The Changes Of Oligodendrocytes In AD And The Effects Of Antipsychotics On Oligodendrocytes

PART ONE The changes of oligodendrocytes in the hippocampus of APP/PS1 transgenic AD miceObjective: This study was designed to quantitatively study the changes of oligodendrocytes?OLG?in the hippocampus of APP/PS1 transgenic AD mice and investigate the expression levels of myelination proteins,OLG development-related and mechanism-related proteins.The findings in the current study would provide scientific basis for subsequent studies and provide an effective target for searching the means to delay AD progress in the future.Methods: Thirteen 10-month-old male APP/PS1 transgenic AD mice?AD group?and thirteen 10-month-old male wild-type littermates?WT group?were randomly selected.The spatial learning and memory abilities of each group mice were tested with Morris water maze.Escape latency was an evaluation indicator of spatial learning and memory ability.The platform-crossing frequency and the percentage of swimming time in the target quadrant were recorded as an evaluation indicator of spatial memory ability.The changes of the total numbers of Olig2⁺ cells and CNPase⁺ cells in the CA1 field,CA2-3 field and dentate gyrus?DG?of the hippocampus of each group mice were quantitatively investigated using immunohistochemistry and unbiased stereological method.The correlation between the total number of OLG of the hippocampus and spatial learning and memory abilities was analyzed.The protein levels of OLG development-related proteins?CNPase,MBP,NG2?and mechanism-related proteins?Lingo1,MAG,MOG,GSK-3?,p-ser9-GSK3??was detected with Western Blot.The content of senile plaques and the level of soluble A? were detected with immunohistochemistry and ELISA.The ratio of P16⁺/Olig2⁺ cells of the CA1 field,CA2-3 field and dentate gyrus of the hippocampus in each group mice was counted with immunofluorescence and laser confocal microscope technique.Results: 1.Morris water maze test results showed that the escape latency of AD group mice was significantly longer than WT group mice?P<0.05?,and the platform-crossing frequency of AD group mice was significantly lower than WT group mice?P<0.05?.There was no significant difference in the percentage of swimming time in the target quadrant between the AD group and WT group mice?P>0.05?.The total numbers of Olig2⁺ cells in the hippocampus of AD group mice were significantly increased compared with WT group mice?P<0.05?,and the total Olig2⁺ cells in the hippocampus were positively correlated with the escape latency and negatively correlated with the platform-crossing frequency.The total numbers of CNPase⁺ cells in the hippocampus of AD group mice were significantly decreased compared with WT group mice?P<0.05?,and the total CNPase⁺ cells in the hippocampus were negatively correlated with the escape latency.2.Western Blot results showed that the protein levels of OLG development-related proteins MBP?21k Da?and CNPase in the hippocampus of AD group mice were significantly lower than that of WT group mice?P<0.05?.There were no significant differences in the protein levels of MBP?17k Da?and NG2 in the hippocampus of WT group and AD group mice?P>0.05?.The protein level of Lingo1 in the hippocampus of AD group mice was significantly higher than that of WT group mice?P<0.05?.There were no significant differences in the protein levels of MAG and MOG between WT group and AD group mice?P>0.05?.Compared with WT group mice,the protein level of GSK-3? in the hippocampus of AD group mice was significantly increased?P<0.05?,while p-ser9-GSK3? was significantly decreased?P<0.05?.3.No amyloid plaques were found in the hippocampus of WT group mice,while many large amyloid plaques were observed in the hippocampus of AD group mice.ELISA result showed that the concentration of A?40 and A?42 in the hippocampus of AD group mice was significantly higher than that of WT group mice?P<0.05,P<0.05?.Immunofluorescence qualitative result showed that the distribution of P16⁺/Olig2⁺ cells in the hippocampus of AD group mice was more dense than that of WT group mice.Quantitative result showed that the ratio of P16⁺/Olig2⁺ cells in the CA2-3 field and DG of the hippocampus of AD group mice was significantly higher than that of WT group mice?P<0.05,P<0.05?.There was no significant difference in the ratio of P16⁺/Olig2⁺cells in the CA1 field of the hippocampus of WT group and AD group mice?P>0.05?.Conclusions: 1.The spatial learning and memory abilities of10-month-old APP/PS1 transgenic AD mice were impaired.2.The number of Olig2⁺ cells increased and the number of CNPase⁺ cells decreased in the hippocampus of AD mice,suggesting that there may be abnormalities in the proliferation,differentiation or maturation of OLG in the hippocampus of AD mice,and that there may be damage to mature OLG.3.The changes of the number of OLG in the hippocampus of AD mice were related to its spatial learning and memory abilities,and may be one of the important causes of spatial learning and memory abilities disorders.4.The protein levels of OLG maturation-related proteins MBP and CNPase in the hippocampus of AD mice were decreased,while OLG proliferation-related protein NG2 was not significantly changed,suggesting that there was mainly maturation obstacle in the hippocampus of AD mice.There was no significant change in the protein levels of MAG and MOG in the hippocampus of AD mice,but there was high expression of Lingo1,suggesting that high expression of Lingo1 might be one of the reasons for OLG maturation obstacles in the hippocampus of AD mice.The increased expression level and activity of GSK-3? further suggested that high expression of Lingo1 may lead to the high expression and high activity of downstream GSK-3?,thus inhibiting the maturation of OLG.5.There were a large amount of depositions of A? senile plaques and a large increase in soluble A? levels in the hippocampus of AD mice,suggesting that A? may cause OLG damage.6.The ratio of P16⁺/Olig2⁺ cells in the hippocampus of AD mice was increased,suggesting that there was excessive senescence phenotypes of OLG in the hippocampus of AD mice,which may be one of the important reasons for the failure in the maturation of OLG.The damage of mature OLG and the abnormal development of OLG may be the initiators for the changes of OLG/myelin sheath in the hippocampus of AD,as well as one of the important structures for the change of AD cognitive function.PART TWO The changes of oligodendrocytes in AD in vitroObjective: To investigate whether OLG are altered at the very early stage of AD and the effect of A? on OLG in AD in vitro.Methods: The brains of APP/PS1 transgenic or C57 newborn mice were taken for primary oligodendrocyte precursor cells?OPCs?culture in vitro,and MOPC cell line were in subculture.The primary OPCs of APP/PS1 transgenic AD mice were divided into APP/PS1?-?group and APP/PS1?+?group after tail genotype identification and purification.The cell cycle and apoptosis of primary OPCs in each group were detected with flow cytometry?FCM?.The protein levels of OLG development-related proteins?CNPase,Olig2,NG2?and mechanism-related proteins?MAG,MOG,GSK-3?,p-ser9-GSK3?,P16,P21?in each group of primary OPCs were detected with Western Blot.The MOPC was divided into three groups: Control group,0.5 ?M A? group,and 1 ?M A? group.The cell cycle and apoptosis of MOPC in each group were detected with FCM.The m RNA level of OLG development-related protein NG2 in the MOPC was detected with real time RT-PCR.The protein levels of OLG development-related proteins?NG2,PDGFR??and mechanism-related proteins?MAG,MOG,P16,P21?in the MOPC of the Control group and 0.5 ?M A? group were detected with Western Blot.The primary OPCs of C57 mice after purification were divided into three groups: C57 + Control group,C57 + 0.5 ?M A? group,and C57 + 1 ?M A? group.The cell cycle and apoptosis of primary OPCs in each group were detected with FCM.Results: 1.For the changes of primary OPCs in the brain of APP/PS1 transgenic AD mice,FCM results showed that compared with the APP/PS1?-?group,G1 phase cell proportion of primary OPCs in the APP/PS1?+?group was significantly reduced?P<0.05?,S phase was significantly increased?P<0.05?,and G2/M phase was not significantly changed?P>0.05?.Compared with the APP/PS1?-?group,the total apoptosis rate of primary OPCs in the APP/PS1?+?group was significantly reduced?P<0.05?,and the proportion of living cells was significantly increased?P<0.05?.Western Blot results showed that the protein level of OLG development-related protein CNPase in the APP/PS1?+?group was significantly lower than that in the APP/PS1?-?group?P<0.05?,and the protein levels of Olig2 and NG2 were significantly higher than those in the APP/PS1?-?group?P<0.05,P<0.05?.Compared with the APP/PS1?-?group,the protein level of OLG mechanism-related protein MAG in the APP/PS1?+?group was significantly increased?P<0.05?,and MOG was not changed?P>0.05?.Compared with the APP/PS1?-?group,the protein level of GSK-3? in the APP/PS1?+?group was significantly increased?P<0.05?,and p-ser9-GSK3? was significantly decreased?P<0.05?.The protein level of senescence protein P16 in the APP/PS1?+?group was significantly higher than that in the APP/PS1?-?group?P<0.05?,and there was no significant difference in the protein level of P21 between each group?P>0.05?.2.For the effects of A? on MOPC cell lines,FCM results showed that compared with the Control group,G1 phase cell proportion of MOPC in the 0.5 ?M A? group was significantly reduced?P<0.05?,and S and G2/M phase were significantly increased?P<0.05,P<0.05?.G1 phase cell proportion of MOPC in the 1 ?M A? group was significantly reduced compared with the Control group?P<0.05?,and S and G2/M phase were not significantly changed?P>0.05,P>0.05?.Compared with the Control group,the late and total cell apoptosis rates of MOPC in the 0.5 ?M A? group were significantly increased?P<0.05,P<0.05?,and there was no significant difference in the early apoptosis rate?P>0.05?.There was no significant difference in the early,late and total cell apoptosis rates of MOPC between the Control group and 1 ?M A? group?P>0.05,P>0.05,P>0.05?.Real time RT-PCR result showed that the m RNA level of NG2 in the MOPC of the 0.5 ?M A? group was significantly lower than that of the Control group?P<0.05?.Western Blot results showed that the protein level of NG2 in the MOPC of the 0.5 ?M A? group was significantly higher than that of the Control group?P<0.05?.There was no significant difference in the protein level of PDGFR? in the MOPC between the Control group and 0.5 ?M A? group?P>0.05?.There was no significant difference in the protein levels of MAG,MOG,P16 and P21 in the MOPC between the Control group and 0.5 ?M A? group?P>0.05,P>0.05,P>0.05,P>0.05?.3.For the effects of A? on primary OPCs in the brain of C57 mice,FCM results showed that there was no significant difference in the G1,S and G2/M phase cell proportion of primary OPCs between the C57 +Control group and C57 + 0.5 ?M A? group?P>0.05,P>0.05,P>0.05?.Compared with the C57 + Control group,G1 phase cell proportion of primary OPCs in C57 + 1 ?M A? group was significantly reduced?P<0.05?,S phase was significantly increased?P<0.05?,and G2/M phase was not significantly changed?P>0.05?.There was no significant difference in the early,late and total cell apoptosis rates of MOPC between C57 + Control group and C57 + 0.5 ?M A? group?P>0.05,P>0.05,P>0.05?.There was also no significant difference in the early,late and total cell apoptosis rates of MOPC between C57 + Control group and C57+ 1 ?M A? group?P>0.05,P>0.05,P>0.05?.Conclusions: 1.Proliferation increase and apoptosis decrease of primary OPCs in the brain of APP/PS1 transgenic AD mice suggested that abnormal proliferation and apoptosis decrease of OPCs may be one of the reasons for abnormal increase of OLG in AD mice.The protein level of OLG maturation marker CNPase of primary OPCs in AD mice was decreased,while the protein levels of OLG proliferation-related proteins Olig2 and NG2 were increased,suggesting that there may be abnormal proliferation and maturation obstacle of primary OPCs in the brain of AD mice.The excessive expressed MAG and increased expression level and enhanced activity of GSK-3? of primary OPCs in AD mice suggested that the maturation of primary OPCs in the brain of AD mice may be inhibited.The increased expression of P16 of primary OPCs in AD mice was consistent with the result in vivo.2.Low concentration of A?_1-42 led to excessive proliferation and apoptosis increase of MOPC,while high concentration of A?_1-42 had no significant effect on the proliferation and apoptosis of MOPC,suggesting that different concentration of A? had different effect on MOPC.However,low concentration of A? was more toxic to MOPC in that it caused excessive activation and increased proliferation of MOPC as well as toxic damage to MOPC.After intervention with low concentration of A?_1-42,there was no difference in the protein content of PDGFR? in the MOPC,but the protein content of NG2 was increased and m RNA level of NG2 was reactively decreased,suggesting that the proliferation of MOPC might increase.Low concentration of A?_1-42 did not affect the expressions of MAG,MOG,P16 and P21 in the MOPC.3.Low concentration of A?_1-42 had no significant effect on the proliferation and apoptosis of primary OPCs in C57 mice,while high concentration of A?_1-42 led to excessive proliferation of primary OPCs in C57 mice,suggesting that different concentrations of A? had different effects on primary OPCs.However,high concentration of A? was more toxic to primary OPCs,indicating that the MOPC was more sensitive to the toxic effect of A? than primary OPCs.High concentration of A?_1-42 had no effect on the apoptosis of primary OPCs in C57 mice,suggesting that the effect of A? on primary OPCs in normal mice was mainly excessive activation rather than inducing apoptosis.PART THREE Preliminary study on the effect of antipsychotics on oligodendrocytes in ADObjective: To preliminarily study the effect of antipsychotics?fluoxetine and quetiapine?on oligodendrocytes in AD in vitro,which might provide an effective scientific basis for finding possible drugs for the prevention and treatment of AD.Methods: The MOPC were in subculture.The MOPC at about 50%confluence were treated with A?_1-42 of two concentrations?0.5 ?mol/L,1?mol/L?for 24 hours.Then,fluoxetine?FLX,5 ?mol/L?and quetiapine?QUE,0.5 ?mol/L?were respectively added to cells in each group.There were nine groups: Control group?0.5 ?M A? group?1 ?M A? group?MOPC + FLX group?MOPC + 0.5 ?M A? + FLX group?MOPC + 1 ?M A? + FLX group?MOPC + QUE group?MOPC + 0.5 ?M A? + QUE group?MOPC + 1 ?M A? + QUE group.The MOPC in each group were collected after 24 h,and the changes of their cell cycle and apoptosis were detected with FCM.The brains of C57 newborn mice were taken for primary OPCs culture in vitro.Purified primary OPCs at about 50%confluence were treated with 1 ?mol/L A?_1-42 for 24 h.Then,FLX?5?mol/L?and QUE?0.5 ?mol/L?were respectively added to cells in each group.There were six groups: C57 + Control group? C57 + 1 ?M A?group?C57 + FLX group?C57 + 1 ?M A? + FLX group?C57 + QUE group?C57 + 1 ?M A? + QUE group.Primary OPCs in each group were collected after 24 h,and the change of their cell cycle was detected with FCM.Results: 1.Compared with the Control group,G1 phase cell proportion of MOPC in MOPC + FLX group was significantly increased?P<0.05?,and S and G2/M phase were significantly decreased?P<0.05,P<0.05?.Compared with the Control group,the early,late and total cell apoptosis rates of MOPC in MOPC + FLX group were all significantly decreased?P<0.05,P<0.05,P<0.05?.Compared with the 0.5 ?M A? group,G1 phase cell proportion of MOPC in MOPC + 0.5 ?M A? + FLX group was significantly increased?P<0.05?,S phase was significantly decreased?P<0.05?,and G2/M phase was not significantly changed?P>0.05?.Compared with the 0.5 ?M A? group,the early,late and total cell apoptosis rates of MOPC in MOPC + 0.5 ?M A? + FLX group were all significantly decreased?P<0.05,P<0.05,P<0.05?.There was no significant difference in the G1,S and G2/M phase cell proportion of MOPC between the 1 ?M A? group and MOPC + 1 ?M A? + FLX group?P>0.05,P>0.05,P>0.05?.Compared with the 1 ?M A? group,the early apoptosis rate of MOPC in MOPC + 1 ?M A? + FLX group was significantly increased?P<0.05?,but the late and total cell apoptosis rates were not significantly changed?P>0.05,P>0.05?.2.Compared with the C57 + Control group,G1 phase cell proportion of primary OPCs in the C57 + FLX group was significantly increased?P<0.05?,G2/M phase was significantly reduced?P<0.05?,and S phase was not significantly changed?P>0.05?.Compared with the C57 + 1 ?M A? group,S phase cell proportion of primary OPCs in C57 + 1 ?M A? +FLX group was significantly reduced?P<0.05?,and G1 and G2/M phase were not significantly changed?P>0.05,P>0.05?.3.Compared with the Control group,G1 and S phase cell proportion of MOPC in MOPC + QUE group were not significantly changed?P>0.05,P>0.05?,and G2/M phase was significantly increased?P<0.05?.Compared with the Control group,the early apoptosis rate of MOPC in MOPC +QUE group was significantly increased?P<0.05?,the late apoptosis rate was significantly decreased?P<0.05?,and total cell apoptosis rate was not significantly changed?P>0.05?.Compared with the 0.5 ?M A? group,G1 phase cell proportion of MOPC in MOPC + 0.5 ?M A? + QUE group was significantly increased?P<0.05?,S phase was significantly decreased ?P<0.05?,and G2/M phase was not significantly changed?P>0.05?.Compared with the 0.5 ?M A? group,the early apoptosis rate of MOPC in MOPC + 0.5 ?M A? + QUE group was significantly increased?P<0.05?,the late and total cell apoptosis rates were significantly decreased?P<0.05,P<0.05?.Compared with the 1 ?M A? group,S phase cell proportion of MOPC in MOPC + 1 ?M A? + QUE group was significantly decreased?P<0.05?,and G1 and G2/M phase were not significantly changed?P>0.05,P>0.05?.Compared with the 1 ?M A?group,the early apoptosis rate of MOPC in MOPC + 1 ?M A? + QUE group was significantly increased?P<0.05?,and the late and total cell apoptosis rates were significantly decreased?P<0.05,P<0.05?.4.There was no significant difference in the G1,S and G2/M phase cell proportion of primary OPCs between the C57 + Control group and C57 + QUE group?P>0.05,P>0.05,P>0.05?.Compared with C57 + 1 ?M A? group,G1 phase cell proportion of primary OPCs in C57 + 1 ?M A? +QUE group was significantly increased?P<0.05?,S phase was significantly reduced?P<0.05?,and G2/M phase was not significantly changed?P>0.05?.Conclusions: The proliferation and apoptosis decrease of MOPC after the intervention of FLX and QUE suggested that FLX and QUE can reduce the overstimulation of A? to MOPC,inhibit the proliferation of MOPC,and to some extent reduce the damage of A? to MOPC.2.The proliferation decrease of primary OPCs in C57 mice after the intervention of FLX and QUE suggested that FLX and QUE can to a certain extent reduce the overstimulation of A? to primary OPCs in normal mice,and inhibit the proliferation of OPCs.3.FLX and QUE may have a protective effect on OLG in the brain of AD.