TNEA Therapy For Alzheimer’s Disease:the Molecular Basis Of Acupoints Combination On Activating TFEB/TFE3

ObjectiveAutophagy dysfunction and neuroinflammation play critical roles in the pathological process of AD.The previous research of our group showed-that the three-needle electroacupuncture(TNEA)could inhibit key kinases like mTORC1,ERK1/2,and AKT that affect the activation of autophagy.TNEA upregulated TFEB for nuclear transcription and thus enhanced lysosomal biosynthesis.The activation of autophagy promoted the degradation of APP/Aβ,which helped to improve the cognitive dysfunction in 5xFAD mice.Based on the previous studies,we intend to further explore the acupoint-specific mechanism for the TNEA treatment.As TFEB/TFE3-mediate ALP may play critical roles in the process of AD,we detected whether TNEA promoted the activation of TFEB/TFE3 depending on the GB13/GV24 acupoints combination.And as we have already found the anti-inflammation effect of TNEA via inhibiting the Aβ-related microglia,we intended to further discuss the link between this anti-inflammatory effect and the TFEB/TFE3-mediated autophagy activated by TNEA.MethodsExperiment 1:6-7 month-old 5xFAD mice(n=44)and wild type mice(n=11)were randomly divided into 5 groups:5xFAD+GB13(n=11),5xFAD+GV24(n=11),5xFAD+TNEA(n=10),5xFAD(n=11),and WT(n=11).The spatial memory of the mice was evaluated via the Morris water maze,and the expression of APP-CTFs in the hippocampus(HI)and prefrontal cortex(PFC)were detected via Western blot.Experiment 2:We used western blot to observe the expression of p-TFEB and LAMP1 in the HI and PFC.And we detect the nuclear TFE3 and cytoplasmic TFE3 expression in the HI of the intervention groups and the model group.The co-localization of TFE3 and nucleus was observed by immunofluorescence staining.We further detected the expression of phosphorylated and total AMPK/AKT/mTORCl/RPS6 in the HI and the PFC.pAMPK/AMPK,p-MTOR/MTOR,p-AKT/AKT,and p-RPS6/RPS6 were calculated to determine the activation/inhibition of the upstream kinases.Experiment 3:Western blot was used to detect the expression of pro-inflammatory cytokine IL-1β in the hippocampus of the mice in the intervention groups and the model group.44 5xFAD mice aged 7-8 months were randomly divided into 4 groups:5xFAD+ShScramble(n=10),Sh-Scramble+TNEA(n=10),Sh-Tfeb+5xFAD(n=12)and ShTfeb+TNEA(n=12).The expression of IL-1βin each group was detected again after Tfeb knockdown.Results1.TNEA improves spatial learning memory and degradation of beta-amyloid precursor protein in 5xFAD mice depending on GB13/GV24 acupoints combination.(1)The results of MWM:In the probe test,compared with wild-type mice,5xFAD mice showed a significant decrease in the proportion of the distance traveled in the target quadrant(P<0.01),and 5xFAD mice have shown obvious spatial memory impairment.After the intervention,the proportion increased significantly in the 5xFAD+TNEA and 5xFAD+GB13 groups(P<0.05),indicating an improvement in spatial memory ability of the mice in these two groups.However,no significant difference was found between the 5xFAD+GV24 group and the model group.(2)The level of APP-CTFs:Western blot showed that after TNEA intervention,the expressions of Fl-APP and CTFs in the hippocampus were significantly reduced(P<0.01,P<0.001).No change was found in the expression of Fl-APP in the prefrontal cortex,but a very significant difference was found in CTFs(P<0.001).For mice that accepted the GB13 electroacupuncture,only CTFs in the PFC were reduced(P<0.01).For mice accepted the GV24 electroacupuncture,EA affected the expression of APP-CTFs neither in the HI nor in the PFC.2.TNEA activates TFEB/TFE3 and lysosomal biogenesis in the hippocampus of 5xFAD mice brains depending on GB13/GV24 acupoints combination.(1)The expression of nucleus TFE3(nTFE3),cytoplasmic TFE3(cTFE3),and total TFE3 in the hippocampus:TNEA significantly increased the nuclear TFE3 expression without affecting its cytosolic levels(P<0.05).The total TFE3 level increased but was without significant difference compared with 5xFAD mice.To further confirm whether TNEA promotes the accumulation of TFE3 in the nucleus,we determined the colocalization of TFE3 and nuclear chromatin in the HI.The results of immunofluorescence staining showed that the area of TFE3+nucleas increased both in the CA1 and CA3 areas of HI,while only in CA1 the difference was statistically significant(P<0.05).We then examined the levels of total,cytosolic,and nuclear TFE3 in the other two single acupoint groups.It showed that GB13 and GV24 electroacupuncture did not seem to affect the expression and activation of TFE3.The results indicate TNEA can promote the translocation of TFE3 to the nucleus in 5xFAD mice.Its effect is based on the synergic effect of the GB13/GV24 acupoints combination.(2)Dephosphorylation of p-TFEB and expression of lysosomal membrane marker LAMP1:TNEA significantly reduced the level of p-TFEB(S142)and increased the expression of LAMP 1 in the HI(P<0.05 and P<0.01),which was the same as our previous report.The tendency in the PFC was the same as those in the HI,but the difference was not statistically significant.As for single acupoint electroacupuncture groups,GB13 electroacupuncture significantly decreased the level of p-TFEB(S142)in the HI(P<0.01),but it did not influence the level of LAMP1.GV24 electroacupuncture still made no difference.The results in the HI suggest that TNEA activated TFEB/TFE3 in the hippocampus of 5xFAD mice depending on the GB13/GV24 acupoints combination.(3)Activation of AMPK/AKT/mTOR:In the hippocampus,TNEA could significantly inhibit the phosphorylation of AKT at S473(P<0.05)and mTORC1 at S2448(P<0.01),and promoted the activation of AMPK at T172(P<0.05).Phosphorylation of RPS6 at S235/236 was not influenced in the HI.In the PFC,there observed an opposite tendency for all those proteins.AMPK was significantly inhibited which might lead to the activated phosphorylation of mTORC1.For mice both in the group of 5xFAD+GB13 and 5xFAD+GV24,the p-RPS6/RPS6 ratio in HI increased significantly(P<0.05),while no other changes were found in both the HI and the PFC.3.TNEA inhibits neuroinflammation based on the GB13/GV24 acupoints combinationEffects on the pro-inflammatory cytokine IL-1β:TNEA could reduce the expression of pro-IL-1β(P<0.01)and mature IL-1β(P<0.01)in the hippocampus.After Tfeb knockdown in HI,there was no significant difference in the expression of mature IL-1β between the ShTfeb+TNEA group and the Sh-Tfeb group.But the obvious difference was found between Sh-Scramble+TNEA and Sh-Tfeb+TNEA groups,in which Scramble+TNEA showed lower expression of mature IL-1β(P<0.01).After Tfeb knockdown,the inhibition effect of TNEA for IL-1β was eliminated.The test for the other two acupoints showed no effect on the inhibition of IL-1β.Conclusions(1)Via regulating the AMPK/AKT/mTORC1 kinases in the hippocampus of 5xFAD mice,TNEA can promote the dephosphorylation of TFEB/TFE3 in the cytoplasm,which helps the biogenesis of lysosome and degradation of Aβ,and thus finally improves the spatial memory of 5xFAD mice.When separated,the GB13 and GV24 can not significantly regulate the AMPK/AKT/mTOR signaling pathway to influence the translocation of TFEB/TFE3,and thus have little impact on lysosomal biogenesis and CNS inflammation.Based on this phenomenon,we assume that the therapeutic effect of TNEA depends on the synergic effect of both acupoints.(2)TNEA can alleviate neuroinflammation by inhibiting the expression and activation of IL-1β.After the Tfeb knockdown,the effect could be blocked.Thus TNEA inhibits the activation of inflammatory factor IL-1β through TFEB-mediated autophagy,thereby improving the AD-related neuroinflammation.Since neither GB13 electroacupuncture nor GV24 electroacupuncture showed any inhibitory effect on IL-1β,the effect of TNEA inhibiting the activation of IL-1β was also based on the combined effect of the acupoints.(3)The effect of TNEA in the hippocampus and the prefrontal cortex may not be consistent.