| Part ? The role of lncRNA-Gbla in depressive-like behaviors induced by chronic social defeat stressObjective: Depression is one of the most common neuropsychiatric diseases.Its pathological mechanism is complex and diverse,which brings great challenges to the development of disease diagnosis and treatment strategies.A series of gene expression is changed by stress,which plays an important role in its pathological process.It has been shown that epigenetics plays an important role in stress response and mediates a wide range of gene expression network regulation.Long chain noncoding RNA is one of the epigenetic regulatory mechanisms,and more and more researches focus on its role in depression.In this study,we screened and verified the role of differential expression of lncRNA in depressive-like behaviors induced by CSDS.Moreover,we investigate the role and mechanism of lncRNA in depressive-like behaviors.Methods:(1)C57BL/6J mice were exposed to CSDS to construct depression model,then we carried out social interaction and sucrose preference test,evaluated the serum corticosterone concentration by ELISA.(2)According to mouse lncRNA microarray data,we screened differential expression of lncRNAs in the mPFC of susceptible mice.(3)The q PCR was used to verify the expression level of lncRNA-Gbla in the mPFC region of susceptible mice.(4)We identified the subcellular location of lncRNA-Gbla through FISH in HT22 cells.(5)LV-sh RNA-Gbla mediated silencing of lncRNA-Gbla in mPFC,then we evaluated its effects on the depressive-like behaviors and the electrophysiological abnormality induced by CSDS.(6)AAV-Gbla mediated the overexpression of lncRNA-Gbla in mPFC,we evaluated the effects on the vulnerability of mice and stress-related electrophysiological abnormality after the mice were exposed to subthreshold stress.Results:(1)C57BL / 6J mice experiencing CSDS were divided into susceptible and resilient group according to social interaction(n = 15,P < 0.001 vs control),sugar preference(SPT : control = 87.20 ± 0.43;susceptible = 44.22 ± 1.58,n = 15,P < 0.001 vs control;resilient = 90.82 ± 0.50)and serum corticosterone level(control: 17.39 ± 0.36 ?g/L,n = 6;susceptible: 21.83 ± 0.26 ?g/L,n =7,P < 0.01 vs control;resilient: 18.86 ± 0.16 ?g/L,n =7).(2)According to mouse lncRNA microarray data,1752 lncRNAs with differential expression were detected from the mPFC of susceptible mice.(3)q PCR verified the results of gene microarray,CSDS significantly increased the expression level of lncRNA-Gbla in mPFC of susceptible mice(control: 1.01 ± 0.01,n = 20;susceptible: 1.62 ± 0.03,n = 20,P < 0.01 vs control;resilient: 1.08 ± 0.02,n = 20).(4)LV-sh RNA-Gbla mediated silencing of lncRNA-Gbla ameliorated the depressive-like behaviors induced by CSDS and restored the decreased frequency(control: 3.24 ± 0.21 Hz,n = 6;LV-GFP: 1.21 ± 0.04 Hz,n = 7;LV-sh RNA-Gbla:2.35 ± 0.21 Hz,n = 7,P < 0.05 vs LV-GFP)and amplitude(control: 14.05 ± 0.68 p A,n = 6;LV-GFP: 8.70 ± 0.21 p A,n = 7;LV-sh RNA-Gbla:14.20 ± 0.70 p A,n = 7,P < 0.01 vs LV-GFP)of m EPSC in mPFC of susceptible mice.(5)AAV-Gbla mediated overexpression of lncRNA-Gbla in mPFC increased the susceptibility of mice to stress and facilitated stress-related damage of synaptic plasticity(frequency: AAV-GFP = 3.49 ± 0.19 Hz;AAV-Gbla = 2.03 ± 0.09 Hz,n = 8,P < 0.05 vs AAV-GFP;amplitude: AAV-GFP = 22.17 ± 0.49 p A;AAV-Gbla = 16.66 ± 0.53 p A,n = 8,P < 0.01 vs AAV-GFP).Conclusion: Through high-throughput screening for differential expression of lncRNA,we found and confirmed the expression change of lncRNA-Gbla in mPFC of susceptible mice.Virusmediated specific silencing and overexpression demonstrated its regulatory effects on synaptic plasticity and depressive-like behaviors.Part ? The mechanism of lncRNA-Gbla in depressive-like behaviors induced by CSDSObjective: We found that the expression level of lncRNA-Gbla was significantly up-regulated in the mPFC of susceptible mice induced by CSDS,and the up-regulation or down-regulation of lncRNA-Gbla mediated by virus could regulate depressive-like behaviors bidirectionally.Therefore,this part will further study the mechanisms of lncRNA-Gbla regulating depressive-like behaviors.Methods: RNA pull-down and mass spectrometry analysis were used to identify the proteins interacting with lncRNA-Gbla.We evaluated the cell location of lncRNA-Gbla and GRP78 with fluorescence in situ hybridization(FISH).The direct interaction between lncRNA-Gbla and GRP78 was evaluated by RNA immunoprecipitation assay(RIP).We constructed missing recombinants for GRP78 predicting fragment to identify the binding region between lncRNA-Gbla and GRP78.The spatial conformational relationship between lncRNA-Gbla and GRP78 was evaluated by fluorescence resonance shift experiment(FRET).The protein level of GRP78 in neurons was regulated to evaluate its effects on the activity of unfolded protein response(UPR)and membrane expression of AMPAR.Then we analysed the effect of decreased lncRNA-Gbla on membrane expression of AMPAR by LV-sh RNA-Gbla-mediated silencing in mPFC.Moreover,AAV-GRP78 mediated the overexpression of GRP78 and we evaluated the effects on vulnerability in CSDS.Patch-clamp recordings were employed to record the effects of virus-mediated regulation of GRP78 on miniature excitatory postsynaptic currents(m EPSCs)in mPFC of mice.Results:(1)The results of RNA pull-down and mass spectrometry showed that several proteins interacted with lncRNA-Gbla,among which GRP78 displayed the important role in synaptic plasticity.(2)Fluorescence in situ hybridization demonstrated the colocalization of lncRNA-Gbla and GRP78 in cell cytoplasm.(3)RNA immunocoprecipitation confirmed the direct interaction between them.Compared with Ig G,the complex recruited by GRP78 antibody displayed higher expression level of lncRNA-Gbla(Input: 1 ± 0,Ig G: 0.064 ± 0.006,GRP78: 0.260 ± 0.001,n = 4 P < 0.001).(4)To identify the binding site between lncRNA-Gbla and GRP78,we constructed recombinant proteins for predicting fragments and compared the results of RNA immunocoprecipitation.Except for GRP78?2,GRP78 full length,GRP78?1 and GRP78?3 could all efficiently precipitate lncRNA-Gbla compared with Ig G(GRP78 full length: Input = 1 ± 0,Ig G = 0.024 ± 0.004,FLAG = 0.462 ± 0.011,n = 3,P < 0.01 vs Ig G;GRP78?1: Input = 1 ± 0,Ig G = 0.024 ± 0.004,FLAG = 0.175 ± 0.012,n = 3,P < 0.05 vs Ig G;GRP78?2: Input = 1 ± 0,Ig G = 0.007 ± 0.001,FLAG = 0.021 ± 0.006,n = 3,P = 0.3418 vs Ig G;GRP78?3: Input = 1 ± 0,Ig G = 0.024 ± 0.010,FLAG = 0.369 ± 0.021,n = 3,P < 0.01 vs Ig G.The results demonstrated the binding region was distributed in the first and third fragment.(5)Fluorescence resonance energy transfer showed the tight interaction of lncRNA-Gbla and GRP78.Fluorescence excitation of lncRNAGbla could induce the excitation of GRP78 and display significant FRET effect(efficiency: 18 S = 0.172 ± 0.002,lncRNA-Gbla = 0.529 ± 0.007,n = 8,P < 0.001 vs 18S;distance: 18 S = 8.845 ± 0.017 nm,lncRNA-Gbla = 6.714 ± 0.038 nm,n = 8,P < 0.001 vs 18S).(6)The increase in GRP78-activated UPR(vehicle: GRP78 = 1 ± 0.06,n = 6;p-IRE-1 = 1 ± 0.06,n = 6;IRE-1 = 1 ± 0.01,n = 6;ATF6 = 1 ± 0.04,n = 6;XBP-1 = 1 ± 0.04,n = 6;Caspase12 = 1 ± 0.04,n = 3,TM: GRP78 = 1.80 ± 0.13,n = 6,P < 0.01;p-IRE-1 = 2.09 ± 0.19,n = 6,P < 0.05;IRE-1 = 1.14 ± 0.03,n = 6,P = 0.1443;ATF6 = 2.18 ± 0.09,n = 6,P < 0.05;XBP-1 = 2.21 ± 0.10,n = 6,P < 0.01;Caspase12 = 1 ± 0.07,n = 3,P > 0.9999 vs vehicle)and promoted the membrane expression of AMPAR in neurons(vehicle: s-Glu A1 = 1 ± 0.06;t-Glu A1 = 0.99 ± 0.01;s-Glu A2 = 1 ± 0.04;t-Glu A2 = 1 ± 0.01,TM: s-Glu A1 = 1.80 ± 0.10,n = 6,P < 0.01;t-Glu A1 = 0.84 ± 0.04,n = 6,P = 0.1662;sGlu A2 = 1.70 ± 0.10,n = 6,P < 0.01;t-Glu A2 = 1.05 ± 0.03,n = 6,P = 0.5590 vs vehicle).LVsh RNA-Gbla mediated silencing of lncRNA-Gbla also increased the membrane expression of AMPAR(control: s-Glu A1 = 1 ± 0.03;t-Glu A1 = 1 ± 0.01;s-Glu A2 = 1 ± 0.02;t-Glu A2 = 1 ± 0.02,LV-GFP: s-Glu A1 = 0.8 ± 0.02;t-Glu A1 = 0.97 ± 0.02;s-Glu A2 = 0.77 ± 0.02;t-Glu A2 = 0.96 ± 0.01,LV-sh RNA-Gbla: s-Glu A1 = 0.97 ± 0.03,n = 6,P < 0.05;t-Glu A1 = 0.98 ± 0.01,n = 6,P = 0.7715;s-Glu A2 = 0.93 ± 0.01,n = 6,P < 0.01;t-Glu A2 = 0.95 ± 0.01,n = 6,P = 0.8316 vs LV-GFP).(7)AAV-GRP78 mediated overexpression of GRP78 ameliorated depressive-like behaviors and restored stress-induced decrease in the frequency(control: 4.02 ± 0.18 Hz;AAVGFP: 1.58 ± 0.03 Hz;AAV-GRP78: 3.60 ± 0.17 Hz,n = 8,P < 0.01 vs AAV-GFP)and amplitude(control: 19.21 ± 0.63 p A;AAV-GFP: 12 ± 0.53 p A;AAV-GRP78: 21.07 ± 0.46 p A,n = 8,P < 0.01 vs AAV-GFP)of m EPSC.(8)LV-sh RNA-GRP78 mediated silencing of GRP78 silencing could not directly alter synaptic plasticity(frequency: LV-GFP = 3.16 ± 0.07 Hz;LV-sh RNA-GRP78 = 3.18± 0.09 Hz,n = 8,P = 0.9478 vs LV-GFP;amplitude: LV-GFP = 24.08 ± 0.28 p A;LV-sh RNAGRP78 = 23.91 ± 0.31 p A,n = 7,P = 0.8846 vs LV-GFP)and induce depressive-like behaviors(LV-GFP: No Target = 44.4 ± 1.4 sec,Target = 70.9 ± 1.5 sec,n = 8;LV-sh RNA-GRP78: No Target = 48.1 ± 1.2 sec,Target = 71.0 ± 0.9 sec,n = 12;SPT: LV-GFP = 83.09 ± 0.42,n = 13,LV-sh RNAGRP78 = 80.85 ± 0.78,n = 13,P = 0.5095 vs LV-GFP),however,knockdown of GRP78 results in increased susceptibility and facilitation stress related electrophysiological abnormalities exposed to subthreshold stress(frequency: LV-GFP = 3.11 ± 0.10 Hz,n =7;LV-sh RNA-GRP78 = 2.29 ± 0.06 Hz,n = 8,P < 0.05 vs LV-GFP;amplitude: LV-GFP = 21.74 ± 0.45 p A,n =8;LV-sh RNAGRP78 = 16.52 ± 0.50 p A,n =8,P < 0.01 vs LV-GFP).These pathological changes finally resulted social avoidance(LV-GFP: No Target = 50.6 ± 1.3 sec,Target = 74.0 ± 1.9 sec,n = 12;LV-sh RNAGRP78: No Target = 53.9 ± 0.9 sec,Target = 40.8 ± 0.9 sec,n = 11,P < 0.05 vs LV-GFP)and decrease of SPT(LV-GFP = 84.38 ± 1.01,n = 13;LV-sh RNA-GRP78 = 64.97 ± 0.92,n = 14,P < 0.001 vs LV-GFP).Conclusion: GRP78 mediates the effects of lncRNA-Gbla on plasticity and depressive-like behaviors.The increase of lncRNA-Gbla decrease the protein level of GRP78,inhibit UPR and the membrane expression of AMPAR,which impair synaptic plasticity and increase the vulnerability to stress. |
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