Roles of the Nedd4 Family E3 Ligases in Glial Function and Nerve Cell Development

Protein ubiquitination is one of the core regulatory post-translational modification in neuronal development. In this study, we used brain specific KO mice to identify novel substrate proteins of several Nedd4 family E3 ligases, i.e. Nedd4-1, Nedd4-2, WWP1 and WWP2. We developed a highly reliable approach to identify transmembrane substrate proteins of Nedd4-1 and Nedd4-2 by combining iTRAQ quantitative mass spectrometry with synaptic membrane purification. We showed that levels of the main inwardly rectifier potassium channel in astrocytes, Kir4.1, and the main gap junction protein in astrocytes, Connexin-43, are upregulated in Nedd4-1 and Nedd4-2 brain specific double KO mice. Furthermore, we showed that Kir4.1 is ubiquitinated in vivo and in vitro by Nedd4-1 and Nedd4-2 whereas Connexin-43 is ubiquitinated by Nedd4-2 in vivo via a K63-linked polyubiquitin chains. In addition, we showed that glial loss of Nedd4-1 and Nedd4-2 leads to the reduced averaged power of gamma oscillatory activity in the CA3 region of the hippocampus in Nedd4-1; Nedd4-2 double KO and Nedd4-2 single KO as compared control mice, indicating that Nedd4-1 and Nedd4-2 play a crucial role in the regulation of neuronal network through astrocytic network in the hippocampus. Moreover, we showed that Prr7 is a novel substrate of Nedd4-2 at postsynapses and Nedd4-2 conjugates K63-linked polyubiquitin chains to cytoplasmic region of Prr7. Such ubiquitination of Prr7 might play a role in spine maturation during the development. Additionally, we identified CAPZA1, VCP and PKM2 as the novel substrate proteins of WWP1 and WWP2 E3 ligases. We showed that genetic deletion of WWP1 and WWP2 conditionally in neurons leads to enhanced dendrite growth in hippocampal neurons, indicating that WWP1 and WWP2 are negative regulator of dendrite development. Overexpression of CAPZA1 in wild type hippocampal neurons phenocopies the enhanced dendrite growth in WWP1; WWP2 double knockout neurons indicating that ubiquitination of CAPZA1 by WWP1 and WWP2 might play a crucial role in regulation of CAPZA1 activity thereby regulation of dendrite development