The Effect And Mechanism Of Gamma-aminobutyric Acid In Regulating Synaptophysin Expression By The Sp1/ATP9B Pathway In Alzheimer’s Disease

Objective: Alzheimer’s disease(AD)is a neurodegenerative disease with cognitive impairment as the main clinical manifestation,and its pathogenesis has not been elucidated.Synaptophysin(SYP)is a phosphoprotein linked to synaptic vesicles and is closely related to synaptic plasticity and cognitive function.Studies have found a decrease in SYP levels in specific regions of the hippocampus in AD patients,which is closely associated with cognitive function.P4 ATPase is a class of turnover enzymes that can actively transport phospholipids in biofilms.The expression of ATP9 B,a type II P4 ATPase,and its potential regulation of SYP expression in AD are not yet clear.Specificity protein 1(Sp1)is highly expressed in AD,and bioinformatics analysis has revealed that Sp1 can bind to the promoter region of ATP9 B and regulate its expression.Sp1 is involved in gene transcription regulation mainly by collecting protein complexes,and the nature of protein complexes determines whether Sp1 is an activator or an inhibitor.Histone acetyltransferases(HATs)and histone deacetylases(HDACs)mediate histone acetylation.Among the reported protein complexes,HATs are mainly p300/CBP and PCAF(transcription activator),whereas HDACs are mainly HDAC1/2/3(transcription inhibitor factor),with HDAC2 most closely related to AD.Evidence suggests that Sp1 may regulate ATP9 B by recruiting HDACs rather than HATs,but this requires further confirmation.Gamma-Aminobutyric acid(GABA)is not only an important inhibitory neurotransmitter but also a functional food factor.Studies have shown that GABA has antagonistic effects on AD and inhibits the expression of HDAC2.This study explored whether GABA can regulate ATP9 B expression in the brain of AD through the inhibition of HDAC2 mediated by Sp1 and subsequently modulate SYP expression.Methods: 1.The 5-month-old mice with APP/PS1 double transgenic AD were randomly divided into AD,AD + GABA group and WT,WT + GABA group.There were 5 male and 5 female rats in each group.AD + GABA,WT + GABA mice drank distilled water containing 0.1% GABA,AD,WT mice drank distilled water.After 6months,the cognitive function of the mice was tested using the T-maze spontaneous alternation test.Subsequently,the mice were euthanized under anesthesia,and the hippocampal tissue was collected.The expression levels of SYP,ATP9 B,Sp1,p300,and HDAC2 in the hippocampus were measured using q RT-PCR and Western blotting.2.Human neuroblastoma cells(SH-SY5Y)were cultured in vitro.The cells were pretreated with GABA at a final concentration of 20 n M for 3 hours,followed by the addition of Aβ at a final concentration of 20 μM.After 24 hours,the cells were harvested,and the expression levels of SYP,ATP9 B,Sp1,p300,and HDAC2 were measured using q RT-PCR and Western blotting.Using the cultured SH-SY5 Y cells,si RNA technology and Ch IP method were employed to analyze whether Sp1 regulates the expression of ATP9 B and SYP by recruiting HDAC2 rather than p300.Results:1.T-maze spontaneous alternation test showed that GABA could antagonize the decrease of alternation accuracy(P<0.01 or P<0.05).Compared with WT group,the expression of SYP and ATP9 B in hippocampus of AD group decreased(P<0.01),while the expression of Sp1,p300 and HDAC2 increased(P<0.01);GABA could reverse the above changes in AD model mice.2.Compared with the control group,the expression levels of SYP and ATP9 B decreased(P<0.01 or P<0.05),while Sp1,p300 and HDAC2 increased(P<0.01);GABA could reverse the above changes of cells in Aβgroup.The expression of SYP in SH-SY5 Y cells of silent ATP9 B decreased(P<0.01).The expression of ATP9 B and SYP in SH-SY5 Y cells were increased(P<0.01).The expression of SYP and ATP9 B in SH-SY5 Y cells were not changed(P>0.05),Sp1decreased(P<0.05)and HDAC2 increased(P<0.01).The expression of SYP and ATP9 B in SH-SY5 Y cells with silent HDAC2 increased(P<0.01),but Sp1 and p300 were not changed(P>0.05).The level of Sp1 in ATP9 B promoter region of Sp1 antibody group was higher than that of Ig G group(P<0.01).The level of H3K9 acetylation in SH-SY5 Y cell promoter region of silent HDAC2 increased(P<0.01),while the level of Sp1 in ATP9 B promoter region did not change(P>0.05).Conclusions: Sp1 regulates ATP9 B through the recruitment of HDAC2 rather than p300.GABA can inhibit HDAC2,thereby reducing the recruitment of HDAC2 by Sp1 and subsequently regulating the expression of ATP9B/SYP,leading to improved cognitive function in the AD model mice.