| Objective:Alzheimer's disease(AD)is a common neurodegenerative disorder and characterized by progressive cognitive dysfunction and behavioral impairment.A? clearance has been considered to be crucial in the development of AD.Polarity distribution of aquaporin-4(AQP4)is important to remove A? from brain.AQP4 polarity can be influenced by ?-syntrophin(SNTA1)and the ratio of two AQP4 isoforms M1 and M23(AQP4-M1/M23),however,it is unknown whether the expression of SNTA1 and AQP4-M1/M23 changes in AD.SNTA1 can be regulated by Sin3/ Histone Deacetylase(HDAC)complex which contains HDAC1 and HDAC2 as core,suggesting that HDAC1 and/or HDAC2 may be involved in the regulation of SNTA1.The transcriptional activity of AQP4-M1 was found to be repressed by micro RNA-130a(mi R-130a),which may result in the reduction of AQP4-M1/M23 ratio,and the expression of mi R-130 a can be inhibited by HDAC3,suggesting mi R-130 a possibly mediates the regulation of HDAC3 on AQP4-M1/M23.Meanwhile,the expression of HDAC1,HDAC2 and HDAC3 was increased and the level of histone acetylation was significantly reduced in the brain of AD.Intermittent fasting results in an increase in ?-hydroxybutyrate(?OHB)level in blood.?OHB,as an endogenous pan HDACs inhibitor,has been reported to significantly inhibit HDAC1,HDAC2 and HDAC3 expression and increase the level of histone acetylation.In this study,we applied APP/PS1 double-transgenic mice and A?-induced human U251 glioma cells as AD model,and aimed to investigate whether intermittent fasting,increasing the level of ?OHB,restores AQP4 polarity in the cerebral cortex of AD via inhibiting HDAC1 and HDAC2 expression to up-regulate SNTA1 expression as well as via inhibiting HDAC3 to up-reguate mi R-130 a expression resulting in the reduction of AQP4-M1/M23.Methods : 1.Animals and Treatment: At 5 months of age,APPswe/PS1 d E9 double-transgenic mice were grouped into AD and AD+ADF,and wild-type(WT)littermates mice were grouped into WT and WT+ADF.Mice in WT and AD groups were fed ad libitum and mice in WT+ADF and AD+ADF groups were fed ad libitum every other day and fasted the following day.After 5 months,mice were sacrificed under ether anesthesia.Their brains were collected and divided into halves,the right of which was stored in 4% paraformaldehyde for immunofluorescence tests,and the left of which was stripped cortex and then used for quantitative reverse transcriptase(q RT)-polymerase chain reaction(PCR)and Western blot analyses.2.Cell Culture: Human U251 glioma cells were incubated in 6-well culture microplates in 2 ml antibiotic-free medium,and cultured for 3 h with ?OHB treatment at a final concentration of 1 m M(?OHB and ?OHB+A? groups)or without ?OHB treatment(control and A? groups).After 3 h,the cells in A? and ?OHB+A? groups were treated with A?(final concentration 2 or 10 ?M),and then cultured for an additional 12 h.3.Measurement of AQP4 distribution: Immunofluorescence was performed for AQP4 and CD31 staining to measure AQP4 distribution in the cerebral cortex of mice.4.Measurement of relative genes m RNA expression level in the cerebral cortex of mice and U251 cells: q RT-PCR was used to measure the m RNA expression levels of AQP4,AQP4-M1,AQP4-M23,SNTA1,HDAC1,HDAC2,HDAC3 and mi R-130 a.5.Measurement of relative gene protein expression levels in the cerebral cortex of mice and U251 cells: Western Blotting was used to measure the protein expression of AQP4,AQP4-M1,AQP4-M23,SNTA1,HDAC1,HDAC2,HDAC3,acetylated histone 3 lysine 9(Ace-H3K9)and Ace-H4K12.6.Measurement of SNTA1 m RNA and expression levels in HDAC1-or HDAC2-silenced U251 cells: Small Interfering RNA(siRNA)was used to silence HDAC1 or HDAC2 expression.q RT-PCR and Western Blotting were respectively used to detect the m RNA and protein expression levels of SNTA1,HDAC1 and HDAC2.7.Measurement of the effect of mi R-130 a on AQP4 m RNA and expression levels in U251 cells: Cells were incubated with mimic and inhibitor for human mi R-130 a.q RT-PCR and Western Blotting were respectively used to detect the m RNA and protein expression levels of AQP4,AQP4-M1 and AQP4-M23.8.Measurement of mi R-130 a and AQP4 m RNA and expression levels in HDAC3-silenced U251 cells: siRNA and HDAC3-specific inhibitor RGFP966 were used to silence HDAC3 expression.q RT-PCR was used to measure the expression level of mi R-130 a,as well as q RT-PCR and Western Blotting were respectively used to detect the m RNA and protein expression levels of AQP4,AQP4-M1,AQP4-M23 and HDAC3.9.Statistical Analyses: Statistical analysis of the data was performed by one-way analyses of variance and Fisher's least significant difference multiple comparison post hoc tests in SPSS 20.0 software for Windows.Data are presented graphically as means ± standard deviations.Probability values(p-values)less than 0.05(P < 0.05)were considered statistically significant.Results:1.AQP4 expression was highly localized to perivascular astrocytic endfeet surrounding the entire cerebral microvasculature,showing a polar distribution,in the cerebral cortex of WT mice with or without ADF treatment.In the cerebral cortex of APP/PS1 mice,AQP4 localization was severely perturbed,exhibiting a loss of polarity to the astrocytic endfeet and an increase of somal labeling.After ADF intervention,the polarity of AQP4 was recovered in APP/PS1 mice brains.2.Compared with WT mice without ADF treatment,the expression of AQP4 and AQP4-M1 m RNA and protein were increased,and the SNTA1 m RNA and protein expression was decreased(P < 0.05;P < 0.01)in the cerebral cortex of APP/PS1 mice.Meanwhile,APP/PS1 mice with ADF treatment had a significant decrease(P < 0.05;P < 0.01)in AQP4 and AQP4-M1 m RNA and protein expression,and a significant increase in SNTA1 m RNA and protein expression in the cerebral cortex relative to APP/PS1 mice.In addition,the increase of AQP4-M1/M23 protein ratio was also observed in APP/PS1 mice,which were reversed by ADF(P < 0.05;P < 0.01).3.Cells exposed to 2 ?M A? had a significant increase in AQP4,AQP4-M1 m RNA and protein expression and a significant derease in SNTA1 m RNA and protein expression compared with those observed in control cells(P < 0.05;P < 0.01).However,AQP4,AQP4-M1,AQP4-M23 and SNTA1 m RNA and protein expression was obviously decreased(P < 0.05;P < 0.01)in cells exposed to 10 ?M A?.Meanwhile,increases in AQP4-M23 and SNTA1 expression was observed in ?OHB pre-treated cells compared with control cells(P < 0.05;P < 0.01),as well in A?-exposed cells with ?OHB pre-treatment compared with cells only treated with A?(P < 0.01).A decrease in AQP4-M1 expression was observed in ?OHB-pre-treated cells exposed to A?(2 ?M)compared with A?-exposed cells(P < 0.05).Furthermore,we also found that the AQP4-M1/M23 ratio was remarkably increased in cells exposed to 2 or 10 ?M A?,which was ameliorated by ?OHB pre-treatment(P < 0.01).4.The results showed that the expression of Ace-H3K9 and Ace-H4K12 was significantly decreased and the expression of HDAC1 and HDAC2 m RNA and protein was significantly increased(P < 0.01)in APP/PS1 mice compared with WT mice.After ADF intervention,a marked increase in Ace-H3K9 and Ace-H4K12 expression and a significant decreasein HDAC1 and HDAC2 expression were found in APP/PS1 mice(P < 0.01).5.Cells exposed to A? had a significant decrease in Ace-H3K9 and Ace-H4K12 expression and a remarkable increase in HDAC1 and HDAC2 expression relative to those observed in control cells(P < 0.05;P < 0.01).The increase of Ace-H3K9 and Ace-H4K12 expression and the decrease of HDAC1 and HDAC2 expression were observed in ?OHB-pre-treated cells compared with control cells(P < 0.01).Meanwhile,?OHB-pre-treated cells exposed to A? had a significant increase in Ace-H3K9 and Ace-H4K12 expression and an obvious decrease in HDAC1 and HDAC2 expression relative to the levels observed in A?-exposed cells(P < 0.01).6.The expression of SNTA1 m RNA and protein was increased in HDAC1 slienced cells compared with control(P < 0.01).However,no differences in SNTA1 m RNA and protein expression were found between HDAC2 silenced cells and control(P > 0.05).7.The results showed that the expression of mi R-130 a was significantly decreased and the expression of HDAC3 m RNA and protein was significantly increased(P < 0.05;P < 0.01)in APP/PS1 mice compared with WT mice.After ADF intervention,a marked increase in mi R-130 a expression and a significant decrease in HDAC3 expression were found in APP/PS1 mice(P < 0.05;P < 0.01).8.Cells exposed to A? had a significant decrease in mi R-130 a expression and a remarkable increase in HDAC3 expression relative to those observed in control cells(P < 0.05;P < 0.01).The increase of mi R-130 a expression and the decrease of HDAC3 expression were observed in ?OHB-pre-treated cells compared with control cells(P < 0.05).Meanwhile,?OHB-pre-treated cells exposed to A? had a significant increase in mi R-130 a expression and an obvious decrease in HDAC3 expression relative to the levels observed in A?-exposed cells(P < 0.05;P < 0.01).9.The expression of AQP4-M1 m RNA was decreased in cells treated with mi R-130 a mimic(P < 0.05)and was increased in cells treated with mi R-130 a inhibitor(P < 0.01).However,no difference in AQP4-M23 m RNA expression was found in cells treated with mi R-130 a mimic or inhibitor.Moreover,the expression of AQP4-M1 and AQP4-M23 protein was decreased in different degrees(P < 0.05;P < 0.01),resulting in the decrease of AQP4-M1/M23 protein ratio(P < 0.01),in cells treated with mi R-130 a mimic,and increased in varying extent in AQP4-M1 and AQP4-M23 protein expression,leading to the increase of AQP4-M1/M23 protein ratio in cells treated with mi R-130 a inhibitor(P < 0.01).10.The expression of mi R-130 a was significantly increased in cells with HDAC3 siRNA or RGFP966(P < 0.01).Interestingly,in HDAC3-silenced cells,we found the expression of AQP4-M1 and AQP4-M23 m RNA and protein were increased in different degrees(P < 0.01),however,the AQP4-M1/M23 protein ratio was decreased compared with the levels in controls(P < 0.01).Conclusions:1.Intermittent fasting alleviates the loss of AQP4 polarity in the cerebral cortex of APPswe/PS1 d E9 double transgenic AD mice by inhibiting the up-regulation of AQP4-M1/M23 ratio and the reduction of SNTA1 expression,which may be partly mediated by ?OHB.2.?OHB may mediate the effect of intermittent fasting on the increase of SNTA1 in the cerebral cortex AD model mice through inhibiting HDAC1 expression.3.?OHB may partly mediate the effect of ADF on the reduction of AQP4-M1/M23 ratio in the cerebral cortex AD model mice,in which the up-regulation of ?OHB on mi R-130 a and the down-regulation of ?OHB on HDAC3 are possibly implicated as a potential mechanism. |
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