Electroacupuncture Ameliorates Beta-Amyloid Pathology And Cognitive Impairment In Alzheimer Disease Via A Novel Mechanism Involving Activation Of Transcription Factor EB

ObjectiveAlzheimer's disease(AD)is the most common dementia that affects millions of people worldwide.The lack of understanding of the pathogenic mechanism hinders the development of effective AD treatment.Recent evidence suggests that misfolded protein aggregation due to impairment of autophagy-lysosomal pathway(ALP)may play a significant role in AD development,and thus making transcription factor EB(TFEB),which orchestrates ALP,as a promising target for treating AD.As a complementary therapy,acupuncture or electroacupuncture(EA)has been commonly used by TCM practitioners in China for the treatment of various neurodegenerative disorders.We previously reported that acupuncturing at GV24 and bilateral GB13 acupoints improved the learning and memory in a rat model of Alzheimer's disease(AD).In this study,we used the 5×FAD mouse model to further confirm the efficacy of three-needle EA(TNEA)in AD and explore the molecular mechanisms.Methods5,5-month-old 5xFAD mice were randomly divided into four groups:wild type group?5×FAD group?three-needle EA group?sham EA group.5xFAD mice were treated with TNEA(GV24 and bilateral GB13)for 4 weeks(five days/week).Spatial memory was determined by Morris water maze and fear conditioning.The changes in APP processing and glial activation were determined by Western blot and immunohistochemistry.The protein levelsof LC3B?SQSTM1?LAMP1?CTSD?TFEB?phosphorylated(p-)and total MTOR,RPS6,AKT and MAPK1 in the prefrontal cortex(PFC)and hippocampus(HI)of mice brains were observed by western blots.Fluorecene intensity of reactive astrocytes and microglia,the change of plaque area were observed by immunofluorescence staining.For chloroquine(CQ)treatment,5×FAD mice were intraperitoneally injected with vehicle(PBS)or CQ(50 mg/kg/day)4 days before the last TNEA treatment and then subjected to behavior tests and biochemical analysis.The injection of AAV-sh-Tfeb(or Scramble)was performed 2 weeks before TNEA treatment,followed by behavior tests and biochemical analysis.Results1.TNEA treatment significantly improved spatial memory and contextual fear memory in 5×FAD mice.5.5-month-old male 55×FAD mice were treated with TNEA or Sham-EA for 4 weeks,then Morris water maze(MWM)and fear conditioning(FC)were used to evaluate spatial learning memory and contextual fear memory respectively.In MWM,the escape latency was significantly increased in 5×FAD mice compared to wild-type(WT)from training day 2 to 5 TNEA treatment significantly reduced the escape latency in 5xFAD mice from training day 2 to 5.For probe trial,the time spent in the target quadrant and the number of platforms crossed was significantly increased for mice treated with TNEA.TNEA treatment significantly increased the freezing time in both contextual and cued test compared with that of 5×FAD mice.2.TNEA promotes the degradation of APP/A? and inhibits microglia activation in 5 xFAD mice brains(1)TNEA treatment promotes the degradation of beta-amyloid precursor protein(APP)fragments in5×FAD mice brains.The overexpression of full-length(F1)-APP and CTFs in the PFC and HI of5 xFAD mice,compared with that of the WT mice was confirmed by western blots.TNEA treatment significantly reduced the levels of F1-APP and CTFs in the PFC and HI.Notably,TNEA did not affect the expression of BACE1(?-secretase 1)in the PFC and HI indicating that TNEA may promote the lysosomal degradation of APP and CTFs,without affecting the amyloidogenic processing of APP.(2)TNEA treatment reduces A? load and inhibits microglia activation in 5 ×FAD mice brains.TNEA significantly reduced the A(3 positive area and plaque size(stained by 6E10 antibody and an A?1-42 specific antibody respectively)in PFC and CA1 regions.Meanwhile,both the total and A?-associated AIF1 intensity was significantly decreased in PFC and CA1 regions of 5×FAD mice treated with TNEA.However,no significant changes in GFAP intensity were found in PFC and CA1 regions of 5×FAD mice treated with TNEA.Together,these results indicate that TNEA treatment promotes the degradation of APP,CTFs and A?,and inhibits microglia activation in 5×FAD mice brains.3.TNEA activates TFEB and promotes lysosomal biogenesis in5×FAD mice brains(1)TNEA treatment activates autophagy-lysosomal pathway in 5×FAD mice brains.The accumulation of the autophagy substrate SQSTM1 in 5xFAD mice brains was reduced by EA treatment.Compared to the CQ alone group,TNEA+CQ group did not further increase the levels of LC3B-II in the TX-100 soluble fractions in the PFC and HI of 5×FAD mice.However,TNEA treatment failed to degrade the insoluble LC3B-? and SQSTM1 in the PFC and HI of 5 xFAD mice co-treated with CQ.TNEA treatment promoted the formation of autolysosomes in 5×FAD mice brains.The results indicate that the conversion of APs to ALs is impaired in 5 xFAD mice brains,which can be restored by TNEA treatment.(2)TNEA treatment promotes lysosomal biogenesisFor lysosomal markers,we found that TNEA treatment accompanied with increased levels of LAMP1,CTSD/cathepsin D and TFEB.(3)TNEA treatment promotes the de-phosphorylation and nuclear translocation of TFEB in 5 xFAD mice brains.TNEA treatment significantly increased the levels of nuclear TFEB,without affecting its cytosolic levels,in the PFC and HI of 5×FAD mice.4.TFEB-induced lysosomal activation is required for memory improvement and APP/A? degradation induced by TNEA(1)TNEA treatment restores the defective recognition and degradation of autophagy substrates in 5×FAD mice brains.TNEA treatment restored the defective autophagy cargos/substrates recognition by LC3B-positive autophagosomes and enhanced degradate APP-associated SQSTM1 in 5×FAD mice brains.(2)TFEB-induced lysosomal activation is required for memory improvement and APP/CTFs degradation induced by TNEA in 5×FAD mice.TNEA treatment significantly reduced the level of CTFs in the hippocampus of 5×FAD mice injected with AAV-sh-Scramble,but not of mice injected with AAV-sh-Tfeb.Meanwhile,the improved spatial learning memory induced by TNEA was also blocked in mice injected with AAV-sh-Tfeb.5.TNEA treatment inhibits MTORC1,AKT and MAPK1 in 5×FAD mice brains.TNEA inhibits the upstream kinases of TFEB in 5×FAD mice brains.TNEA activated TFEB via inhibiting AKT-MAPK1-MTORC1 pathway,thus promoting ALP in the brains.ConclusionOur results indicate that TNEA treatment can activate the autophagy-lysosomal system in the brain,which facilitates the degradation of A?,reduces neuroinflammation,and improves cognition in AD mice.We developed a novel TNEA therapy which activates TFEB-mediated lysosomal biogenesis to attenuate A? pathology and improve memory in an AD animal model via inhibiting AKT-MAPK1-MTORC1 pathway.