Effects And Mechanism Of NEDD4L Down-regulating NRG1 On The Depression-like Behaviour Of Mice Induced By Chronic Social Defeat Stress

Background: Major depressive disorder(MDD),which has been increasing in the incidence of global diseases,is a chronic multifactorial neuropsychiatric disease involving the joint effects of individual genetics and living environment.The stress of social life related to social and interpersonal relationships is an important risk factor for depression.The most common time of onset is in a person's 20 s and 30 s.Therefore,depression is becoming a serious problem affecting young people's health.Basic and clinical studies demonstrate that depression is associated with reduced size of brain regions that regulate mood and cognition,including the prefrontal cortex and the hippocampus,and decreased neuronal synapses in these areas.The medial prefrontal cortex(m PFC)is one of the critical brain regions that involved in the regulation of pathological reaction to stress,changes in affective behaviour,the neuroendocrine response,and executive and cognitive functions that are adversely impacted by stress.Neuroregulin 1(NRG1)is an intercellular signal transduction protein produced by glial cells and neurons.It is widely distributed in the frontal cortex,midbrain,and cerebellum.NRG1 is a member of a family of epidermal growth factor-like proteins,which interact with the Erb B family of receptor tyrosine kinases to play a role in neurodevelopment,synaptic plasticity,neuronal migration,ect.It has been shown that a loss of NRG 1 within cortical projection neurons results in increased inhibitory connections and reduced synaptic plasticity.The effect of social stress on depression-like behaviour has been investigated with the chronic social defeat stress(CSDS)paradigm in mice.However,related studies have not shown how chronic stress affects m PFC damage and leads to neurobehavioral changes.It is unclear whether NRG1 in the m PFC is involved in stress susceptibility.Ubiquitination has been shown to regulate the levels of EGFR family receptors by targeting these receptors to proteasomes or lysosomes.Neural precursor cell expressed developmentally downregulated 4-2(Nedd4-2,also known as Nedd4l)belongs to the Nedd4 family of E3 ubiquitin ligases.At present,there are few studies on Nedd4 l,especially in the field of depression or stress.The relationship between Nedd4 l and NRG1 has not been reported.Therefore,we want to study the molecular mechanisms of stress-induced behavioural responses and potential behavioural vulnerability,and to conduct more research on the occurrence and development of depression.Objective: By establishing a chronic social defeat stress model in mice,detecting changes in NRG1 protein and gene expression in brain regions related to depressive behaviour,to determine whether the possible regulation of Nedd4 l on NRG1 is involved in the mechanism of stress-induced depression-like behaviour.To provide new molecular targets for the treatment of depression.Methods: The CSDS paradigm was used to construct a mouse depression model.Western blotting(WB)was used to detect changes in the expression level of the target proteins.Quantitative real-time PCR(Q-PCR)was used to quantitatively analyze the m RNA expression of the target gene.In primary cortical neuron culture experiments,dexamethasone(Dex),a glucocorticoid receptor agonist,was used to mediate chronic stress at the cellular level to test its effect on gene expression.Co-IP and brain slice immunofluorescence were used to determine the interaction and co-localization of Nedd4 l and NRG1.In the m PFC brain region of mice,adeno-associated virus over-expressing NRG1,over-expressing and knocking down Nedd4 l were performed to detect the effects of NRG1-Nedd4 l signaling in stress-mediated behaviours related to anxiety and depression in mice.Golgi staining was used to observe dendritic spine changes in the m PFC brain regions.Results: By analyzing the SI ratio in the CSDS paradigm,we found that 60% of the mice in the experimental group exhibited stress-sensitive behaviour,defined as stress-susceptible(SS)mice,and the rest corresponded Non-susceptible mice are defined as resistant mice(Resilient,RES).A series of behavioural tests revealed that SS mice exhibited significant anxiety and depression-like behaviours.Golgi staining showed reduced dendritic spines in the m PFC brain region of SS mice.Western blot experiments were performed on mouse brain tissue samples,and we found that the protein level of NRG1 in the m PFC in SS mice was reduced.However,Q-PCR results showed no changes in NRG1 m RNA levels in the m PFC in SS mice.In cell experiments,treatment of primary cortical neurons with Dex caused cellular stress and reduced NRG1 protein levels,but NRG1 m RNA levels remained unchanged.In cells treated with Dex,the protein biosynthesis inhibitor actinomycin(CHX)was used for 4 h,8 h,or 12 h.Compared with the control group,Dex treatment significantly promoted the degradation of NRG1.For Dex-treated neurons,the use of the proteasome inhibitor MG132 can prevent the reduction of NRG1 caused by Dex,but it does not affect the NRG1 m RNA levels.The above results suggest that NRG1 degradation increases and protein levels decrease during chronic stress.Using the virus to overexpress NRG1 in the m PFC brain region,it was found that upregulation of NRG1 in the m PFC region reduces CSDS-induced depression-like behaviour compared to control mice that experienced CSDS.RNA sequencing results of mouse m PFC brain tissue showed that compared with CTR mice,SS mice had 670 genes up-regulated and 462 genes down-regulated.We analysed the signaling pathways associated with increased protein degradation of NRG1 and found that the m RNA level of E3 ubiquitin ligase Nedd4 l was up-regulated and the level of Nedd4 l protein in the m PFC region was increased.In the m PFC brain region of SS mice,there is a significant protein interaction between Nedd4 l and NRG1,and the ubiquitination of NRG1 is significantly increased in SS mice.Immunofluorescence showed co-localization of Nedd4 l and NRG1 in the cytoplasm of the m PFC brain region of SS mice.Chronic stress of cultured primary cortical neurons treated with Dex can down-regulate NRG1 expression,while Nedd4 l expression is up-regulated,and NRG1 ubiquitination levels increase.Down-regulation of Nedd4 l by simultaneous administration of LV-Si Nedd4 l virus in Dex-treated primary cortical neurons can rescue NRG1 expression,suggesting that Nedd41 is closely related to the degradation of NRG1.In animal experiments,the subthreshold social defeat stress(SSDS)paradigm increases the sensitivity of animals to stress without causing depression in social avoidance.After administration of Nedd4l-expressing virus,in mice without SSDS,overexpression of Nedd4 l did not change its behavioural phenotype,while overexpression of Nedd4 l could significantly increase the depression-like behaviour of SSDS mice,and lead to NRG1 in mouse m PFC.Significantly increased ubiquitination,decreased NRG1 protein levels,and accompanied dendritic spine injury.After administering the Nedd4 l knockdown virus,we found that in unstressed mice,the knockdown of the Nedd4 l gene did not change the behavioural phenotype.Down-regulation of Nedd4 l can alleviate CSDS-induced depression-like behaviour.Compared with control mice that underwent CSDS,in the m PFC brain region of Sh-Nedd4 l mice,the ubiquitination level of NRG1 decreased significantly,and the NRG1 protein increased significantly.Golgi staining showed that down-regulation of Nedd4 l reversed CSDS-induced dendritic spine reduction.Conclusion: Increased degradation of NRG1 in the m PFC region leads to a decrease in NRG1 protein levels and is involved in the occurrence of depression-like behaviour in mice induced by CSDS.Overexpression of NRG1 can reverse depression-like behaviour.Upregulation of Nedd4 l may be involved in promoting the degradation of NRG1.In the m PFC brain region of stress-susceptible mice,Nedd4 l up-regulates the increase in ubiquitination of NRG1,leading to a decrease in NRG1 levels,synaptic damage,and mediating depression-like behaviour in mice induced by stress.And down-regulate Nedd4 l,alleviate the above pathological and behavioural abnormalities.These data provide new clues to the pathogenesis of depression.