NEDD4-2 Regulates Endoplasmic Reticulum Stress And Seizures Through Ubiquitination Of RER1

Objective:The neural precursor cell expressed developmentally down-regulated gene 4-2（NEDD4-2）encodes an E3 ligase which is associated with epilepsy through ubiquitination of neuroactive substrates.The endoplasmic reticulum retention protein 1（RER1）that recognizes transmembrane domain（TMD）based ER retention signals of abnormal proteins and retrieves them back to endoplasmic reticulum（ER）for further assembly or degradation is mainly expressed in the cis-Golgi,which affects the cell membrane expression level of substrate proteins or causes ER stress.We had built an epileptic Nedd4-2^+/-mouse model and had found up-regulated expression of Rer1 in the hippocampus through protein mass spectrometry.However,the molecular mechanism of the over-expressed Rer1 and its potential ER-retention substrates involved in epilepsy need further study.In this study,thapsigargin（Tg）and pentylenetetrazole（PTZ）were used to induce endoplasmic reticulum stress and seizures in Nedd4-2^+/-mice.The motif and molecular mechanism of NEDD4-2-mediated ubiquitination degradation of RER1 were identified by constructing wild-type and mutant expression vectors of RER1.Proteins that bind differently to Rer1 in the hippocampus of Nedd4-2^+/-mice and wild-type mice were screened by immunoprecipitation-mass spectrometry（IP-MS）to reveal the potential substrates of Rer1 that may be involved in epilepsy.This research may be have potential applications in the treatment of epilepsy.Methods:1.Nedd4-2^+/-and wild-type mice（n=4）were intraperitoneally injected with ER stress inducer Tg（2mg/kg）or normal saline at one time.After 6 hours,we sacrificed mice and dissected the cerebral cortex and hippocampus.To analyze whether Nedd4-2^+/-mice were susceptible to ER stress,we used Western blot to detect the level of ER stress marker CHOP,as well as immunohistochemistry and Nissl staining to analyze the level and distribution of CHOP.2.Nedd4-2^+/-and wild-type mice were divided into three groups（n=4）for daily intraperitoneal injection with ER stress inducer Tg（1mg/kg）,ER stress inhibitor salibrinal（1mg/kg）or saline vehicle,followed by seizure inducer PTZ（35mg/kg）1hour later.The mice were observed for 1 hour to evaluate the seizure scores according to the Racine scale to analyze the effect of ER stress on epilepsy susceptibility of Nedd4-2^+/-mice.3.The mice given daily intraperitoneal injection as described above were sacrificed when the first mouse in each group reached grade 7（convulsive death）.Then we detected the CHOP level in mice brains by Western blot to analyze the level of chronic ER stress in Nedd4-2^+/-mice.4.Western blot was used to detect the expression of Nedd4-2 and Rer1 in the cortex and hippocampus of mice.Co-immunoprecipitation（Co-IP）was used to detect the interactions between Rer1 and Nedd4-2 or Ubiquitin（Ub）in mouse brain.These were used to analyze the Nedd4-2-mediated ubiquitination of Rer1 and the effect of ER stress on Nedd4-2-mediated ubiquitination in vivo.5.Human neuroblastoma cells SH-SY5Y and human glioma cells U251 were cultured and treated with three si RNAs（0.5μg）to knokdown human NEDD4-2 for 48 hours.The expression of NEDD4-2 and RER1 were determined by Western blot to verify the up-regulation role of knockdown NEDD4-2 on the expression of RER1 in vitro.6.The expression vectors of NEDD4-2 and RER1 were constructed with different tags and co-transfected into SH-SY5Y and U251 cells.The expression of NEDD4-2 and RER1was detected by Western blot with tag antibodies.The interactions between RER1 and NEDD4-2 or Ub were detected by Co-IP.These were used to analyze the down-regulation role of overexpression NEDD4-2 on the expression of RER1 to verify NEDD4-2-mediated ubiquitination of RER1 in vitro.7.SH-SY5Y and U251 cells co-transfected with NEDD4-2 and RER1 expression vectors were treated with ER stress inducer Tg（0.5μM）for 24 hours.Co-IP was used to detect the interaction between NEDD4-2 and RER1 to analyze the effect of ER stress on NEDD4-2-mediated RER1 ubiquitination.8.Wild-type and TP37＿38AA mutant RER1 expression vectors were constructed.Then wild-type/mutant RER1 and NEDD4-2 expression vectors co-transfected into SH-SY5Y and U251 cells.The interaction between NEDD4-2 and RER1 was detected by Co-IP to analyze the binding motif of RER1 and NEDD4-2.9.SH-SY5Y and U251 cells co-transfected with wild-type/mutant RER1 and NEDD4-2expression vectors for 36 hours were treated with protein synthesis inhibitor actinomycin CHX（100μg/ml）for 0 hour,3 hours and 6 hours.Western blot was used to detect the change of RER1 expression to evaluate the stability of mutant protein.10.SH-SY5Y and U251 cells co-transfected with wild-type NEDD4-2 and RER1expression vectors for 36 hours were treated with proteasome inhibitor MG132（10μM）or lysosomal inhibitor leupeptin（10μM）for 12 hours.Western blot was used to detect the RER1 level to analyze the proteasomal or lysosomal degradation pathway of RER1.11.The hippocampaus of the chronic PTZ-induced epileptic Nedd4-2^+/-and wild-type mice（n=4）were dissected.To compare differentially expressed proteins,we used IP-MS by Rer1 antibody and then analyzed proteins by GO and KEGG and so on.12.The differentially expressed proteinsα1 subunit of GABA_Areceptor（Gabra1）screened by IP-MS was chosen for validation of protein interaction between Rer1 and Gabra1 by Co-IP in mice brains to verify that Gabra1 is ER retention cargo of Rer1.13.The brain protein of Nedd4-2^+/-and wild-type mice was digested with ER specific glycosidase Endo-H（0.5U/ml）at 37℃for 1 hour.The lower molecular weight Gabra1was supposed to represent the ER-retained Gabra1.The changes in molecular weight of Gabra1 was detected by Western blot to analyze ER retention of Gabra1 in the Nedd4-2^+/-mice brains.Result:1.The expression of CHOP was not detected in the cortex and hippocampus of Nedd4-2^+/-and wild-type mice under basic conditions（intraperitoneal injection of normal saline）.While the CHOP was detected in both groups that treated with Tg,CHOP was significantly more expressed in Nedd4-2^+/-mice than in wild-type mice,suggesting the vulnerability of ER stress in Nedd4-2^+/-mice.2.Immunohistochemistry of mouse brain showed stronger staining signals of CHOP in the cortex and hippocampus of Nedd4-2^+/-mice than wild-type mice,and the CHOP was mainly localized to the neurons according to the Nissl staining.3.The seizure Racine scores in Nedd4-2^+/-mice were higher than in wild-type mice in all the three groups in the research of Tg,salubrinal or normal saline pretreatment with PTZ induced chronic epilepsy in mice.Comparatively,the scores were significantly higher in Tg pretreatment group than in salubrinal and normal saline pretreatment groups（P<0.05）.Meanwhile,the first mouse died at grade 7 on the 6th day in Tg pretreatment group as well as on the 13th and 12th days in salubrinal and normal saline pretreatment groups with lower seizure Racine scores,respectively.These data suggested that ER stress aggravates the epileptic phenotype of Nedd4-2^+/-mice.4.The expression of CHOP in the above mice brains was positive by Western blot,but the CHOP level relatively higher in Nedd4-2^+/-mice than in wild-type mice in all the three groups and the most significant in Tg pretreatment group.5.The expression of Nedd4-2 in cortex and hippocampus of PTZ-induced Nedd4-2^+/-mice decreased by approximately half,but the expression of Rer1 increased by about1.7-flod compared with wild-type mice.The ubiquitination level of Rer1 in the brains of Nedd4-2^+/-mice compared with wild-type mice was down-regulated（P<0.01）.The interaction between Nedd4-2 and Rer1 was decreased in Nedd4-2^+/-mice to about 60%of wild-type mice（P<0.01）,which suggested that Nedd4-2 hippoinsufficiency in mice leaded to the decreased ubiquitnation degradation of Rer1 and the up-regulated expression of Rer1.6.The interaction between Nedd4-2 and Rer1 in Tg induced ER stress mice was enhanced,which was more obvious in wild-type mice than in Nedd4-2^+/-mice.This suggested that Nedd4-2^+/-mice had relatively low reactivity to Tg.7.Knockdown NEDD4-2 in SH-SY5Y and U251 cells by si RNAs increased the expression of RER1.Compared with the control group,the higher knockdown efficiency of si RNA,the more increased expression of RER1.8.Overexpression of NEDD4-2 in SH-SY5Y and U251 cells that co-transfected expression vectors of NEDD4-2 and RER1 significantly reduced the expression of RER1by Western blot and increased the ubiquitination level of RER1 by Co-IP.The interaction between NEDD4-2 and RER1 was dected by Co-IP.This interaction was enhanced in co-transfected cells treated with Tg,which suggested that NEDD4-2-mediated ubiquitination of RER1 showed ER stress reactivity.9.The interaction between TP37＿38AA mutant RER1 and NEDD4-2 in SH-SY5Y and U251 cells that co-transfected wild-type NEDD4-2 and wild-type or mutant RER1expression vectors was significantly reduced by Co-IP（P<0.01）.Blocking protein synthesis by CHX,the expression level of mutant RER1 was higher than wild type RER1by Western blot（P<0.01）.These data suggested that NEDD4-2-mediated ubiquitination of RER1 through ³⁶STPY³⁹motif.10.NEDD4-2 decreased the expression of RER1 in SH-SY5Y and U251 cells that co-transfected wild-type NEDD4-2 and RER1 expression vectors by Western blot,which was partially reversed by MG132,but not by leupiptin.These data suggested that NEDD4-2 mediated RER1 ubiquitination to elict proteasomal degradation.11.The interacting cargoes of Rer1 screened by IP-MS showed 127 and 133 proteins in the hippocampus in PTZ-induced epileptic Nedd4-2^+/-and wild-type mice,respectively.There were 72 common interaction proteins in both groups,and GO analysis suggested that they were related to“protein binding”,“membrane”,“neurotransmitter”and“synapse”.Among the differential interaction proteins of the two groups,there were 55unique proteins of Nedd4-2^+/-mice.GO analysis suggested that they are related to“synapse”,“neurotransmitter receptor”and“transmembrane transporter”.12.The differential interaction protein Gabra1 was verified to interact with Rer1 in mouse brain tissue by Co-IP,which was stronger in Nedd4-2^+/-mice brains than in wild-type mice brains.13.The molecular weight of Gabra1 was reduced in mice brains digested by Endo-H which is known to remove the ER-modified high mannose N-linked carbohydrates.The undigested Gabra1 was slightly lower and the digested Gabra1 was significantly higher in Nedd4-2^+/-mice than in wild-type mice by Western blot,which indicated that Gabra1was retained in ER more heavily in Nedd4-2^+/-mice brains.Conclusions:1.Nedd4-2 haploinsufficiency in Nedd4-2^+/-mice decreased the protective effect on ER stress and PTZ-induced epilepsy.2.NEDD4-2 mediated the ubiquitination of RER1 by interacting with the³⁶STPY³⁹motif of RER1 and RER1 undergoes the proteasomal degradation.3.A variety of potential Rer1 cargoes that mediate neuroexcitability were screened by IP-MS in the hippocampus of Nedd4-2^+/-mice,among which Gabra1 was verified to interact with Rer1 and retained in ER more heavily in Nedd4-2^+/-mouse brains.