The Correlation Between MCT1 Expressed In Oligodendrocytes And Neuronal Damage In Alzheimer's Disease

Alzheimer's disease?AD?is a neurodegenerative disease,and its main clinlical symptom is progressively cognitive dysfunction.The deposit of amyloid beta?A??and neurofibrillary tangles?NFTs?are the main molecular pathology of AD.Actually,the formation of A?plaques and NFTs,inflammation and oxidative stress are related with neuronal energy deficiency.Clinical evidence proves that the alteration of glucose metabolism and reduction of glucose utilization take place at the early stage of AD.Particularly,the decrease of glucose ultilization in AD brains is correlated with cognitive dysfunction at the later stage of AD,and insufficient energy supplement can further accelerate AD progression.In AD brains,the decrease of glucose,the reduced activity of mitochondrial electron transport and the increase of oxidative products are the main causes of neuronal damage.At this moment,timely energy supplement in neurons is necessory for neuronal activity.On the other hand,mitochondrial function is impaired.Glycolysis is compensatorily enhanced and becomes the dominant pathway of energy metabolism.Energetic metabolism in neural cells convert from aerobic oxidation to glycolysis,and the produced lactate is significant for brain activity.Especially,when glucose is deficit,lactate becomes the main energetic substrate of the brain.In the central nervous system?CNS?,neuronal lactate is mainly derived from the catabolization of glucose in oligodendrocytes and astrocytes.Monocarboxylate transporters?MCTs?assist lactate transport from oligodendrocytes and astrocytes to neurons.Monocarboxylate transporter 1?MCT1?,monocarboxylate transporter 2?MCT2?and monocarboxylate transporter 4?MCT4?are the members of MCTs,which are expressed in the CNS.MCT1 is mainly localized to oligodendrocytes and myelin sheath.Neurons dominantly express MCT2,and MCT4 is mostly expressed in astrocytes.MCT1 takes part in lactate transport in oligodendrocytes and myelin.MCT4 mediates lactate transport in astrocytes.MCT2 assists MCT1 and MCT4 in lactate supplement from glial cells to neurons.Most importantly,the role of oligodendroglial MCT1 in lactate transport from myelin to neuronal axons is significant for the maintenance of neuronal activity and physiological function.On the other hand,myelin,formed by oligodendrocytes,is an important structure,which can sustain electrical transduction of neurons.Therefore,this study emphatically analyzes the correlation between the changeably expression of MCT1 in oligodendrocytes and neuronal damage in AD,and further explores the relation between the injury of myelin formed by oligodendrocytes and neuronal impairment in AD.Primarily,this work explored the association between the changeably expression of MCT1 in the medial prefrontal cortex?mPFC?and the alteration in neuronal amounts under physiological status.Then,it analyzed the relation between the changed lactate metabolism in the participance of MCT1and neuronal damage in AD.Lastly,it identified the underlying correlation between the impairment of myelin formed by oligodendrocytes and neuronal damage in AD.The main results are listed as follows:1.The changed expression of MCT1 is associated with neuronal amountsAs the results of western blot?WB?showed,MCT1 expression in the mPFC is gradually increased from a low level at the 1^st week to a high level at the 3^rd week?n=3,*P<0.05?.MCT1 expression keeps at a high level between the 3^rd week and the 6^th week,higher than that of the 1^st week?n=3,*P<0.05?.At the 7^th week and the 8^th week,MCT1expressions are gradually decreased to a level closing to those of the 1^st week and the 2ⁿdd week?n=3,P>0.05?.MCT1 expression in the mPFC is gradually decreased from a high level at the 1^st month to a low level at the 8^th month?n=3,*P<0.05?.Besides,MCT1expressions keep at a low level between the 8^th month and the 12^th month,which are lower than that of the 1^st month?n=3,*P<0.05?.In addition,no significant differences in MCT1expressions can be found from the 1^st month to the 7^th month?n=3,P>0.05?.Immunofluorescent staining of MCT1 was statistically analyzed.The quantity of MCT1 positive cells in the mPFC is gradually increased from a low level at the 1^st week to a high level at the 3^rd week?n=3,*P<0.05?,which keeps stable between the 3^rd week and the 6^th week.In addition,the amounts of MCT1 positive cells from the 3^rd week to the 6^thh week are more than that of the 1^st week?n=3,*P<0.05?.At the 7^th week and the 8^th week,the quantities of MCT1 positive cells are gradually decreased to a low level,which have no differences in comparison with those of the 1^st week and the 2^nd week?n=3,P>0.05?.The number of MCT1 positive cells is gradually reduced from a high level at the 1^st month to a low level at the 8^th month?n=3,*P<0.05?,and keeps at a low level between the 8^th month and the 12^th month,lower than that of the 1^st month?n=3,*P<0.05?.There are no significant differences in MCT1 expressions from the 1^st month to the 7^th month?n=3,P>0.05?.These results signify that MCT1 expression at the 6^th week is higher than those of the1^st week and the 12^th month.Here,double staining of MCT1 and neuronal neucli?Neu N?in1-week-old,6-week-old and 12-month-old C57BL/6 mice were used to identify the association between the changed MCT1 expressions and neuronal amounts.As results showed,in 1-week-old,6-week-old and 12-month-old C57BL/6 mice,the changing trend of neuronal amounts is in accordance with the altered quantities of MCT1 positive cells,despite that MCT1 can not be colabeled with neurons.In detail,neuronal quantity at the 6^thh week is more than those of the 1^st week and the 12^th month?n=3,*P<0.05?.There are no differences in neuronal amounts between the 1^st week and the 12^th month?n=3,P>0.05?.On the other hand,the level of MCT1 expression at the 6^th week is higher than those of the1^st week and the 12^th month?n=3,*P<0.05?.No significant differences can be found in MCT1 expressions between the 1^st week and the 12^th month?n=3,P>0.05?.Importantly,the correlation analysis found that the alteration of MCT1 expressions in the mPFC of C57BL/6 mice aged at 1-week-old,6-week-old and 12-month-old is correlated with neuronal amounts changed at these periods?R=0.86,***P<0.001?.2.MCT1 is developmentally expressed in oligodendrocytesBased on the analysis of MCT1 and Neu N expressions in the mPFC,it is found that MCT1 isn't colocalized with neurons marked by Neu N.Then,we detected the expression of MCT1 in neuronal axons.MCT1 and non-phosphorylated neurofilament?SMI-32?,the marker of neuronal axons,were double stained in 1-week-old,6-week-old and12-month-old C57BL/6 mice.As results showed,MCT1 does not completely localize to axons,while MCT1 partly spreads around axons.Moreover,through the double staining of MCT1 and MBP in 6-week-old C57BL/6 mice,it is recognized that MCT1 is dominantly expressed in oligodendrocytes.To clearly recognize MCT1 expressions in oligodendrocytes during 12 months,we conducted double staining of MCT1 and MBP in the mPFC of C57BL/6 mice?aged from1-week-old to 8-week-old and from 1-month-old to 12-month-old?.As results showed,MCT1 is highly colocalized with oligodendrocytes.Especially,oligodendrocytic numbers change in a similar trend as MCT1 expressions altered in 12 months.The quantity of oligodendrocytes is increased from a low level at the 1^st week to a high level at the 3^rd week.Then,oligodendrocytic amount keeps at a high level between the 3^rd week and the 6^th week,higher than that of the 1^st week?n=3,*P<0.05?.At the 7^th week and the 8^th week,there are no significant differences in the amounts of oligodendrocytes compared with the 1^stt week and the 2^nd week?n=3,P>0.05?.The analysis of MCT1 and MBP expressions in C57BL/6 mice aged from 1-month-old to 12-month-old proves that MCT1 expressions change in a similar trend as oligodendrocytic quantities from the 1^st month to the 12^th month.In comparison with the 1^st month,there is a decrease in oligodendrocytic amounts from the8^th month to the 12^th month?n=3,*P<0.05?.From the 1^st month and the 7^thh month,no significant differences can be found in the quantities of oligodendrocytes?n=3,P>0.05?.Correlation analysis clarifies that the the changeably expression of MCT1 is associated with the alteration of oligodendrocytic amounts in 12 months?R=0.88,***P<0.001?.3.Astrocytes and microglias do not express MCT1,quantities of which keep stable during 12 monthsWB was used to identify changes of astrocytic and microglial quantities in 12 months.Double immunofluorescent staining was applied to recognize MCT1 expressions in astrocytes and microglias.Glial fibrillary acidic protein?GFAP?and anti-CD11b/c antibody?OX42?are the markers of astrocytes and microglias respectively.There are no significant differences in the expressions of GFAP and OX42 in the mPFC of C57BL/6 mice?aged from 1 week-old to 8-week-old and from 1-month-old to 12-month-old??n=3,P>0.05?.Double staining of MCT1 and GFAP or OX42 displays that MCT1 does not localize to astrocytes and microglias.4.The amounts of A?plaques and astrocytes are increased,and the quantities of neurons and oligodendrocytes are reduced in ADAs the results of immunofluorescent staining showed,the numbers of A?plaques are increased in the cortex and hippocampal CA1 area of APP/PS1 mice compared with wild type C57BL/6 mice,?n=4,**P<0.01?.WB test reveals that relative level of A?in APP/PS1 mice is obviously higher than that of wild type C57BL/6 mice?n=4,**P<0.01?.Moreover,in comparison with wild type C57BL/6 mice,neuronal and oligodendrocytic amounts in the cortex and hippocampal CA1 area of APP/PS1 mice are reduced?n=4,*P<0.05?,in accompanying with the increased quantity of astrocytes?n=4,*P<0.05?.5.Cerebral lactate level and positive expressions of MCT1,2,4 are reduced in ADThen,lactate assay kit and WB experiment were used to detecte cerebral lactate content and relative expressions of MCT1,2,4 in the brain.In comparison with wild type C57BL/6 mice,cerebral lactate content is reduced in APP/PS1 mice?n=4,*P<0.05?.Relative expressions of MCT1,2,4 are decreased in the brains of APP/PS1 mice when compared with wild type C57BL/6 mice?MCT1:n=4,**P<0.01;MCT2:n=4,*P<0.05;MCT4:n=4,*P<0.05?.To confirm MCT1,2,4 expressions and cellular distribution of them in the cortex and hippocampus,MCT1 and MBP,MCT2 and Neu N,MCT4 and GFAP were double stained in wild type C57BL/6 mice and APP/PS1 mice.As results showed,MCT1 and MBP,MCT2 and Neu N,MCT4 and GFAP are colocalized in the cortex and hippocampal CA1 areas of APP/PS1 mice and wild type C57BL/6 mice.Statistically,it is found that integrated optical density?IOD?values of MCT1,2,4 expressions in the cortex and hippocampal CA1 region of APP/PS1 mice are less than those of wild type C57BL/6mice?MCT1:n=4,*P<0.05;MCT2:n=4,*P<0.05;MCT4:n=4,*P<0.05?.6.The alterations of neuronal lactate enzymes LDHA and LDHB in ADLDHA and LDHB are the enzymes that take part in cellular lactate metabolism.Here,double stainings of Neu N and LDHA or LDHB were used to label neuronal LDHA and LDHB expressions in the cortex and hippocampal CA1 areas of APP/PS1 mice and wild type C57BL/6 mice.The results display that in comparison with wild type C57BL/6 mice,relative levels of neuronal LDHA and LDHB are downregulated,and neuronal LDHB is reduced more obvious than neuronal LDHA in the cortex and hippocampal CA1 area of APP/PS1 mice?Cortex:n=4,*P<0.05;Hippocampal CA1:n=4,*P<0.05?.Moreover,it is displayed that the ratio of neuronal LDHA and LDHB in APP/PS1 mice is increased in the cortex and CA1 area of hippocampus when compared with wild type C57BL/6 mice?Cortex:n=4,*P<0.05;Hippocampal CA1:n=4,**P<0.01?.7.Demyelination takes place at the 3^rd week and neurons are impaired at the 8^thh week in A?O groupDouble staining of MBP and neurofilament 200?NF200?was used to identify the damages of myelin and neuronal fibers in corpus callosum in Control,Sham and A?O groups at the 3^rd week and the 8^th week.At the 3^rd week,MBP expression in corpus callosum is downregulated in A?O group in comparison with Control group?n=5,*P<0.05?.There are no differences in MBP expressions between Control group and Sham group at the 3^rd week?n=5,P>0.05?.On the other hand,no significant differences can be found in NF200 expressions among Control,Sham and A?O groups at the 3^rd week?n=5,P>0.05?.At the 8^th week,MBP and NF200 expressions in corpus callosum are reduced in A?O group compared with Control group?n=5,*P<0.05?.No differences in MBP and NF200expressions can be found in corpus callosum between Control group and Sham group at the8^th week?n=5,P>0.05?.8.Neurons are damaged in hippocampus,corpus callosum and cortex at the 8^thh week in A?O groupNissl staining and immunostaining were used to assess the changes of neuronal amounts and neurofilaments in Control,Sham and A?O groups at the 8^th week.It is showed that there are no significant differences in neuronal quantities in hippocampal CA1 and CA3 areas between Control group and Sham group?CA1:n=5,P>0.05;CA3:n=5,P>0.05?.In A?O group,neuronal amounts in hippocampal CA1 and CA3 regions are less than those of Control group?CA1:n=5,*P<0.05;CA3:n=5,*P<0.05?.The results of NF200 staining in corpus callosum display that optical density?OD?values of NF200 in Control and Sham group have no significant differences?n=5,P>0.05?.OD value of NF200 in A?O group is lower than that of Control group in corpus callosum?n=5,*P<0.05?.In the cortx,OD values of Neu N positive expressions in Control,Sham and A?O groups were analyzed.No significant differences can be found in OD values of Neu N in the cortex between Control group and Sham group?n=5,P>0.05?.Compared with Control group,a decrease in OD value of Neu N in the cortex can be identified in A?O group?n=5,*P<0.05?.9.Astrocytic and microglial quantities are increased at the 8^th week in A?O groupGFAP labels astrocytes,and allograft inflammatory factor 1?AIF-1?is the marker of microglias.In statistic,expressions of GFAP and AIF-1 in the cortex have no significant differences between Control group and A?O group at the 3^rd week?n=5,P>0.05?.All the same,no significant differences can be found in GFAP and AIF-1 expressions in the cortex between Control group and Sham group at the 3^rd week?n=5,P>0.05?.At the 8^th week,expressions of GFAP and AIF-1 in the cortex are upregulated in A?O group compared with Control group?n=5,*P<0.05?.There are no significant differences in GFAP and AIF-1expressions in the cortex between Control group and Sham group at the 8^th week?n=5,P>0.05?.10.A?O induces the reduction of oligodendroglial quantity and the restriction of MBP positive membrane area in vitroIn vitro,experiments observed the impact of A?O on the differentiation of OPCs into mature oligodendrocytes.As results showed,oligodendrocytic amount of A?O group is less than that of Control group?n=5,*P<0.05?.There are no differences in the quantities of oligodendrocytes between Control group and Sham group?n=5,P>0.05?.Besides,the area of MBP positive expression in oligodendrocytes of A?O group is restricted in comparison with Control group?n=5,*P<0.05?.In Control group and Sham group,no differences can be found in the region of MBP positive expression?n=5,P>0.05?.Conclusively,this study primarily proves that,under physiological status,MCT1 is developmentally expressed in oligodendrocytes,and the changeably expression of MCT1 is associated with the alteration of neuronal amounts.Then,it is recognized that the change of MCT1 involved in lactate metabolism is related with neuronal injury in AD.At last,it is clarified that the injury of myelin sheah formed by oligodendrocytes takes place prior to neuronal impairment and the increase of astroglial and microglial quantities in AD.It is signified that early demyelination is correlated with neuronal damage in AD.Especially,the direct toxicity of A?O on OPCs is the main cause of early demyelination in AD.Based on these findings,this study proposes the following inference:In the course of AD,the downregulation of MCT1 and the disruption of myelin formed by oligodendrocytes can disturb lactate transport from oligodendrocytes to neurons.As a reult,lactate,an energetic substrate,is insufficiently supplied in neurons,which can aggravate neuronal damage.Therefore,in the course of AD,especially at the early stage of AD,the upregulation of MCT1 expression and the promotion of remyelination can ameliorate neuronal energy deficiency,and then alleviate neuronal damage,postponing AD progression.