Effects Of Treadmill Exercise On Mitophagy In Hippocampus Of APP/PS1 Transgenic Mice

Background: Alzheimer’s disease(AD)is the age-related neurodegenerative disease,and neurotoxic of β-amyloid(Aβ)peptides is main inducement of AD.Recently some studies have shown that the mitochondria dysfunction induced by Aβ entering the mitochondria in the hippocampus,and mitophagy process was inhibited,which cause damaged mitochondria and Aβ cluster in neurons.At present,mounts number of studies have shown that exercise can reduce the deposition of Aβ and improve the ability of learning and memory,which can prevent and alleviate AD.However,the mechanism has not been fully elucidated.Recent studies have found that exercise can significantly improve the mitophagy activity and the learning and memory ability in the brain of wild-type mice,which suggests that mitophagy may be an important mechanism for exercise to improve AD.However,whether exercise can prevent and improve AD is associated with improving the activity of mitophagy has not been elucidated,which needs to be explored.Objects: In this study,3-month-old APP/PS1 transgenic mice were used subjects,we observe the effect of 12-week treadmill exercise on learning and memory ability,confirm whether exercise can reduce deposition of Aβ in the hippocampal mitochondria and improve mitochondria function,investigate whether exercise can improve mitophagy activity,and explore whether exercise can improve mitophagy activity are associated with PINK1/Parkin signal pathway and lysosomal function,so as to provide a target for the prevention and treatment of AD.Methods: Thirty 3-month-old male APP/PS1 mice and thirty littermates male widetype mice were divided into control group and exercise group.APP/PS1 mice were divided into two groups,AD control group(ADC,n=15)and AD exercise group(ADE,n=15).Wild-type mice were divided into two groups,wild-type control group(WTC,n=15)and wild-type group(WTE,n=15).ADE and WTE group have 12-week treadmill exercise from the age of 3-month-old.After 12 weeks of treadmill exercise,Morris water maze experiment was conducted on all mice to evaluate their learning and memory ability.After anesthesia,the hippocampus was sacrificed,make paraffin sections,and extracted mitochondria.The co-localization of mitochondria and Aβ as well as autophagosome was measured by double-labelling immunofluorescence.The m RNA express level of Cathepsin D、Rab7 in the hippocampus were assessed by Realtime PCR.The mitochondria-related protein level of ABAD、PGC-1α、Drp1、Mfn2 and mitophagy related protein PINK1、Parkin、LC3II/I、P62、Cathepsin D、Rab7 were measured by Western Blot.Results:(1)Exercise can delay the decline of learning and memory of ability of APP/PS1mice: the results of Morris water test reveal: in the positioning navigation experiment,comparing with WTC group,the escape latency of ADC group was significant increased(P<0.05),and the time percent in platform quadrant of ADC mice was significant reduced(P<0.05).Compared with ADC group,the escape latency of ADE group was significant decreased(P<0.05),and the time percent in platform quadrant of ADE mice was significant increased(P<0.05).In the experiment of exploration,comparing with WTC group,the crossing time and the pathlength percent in platform quadrant and time percent in platform quadrant of ADC were significant decreased(P<0.05).Compared the ADC group,the crossing time and swimming time and pathlength percent in platform quadrant of ADE were significant increased(P<0.05).(2)Exercise can decrease the deposition of Aβ in mitochondria of APP/PS1 mice: the results of double-labelling immunofluorescence revealed: compared to WTC group,the co-location area of the mitochondria(COXIV)and Aβ were increased in ADC group(P<0.01).Compared with ADC group,the co-location area of the mitochondria(COXIV)and autophagosome were decreased(P<0.01).The results of Western Blot shown that the Aβ oligomers protein level were significantly increased in the hippocampal mitochondria of ADC compared to WTC group(P<0.01);Compared with ADC group,the protein level of Aβ oligomers were significantly decreased in the hippocampal mitochondria of ADE group(P<0.01).(3)Exercise can improve the mitochondria function in the hippocampus of APP/PS1 mice: the results of Western Blot revealed: compared to WTC group,the ABAD(P<0.01)and Drp1(P<0.05)protein level was increased in ADC group;Compared to ADC group,the ABAD and Drp1 protein level were decreased in ADE group(P<0.01).Compared to WTC group,the PGC-1α and Mfn2 protein level was reduced in ADC group(P<0.05);Compared to ADC group,the PGC-1α and Mfn2 protein level were increased in ADE group(P<0.01).(4)Exercise can increase the mitophagy activity in the hippocampus of APP/PS1 mice: the results of double-labelling immunofluorescence revealed: compared to WTC group,the co-location of the mitochondria(COXIV)and autophagosome(LC3II)were increased(P<0.01).Compared with ADC group,the co-location of the mitochondria(COXIV)and autophagosome(LC3)were increased(P<0.01).The results of Western Blot revealed,compared to WTC group,the protein level of LC3II/I were significantly increased(P<0.01),and P62 was significantly increased(P<0.05)in the hippocampal mitochondria of ADC group.Compared with ADC group,the protein level of LC3II/I were significantly increased(P<0.05)and P62 were significantly decreased(P<0.01)in the hippocampal mitochondria of ADE group.(5)Exercise can increase the recruitment of Parkin of mitochondria to induce mitophagy in the hippocampus of APP/PS1 mice: the results of Western Blot revealed: the protein level of PINK1(P<0.01)and Parkin(P<0.05)were significantly increased in the hippocampus of ADC group compared to WTC group;Compared with ADC group,the protein level of PINK1 was significantly decreased(P<0.01),however,the protein level of Parkin was significantly increased(P<0.01).(6)Exercise can increase the lysosomal function in the hippocampus of APP/PS1 mice: the results of Real-time PCR that the m RNA level of Cathepsin D(P<0.01)and Rab7(P<0.01)were significantly increased in the hippocampus of ADC group compared to WTC group.The m RNA level of Cathepsin D(P<0.05)and Rab7(P<0.01)were significantly decreased in the hippocampus of ADC group.The m RNA level of Cathepsin D(P<0.05)and Rab7(P<0.01)were significantly increased compared with ADC group.The results of Western Blot revealed: Compared to WTC group,there was no difference on the protein level of Cathepsin D in ADC group(P>0.05),the protein level of Rab7 was decreased in ADC group(P<0.01).Compared with ADC group,the protein level of Cathepsin D and Rab7 were significantly increased(P<0.01).Conclusions: Exercise can prevent and improve learning and memory dysfunction in AD mice by improving the activity of mitophagy.The mechanism may be that exercise enhances the ability of mitochondria to recruit Parkin,and up-regulates the initiation level of mitophagy,thus accelerating the degradation and clearance of Aβ and damaged mitochondria,improving mitochondrial dysfunction,and preventing and delaying the occurrence of AD.