| Part one: Neuronal Zinc-?2-glycoprotein(ZAG)expression was decreased by seizures both in vivo and in vitroObjective:To detect the location of ZAG in neuron and effect of epilepsy or seizure on ZAG expression in PTZ-kindled epileptic rat and Mg2+-free artificial cerebrospinal fluid(ACSF)-induced neuronal seizure model.Methods:1.Adult male Sprague-Dawley rats weighing 200 to 300 g received daily intraperitoneal injections of PTZ(35mg/kg)for 28 days to establish an epileptic model.2.Cultured neurons were incubated with Mg2+-free ACSF to establish the neuronal seizure model.3.The double-labeled immunofluorescence staining to testify the location of ZAG in brain tissues and cultured rat cortical neurons.4.Western blot and q RT-PCR was used to determine the ZAG protein and m RNA level.Results:1.Double immunofluorescence staining of rat brain sections revealed the colocalization of ZAG with microtubule-associated protein 2(MAP2).ZAG also colocalized with Neu N in primary cultures of rat cortical neurons.2.In the cortex of PTZ-kindled rats,levels of both the ZAG protein(0.235±0.033 vs.0.435±0.031,p<0.001,n=5)and AZGP1 m RNA(0.358±0.081 vs.1,p=0.001,n=4)were decreased compared to the control.3.In the Mg2+-free ACSF-induced neuronal seizure model,levels 4of the ZAG protein(0.577±0.065 vs.0.172±0.073,p=0.002,n=3)and AZGP1 m RNA(0.357±0.098 vs.1,p<0.001,n=3)were also decreased compared to the control.Conclusions:1.ZAG is localized in cortical neurons.2.Both ZAG protein and m RNA are decreased by epilepsy or seizure.Part two: Seizures decreased ketogenesis in neurons,which was reversed by ZAG overexpressionObjective: To detect whether ketogenesis could be decreased by epilepsy or seizure,and reversed by ZAG overexpression.Methods:1.Adult male Sprague-Dawley rats weighing 200 to 300 g received daily intraperitoneal injections of PTZ(35mg/kg)for 28 days to establish an epileptic model.2.Cultured neurons were incubated with Mg2+-free ACSF to establish the neuronal seizure model.3.AAV containing the full-length AZGP1 c DNA(AAV-AZGP1)or an AAV-vector were stereotaxically injected into bilateral hippocampus to overexpress ZAG.Lentivirus containing the full-length AZGP1 c DNA(LV-AZGP1)or LV-AZGP1-RNAi was used to overexpress ZAG or knockdown ZAG level,and Vector-AZGP1 or Vector-RNAi containing GFP alone was used as controls,respectively.4.Rats were divided into 4 groups: Vector-AAV,Vector-AAV+PTZ,AAV-AZGP1,and AAV-AZGP1+PTZ.5.Cultured neurons were divided into 5 groups: Vector-AZGP1,Vector-RNAi,Vector-AZGP1+seizure,LV-AZGP1+seizure,LV-AZGP1-RNAi6.A 100?M Palmitic acid(PA)and 1m M L-carnitine(LC)were used to treat culture neurons further keton body production.7.Modified Racine scale was used to grade the seizure severity.8.Scalp electroencephalography(EEG)was performed to evaluate the epileptic discharges.9.The cyclic thio-NADH method was used to measure the level of Ac Ac and BHB in brain homogenate and culture media.The results were normalized by protein level and duration treatment time of PA.10.Western blot and q RT-PCR was used to determine the ZAG protein and m RNA level.Results:1.Rats in Vector-AAV+PTZ group presented significantly severer seizure compared to rats in AAV-AZGP1+PTZ group.2.Scalp EEG showed that AAV-AZGP1 treatment decreased the frequency and amplitude of seizure spike wave after PTZ kindling compared to Vector-AAV.3.AAV-AZGP1 group presented increased acetoacetate acid(Ac Ac)level compared to Vector-AAV group(66.935±2.588pmol/?g vs.56.000±5.869pmol/?g,p=0.010,n=4).A significantly lower Ac Ac level was observed in the Vector-AAV+PTZ group than in Vector-AAV group(31.334±3.448pmol/?g vs.56.000±5.869pmol/?g,p<0.001,n=4),while a significantly higher Ac Ac level was detected in the AAV-AZGP1+PTZ group than in the Vector-AAV+PTZ group(55.866±2.504pmol/?g protein vs.31.334±3.448pmol/?g protein,p<0.001,n=4).However,the?-hydroxybutyric acid(BHB)level was not different between groups.4.Ac Ac levels were significantly decreased in the Vector-AZGP1+seizure group(1.919±0.483nmol/?g/8hours vs.3.474±0.766nmol/?g/8hours,p=0.012,n=5)and LV-AZGP1-RNAi group(1.592±0.595nmol/?g/8hours vs.3.428±0.402nmol/?g/8hours,p=0.002,n=5)compared to the Vector-AZGP1 group and Vector-RNAi group,respectively.The seizure-induced decrease in the Ac Ac level was rescued by ZAG overexpression using LV-AZGP1(1.919±0.483nmol/?g/8hours vs.4.453±0.887nmol/?g/8hours,p<0.001,n=5).The BHB level was not different between groups.Conclusions:1.Epilepsy or Seizure decreased ketogenesis in neurons,which was reversed by ZAG overexpression.2.ZAG overexpression alleviated seizure severity induced by PTZ-kindling.Part three: PPAR? promoted neuronal ketogenesis in cultured neurons via increasing neuronal ZAG expression by binding to the promoter region of the AZGP1 geneObjective: To testify whether PPAR? promoted neuronal ketogenesis in cultured neurons via increasing neuronal ZAG expression by binding to the promoter region of the AZGP1 gene.Methods:1.Cultured primary rat cortical neurons were treated with following drugs: PPAR? agonist pioglitazone(10?M);PPAR? inhibitor GW9662;PA(100?M)and LC(1m M)and transfected with LV.2.To confirm whether PPAR? regulated ZAG transcription and neuronal ketogenesis,cultured primary rat cortical neurons were divided into 3 groups: pioglitazone(10?M),GW9662(10?M)and DMSO).3.Chromatin immunoprecipitation(Ch IP)was conducted to examine whether PPAR? regulates ZAG by binding to the promoter region of the AZGP1 gene.4.To confirm whether PPAR? promoted neuronal ketogenesis via increasing ZAG expression,neurons were divided into 6 groups:Vector-AZGP1+DMSO,Vector-RNAi+DMSO,LV-AZGP1+DMSO,LV-AZGP1+GW9662,LV-AZGP1-RNAi+DMSO,and LV-AZGP1-RNAi+pioglitozole.5.The cyclic thio-NADH method was used to measure the level of Ac Ac and BHB in culture media.The results were normalized by protein level and duration treatment time of PA.6.Western blot and q RT-PCR was used to determine the ZAG protein and m RNA level.Results:1.Compared to the DMSO group,pioglitazone increased the levels of the ZAG protein(1.013±0.135 vs.0.654±0.090,p=0.004,n=4)and AZGP1 m RNA(1.474±0.078 vs.1,p=0.003,n=3),while GW9662 significantly decreased the levels of the ZAG protein(0.361±0.097 vs.0.654±0.090,p=0.013,n=4)and AZGP1 m RNA(0.482±0.153 vs.1,p=0.002,n=3).2.Ac Ac level was increased by pioglitazone(3.609±0.298nmol/?g/8hours vs.2.543±0.402 nmol/?g/8hours,p=0.003,n=4)and decreased by GW9662(1.676±0.200nmol/?g/8hours vs..543±0.402nmol/?g/8hours,p=0.010,n=4)compared to DMSO.However,the BHB level was not altered.3.Ch IP-q RT-PCR revealed that the sequence in the ZAG promoter region was enriched in the Ch IP group,but not in the Ig G group(Inupt%:0.044±0.011 for Ig G vs.0.622±0.198 for anti-PPAR? antibody,p=0.007,n=3).4.Ac Ac levels were significantly increased after LV-AZGP1 treatment(5.725±0.589nmol/?g/8hours vs.3.850±0.486nmol/?g/8hours,p<0.001,n=4)compared to the Vector-AZGP1+DMSO group;this increase in the Ac Ac level was not abolished by GW9662(5.635±0.656nmol/?g/8hours vs.5.725±0.589nmol/?g/8hours,p=1.000,n=4).In contrast,the Ac Ac level was significantly decreased after LV-AZGP1-RNAi treatment(2.366±0.177nmol/?g/8hours vs.3.603±0.244nmol/?g/8hours,p=0.013,n=4)compared to the Vector-RNAi+DMSO group.This decrease in the Ac Ac level was not rescued by pioglitazone(2.547±0.220nmol/?g/8hours vs.2.366±0.177nmol/?g/8hours,p=1.000,n=4).Conclusions:1.PPAR? promoted neuronal ketogenesis in cultured neurons.2.PPAR? promoted ZAG expression by binding to the promoter region of the AZGP1 gene.3.PPAR? promoted neuronal ketogenesis via increasing neuronal ZAG expression by binding to the promoter region of the AZGP1 gene.Part four: ZAG was located in the mitochondria and possibly enhanced HADHB activity by bindingObjective: Immunoprecipitation and mass spectrometry(IP/MS)was used to screen potential proteins that interacted with ZAG in primary cultures of cortical neurons to further explore the mechanism by which ZAG affected ketogenesis.Methods:1.Cultured neurons were transfected with LV to modulate ZAG protein level.2.IP/MS was used to screen potential proteins that interacted with ZAG in primary cultures of cortical neurons.3.Co-immunoprecipitation was used to confirm interaction between ZAG and HADHB.4.Immunofluorescence staining to testify the location of ZAG in mitochondria of cultured rat cortical neurons.5.Mitochondrial proteins were extracted from neurons and separated by western blot to detect the presence of ZAG in mitochondrial.6.Neurons were divided into 5 groups: Control group;Vector-AZGP1group;Vector-RNAi group;LV-AZGP1 group;LV-AZGP1-RNAi group.7.HADHB activity was measured to investigated the effect of ZAG on HADHB.Results:1.114 proteins precipitated by the anti-ZAG antibody were identified,including cytosolic proteins,nuclear proteins,ribosomal proteins and mitochondrial proteins and were involved in metabolism,translocation,immunology and transcriptional regulation.2.Co-IP assay found that ZAG coimmunoprecipitated with HADHB.3.Double-labeled immunofluorescence staining confirmed the localization of ZAG in mitochondria4.Western blots also revealed the presence of ZAG in mitochondrial protein extracts.5.LV-AZGP1-RNAi treatment in neurons significantly decreased the3-ketoacyl-Co A thiolase activity of HADHB(135.005±3.787 IU/mg protein vs.157.360±4.216 IU/mg protein,p<0.001,n=5)compared to Vector-RNAi-transfected neurons,while LV-AZGP1 treatment in neurons significantly increased the 3-ketoacyl-Co A thiolase activity of HADHB compared with the Vector-AZGP1(169.341±5.076 IU/mg protein vs.156.755±7.122 IU/mg protein,p=0.031,n=5).Conclusions:1.ZAG was located in the mitochondria.2.ZAG enhanced HADHB activity by binding to HADHB.Part five: ZAG translocation into the mitochondria was mediated by a HSC70-dependent mechanism that affected ketogenesisObjective: The IP/MS identified a protein HSC70 which was reported to mediate protein translocation into mitochondria.We intended to investigate whether HSC70 was involved in the translocation of ZAG into mitochondria.Methods:1.Cultured primary rat cortical neurons were treated with following drugs: apoptozole(5?M),PA(100?M)and LC(1m M)and transfected with LV.2.Co-immunoprecipitation was used to confirm interaction between ZAG and HSC70.3.Neurons were divided into 2 groups: apoptozole(5?M)or DMSO,and mitochondrial proteins were extracted to examine the effect of HSC70 on mitochondrial ZAG levels.4.Quantitative Co-IP was also used to determine the interaction between ZAG and HSC70 was inhibited by apopotozole.5.cultured cortical neurons were divided into 4 groups to detect the effect of HSC70 inhibition on neuronal ketogenesis:Vector-AZGP1+DMSO,LV-AZGP1+DMSO,Vector-AZGP1+apoptozole,and LV-AZGP1+apoptozole.6.The cyclic thio-NADH method was used to measure the level of Ac Ac in culture media.The results were normalized by protein level and duration treatment time of PA.Results:1.ZAG coimmunoprecipitated with HSC70,and a subsequent reciprocal experiment using an anti-HSC70 antibody also validated this result.2.Compared to the control,mitochondrial ZAG levels were significantly decreased in the apoptozole-treated group(0.275±0.035 vs.0.579±0.051,p<0.001,n=5).3.The amount of ZAG bound to HSC70 was significantly decreased by the apoptozole treatment compared to DMSO(0.091±0.009 vs.0.155±0.026,p=0.015,n=3),while apoptozole did not change the total ZAG level compared to DMSO.4.Significantly lower neuronal Ac Ac levels were observed in the Vector-AZGP1+apoptozole group compared to the Vector-AZGP1+DMSO group(1.865±0.242nmol/?g/8hours vs.2.837±0.178nmol/?g/8hours,p=0.005,n=4).LV-AZGP1 significantly increased Ac Ac levels compared to the Vector-AZGP1+DMSO group(4.087±0.261nmol/?g/8hours vs.2.837±0.178nmol/?g/8hours,p=0.001,n=4),while this increase was abolished by apoptozole(2.898±0.487nmol/?g/8hours vs.4.087±0.261nmol/?g/8hours,p=0.001,n=4).Conclusions:HSC70 regulated the mitochondrial translocation of ZAG and subsequently affected ketogenesis. |
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