Effects And Mechanisms Of Death-associated Protein Kinase 1 On Interleukin-1? Production In Microglia

Part I DAPK1 is required for ?-amyloid-induced interleukin-1? production in microgliaObjective Investigate whether P-amyloid(A(3)could induce the production of interleukin-1?(IL-1?),and the activation of NLRP3 inflammasome and DAPK1 in microglia,as well as explore the role of DAPK1 in the process of inflammasome activation.Methods The experiment was divided into A,B and C three parts.The A part was to investigate whether A(3 treatment could result in IL-1? secretion,NLRP3 inflammasome and DAPK1 activation.Bv2 cells were primed with 100 ng/mL LPS for 6 h and stimulated with A?25-35(25 ?M)/fA?1-42(10 ?M)/oA?1-42(10 ?M)for 6 h,24 h and 48 h respectively.Culture supernatants and whole cell lysates were collected.The concentration of IL-1(3 in supernatant was measured by ELISA.Caspase-1 activity in cells were determined by a commercial caspase-1 activity kit.The expression levels of caspase-1,NLRP3,DAPK1 and p-MLC were measured by western blotting.The B part was to investigate whether the expression of DAPK1 had an effect on A?25-35-induced NLRP3 inflammasome activation.The experimental groups:?scrambled shRNA+LPS group:cells were transfected with scrambled shRNA and primed with LPS;?scrambled shRNA+LPS+A?25-35 group:scrambled shRNA transfected cells were primed with LPS and stimulated with A?25-35;?DAPK1-shRNA+LPS group:DAPK1 silencing cells were generated using DAPK1-shRNA containing lentiviral vector,and primed with LPS;?DAPK1-shRNA+LPS+A(325-35 group:DAPK1 silencing cells were primed with LPS and treated with AP25-35;?cDAPKl+LPS group:DAPK1 silencing cells were transfected with cDAPK1 and primed with LPS;?cDAPK1+LPS+A?25-35 group:cDAPKl transfected cells were primed with LPS and treated with A?25-35.Culture supernatants and whole cell lysates were collected after 24 h of AP25-35 treatment.The concentration of IL-1?in supernatant was measured by ELISA.The expression of NLRP3 inflammasome components(NLRP3,pro-caspase-1,caspase-1 p20 and ASC),pro-IL-1?,DAPK1 and p-MLC was measured by western blotting.The C part was to investigate whether the inhibition of DAPK1 activity could affect AP25-35-induced NLRP3 inflammasome activation.The experimental groups:?LPS group:cells were primed with LPS;?LPS+DAPK1 inhibitor group:cells were treated with 20?M DAPK1 inhibitor after LPS priming for 5 h;?LPS+A?25-35 group:cells were primed with LPS for 6 h and treated with AP25-35;?LPS+A?25-35+DAPK1 inhibitor group:cells were treated with varying doses of DAPK1 inhibitor(5,10,20 ?M)1 h prior to A?25-35 treatment.Culture supernatants and whole cell lysates were collected after 24 h of A?25-35 treatment.The concentration of IL-1? in supernatant was measured by ELISA.The expression of NLRP3 inflammasome components(NLRP3,pro-caspase-1,caspase-1 p20 and ASC),pro-IL-1?,DAPK1 and p-MLCwas measured by western blotting.Results In the A part,compared with control group,stimulation with LPS plus A?25-35 markedly increased IL-1? in supernatants(P<0.05 at 6 h;P<0.001 at 24 h;P<0.001 at 48 h).IL-1? concentration in the supernatant was highest at the 24 h time point.However,the concentration of IL-1? was slightly increased with no statistical significance at any time point in LPS+fA?1-42 and LPS+oA?1-42 group(P>0.05).Caspase-1 activity and caspase-1p20 expression were significantly increased in LPS+AP25-35 group at the 24 h time point(P<0.001).The level of NLRP3 was markedly increased in LPS group(P<0.001)and LPS+A?25-35 group(P<0.001).The expression of p-MLC was significantly up-regulated in LPS+A?25-35 group(P<0.001),nevertheless,the expression of DAPK1 was comparable among all groups.In the B part,compared with Scr-LPS+A?25-35 group,IL-1? concentration in the supernatant was significantly reduced in DAPKl-shRNA+LPS+A?25-35 group(P<0.001);the expression of caspase-1 p20 and ASC was markedly decreased in DAPK1-shRNA+LPS+A?25-35 group(P<0.001).Compared with DAPK1-shRNA+LPS+A?25-35 group,IL-1(3 concentration in supernatants was significantly increased in cDAPKI+LPS+A?25-35 group(P<0.001);the levels of caspase-1 p20 and ASC were markedly increased in cDAPK1+LPS+A?25-35 group(P<0.001).The expression of NLRP3,ASC and pro-IL-1?was comparable among all groups.In the C part,compared with LPS+A?25-35 group,IL-1? concentration in the supernatant was reduced in LPS+A?25-35+DAPK1 inhibitor group in a dose-dependent manner(P<0.05 at 5 ?M;P<0.05 at 10 ?M;P<0.001 at 20 ?M).The expression of p-MLC and caspase-1 p20 was significantly down-regulated in LPS+A?25-35+DAPK1 inhibitor group(P<0.01).The levels of NLRP3,ASC and pro-IL-1? were comparable among all groups.Conclusion(1)LPS plus A?25-35 treatment induces IL-1? secretion,as well as NLRP3 inflammasome and DAPK1 activation in Bv2 cells.(2)DAPK1 mediates the process of A?25-35-induced NLRP3 inflammsome assembly and activation.(3)The catalytic activity of DAPK1 is required for NLRP3 inflammasome assembly and activation.Part ? The interaction between cathepsin B and DAPK1Objective Investigate the role of cathespin B in DAPK1 activation.Methods The experiment was divided into A,B and C three parts.The A part was to investigate whether A?25-35 treatment could induce lysosomal disruption and cathepsin B release.The experimental groups:?control group;?LPS group:cells were primed with 100 ng/ml;?LPS+A?25-35 group:cells were treated with 25 ?M A?25-35 after 6 h of LPS priming.To monitor lysosome rupture and cathepsin B activity,cells were incubated with acridine orange(AO)or with a fluorogenic cathepsin B substrate after 24 h of A?25-35 treatment.The B part was to investigate whether inhibition of cathepsin B had an effect on A?25-35-induced inflammasome and DAPK1 activation.The experimental groups:?LPS group:cells were primed with LPS;?LPS+cathespin B inhibitor group:cells were treated with 5 ?M cathepsin B inhibitor after 5 h of LPS priming;?LPS+A?25-35 group:cells were treated with 25 ?M A?25-35 after 6 h of LPS priming;?LPS +A?25-35+cathepin B inhibitor group:cells were treated with varying doses of cathepsin B inhibitor(1,2.5,5 ?M)1 h Prior to A?25-35 treatment.Culture supernatants and cell lysates were collected after 24 h of A?25-35 treatment.The concentration of IL-1? in supernatant was measured by ELISA.The expression of pro-caspase-1,caspase-1 p20,DAPK1 and p-MLC was measured by western blotting.The C part was to investigate whether inhibition of DAPK1 had an effect on A?25-35-induced cathepsin B leakage.The experimental groups:?LPS group:cells were primed with LPS;?LPS+DAPK1 inhibitor group:cells were treated with 20 ? DAPK1 inhibitor after 5 h of LPS priming;?LPS+A?25-35 group:cells were treated with 25 ?M A?25-35 after 6 h of LPS priming;?LPS +A?25-35+DAPK1 inhibitor group:cells were treated with DAPK1 inhibitor 1 h prior to A?25-35 treatment.Culture supernatants and cell lysates were collected after 24 h of A?25-35 treatment.The concentration of IL-1? in supernatant was measured by ELISA.After 24 h of A?25-35 treatment,the expression of active cathepsin B in the cytoplasm by western blotting.To measure lysosome rupture and cathepsin B activity,cells were incubated with AO and a fluorogenic cathepsin B substrate.Results In the A part,compared with control group,LPS plus A?25-35 treatment induced lysosomal disruption and cathepsin B leakage to the cytosol.In the B part,compared with LPS+A?25-35 group,IL-1? concentration in the supernatant was reduced in LPS+A?25-35+cathepsin B inhibitor group in a dose-dependent manner(P<0.001 at 2.5 ?M;P<0.001 at 5 ?M).The expression of p-MLC and caspase-1 p20 was significantly down-regulated in LPS+ A?25-35+cathepsin B inhibitor group(P<0.05).The levels of DAPK1 were comparable among all groups.In the C part,compared with LPS+A?25-35 group,the expression of active cathepsin B in the cytoplasm,lysosomal disruption and cathespin B leakage were unchanged in LPS+A?25-35+DAPK1 inhibitor group.Conclusion A?25-35 treatment induces lysosomal rupture and cathepsin B leakage in microglia which is necessary for the activation of NLRP3 inflammasome.Furthermore,cathepsin B acts upstream of DAPK1.Part ? DAPK1 mediates NLRP3 inflammasome activtaion in the hippocampus of miceObjective Investigate the role of DAPK1 in A?25-35-induced interleukin-1?(IL-1?)production in the hippocampus and memory deficits of mice.Methods The experiment was divided into A,B and C three parts.The A part was to investigate whether A?25-35 could induce the activation of microglia,NLRP3 inflammasome and DAPK1 in the hippocampus of mice.Twenty male C57BL/6 mice,8-10 weeks old,body weight 23-25g,were used in this part.Mice were randomly divided into two groups(n=10):?sham group:mice were intracerebroventricularly(i.c.v.)injected with 3 ?lof PBS;?AP25-35 group:mice were i.c.v.injected with 3 ?lof A?25-35.After 48 h of A?25-35 treatment,mice brains were collected.The expression of microglial marker Ibal,NLRP3 and DAPK1 was measured by immunofluorescence analyses.The levels of NLRP3 inflammasome components(NLRP3,pro-caspase-1,caspase-1 p20 and ASC),pro-IL-1?,DAPK1 and p-MLC were determined by western blotting.The B part was to investigate whether inhibition of DAPK1 activity could reduce A(325-35-induced NLRP3 inflammasome activation in the hippocampus.Fifty male C57BL/6 mice,8-10 weeks old,body weight 23-25g,were used in this part.For the acute treatment of DAPK1 inhibitor,mice were received a single i.c.v.injection of DAPK1 inhibitor(2.5 or 5 nmol/mouse at 2 ?l)1 h before A?25-35 administration.Mice were randomly divided into five groups(n= 10):?sham+vehicle group:mice were i.c.v.injected with 2 ?lof 10%DMSO 1 h prior to PBS injection;?sham+DAPK1 inhibitor group:mice were i.c.v.injected with 5 nmol of DAPK1 inhibitor(2 ?l)1 h prior to PBS injection;?A?25-35+vehicle group:mice were i.c.v.injected with 10%DMSO and 3 nmol of A?25-35(3?l);?A?25-35+ low dose DAPK1 inhibitor group:mice were i.c.v.injected with 2.5 nmol of DAPK1 inhibitor(2 ?l)and A?25-35;?A?25-35+high dose DAPK1 inhibitor group:mice were injected with 5 nmol of DAPK1 inhibitor and AP25-35.Mice were sacrificed and collected samples after 48 h of A?25-35 treatment.Hippocampal contents of IL-1? were analyzed by ELISA.The protein levels of NLRP3 inflammasome components,DAPK1 and p-MLC were examined by western blotting.The Part C was to investigate the involvement of DAPK1 in A?25-35-induced memory decline of mice.Forty-four male C57BL/6 mice,8-10 weeks old,body weight 23-25g,were used in this part.For the subchronic treatment of DAPK1 inhibitor,mice were intraperitoneally(i.p.)injected with the DAPK1 inhibitor(1 mg/kg/day at 100 ?l)for 8 consecutive days.Mice were randomly divided into four groups(n=11):?sham+vehicle group:mice received i.c.v.injection of PBS after 8 consecutive days of i.p.injection with 10%DMSO(100 ?l);?sham+DAPK1 inhibitor group:mice received i.c.v.injection of PBS after DAPK1 inhibitor treatment;? A?25-35+vehicle group:mice received i.c.v.injection of A?25-35 after DMSO treatment;?A?25-35+DAPK1 inhibitor group:mice received i.c.v.injection of A?25-35 after DAPK1 inhibitor treatment.The open field test(OFT)and object recognition test(ORT)were performed on day 7 and 8 after peptide injection.On the days 9-10 after AP25-35 administration,fear conditioning tests(FCTs)were carried out.Results In the A part,compared with sham group,increased Ibal and NLRP3 immunofluorescent staining were observed in the CA area of hippocampus from A?25-35-injected mice.At the same time,the Protein levels of NLRP3,pro-IL-1?,caspase-1 p20 and p-MLC of hippocampus were significantly increased(P<0.05).In the B part,compared with A?25-35+vehicle group,IL-1? concentration in the hippocampus was reduced in AP25-35+DAPK1 inhibitor group(P<0.001 at 5 ?M).The expression of p-MLC and caspase-1 p20 were also significantly decreased in AP25-35+DAPK1 inhibitor group(P<0.05).In the C part,compared with sham+vehicle group,the recognition index as well as contextual and cue freezing responses were significantly decreased in AP25-35+vehicle group(P<0.05).DAPK1 inhibitor administration significantly increased the recognition index(compared with A?25-35+vehicle group,P<0.01)and attenuated the impairment of contextual and cue freezing responses(compared with A?25-35+vehicle group,P<0.001)in A?25-35-injected mice.Conclusion A?25-35 treatment induces the activation of NLRP3 inflammasome and DAPK1 in the hippocampus of mice.However,DAPK1 inhibitor administration reduces IL-1? production through inhibiting NLRP3 inflammasome activation and ameliorates memory deficits in A?25-35-injected mice.