The authors showed that both membrane depolarization and glutamate receptor activation increased the levels of UBE3A mRNA and protein in cultured neurons and in the brains of mice housed in an enriched environment. Binding sites for myocyte enhancer factor 2, an activity-regulated transcription factor that controls synapse development, were found in the promoter region of the Ube3a gene. Using a transgenic mouse model expressing a haemagglutinin epitope-tagged version of ubiquitin, the authors identified candidate substrates of UBE3A in neurons. These candidates shared a region of similarity 75 amino-acids in length that could serve as a UBE3A-binding domain.
A search for sequence similarities in mammalian genome databases identified the synaptic protein ARC as a potential UBE3A substrate. ARC regulates AMPAR trafficking, providing a possible link with the role of UBE3A in synaptic function. To test whether UBE3A influences ARC levels, the authors co-expressed UBE3A and ARC in vitro and observed a decrease in the level of ARC that was prevented by the addition of a proteasome inhibitor to the culture medium. Moreover, ARC protein levels were increased in the brains of UBE3A-knockout mice. Blocking UBE3A expression in hippocampal neurons using RNA interference (RNAi) or by knocking out UBE3A in vivo decreased AMPAR levels at the plasma membrane and impaired AMPAR recruitment to the postsynaptic region. Furthermore, inhibition of UBE3A expression led to a reduction in the frequency (but not the amplitude) of miniature excitatory postsynaptic currents, an effect that was attributed to the elimination of AMPAR expression from a subset of synapses. These changes were dependent on ARC ubiquitylation and degradation, as overexpression of a version of ARC lacking the UBE3A-binding domain mimicked the effect of UBE3A expression blockade, and ARC-specific RNAi increased surface AMPAR expression.
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