Activity-dependent Regulation of Extracellular UBE3A in a Novel Angelman Syndrome Rat Model

Angelman Syndrome (AS) is a rare genetic and neurologic disorder affecting approximately 1 in every 15,000 people. This disorder is characterized by seizures, absence of speech, motor and cognitive deficits, ataxia, and notably an abnormal happy demeanor with frequent smiling and laughter. A neuron-specific loss of function of the maternal allele of UBE3A, encoding for an E3 ubiquitin ligase, leads to the manifestation of AS. There are currently no cures for AS and few therapeutic options to abate symptoms. Although much investigation is required, research using the null mutation AS mouse model suggests a plausible case for pharmacological intervention. Major caveats of current mouse models includes strain influences and phenotypic inconsistencies. There is a high demand for an AS model that consistently recapitulates the majority of human phenotypes. With advancements in CRISPR/Cas9 technology, we have successfully created a novel AS rat model comprised of a full deletion of maternal UBE3A. The mouse model only contains a deletion in exon, which means it could potentially still generate a small N-terminal fragment of UBE3A. Western blotting of our rat model brain and peripheral tissues indicates the UBE3A deletion is pervasive with almost a complete loss in the CNS and about a 50% reduction in peripheral tissues. The AS rat model displays normal breeding ability and Mendelian distribution with no significant weight gain, unlike the mouse model which has significant weight gain compared to litter mate controls. The AS rat recapitulates many aspects of AS, including significant deficits in motor coordination (increased hind limb clasping, rotarod, and DigiGait) learning and memory (fear conditioning and digital touchscreen), and deficits in social approach. These deficits in learning and memory tasks, are most likely due to significant deficits in synaptic plasticity. Using hippocampal electrophysiology (CA1-CA3) we demonstrated that this AS model has deficits in their input/output curve, long-term potentiation (LTP) and well as long-term depression (LTD). These data are consistent with the existing mouse model and further supporting for this new AS rat as a good model for human AS. Lack of an established AS biomarker has been detrimental in patient diagnosis as well as testing the efficacy of novel therapeutics. With the creation of this rat model, biomarker research has garnered significant interest, in part due to the rat’s larger size. Here we report for the first time that Ube3a is present within cerebrospinal fluid (CSF) of wild type rats but is absent in the AS rat. This observation was largely due to easier sampling of sufficient CSF from a rat compared to mouse. We subsequently observed similar results in the mouse model and human CSF. Following the discovery that Ube3a is located in the CSF, we next investigated if Ube3a is within the extracellular space of the hippocampus. Due to the deficits in learning and memory paradigms as well as synaptic plasticity, we focused on the hippocampus. Hippocampal microdialysis revealed Ube3a is indeed located within the extracellular space. Utilizing the ubiquitination assay that involves a well-known Ube3a substrate, S5A, we demonstrated that extracellular Ube3a maintains its E3 ubiquitin ligase activity. This activity also included its ability to self-ubiquitinate. Although the function of Ube3a ligase activity extracellularly is unknown, we speculate that it may have a functional role in synaptic strength and stability. Previous reports have demonstrated intracellular Ube3a is under activitydependent regulation with a significant increase in mRNA levels after exposure to the adverse learning task, fear conditioning. This increase suggests a dynamic involvement of Ube3a in learning and memory. This lead us to hypothesize that secretion of Ube3a into the extracellular environment may also be a dynamic process in learning and memory. To test this idea, we used microdialysis on wild-type rats in conjunction with fear conditioning exposure. Our results demonstrate that extracellular Ube3a is under activity-dependent regulation with a significant increase in secretion of Ube3a at ~2 h after the adverse learning task. This increase was maintained for at least 3 h after induction, which may indicate a role in memory consolidation. To further support the idea that extracellular Ube3a plays a role in learning and memory, we applied exogenous Ube3a to AS rat hippocampal slices during LTP recordings. We demonstrate that the deficits in LTP observed in the AS rat are rescued to WT levels with exogenous Ube3a application. This is a significant finding, because Ube3a appears to have multiple roles in different cellular compartments, but here we demonstrate for the first time that Ube3a’s activity at the synapse is likely the most important role of Ube3a in synaptic plasticity and learning and memory. In summary, we have developed a novel rat model of AS which recapitulates many important aspects of the human condition, including motor deficits, learning deficits and electrophysiology deficits. Utilizing this novel AS rat model we are the first to report the presence of Ube3a in the extracellular space and demonstrate its potential as an AS biomarker. This is the first report to show extracellular Ube3a is under activitydependent regulation and has the ability to recover LTP deficits. This new AS rat model should offer avenues for increased exploration of AS and advance our understanding of molecular targets of Ube3a, thus expanding our knowledge of the disease. This model offers a high potential utility for drug evaluation, biomarker discovery, and will aid in the development and testing of novel therapeutic treatments