Effects Of Aquaporin-4 Knockout On The Expression And The Function Of Cytochrome P4502e1 In Astrocyte

Aquaporins(AQPs)areintegralmembraneproteinsthatserveaschannelsinthetransferofwater,andinsomecases,smallsolutesacrossthemembrane.Sofar,13aquaporinisoforms(AQP0-AQP12)havebeenidentifiedinmammalianspecies.AQP4isthepredominantisoformwhichislocalizedonependymalcellsliningtheventriclesandastrocytesmembrane.Itsexpressioninneurovascularunitisrestrictedtotheperivascularglialprocesses,suggestingAQP4involvementinthemaintenanceofcerebralhomeostasisandmaybethekeypointinastrocytesmodulationtocontroldiversephysiologicalprocesses.Astrocytesplayacrucialroleinmaintainingaproperenvironmentforneuronalactivity.Thisincludes:removal,metabolism,andreleaseofneurotransmitters;maintenanceofextracellularK+andH+levels;secretionofneurotrophicfactor,promotingneurogeneis,andprovidingenergysubstratesfortheirdemandingneighborstheneurons.AccumulatingdatahaveshownthatAQP4depletionordown-regulationcausedastrocytesdysfunctionsuchasreducedmembranewaterpermeability,impairedcellgrowthormigration,attenuatedpostassiumbuffering,andalteredcytoskeletonrearrangement.Italsoservesasacomponentinmicrogliaactivationorblood-brainbarrier(BBB)integritywithexperimentalsupport.Byfar,AQP4hasgainedsufficientattractioninnumerouscerebraldisordersincludingedema,traumaticinjury,tumor,infection,andepilepsy. CytochromeP450representsafamilyofhemoproteinsthatparticipateinthebiotransformationofseveralendogenous(e.g.,steroids,fattyacids,biogenicamines)andexogenous(e.g.,chemicalcarcinogens,solvents,alcohols)compounds.Inmammals,atleast12differentcytochromeP450familiesand22subfamilieshavebeenidentified.Althoughtheseenzymesarefoundprincipallyintheendoplasmicreticulumofhepatocytes,significantlevelsarealsofoundinmanyextrahepatictissues,includingbrain.Oneoftheisoformsthathasreceivedmuchattentionduringrecentyearsistheethanol–induciblecytochromeP4502E1(CYP2E1),notonlybecauseofthecapacityofthisisoenzymetometabolizeethanoltoacetaldehyde,butalsobecauseofitsroleinthemetabolicactivationofalargenumberoftoxicologicalcompounds,includingacetaminophen,varioussolvents,andnitrosamines.Inaddition,CYP2EIhasanapparentlyhighrateofoxidaseactivitythatcausestheformationofreactiveoxygenspecies(ROS)duringitscatalyticcycle,andtheseareabletoinitiatelipidperoxidationanddamagecellmembranes.ThepresenceofCYP2E1inthebrainhasbeenreported,anditsinductionbyethanolhasbeendemonstratedinthisorgan.Moreover,wealsofoundacorrelationbetweentheinductionofCYP2E1andtheincreaseintheformationofoxygenradicalspeciesinthebrainofchronic-alcohol-fedrats.IthasbeensuggestedthatLPSincreaseCYP2E1andinduceoxidativestressinastrocytes.IncreasingevidenceshowsthatcytochromeP4502E1isinvolvedtheMPTP-inducedmousemodelofPD.However,whetherMPTPinfluencetheexpressionofCYP2E1inastrocytesremainsunclear.Oxidativestresshasbeenimplicatedinarangeofdegenerativedisease.Astrocytesplayanimportantroleinclearingproductionofreactiveoxygenspecies(ROS)inthebrain,AstrocytesmayhaveacloserelationwiththeselectivevulnerabilityofneuronsbyscavengingROSandreleasingtheprecursorofγ-L-glutamyl-L-cysteinyglycine(GSH)synthesisinneurons,Furthermore,alterationinastrocyteGSHlevelmaybeanimportantcontributortothepathogenesisof neurodegenerativedisease.Inthepresentstudy,firstweinvestigatetheeffectoftheexpressionoftheCYP2E1inducedbytheMPP+(theionformofMPTP);second,weinvestigatetheroleofAQP4intheexpressionandthefunctionofCYP2E1invivobyusingAQP4geneknockoutmice,especiallytheproductionofROS.TheresultsrevealedherewillnotonlyprovethenewevidenceofthepathogenesisandthetreatmentofParkinson'sdiseaseintherespectofthedrugmetabolism;butalsoimproveourunderstandingontheNeurobiologyofAQP4andprovidethenewtargetfordevelopingthetherapeuticoptionforneurodegenerativediseases.AIM:ToinvestigatetheimpactofAQP4deletionontheexpressionandthefunctionoftheMPP+andLPS-inducedCYP2E1inastrocyte.METHODS:Primaryastrocytecultureswerepreparedfromwildtypeandhomozygousmutantmice.CYP2E1,glialfibrillaryacidicprotein(GFAP)immunocytochemistrydetecttheexpressionoftheCYP2E1inastrocyte.MTTanalysiswasusedtoassesstheMPP+,LPSandethanol-inducedcytotoxicity.RT-PCRandimmunocytochemistryweretakenforCYP2E1expression.ROSproductionofastrocytewasmeasuredusing2'7'-dichlorofluoresceindiacetate(DCFH-DA)byflowcytometer.RESULTS:1)AQP4knockoutincreasedcytotoxicityandtheROSproductioninducedbyMPP+(100μM),LPS(1μg/ml)andethanol(200mM)inastrocytes.2)Astrocytesexhibitstainingwithanti-GFAPaswellasCYP2E1.3)AQP4knockoutenhancedtheup-regulationoftheCYP2E1expressionafterexposuretoMPP+,LPSandethanol.4)CYP2E1inhibitorDAS(500μM)amelioratedthecelldamageandtheROSproductionoftheastrocytesinducedbyMPP+andLPS.CONCLUSION:AQP4knockoutincreasedcytotoxicityandtheROSproductioninducedbyMPP+,LPSandethanolinastrocytes.Andthesepartly becauseoftheincreasedexpressionoftheCYP2E1whichindicatesthatAQP4playsanimportantroleintheregulationofdrugmetabolismenzymesinbrain.Themajor contributions of the present study lie in:WereportforthefirsttimethatMPP+inducestheCYP2E1expressionintheprimaryastrocytes.AQP4knockoutenhancesthecelldamageinastrocytesmediatedbyMPP+andLPSthroughinducingtheCYP2E1expressionwhichprovidesnewinsightintotheunderstandingofAQP4inthedrugmetabolismenzymesintheneuroinflammationandtheneurodegenerativediseases.