Nedd4-2 Haploinsufficiency in Mice Impairs the Ubiquitination of Rer1 and Increases the Susceptibility to Endoplasmic Reticulum Stress and Seizures.

Neural precursor cell expressed developmentally downregulated gene 4-like (NEDD4-2) is an epilepsy-associated gene encoding an E3 ligase that ubiquitinates neuroactive substrates. An involvement of NEDD4-2 in endoplasmic reticulum (ER) stress has been recently found with mechanisms needing further investigations. Herein, Nedd4-2 +/- mice were found intolerant to thapsigargin (Tg) to develop ER stress in the brain. Pretreatment of Tg aggravated the pentylenetetrazole (PTZ)-induced seizures. Retention in endoplasmic reticulum 1 (Rer1), an ER retrieval receptor, was upregulated through impaired ubiquitination in Nedd4-2 +/- mouse brain. Nedd4-2 interacted with Rer1 more strongly in mice with Tg administration. The negative regulation and NEDD4-2-mediated ubiquitination on RER1 were evaluated in cultured neurocytes and gliacytes by NEDD4-2 knockdown and overexpression. NEDD4-2 interacted with RER1 at higher levels in the cells with Tg treatment. Disruption of the 36STPY39 motif of RER1 attenuated the interaction with NEDD4-2, and the ubiquitinated RER1 underwent proteasomal degradation. Furthermore, the interactome of Rer1 was screened by immunoprecipitation-mass spectrometry in PTZ-induced mouse hippocampus, showing multiple potential ER retrieval cargoes that mediate neuroexcitability. The α1 subunit of the GABA A receptor was validated to interact with Rer1 and retain in ER more heavily in Nedd4-2+/- mouse brain by Endo-H digestion. In conclusion, Nedd4-2 deficiency in mice showed impaired ubiquitination of Rer1 and increased ER stress and seizures. These data indicate a protective effect of NEDD4-2 in ER stress and seizures possibly via RER1. We also provided potential ER retention cargoes of Rer1 awaiting further investigation.