RESUMO
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.
RESUMO
Neural precursor cell expressed developmentally down-regulated gene 4-like (NEDD4-2) encodes a ubiquitin E3 ligase that is involved in epileptogenesis with mechanisms needing further investigation. We constructed a novel Nedd4-2+/- mouse model with half level of both Nedd4-2 long and short isoforms in the brain. Nedd4-2 haploinsufficiency caused increased susceptibility and severity of pentylenetetrazole (PTZ)-induced seizures. Of the 3379 proteins identified by the hippocampal proteomic analysis, 55 were considered altered in Nedd4-2+/- mice compared with wild-type control, among which the inwardly rectifying K+ channel Kir4.1 was up-regulated by 1.83-fold. Kir4.1 was subsequently confirmed to be less ubiquitinated in response to comprised Nedd4-2 in mouse brains and C6 cells. Kir4.1 associated with Nedd4-2 through the threonine312-proline motif in the intracellular domain by target mutagenesis. Adaptor protein 14-3-3 facilitated Nedd4-2-mediated ubiquitination of Kir4.1. Our data consolidate the detailed molecular mechanism of Nedd4-2-mediated Kir4.1 ubiquitination, and provide a possible relationship between increased seizure susceptibility and impaired Kir4.1 ubiquitination in the brain.
