CK2 Deficiency Induces The Dysfunction Of NRG1-ErbB4 Pathway In Depression

Background:Depression is a type of mood disorder characterized by a significant and persistent depression,slow thinking,cognitive impairment.will decreasing and physical symptoms.Its pathogenesis is mainly concentrated in the neural circuits,monoamine neurotransmitters.neuroendocrine and neuroimmunotic functions,genetic factors and epigenetic mechanisms,neurotrophic factors and neurogenesis and other doctrine,and each theory has a corresponding limitation.Postpartum depression,as a major branch of depression.has been shown to be associated with neural plasticity damage?Neuro-plasticity-related protein NRG 1 plays an important role in neurodevelopmental and neural plasticity regulation.At the same time,the signal of NRG1-ErbB was attenuated in chronic schizophrenia patients with depression symptom,and the level of NRG1 mRNA was significantly increased in sera of patients with severe depression.The injection of NRG1 to hippocampus increased neurogenesis and induced antidepressant-like effects.The genome-wide association analysis of antidepressant response found that NRG1 genetic variation could affect the antidepressant effect of SSRIs.These all show that NRG1 may play an important role in the pathogenesis of depression disorder,but the role of NRG1 in the depression pathogenesis and its regulatory mechanism are still unknown.Purpose:To explore whether NRG1 is involved in the pathogenesis of postpartum depression and its mechanism.to provide new clues for the pathogenesis of depression.Method:To investigate whether NRG 1 was involved in the development of postpartum depression disorder,we used Open Field Test,Sucrose Preference Test and Forced Swimming Test to screen out rats with depressive-like behavior as postpartum depression rat model in postnatal SD rats and to judge the success of screening,we used immunoblotting and immunofluorescence to measure the expression of partly depressive-related protein.The level and expression of NRG1 in the brain PPD rats was measured by immunoblotting,immunofluorescence and quantitative real-time PCR.The expression of NRG1 was increased and the level of NRG1 was reduced,showed that the degradation of NRG1 was increased.To investigate the mechanism of NRG1 degradation,we used immunoprecipitation to measure the ubiquitination and phosphorylation of NRG1 in PPD rats,and found that its ubiquitination was increased and phosphorylation was reduced.To investigate the mechanism of NRG1 phosphorylation reducing in PPD rats,We measured the level and activity of partly kinase by immunoblotting and found that the GSK3? activity CK2 level was decreased.To explore which kinase can regulate NRG1 phosphorylation,we used GSK3? activator and inhibitor and CK2 inhibitor to treat HEK293 cells,and transfected GSK3? and GSK3P-DN and CK2 plasmids into HEK293 cells,and then used immunoprecipitation to measure NRG1 phosphorylation.we found that only CK2 can regulate NRG1 phosphorylation.In order to search the specific CK2-regulated NRG1 phosphorylation position and explore the relationship of the ubiquitination and phosphorylation of NRG1,We used Scansite3 to predict and found that CK2 maybe can phosphorylate NRG1 at T582 site and then we used site-directed mutagenesis to structure NRG1 T582E T582A mutant plasmids as phosphorylation and non-phosphorylation NRG1 CK2 and NRG 1 wt and mutant plasmids were co-transfected into N2A cells,and NRG1 phosphorus was detected by immunoprecipitation,It was found that CK2 can increase the phosphorylation of NRG1 wt but can not increase the phosphorylation of the mutants,indicating that T5 82 was the phosphorylation site of NRG 1.Transfecting NRG1 wt and mutantplasmid into N2A cells,and the ubiquitination was measured by immunoprecipitation,at the same time,the NRG1 level was measured by immunoblotting at different time point after actinone treat.It was found that the non-phosphorylation of NRG 1 could lead to the increase of NRG1 ubiquitination and degradation,which indicated that the decrease of NRG 1 phosphorylation could lead to the increase of NRG 1 degradation.To explore the mechanism of NRG1 expression increase,we used Elisa kit to measure the levels of progesterone and estradiol in the serum of PPD rats.Meanwhile,we used immunoblotting to measure the level of estrogen-related transcription factor in PPD rats,and used chip-PCR to investigate whether NRG1 promoter region can bind to them in the cortical of rats and found that the changes in transcription factor levels resulting from hormone disorders may be the reason of NRG 1 expression increase.To explore the specific mechanism of NRG1 involvement in the development of depression,we transfected NRG1 wt and mutant plasmids into N2A cells,and their binding with ErbB4 was measured by immunoprecipitation.ErbB4 and p-ErbB4 level were measured by immunoblotting.In the rat cortex,ErbB4 and p-ErbB4 were measured by immunoblotting,the binding of ErbB4 and PSD95 were measured by immunoprecipitation.It was found that the decrease of synaptic plasticity caused by attenuated NRG1-ErbB signal maybe participate in the pathogenesis of depression.To investigate the role of CK2 reducing in Depression,we treated C57 mice with CK2 inhibitor TBB for one week,used behavioral experiment to measure the depressive-like behavior changes,and used immunoblotting and immunoprecipitation to measure NRG1-ErbB and its downstream signals,used Golgi staining to measure the synaptic plasticity in miceResults:1.Abou t 14%of postpartum rats showed depressive-like behavioral changes.2.The levels of GAD65 and PSD95 in the brain of PPD rats were reduced,showed partly pathological changes of depression.3.The level of NRG1 in the cortex of PPD rats was decreased and the mRNA level was increased,suggesting that NRG1 may be involved in the development of depression.4.The ubiquitination of NRG1 in the depressive-like rat was increased and the phosphorylation was decreased,while the CK2 level and GSK3P activity were decreased.In the cell experiments.CK2 inhibition could reduce the phosphorylation of NRG1 at T582 site and increase its ubiquitination,but GSK3? did not do this,suggesting that NRG1 phosphorylation decrease mediated by CK2 reducing can lead to the increases of its ubiquitination and degradation,and it may be the possible mechanism of NRG1 level decrease in PPD rats.5.The proportion of estradiol and progesterone in the serum of PPD rats was disorganized and the levels of transcription factors FOXO1 and E4BP4 were reduced,and they both could bind to the promoter region of NRG1,suggesting that hormone disorder mediated the changes of partly transcription factor level may be the mechanism ofNRG1 expression increase in the brain of PPD rats.6.The cell experiments proved that non-phosphorylation mutation of NRG1 at T582 site can make it more difficult to bind with ErbB4.and more difficult to phosphorylate ErbB4.And in the cortex of PPD rats,the combination of NRGl and ErbB4,the level of p-ErbB4 and the combination of ErbB4 and PSD95 were all reduced.It suggest that the damage of synaptic plasticity caused by the weakening of NRG1-ErbB signal may be the mechanism of its participation in the pathogenesis of depression.7.The using of CK2 inhibitor TBB caused depressive-like behavioral changes in mice.and was resulted in a decrease of NRG1-ErbB and its downstream signaling and dendritic spine number in mouse cortical,suggesting that CK2-mediated NRG1 phosphorylation decrease attenuated NRGl-ErbB signaling and induced depression by damaging synaptic plasticity.Conclusion:CK2-mediated NRG1 phosphorylation decrease increases its ubiquitination and leads to an increase in its degradation and thus inhibits NRGl-ErbB and its downstream signaling,causes synaptic plasticity damage,participates in the development of depression disorder.Ginkgo biloba extract EGb761 has shown the neuroprotective effects on Alzheimer's disease(AD)through the protection against the A?-induced neurotoxicity.However,it is not completedly clear whether EGb761 attenuates tau hyperphosphorylation,another of the most prominent mechanisms underlying the pathology of AD.Here.we employed hyperhomocysteinemia(HHcy),a strong and independent risk factor of AD.to mimic AD like pathological alterations and memory deficits in rats.We found that EGb761 could significantly attenuate HHcy-induced oxidative damage,recover PP2Ac and GSK3P activities deregulated by HHcy.Furthermore,tau was hyperphosphorylated at Thr231,Ser262,Ser396,and Ser404.most common PP2Ac and GSK3? targeted sites in the hippocampus and prefrontal cortex of HHcy rats,whereas EGb761 blocked the tau phosphorylation.Behavioral tests revealed that EGb761 rescued HHcy-induced spatial reference memory deficit and upregulated the expression of synapse-associated protein PSD95 and synapsin-1.Our data further suggest that EGb761 could treat AD through decrease in tau hyperphosphorylation besides the protection against the A?-induced neurotoxicity.