The Effect And Regulation Of α-DG Glycosylation On Chronic Social Defeat Induced Depressive-like Behaviors Of Mice

Part I The effect of a-DG glycosalation on depressive-like behaviors induced by chronic social defeat stress of miceObjective:a-dstroglycan is a prodominant component in the dystrophin-glycoprotein complex(DGC)and a recently characterized receptor for several extracellular matrix components with high affinity.The precursor protein of a-DG is about 72 kDa and contains a mucin-like structure.This structure can be extensively glycosylated by post translational modification and formed into the glycosylated α-DG(GLYα-DG).The glycosylation amounts are significantly different among the tissues,for example GLY-α-DG in skeletal muscle is 156 kDa,140 kDa in cardiac muscle and 120 kDa in brain and peripheral nerve.The N-terminal of α-DG can be cleavaged into α-DG-N,a 39 kDa protein,which can be secreted into cerebrospinal fluid and plasma.Recent researches have reported that hypoglycosylation of α-DG is associated with the pathophysiology of diseases,especially on muscular dystrophy,but little is known about MDD.Mdx mice are transgenetic models for the investigation of Duchenne muscular dystrophy.Although the main knockout gene of mdx mice is the expression of dystrophin among the DGC complex,the expression of GLY-α-DG is also significantly decreased.Mice with depression often exhibit cognitive deficit and increased anxiety-like behaviors.Recent researches demonstrate that patients with MDD display cognitive impairment,accompanied by symptoms of anxiety.In this study,we employed chronic social defeat stress(CSDS)to establish an animal model of depression.We detected the expression of GLY-α-DG among the brain areas associated with the pathophysiology of depression,further to explore its role in stress-induced depressive-like behaviors.Methods:CSDS was used as an animal model for depression;western blot was used to examine the protein level of α-DG in CSDS susceptible mice and resilient mice among the brain areas associated with the pathophysiology of depression,including prefrontal cortex,(PFC),caudate putamen(CPu),nucleus accumbens(NAc),hippocampus(Hip),habenular nuclei(Hb),lateral amygdala(LA)and central amygdala(CeA).After subdivision of hippocampus into dorsal hippocampus and ventral hippocampus,western blot was used to examine a-DG expression in these subfields;Immunofluorescence was used to detect the expression of a-DG in the ventral hippocampus subfield,including CA1,CA3 and DG Western blot was used to detect glycolysated α-DG(GLY-α-DG)in ventral hippocampus of CSDS susceptible mice.Mdx mice were validated by immunofluorescence;Western blot was used to detect the expression of GLY-α-DG in mdx mice.The depressive-like and anxiety-like behaviors were detected in mdx mice.Subthreshold social defeat stress was used to explore the susceptibility to stress in mdx mice.After in vivo identification on the downregulation effect of si-DAG1,subthreshold social defeat stress was used to explore the susceptibility to stress in mice after virus injection.Results:(1)Post 10 consecutive days of social defeat stress,CSDS susceptible mice exhibited depressive-like behaviors,social interaction ratio was decreased(control:1.57 ±0.17,n = 22;susceptible:0.51 ± 0.06,n = 30,p<0.01 vs control),sucrose preference was decreased(control:0.88 ± 0.01,n = 22;susceptible:0.48 ± 0.03,n = 30,p<0.01 vs control),while CSDS resilient mice showed no difference compared with control(1.46 ±0.07 and 0.84 ± 0.03,n = 21).(2)The expression of α-DG was detected in brain areas associated the pathophysiology of depression,and only the hippocampus showd a decreased expression of α-DG(control:1.00 ± 0.03;susceptible:0.69 ± 0.08,n = 12-13 mice/group,p<0.01 vs control).(3)The hippocampus was subdivided into ventral hippocampus and dorsal hippocampus,only the ventral hippocampus showd decreased expression of α-DG in CSDS susceptible mice(control:0.99 ± 0.02;susceptible:0.79 ±0.03,n = 11-13 mice/group,p<0.05 vs control),while the expresson in the dorsal hippocampus showed no difference(control:0.90 ± 0.03;susceptible:0.99 ± 0.03,n = 8-9 mice/group).(4)The expression of GLY-α-DG was significantly decreased in ventral hippocampus in CSDS susceptible mice(control:1.00±0.03;susceptible:0.80 ± 0.04,n =11-12 mice/group,p<0.05 vs control).(5)The mdx transgenetic mice were further demonstrated by immunofluorescence staining of 426 kDa dystrophin.Mdx mice showed decreased expression of GLY-α-DG(WT:1.00 ± 0.03;susceptible:0.77 ± 0.04,n=4 mice/group,p<0.01 vs WT).(6)Mdx mice showed increased anxiety-like behaviors.In the open field test,the total distance in mdx mice was decreased compared with WT mice(WT:3757.00 ± 231.10 cm,n = 10;mdx:2855.00 ± 196.70 cm,n = 18,p<0.05 vs WT).The center distance,center time and zone crossing in mdx(center distance:481.40 ± 62.88 cm;center time:54.01 ± 7.08 s,n = 18)were significantly decreased compared with WT mice(center distance:748.9 ± 95.97 cm;center time:78.86 ± 8.43 s,n = 10).The average speed of mdx mice was lower than that of WT mice(WT:7.880 ± 0.2375 cm/s;mdx:7.011 ±0.1881 cm/s,p<0.01 vs WT).In the elevated plus maze test,the time spent in open arms was significantly lower than that of WT mice(WT:7.88 ± 0.24 cm/s,n = 10;mdx:7.01 ±0.19 cm/s,n = 18,p<0.01 vs WT).The time spent in closed arms was significantly higher than that of WT mice(WT:175.80 ± 10.31 s,n = 10;mdx:227.40 ± 9.94 s,n = 16,p<0.01 vs WT).The total distance was significantly higher compared with WT mice(WT:971.70 ± 73.03 cm,n = 10;mdx:1182.00 ± 61.68 cm,n = 16,p<0.05 vs WT).(7)Mdx mice displayed some depressive-like behaviors,the latency to feed was increased(WT:166.80 ± 34.14 s,n = 10;mdx:336.2 ± 41.60 s,n = 19,p<0.05 vs WT).The immobility time in FST(WT:90.78 ± 11.17 s,n = 9;mdx:131.60 ± 11.27 s,n = 19,p<0.05 vs WT)and TST(WT:88.80 ± 13.44 s,n = 10;mdx:122.40 ± 6.18 s,n = 12,p<0.05 vs WT)were increased.Mdx mice showed cognitive deficit in the NOR test(discrimination index:WT:0.66 ± 0.03,n = 9;mdx:0.52 ± 0.02,n = 19,p<0.01 vs WT).After CSDS,mdx mice showed decreased social interaction ratio(WT:1.86 ± 0.18,n= 7;mdx:0.91 ± 0.27,n = 8,p<0.05 vs WT)and sucrose preference(WT:0.75 ± 0.04,n = 8;mdx:0.50 ± 0.07,n = 9,p<0.05 vs WT).(8)The lentivirus si-DAG1 decreased the expression of GLY-α-DG(si-GFP:1.01 ± 0.01,n = 16;si-DAG1:0.80 ± 0.02,n = 16,p<0.01 vs si-GFP).(9)The mice were subjected to subthreshold social defeat stress after virus injection,mice with si-DAG1 showed decreased social interaction ratio(si-GFP:1.61 ± 0.10;si-DAG1:1.18 ± 0.12,n =31-32 mice/group,p<0.01 vs si-GFP),prolonged immobility time in FST(si-GFP:41.29 ±3.811 s;si-DAG1:71.72 ± 5.40 s,n = 31-32 mice/group,p<0.01 vs si-GFP)and TST(si-GFP:131.60 ± 7.24 s;si-DAG1:167.40 ± 6.81,n = 32 s,p<0.01 vs si-GFP),and decreased sucrose preference(si-GFP:0.72 ± 0.03;si-DAG1:0.59 ± 0.04,n = 31-33 mice/group,p<0.01).Conclusion:CSDS induced depressive-like behaviors in C57 mice.The expression ofα-DG and GLY-α-DG were significantly decreased in ventral hippocampus of CSDS susceptible mice.Mdx mice showed decreased expression of GLY-α-DG and increased anxiety-like behaviors.Mdx mice displayed some depressive-like behaviors,and the susceptiblity to stress was significantly increased.Downregulation of the expression α-DG in ventral hippocampus by lentivirus increased the susceptibility to stress.Part Ⅱ The regulation of α-DG glycosylation on depressive-like behaviors induced by chronic social defeat stress of miceObjective:Glycosylated α-DG(GLY-α-DG)can bind with high affinity to extracellular matrix(ECM)moleculars that contain laminin globular(LG)domains,including perlecan,agrin and neurexin.In inhibitory neurons,neurexin can interact with postsynaptically expressed neuroligin and neurexophilins,the binding with GLY-α-DG is not specific.Rencent researches have demonstrated that agrin could be expressed in both neurons and astrocytes.The concentration of neuronal agrin is more than 1,000-fold higher in cultured hippocampal neurons than that in cultured astrocytes,suggesting that neurons are a major source of agrin in the brain.The researches on agrin focus mainly on the cultured neurons,but little is known in the brain.In our research,we will screen the antidepressant dose of agrin in the normal mice and investigate the effect of the agrin on depressive-like behaviors in CSDS susceptible mice.Large is an enzyme associated with the glycosylation of α-DQ thus,we investigated the antidepressant effect of Large by using the adeno-associated virus mediated overexpression.Meanwhile,recent research reports that GLY-α-DG can take part in the regulation of homeostatic plasticity,and GLY-α-DG inhibits the GABAergic neurons mediated scaling up of synapse.Overexpression of Large enhances the synaptic strength and increases the clustering of GABAAR.The researches on the MDD also report that the activity of GABAergic neurons was significantly decreased.The expression of GAD65 and GAD67,the enzymes which are responsible for the synthetization of inhibitory neurotransmitter GABA were decreased in the hippocampus,the synaptic transmission of inhibitory neurons was impaired.According to this evidence,we investigated the role of GLY-α-DG on the regulation of inhibitory synaptic transmission using electrophysiological method,trying to clarify the mechanism of GLY-α-DG mediated depressive-like behaviors.Methods:Stereotactic implanting of cannulas into ventral hippocampus of normal mice,behavior tests were conducted 1 d and 3 d after administration of agrin.Administration of agrin through the cannulas in CSDS susceptible mice,behavior tests were conducted 1 d,1 w and 2 w after administration of agrin.Western blot was used to detecte the expression of Large.Confocal fluorescence,western blot and qPCR were used to validate the effect of AAV-Large.The behavior tests were conducted after virus injection in ventral hippocampus,whole-cell patch-clamp technique was used to record miniature inhibitory postsynaptic current(mIPSC)in ventral CA1 area.Results:In normal mice,the immobility time in TST showed a trend of dcrease after a single dose of agrin,but without significance.After 3 consecutive days of administration,agrin at low and medium concentration decreased the immobility time significantly compared with the mice treated with vehicle(vehicle:156.70 ± 18.51 s;10 μg/ml:87.06 ±13.97 s;20 μg/ml:88.83 ± 21.90 s;10 μg/ml:112.30 ± 25.34 s,n = 5-7 mice/group,p<0.05 vs vehicle).(2)CSDS susceptible mice were administrated with agrin(20 μg/ml)and then subjected to behavior test.In TST test,the immobility time was significantly decreased 1 d after administration,compared to pre-administration.No difference was found 1 and 2 w after administration(CSDS:160.7 ± 9.266 s;1 d:129.10 ± 13.53 s,n = 5-15 mice/group,p<0.05 vs CSDS).In FST test,the immobility time was significantly decreased 1 d and 1 w after agrin adniminstration compared to before administration.No difference was found 2 w after agrin administration(CSDS:59.83 ± 7.170 s;1 d:36.08 ±7.70 s;1 w:19.00±6.95 s,n = 5-15 mice/group,p<0.05 vs CSDS).(3)Large was the vital enzyme,which mediates the glycosylation of α-DG.It was significantly decreased in CSDS susceptible mice(control:1.00 ±0.02,n = 10;susceptible:0.85 ± 0.05,n = 10,p<0.05 vs control).(4)The adeno-associated virus AAV-Large increased the expression of Large(AAV-GFP:1.00± 0.05,n = 4;AAV-Large:1.72 ± 0.12,n = 7,p<0.01 vs AAV-GFP)and enhanced the glycosylation of a-DG(AAV-GFP:1.00 ± 0.01,n = 6;AAV-Large:1.12 ± 0.05,n = 6).(5)Microinjection of AAV-Large into susceptible mice decreased the immobility time in TST(control + AAV-GFP:142.20 ± 8.133 s;control + AAV-Large:155.60 ± 7.43 s;susceptible+AAV-GFP:170.60 ± 6.50 s;susceptible + AAV-Large:141.50 ± 6.46 s)and FST(control+ AAV-GFP:31.41 ± 6.14 s;control + AAV-Large:36.64 ± 10.22 s;susceptible +AAV-GFP:59.95 ± 6.15 s;susceptible + AAV-Large:34.06 ± 7.27 s),alleviated the social avoidance(control + AAV-GFP:1.39 ± 0.29;control + AAV-Large:1.17 ± 0.15;susceptible+AAV-GFP:0.61 ± 0.06;susceptible + AAV-Large:1.60 ± 0.50;n = 10-20 mice/group;p<0.05,p<0.01 vs control + AAV-GFP,p<0.05,p<0.01 vs susceptible + AAV-GFP).(6)Overexpression of Large in ventral hippocampus reversed the decrease in mIPSC frequency(control + AAV-GFP:5.01± 0.36 Hz;control + AAV-Large;4.48 ± 0.64 Hz;susceptible +AAV-GFP:3.34 ± 0.37 Hz;susceptible + AAV-Large:5.71 ± 0.37 Hz,p<0.01 vs control +AAV-GFP,p<0.01 vs susceptible + AAV-GFP)and amplitude(control + AAV-GFP:11.21± 0.29 pA;control + AAV-Large:8.65 ± 0.44 pA;susceptible+AAV-GFP:9.52 ± 0.29 pA;susceptible + AAV-Large:11.24 ± 0.42 pA,p<0.01 vs control + AAV-GFP,p<0.01 vs susceptible + AAV-GFP).Conclusion:By stereotactic injection of agrin directly into vHip in normal mice for three consecutive days,the low and medium dose of agrin could decrease the immobility time in TST.Administration of agrin to CSDS susceptible mice exerted antidepressant effects,and this effect could partially sustain for a week.The expression of Large was decreased in vHip.Overexpression of Large through AAV-Large reversed the depressive-like behaviors and restored the decreased frequency and amplitude of mIPSC.