of PEG10 during mouse brain development alters neuronal migration, suggesting that it can affect brain development. These findings imply that PEG10 is a secreted human UBE3A target involved in AS pathophysiology.

Antisense oligonucleotide treatment rescues UBE3A expression and multiple phenotypes of an Angelman syndrome mouse model.

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.

CRISPR/Cas9 directed to the Ube3a antisense transcript improves Angelman syndrome phenotype in mice.

Gene editing holds the potential to correct mutations and cure devastating genetic disorders. The technology has not yet proven efficacious for therapeutic use in CNS diseases with ubiquitous neuronal defects. Angelman syndrome (AS), a severe neurodevelopmental disorder, is caused by a lack of maternal expression of the UBE3A gene. Because of genomic imprinting, only neurons are affected. One therapeutic approach focuses on the intact paternal UBE3A copy in patients with AS that is silenced by an antisense transcript (UBE3A-ATS). We show here that gene editing of Ube3a-ATS in the mouse brain resulted in the formation of base pair insertions/deletions (indels) in neurons and the subsequent unsilencing of the paternal Ube3a allele in neurons, which partially corrected the behavioral phenotype of a murine AS model. This study provides compelling evidence to further investigate editing of the homologous region of the human UBE3A-ATS because this may provide a lasting therapeutic effect for patients with AS.

Bioinformatics analyses show dysregulation of calcium-related genes in Angelman syndrome mouse model.

BACKGROUND: Angelman syndrome (AS) is a genetic neurodevelopmental disorder caused by the loss of function of the UBE3A protein in the brain. In a previous study, we showed that activity-dependent calcium dynamics in hippocampal CA1 pyramidal neurons of AS mice is compromised, and its normalization rescues the hippocampal-dependent deficits. Therefore, we expected that the expression profiles of calcium-related genes would be altered in AS mice hippocampi. METHODS: We analyzed mRNA sequencing data from AS model mice and WT controls in light of the newly published CaGeDB database of calcium-related genes. We validated our results in two independent RNA sequencing datasets from two additional different AS models: first one, a human neuroblastoma cell line where UBE3A expression was knocked down by siRNA, and the second, an iPSC-derived neurons from AS patient and healthy donor control. FINDINGS: We found signatures of dysregulated calcium-related genes in AS mouse model hippocampus. Additionally, we show that these calcium-related genes function as signatures for AS in other human cellular models of AS, thus strengthening our findings. INTERPRETATION: Our findings suggest the downstream implications and significance of the compromised calcium signaling in Angelman syndrome. Moreover, since AS share similar features with other autism spectrum disorders, we believe that these findings entail meaningful data and approach for other neurodevelopmental disorders, especially those with known alterations of calcium signaling. FUNDING: This work was supported by the Angelman Syndrome Foundation and by the Israel Science Foundation, Grant Number 248/20.

Adenosine A2A receptors format long-term depression and memory strategies in a mouse model of Angelman syndrome.

Angelman syndrome (AS) is a neurodevelopmental disorder caused by loss of function of the maternally inherited Ube3a neuronal protein, whose main features comprise severe intellectual disabilities and motor impairments. Previous studies with the Ube3am-/p+ mouse model of AS revealed deficits in synaptic plasticity and memory. Since adenosine A2A receptors (A2AR) are powerful modulators of aberrant synaptic plasticity and A2AR blockade prevents memory dysfunction in various brain diseases, we tested if A2AR could control deficits of memory and hippocampal synaptic plasticity in AS. We observed that Ube3am-/p+ mice were unable to resort to hippocampal-dependent search strategies when tested for learning and memory in the Morris water maze; this was associated with a decreased magnitude of long-term depression (LTD) in CA1 hippocampal circuits. There was an increased density of A2AR in the hippocampus of Ube3am-/p+ mice and their chronic treatment with the selective A2AR antagonist SCH58261 (0.1 mg/kg/day, ip) restored both hippocampal-dependent learning strategies, as well as LTD deficits. Altogether, this study provides the first evidence of a role of A2AR as a new prospective therapeutic target to manage learning deficits in AS.

PKA and Ube3a regulate SK2 channel trafficking to promote synaptic plasticity in hippocampus: Implications for Angelman Syndrome.

The ubiquitin ligase, Ube3a, plays important roles in brain development and functions, since its deficiency results in Angelman Syndrome (AS) while its over-expression increases the risk for autism. We previously showed that the lack of Ube3a-mediated ubiquitination of the Ca2+-activated small conductance potassium channel, SK2, contributes to impairment of synaptic plasticity and learning in AS mice. Synaptic SK2 levels are also regulated by protein kinase A (PKA), which phosphorylates SK2 in its C-terminal domain, facilitating its endocytosis. Here, we report that PKA activation restores theta burst stimulation (TBS)-induced long-term potentiation (LTP) in hippocampal slices from AS mice by enhancing SK2 internalization. While TBS-induced SK2 endocytosis is facilitated by PKA activation, SK2 recycling to synaptic membranes after TBS is inhibited by Ube3a. Molecular and cellular studies confirmed that phosphorylation of SK2 in the C-terminal domain increases its ubiquitination and endocytosis. Finally, PKA activation increases SK2 phosphorylation and ubiquitination in Ube3a-overexpressing mice. Our results indicate that, although both Ube3a-mediated ubiquitination and PKA-induced phosphorylation reduce synaptic SK2 levels, phosphorylation is mainly involved in TBS-induced endocytosis, while ubiquitination predominantly inhibits SK2 recycling. Understanding the complex interactions between PKA and Ube3a in the regulation of SK2 synaptic levels might provide new platforms for developing treatments for AS and various forms of autism.