The Role And Mechanism Of AKAP150 In Chronic Restraint Stress-induced Depressive-like Behaviors

Part I The role of AKAP150 in chronic restraint stress-induced depressive-like behavior in miceObjective:A-kinase anchoring proteins(AKAPs)are known as a family of special anchoring proteins that are expressed ubiquitously in many tissues.AKAPs have the common amphiphilic helical structural,binding to the regulatory subunits of protein kinase A(PKA).By this way,AKAPs confine the PKA discrete intracellular locations.AKAP79/150(human 79/rodent 150),is the best-characterized brain isoform.AKAP79/150 is identified as postsynaptic scaffold protein that regulates the synaptic plasticity and memory formation and consolidation processes.Some genetic studies have indicated that DNA copy number of AKAP79 is relevant with pathophysiological process of bipolar disorder(BD)and schizophrenia.However,it remains unknown whether AKAP79/150 participates in the development of depression.In present study,we subjected mice to chronic restraint stress(CRS)to develop depressive-like behaviors,aiming to evaluate the role of AKAP150 signaling in the pathophysiology of major depression disease.Methods:Restaint stress paradigm was utilized to induce mice depressive-like behaviors,including:for acute stress,restraint once for 2 h(ARS)and for chronic stress,restraint 2 h once daily for consecutive 7,14 and 21 days(CRS 7,CRS 14 and CRS 21).Depressive-like behaviors were evaluated with the sucrose preference test(SPT),tail suspension test(TST)and forced swim test(FST).Locomotor activity was measured with the open field test(OFT)in control and stress-exposed mice.Quantitative real-time polymerase chain reaction(qRT-PCR)was used to measure the richment of AKAP150 mRNA in different brain regions.Western blotting examined AKAP150 protein expression in BLA of mice exposed to ARS and CRS.With lentivirus(LV)expressing constructs encoding enhanced green fluorescent protein(EGFP)and short hairpin RNAs(shRNAs)targeting AKAP150 into the BLA,we evaluated the role of AKAP150 signaling in stress-induced depressive-like behaviors.Results:(1)Compared to controls(CON),mice exposed to CRS 14 and CRS 21 developed depressive-like behaviors,however,CRS 7 and ARS exposure had no effect on depressive-related behaviors(SPT:CON:0.81 ± 0.02,ARS:0.74 ± 0.03,CRS 7:0.75 ±0.03,CRS 14:0.62 ± 0.05,CRS 21:0.63 ± 0.05.TST:CON:90.80 ± 4.29 s,ARS:90.36 ±5.09 s,CRS 7:70.20 ± 5.89 ss CRS 14:128.73 ± 9.88 s,CRS 21:133.00 ± 10.60 s.FST:CON:73.20 ± 6.01 s,ARS:68.62 ± 7.32 s,CRS 7:60.07 ± 9.42 s,CRS 14:133.78 ± 7.11 s,CRS 21:124.6 ± 9.60 s).(2)AKAP150 mRNA had much higher expression enrichment in the medial prefrontal cortex(mPFC),basolateral amygdala(BLA)and hippocampus(HIP).Among these,the enrichment of AKAP150 in BLA was the second position,following mPFC.(3)CRS 14 and CRS 21,not CRS 7 and ARS,increased AKAP150 expression in BLA(CON:1.00 ± 0.02,ARS:0.85 ± 0.04,CRS 7:0.98 ± 0.02,CRS 14:1.37 ± 0.07,CRS 21:1.29 ± 0.07).CRS(aslo previously referred as CRS 14)failed to change AKAP150 expression in mPFC and HIP.(4)Compared with control group,CRS promoted AKAP150 to assemble into synaptosomes(CON:1.00 ± 0.04,CRS:1.31 ± 0.07).(5)Expression of AKAP150 shRNA in BLA prevented stress-induced depressive-like behaviors(SPT:LV-GFP:0.54 ± 0.06,LV-AKAP150 shRNA:0.76 ± 0.03.TST:LV-GFP:122.42 ± 5.94 s,LV-AKAP150 shRNA:76.33 ± 7.00 s.FST:LV-GFP:120.75 ± 7.70 s5 LV-AKAP150 shRNA:73.00 ± 6.76 s).Conclusion:Mice develop depressive-like behaviors when are subjected with CRS.Knockdown of AKAP150 in BLA prevents depressive-like behaviors induced by CRS.These data indicate that scaffold AKAP150 in the BLA may be responsible for stress-associated behavioral changes.Part ? The mechanism of AKAP150 in chronic restraint stress-induced depressive-like behaviorsObjective:In central nervous system,AKAP150 directs protein kinase A(PKA)and phosphatase(PP2B/Calcineurin)toward selected substrates to control the content,efficacy and duration of phosphorylation events.Excitatory synaptic transmission is dependent on glutamatergic system,which is regulated by AKAP150-PKA complex signaling mediated phosphorylation events.Furthermore,accumulating evidence has both linked glutamatergc dysfunction to the pathophysiological process of depression.However,at present,the role of AKAP150 signaling mediated glutamate receptors phosphorylation in stress-induced depression remains unclear.In this part of study,we explored the role of AKAP150 mediated phosphorylation modification in depressive-like behaviors in mice.Methods:Western blotting measured the expression of PKA catalytic subunit a(PKA c-a)and regulatory subunit 2(PKA R2)in total and synaptic protein that were prepared from BLA of stressed mice and controls.The association of AKAP150 and PKA was evaluated using co-immunoprecipitation(Co-IP)assay.We next used a well-characterized interference peptide Ht-31 that blocked the interaction of AKAP150 and PKA to evaluate the role of AKAP150-PKA in depressive-like behaviors.Surface membrane protein was obtained using sulfo-NHS-LC-biotin and NeutrAvidin coupled-agarose breads.Western blotting measured phosphorylation and surface expression of GluA1 and GluA2 subunits of AMPA receptor in BLA of control and stress-exposed mice.Parallel studies were performed on GluN2A and GluN2B subunits of NMDA receptor.Further,we explored changes in GluAl/2 and GluN2A/2B with expression of LV-AKAP150 shRNA and infusion of Ht-31 peptide.Patch-clamp electrophysiological recording method was employed to record AMPA receptor mediated miniature excitatory postsynaptic currents(AMPAR-mEPSCs)in BLA in control and stressed mice with injection of scramble vector or AKAP150 shRNA,also with infusion of CP or Ht-31 peptide.Results:(1)CRS increased AKAP150-anchored PKAc-a(CON:0.98 ± 0.03,CRS:1.20 ±0.09)and PKA R2(CON:1.00 ± 0.06,CRS:1.16:± 0.04).CRS failed to change phosphatase calcineurin level.(2)CRS increased the interaction of AKAP150 and PKA(CON:1.00 ± 0.04,CRS:1.68 ± 0.20),and promoted PKA to assemble into postsynaptic area(PKA c-a:CON:1.00 ± 0.05,CRS:1.57 ± 0.10.PKA R2:CON:1.00 ±0.04,CRS:1.45 ± 0.06).(3)Ht-31,which blocks the association of AKAP150 and PKA,reversed depressive-like behaviors induced by CRS(SPT:CP:0.56 ± 0.07,Ht-31:0.82 ± 0.04.TST:CP:136.38 ± 13.97 s,Ht-31:86.50 ± 8.33 s.FST:CP:112.63 ± 13.09 s,Ht-31:50.50 ±6.78 s.(4)Knockdown of AKAP150 in BLA prevented CRS-induced excessive phosphorylation(LV-GFP:1.45 ± 0.11,LV-AKAP150 shRNA:0.96 ± 0.06)and surface expression of GluAl(LV-GFP:1:37 ± 0.11,LV-AKAP150 shRNA:1.09 ± 0.04).(5)Infusion of Ht-31 rescued stress-induced excessive phosphorylation(CP:1.72 ± 0.19,Ht-31:0.96 ± 0.14)and surface expression of GluA1(CP:1.45 ± 0.14,Ht-31:0.90±0.16).(6)AKAP 150 signaling had no effect on GluN2A/B.(7)CRS increased the amplitude of AMPA receptor mediated mEPSCs,which were normalized with AKAP150 shRNA expression(LV-GFP:17.03± 0.67 pA,LV-AKAP150 shRNA:11.01± 0.62 pA)and Ht-31 infusion(CP:18.06±1.10 pA,Ht-31:12.31 ± 0.46 pA).CRS exposure,AKAP150 shRNA expression and Ht-31 infusion in BLA had no effect on frequency of AMPAR-mEPSCs.Conclusion:AKAP150-PKA complex signaling contributes to development of depressive-like behaviors induced by chronic restraint stress,via regulation of AMPA receptor mediated glutamatergic transmission.Inhibition of AKAP150 and PKA interaction restores stress-induced behavioral abnormalities.Thus AKAP150-PKA signaling could be a novel target for therapeutic intervention of depression with fewer off-target effects.