Impact Of Different Apoe On Secondary Injury Of Neuron/astrocyte After Trauma

Traumatic brain injury (TBI) is one of the most common central nervoussystem disorders with high morbidity and mortality, and often bringsdisastrous consequences and heavy economic burden to patients' familiesand the society.Apolipoprotein E (ApoE), a34-kDa protein with299aa, is the majorapolipoprotein in the central nervous system. ApoE has three isoforms inhumans: ApoE2, ApoE3and ApoE4, respectively encoded by threedifferent alleles (APOEε2, APOEε3, APOEε4). There are some differencesamong three subtypes of ApoE, and they differ at residues112and158:ApoE3has cysteines (Cys) at112and arginine (Arg) at158, whereasApoE4has arginine at both sites, and ApoE2has cysteines. APOEε4isbelieved to be a negative factor that can result in an unfavorable outcome,and ApoE4, encoded by APOEε4, is considered as a dysfunctional orharmful protein. As the major extracellular lipid carrier in the centralnervous system, ApoE plays a key role in pathological and physiologicalpathways, such as lipid transportation, oxidative stress and inflammatoryresponse.As shown by many studies, APOE polymorphism can influence theoutcome of TBI, but the exact mechanism remains unclear, and so does theexact mechanism through which ApoE4deteriorates the outcome of TBI. By regulating numerous enzyme systems that affect neuronal physiologyand gene transcription and by influencing the functions of organelles,calcium plays a vital role in regulation of cellular functions. Therefore,variation of intracellular calcium concentration can dramatically influencethe function and metabolism of cells, and sometimes even directly induceapoptosis. Furthermore, intracellular calcium level is believed to play a keyrole in the secondary injury after TBI, so maintenance of intracellularcalcium homeostasis is of critical importance to cells.Intracellular calcium level may be influenced by many factors after TBI,including increase of extracellular glutamate level, dysfunction of organellesuch as endoplasmic reticulum (ER) and mitochondria, all of which havebeen proved to be related with ApoE. In the present study, we investigatedthe effects of different ApoE on the intracellular calcium level, extracellularglutamate level and mitochondria function in cultured neurons andastrocytes of APOE knock-out mouse after mechanical injury, and studiedeffects of different ApoE on repairing capability of astrocytes, and affinitiesof different ApoE for ApoER2. We also discussed the possible mechanismthrough which APOE polymorphism influence the outcome of TBI.1. Primary neuron and astrocyte culture and foundation of mechanicalinjury modelMethods: Astrocytes and neurons were obtained from postnatal (24hours)APOE knock-out mouse (APOE-/-) by a standard enzyme treatmentprotocol. The astrocyte and neuron purity were determined by glialfibrillary acidic protein (GFAP) and tubulin Ⅲpositivity respectivelythrough immunofluorescence. Mechanical injury model was produced byscraping adherent cells from a culture dish. All primary astrocytes wereprepared from APOE knock-out (APOE-/-) mouse to avoid the interferenceof ApoE generated by wild-type mouse. Results: Cells can be recognized by GFAP-antibody and tubulin Ⅲ-antibody respectively. The mechanicalinjury area can be observed under microscope. Conclusion: The primaryneuron and astrocyte culture and neuron/astrocyte co-culture weresuccessfully obtained, and mechanical injury model was successfullyestablished, providing foundation for next experiment.2. Effects of different ApoE on intracellular calcium level, extracellularglutamate level and mitochondria function after mechanical injuryMethods: Three full-length exogenous recombinant human ApoE, i.e.,ApoE2, ApoE3and ApoE4, were obtained from Peprotech (USA). Neuronsand astrocytes were divided into four groups, among which, Group-ApoE4,Group-ApoE3and Group-ApoE2were respectively treated with full-lengthrecombinant human ApoE4, ApoE3and ApoE2, while astrocytes nottreated with ApoE was included as a control group. Neurons and astrocyteswere loaded with Fluo-3/AM, a calcium fluorescent probe, and thefluorescent intensity of cells was individually measured under laserscanning confocal microscope after injury. Mitochondria in neurons waslabeled by JC-1, a fluorescent probe for mitochondria, and the fluorescentintensity was determined by flow cytometry (FCM). Extracellular fluid ofastrocyte was collected, and extracellular glutamate level was analyzed byautomatic amino-acid analyzer. Results: Data of laser scanning confocalmicroscope showed that, the fluorescence intensity of neurons andastrocytes in Group-ApoE4were significantly higher than those inGroup-ApoE2and Group-ApoE3after injury. FCM showed that, thefluorescence intensity of neurons in Group-ApoE4was significantly higherthan that in Group-ApoE2and Group-ApoE3after mechanical injury.Moreover, glutamate level in extracellular fluid of Group-ApoE4wassignificantly higher than that in Group-ApoE2and Group-ApoE3afterinjury. Conclusion: ApoE4may cause higher intracellular calcium level in neurons and astrocytes after mechanical injury, which was associated withextracellular glutamate level and mitochondria function.3. Possible mechanisms through which different ApoE participate in theprocess after mechanical injuryMethods: Neurons and astrocytes were divided into four groups, amongwhich, Group-ApoE4, Group-ApoE3and Group-ApoE2were respectivelytreated with full-length recombinant human ApoE4, ApoE3and ApoE2,while astrocytes not treated with ApoE was included as a control group.Repairing capability of astrocytes in different groups after mechanicalinjury was measured under microscope. Meanwhile atrocytes survival wasestimated by MTT. Affinities of different ApoE for ApoER2weredetermined by double-immunofluorescent under laser scanning confocalmicroscope. Results: Repairing capability of astrocytes in Group-ApoE4was significantly lower than that in Group-ApoE3and Group-ApoE2, andsurvived astrocytes in Group-ApoE4were significantly less than those inGroup-ApoE3and Group-ApoE2. In addition, no statistically difference offluorescent intensity was found among neurons in different groups.Conclusion: Repairing capability and survival of astrocytes may beimpaired by ApoE4, while different ApoE may have similar affinities whenbinding to ApoER2.