Impact Of APOE Protein And Gene Polymorphism On Apoptosis Of Astrocytes After Hypoxic Injury

In the brain, apolipoprotein E (APOE: gene; apoE: protein) ispredominantly synthesized by astrocytes. Several lines of evidence point toa direct role of apoE in modulating neuronal response to injury. Recently，mounting evidence have indicated that apoE genotype influences theoutcome from TBI. APOEε4is believed to be a negative factor that canresult in an unfavorable outcome from TBI，but the related mechanismsremain unclear.Hypoxia is the most common cause of cell damage in the brain. It canoccur during brain trauma, ischemic strokes. Astrocyte is the predominantcell type in the brain. Hypoxia-activated astrocytes can kill neighboringneurons through the realease of cytotoxic mediators and mounting evidencehas indicated that alterations in astrocyte played a crucial role in thepathogenesis of other disorders, such as brain edema, inflammatorydemyelinating diseases, neurodegenerative disorders and so on. While mostresearch is focused on the direct protection of neuronal cells, the role of non-neuronal cells, such as astrocytes that may be functional in thepathogenesis of these disorders, is relatively unclear. Our current study wasperformed from four aspects as follows.Part I Primary astrocyte cultivation and foundation of hypoxic injurymodelAstrocytes were separated from APOE wild type (WT) mice and APOEknockout (KO) mice and were determined by glial fibrillary acidic protein(GFAP) through immunofluorescence. Hypoxic injury models wereestablished by putting cells in the setting of hypoxia for6h. The changes ofcells and its mitochondria were observed by transmission electronmicroscope. Results: Cells can be recognized by GFAP-antibody. At6h ofischemia, swollen mitochondria were shown in the cytoplasm and cellswere shrunken and pyknotic; Apoptotic bodies were also observed.Conclusion: Astrocytic hypoxia injury model was successfully founded,providing foundation for next experiment.Part II Protective effects of apoE on astrocytes after hypoxic injury1. The APOE WT astrocytes and APOE KO astrocytes were dividedinto four groups: normoxia group, hypoxia group，normoxia+T0901317group and hypoxia+T0901317group. we added T090131748h prior tohypoxia and maintained in the culture media throughout the experiment.The apoptosis rate of astrocytes was determined6h later by flowcytometry(FCM).2. The APOE KO astrocytes were divided into six groups, among which, Control group,100nM group,300nM group,1000nMgroup,2000nM group,3000nM group were pretreated with full-lengthrecombinant human apoE30-3000nM30min before exposure to hypoxiarespectively. The apoptosis rate of astrocytes was determined6h later byFCM.3. The APOE WT astrocytes and APOE KO astrocytes were dividedinto four groups separately: normoxia group, hypoxia group，normoxia+T0901317group and hypoxia+T0901317group. we added T090131748hprior to hypoxia and maintained in the culture media throughout theexperiment. The culture of astrocytes exposed to hypoxia was collected.The concentration of glutamate in the culture was determined using theHitachi L-8800automatic amino acid analyzer.4. ApoE KO astrocyteswere divided into five groups: normoxia group, hypoxia group, hypoxia+apoE3group, hypoxia+MK801group and hypoxia+apoE3+MK801group, we pretreated apoE KO astrocytes with the recombinant humanapoE3(1μM) or the NMDA receptor antagonist, MK-801(10μM) or theboth,30min before hypoxia treatment. The apoptosis rate of astrocytes wasdetermined6h later by FCM.5. The APOE WT astrocytes and APOE KOastrocytes were divided into four groups separately: normal control (NC)group, glutamate group，NC+T0901317group and glutamate+T0901317group. we pretreated astrocytes with T0901317(300nM)48h prior toglutamate (50mM) treatment. The apoptosis rate of astrocytes weredetected by FCM. Results:1. The apoptosis rate in the WT hypoxia group was significantly lower than that in apoE KO group. No effect of T0901317was observed for hypoxia-treated apoE KO astrocytes.2. Pretreatment with300nM to3μM of apoE3resulted in partial protection compared to thecontrol group.3. The glutamate concentration in the culture of astrocyteswas increased significantly after being exposed to hypoxia for6h,especially in the apoE KO astrocytes. Under hypoxic condition,pretreatment with T0901317dramatically reduced the hypoxia-inducedincrease of extracelluar glutamate around WT astrocytes. However, thiseffect was not observed in apoE KO astrocytes.4. MK-801was sufficientto protect astrocytes from hypoxia-induced astrocyte apoptosis. Theprotective effect of MK801was similar to that observed in sister culturesexposed to hypoxia after pretreatment with1μM apoE3. Moreover, theeffects of MK-801and apoE3were not additive, that is, no furtherprotection was observed in cultures pretreated with both MK-801and apoEprior to hypoxia exposure.5. A significant excitotoxic response wasobserved in cultures exposed to50mM glutamate for24h both in WTastrocytes and apoE KO astrocytes, but the apoptosis rate in WT astrocyteswas significantly decreased compared with apoE KO astrocytes.Pretreatment with T0901317conferred partial protection againstglutamate-induced apoptosis in WT astrocytes. However, no T0901317protection was observed in apoE KO astrocyte cultures. Conclusion: apoEprotected astrocytes from hypoxia induced apoptosis in a dose-dependent manner. apoE exerts its protective effects both by reducing the level ofextracelluar glutamate around the astrocytes and decreasing the activationof NMDA receptors triggered by hypoxia. Approaches that elevate apoEsecretion in astrocytes might provide a novel strategy in the protection ofneuronal ischemic injury.Part III The changes of apoE expression in astrocytes exposed to hypoxicinjuryThe astrocytes of APOE WT mice were divided into normoxia group,hypoxia group and hypoxia+ERK inhibitor U0126group. The expressionof apoE of three groups cells were detected by Western blotting. Results:Compared to normoxia group and hypoxia+ERK inhibitor U0126group,the expression of apoE was highest in hypoxia group. Conclusion: Hypoxicinjury triggered the astrocytes to increasingly synthesize apoE, whichthrough the ERK signaling pathway.Part IV Effects of APOE polymorphisms on apoptosis of astrocytes afterhypoxic injuryAstrocytes were separated from APOE WT mice and APOE transgenicmice(ε3, ε4). Hypoxic injury models were established by putting cells inthe setting of hypoxia for6h. The apoptosis rate and mitochondrialmembrane potential were detected by flow cytometry. Results: Theapoptosis rate and decreasing degree of mitochondrial membrane potentialof ε4allele were significantly higher than the other two types(p<0.05). Conclusion: The higher of decreasing degree of mitochondrial membranepotential of APOEε4may induce more astrocytes apoptosis after hypoxia.