The Role Of Long Noncoding RNA-Gm26543 In Chronic Social Defeat Stress-induced Depression-like Behavior Of Mice And The Underlying Mechanisms

Part Ⅰ The role of lncRNA-Gm26543 in chronic social defeat stress-induced depression-like behavior in miceObjective:Depression is a common,devastating illness,which is associated with enormous personal suffering and social economic burden.Long noncoding RNAs(IncRNAs)regulation is one of the epigenetic modifications.Recent studies demonstrate that lncRNAs paly important roles in synaptic plasticity,biochemical process and nervous system disease.Using microarrays,we findthe expression of lncRNA-Gm26543 is increased in mPFC of chronic social defeat stress(CSDS)susceptible mice.On this basis,we attempt to investigate the potential effect of lncRNA-Gm26543 on depression-like behavior in mice.Methods:CSDS was used to establish the C57BL/6J mouse models of depression;Quantitative real-time polymerase chain reaction(qRT-PCR)experiments were performed to analyze the levels of lncRNA-Gm26543 in mPFC and other brain regions of CSDS susceptible mice;Adenovirus(ADV)-and adeno-associated virus(AAV)-mediated overexpression in mPFC were used to investigate the effect of overexpression of lncRNA-Gm26543 on depression-like behavior of mice;Antisense oligonucleotides(ASO)were designed to knockdown the expression of lncRNA-Gm26543,subthreshold social defeat stress were used to investigate the effect of lncRNA-Gm26543 knockdown in mPFC on susceptiblility of mice to social defeat stress.Results:(1)Compared with non-defeated control mice,susceptible mice displayed significant depression-like behavior.qRT-PCR confirmed that the expression of lncRNA-Gm26543 was significant decreased by 57.10 ± 6.45%in mPFC of CSDS susceptible mice,while there was no difference between CSDS resilient mice and control mice.(n = 8-11,P<0.01 vs.control).(2)The change of lncRNA-Gm26543 expression in mPFC was most significant than the other brain regions of CSDS susceptible mice.(3)The depression-like behavior of CSDS susceptible mice was reversed after overexpression lncRNA-Gm26543 in mPFC.In the social interaction test,ADV-mediated lncRNA-Gm26543 overexpression in mPFC led to an increase in the interacting time from 1.69 ± 0.96%to 21.50 ± 4.20%(n = 7-10,P<0.01 vs.susceptible-ADV-GFP group).In the sucrose preference test,ADV-mediated lncRNA-Gm26543 overexpression in mPFC led to an increase in the sucrose preference levels from 42.12 ±7.64%to 75.17 ± 4.72%(n = 7.10,P<0.01 vs.susceptible-ADV-GFP group).Similarly,AAV-mediated lncRNA-Gm26543 overexpression in mPFC led to an increase in the interacting time from 14.96 ± 3.68%to 48.80±4.76%(n = 11-12,P<0.01 vs.susceptible-AAV-GFP group),and an increase in the interacting ratio from 35.99 ± 4.84%to 145.57 ± 14.97%(n = 11-12,P<0.01 vs.susceptible-AAV-GFP group).(4)Knockdown of IncRNA-Gm26543 directly led to greater behavioral susceptibility to subthreshold social defeat stress.In the social interaction test,knockdown of lncRNA-Gm26543 in mPFC resulted in a decrease in the interacting time from 46.82 ± 3.74%to 17.53 ± 3.52%(n = 12,P<0.01 vs.ASO-NC group),and a decrease in the interacting ratio from 147.10±9.03%to 51.84 ± 8.89%(n = 12,P<0.01 vs.ASO-NC group).In the sucrose preference test,knockdown of lncRNA-Gm26543 in mPFC resulted in a decrease in the sucrose preference levels from 86.55 ±1.79%to 57.57±3.52%(n = 12,P<0.01 vs.ASO-NC group).Conclusion:CSDS induces the decreased expression of lncRNA-Gm26543 in mPFC of susceptible mice,upregulation of lncRNA-Gm26543 in mPFC reverses depression-like behavior of CSDS susceptible mice,whereas downregulation of lncRNA-Gm26543 in mPFC increases behavioral susceptibility to subthreshold social defeat stress.These results suggest that lncRNA-Gm26543 in mice mPFC plays a critical role in the development of depression,and upregulation of lncRNA-Gm26543 could be a new strategy for depression treatment.Part Ⅱ The mechanism of lncRNA-Gm26543 in chronic stress-induced depressive behavior of miceObjective:CSDS induced the decrease in the expression of lncRNA-Gm26543 in mPFC of susceptible mice;upregulation of lncRNA-Gm26543 in mPFC reversed depression-like behavior of CSDS susceptible mice.Therefore,we tested the mechanism of lncRNA-Gm26543 in CSDS-induced depression-like behavior of CSDS susceptible mice and synaptic plasticity in mPFC.Methed:Fluorescent in situ hybridization(FISH)was used to determine subcellular localization of lncRNA-Gm26543;The putative mRNA or protein targets were explored by bioinformatics analysis and qRT-PCR experiments were performed to analyze the levels of putative mRNA in mPFC of CSDS susceptible mice;Mouse RNA microarray(8 × 60K)was designed by Agilent to screen mRNAs which were differential expression in the mice with ADV-mediated lncRNA-Gm26543 overexpression in mPFC;qRT-PCR experiments wereperformed to determine the relationship between lncRNA-Gm26543 and putative target mRNA and the levels of putative target mRNAs in mPFC of CSDS susceptible mice;RNA pulldown experiment were used to obtain the proteins which directly connected with lncRNA-Gm26543 and formed IncRNA-protein complexes;Mass spectrometrywas used to identify the specific proteins binding to IncRNA-Gm26543;RNA binding protein Immuno-preciptitation(RIP)experiment was used to verify specific proteins binding to IncRNA-Gm26543 identified by RNA pulldown.Results:(1)lncRNA-Gm26543 was detected in both nuclear and cytoplasmic fractions of N2a cell by qRT-PCR analysis,and subcellular localization of lncRNA-Gm26543 was found in both nucleus and cytoplasm by FISH(n = 5).(2)According to the bioinformatics analysis,lncRNA-Gm26543 located at Chromosome 10:49,219,422-49,225,653 and belonged to intronic lncRNAs,whose proximal protein-coding genes is glutamate ionotropic receptor kainate type subunit 2(Grik2).There was no significant difference inthelevel of Grik2 both at mRNA and protein level in mPFC between CSDS susceptible mice and control mice(n = 4-9).(3)According tobioinformatics analysis,IncRNA-Gm26543 regulated Prss35,Angptl4,Rgs9,Traf4 and Myd88 mRNA by trans.No significant difference was found in the mRNA level of Prss35,Angptl4,Rgs9,Traf4 and Myd88between CSDS susceptible mice and control mice.There was no difference in Myd88 protein expression in mPFC between control mice and CSDS susceptible mice.(4)We established ADV-mediated lncRNA-Gm26543 overexpression in mPFC of mice,By Mouse RNA Microarray,we detected different expression of 39430 RNAs and 16251 IncRNAs between ADV-Gm26543 group and ADV-GFP group,and found 1,071 genes were expressed abnormally,among which 1030 genes increased significantly,and 41 genes decreasedsignificantly.GO analysis showed that the differentially expressed genes may related closely to fear,neuron projection development,neuron migration,protein transport and phosphory-lation.These genes were mainly located at cytoplasm,plasma membrane,cell organelle,cytoskeleton and synapse.(5)We chose synaptic plasticity related gene CASK,DLGAP2 and NSF for subsequent study.qRT-PCR experiments were performed to analyze the levels of CASK,DLGAP2 and NSF,the RNA and protein expression of CASK were increased by 30.51 ± 5.73%(n = 9-10,P<0.01)and 22.98 ± 5.84%(n = 7-8,P<0.01),respectively;The RNA and protein expression of DLGAP2 were increased by 30.70 ±5.73%(n = 9-10,P<0.01)and 26.55 ± 4.31%(n = 10-14,P<0.01),respectively;The RNA and protein expression of NSF were increased by 34.54±5.45%(n = 10-11,P<0.01)and 25.65 ± 4.71%(n = 14-18,P<0.01),respectively.(6)After overexpression of IncRNA-Gm26543 in cultured N2a cell by adenovirus,qRT-PCR and western blot analysis confirmed that the mRNA and protein expression of CASK,DLGAP2 and NSF were significantly upregulated in ADV-Gm26543 group compared with ADV-GFP group.The mRNA and protein expression of CASK were upregulated by 65.24±16.75%(n = 6-7,P<0.01)and 75.93±19.20%(n = 7,P<0.01),respectively;The mRNA and protein expression of DLGAP2 were upregulated by 221.60 ± 70.62%(n =6-7,P<0.01)and 19.81 ± 9.51%(n = 3),respectively;The mRNA and protein expression of NSF were upregulated by 60.14±15.70(n = 6-7,P<0.01)and 31.00 ±7.94%(n = 8,P<0.01),respectively.(7)We found that knockdown of IncRNA-Gm26543 in cultured N2a cell led to significant decrease in mRNA and protein expression of CASK,DLGAP2 and NSF.The mRNA and protein expression of CASK were downregulated by 58.19 ± 18.44%(n = 3,P<0.05)and 24.66 ± 9.81%(n = 8,P<0.05),respectively;The mRNA and protein expression of DLGAP2 were downregulated by 58.19 ± 18.44%(n = 3,P<0.05)and 24.66± 9.81%(n = 6-7,P<0.01),respectively;The mRNA and protein expression of NSF were downregulated by 58.19 ± 18.44%(n = 3,P<0.01)and 24.66±9.81%(n＝10,P<0.05),respectively.(8)We used immunostaining experiments and found an decrease in the proportion of NSF colocalized with GluA2 in mPFC neurons of CSDS susceptible mice compared with control mice.Moreover western blot experiments showed that the the surface protein expression of GluA2 was significant decreased in mPFC of susceptible micecompared with control mice,whereas total protein expression was not significantly different between susceptible and control mice(S-control:100.00 ±5.84%;S-susceptible:68.75±6.55%;T-control:100.00 ± 1.53%;T-susceptible:105.80 ±3.31%;n = 4-8,P<0.01 S-susceptible vs.S-control).(9)Our coimmuno-precipitation results revealed that NSF-GluA2 interaction was significantly higher in ADV-Gm26543 group compared with ADV-GFP group in N2a cell(ADV-GFP:100.0 ± 18.12%,n = 2;ADV-Gm26543:140.50 ± 9.52%,n=2).(10)AAV-Gm26543 injected into mPFC could reverse the loss of dendritic spine in mPFC of susceptible mice(susceptible+GFP group:7.06 ± 0.18;susceptible+Gm26543 group:9.50±0.68,n = 5,P<0.01).(11)Using immunostaining experiments we found that CSDS led to a significant decrease of postsynaptic density protein-95(PSD95)in PFC of susceptible mice.(12)RNA pulldown and mass spectrometryidentified the specific proteins binding to lncRNA-Gm26543,including microtubule-associated protein family member Map1a.Conclusion:lncRNA-Gm26543 presentes a multi-target and network-based regulation mechanism on CSDS-induced depressive behaviors of mice.lncRNA-Gm26543 regulates synaptic plasticity related genes,such as CASK,NSF,DLGAP2,promotes colocalization of NSF with GluA2,restoresCSDS-induced dendritic spine loss,and directly interactes with Mapla,which also regulates synaptic plasticity in the central nervous system.lncRNA-Gm26543may be a highlight for the treatment of depression and an important target for antidepressants.