Activity-dependent ubiquitination of GluR1 mediates a distinct AMPAR endocytosis and sorting pathway in hippocampal neurons

The accurate trafficking of AMPA receptors (AMPARs) to and from excitatory glutamatergic synapses in the hippocampus is a critical component of mammalian learning and memory. In conjunction, recent research suggests that dysfunction of AMPAR trafficking in the hippocampus may be an underlying mechanism of Alzheimer's disease. Previous work has shown that ubiquitination of integral membrane proteins is a common post-translational modification used to mediate endocytosis and endocytic sorting of surface proteins in eukaryotic cells. Therefore, we hypothesized that mammalian AMPARs may become ubiquitinated to regulate their synaptic stability in neurons. Here we report that mammalian AMPARs become ubiquitinated in response to their activation. Using a mutant of GluR1 that is unable to be ubiquitinated at lysines on its carboxy-terminus, we demonstrate that these ubiquitination sites are required for internalization of surface AMPARs and their trafficking to the lysosome in response to the AMPAR agonist AMPA, but not for internalization of AMPARs in response to NMDA receptor (NMDAR) agonist NMDA. Through over-expression or RNAi-mediated knockdown, we identify that a specific E3 ligase, Nedd4-1, is necessary for this process. Finally, we show that ubiquitination of GluR1 by Nedd4-1 is much more prevalent in aged neurons. Together, these data show that ubiquitination of GluR1-containing AMPARs by Nedd4-1 mediates their endocytosis and trafficking to the lysosome. Furthermore, these results provide insight into how hippocampal neurons regulate AMPAR trafficking and degradation with high specificity in response to differing neuronal signaling cues, and suggest that changes to this pathway may occur with ag