Study On The Mechanism Of Mitochondrial Fission Fusion And Transport In Exercise Prevention Of AD

Alzheimer's Disease(AD)is a common progressive neurodegenerative Disease among the elder,characterized by memory loss and cognitive decline,as well as changes in behavior and personality.With the increasing aging population in the world,the incidence of AD is increasing in recent years,which has become a public health security issue that cannot be ignored.The pathological features of AD are mainly extracellular senile plaques formed by?-amyloid(A?)protein and intracellular neuronal fiber tangles formed by hyperphosphorylated Tau protein,accompanied by loss of synaptic function and abnormal apoptosis of neuronal cells.However,there are no effective drugs to prevent or treat for AD currently.Mitochondria is the main organelles for the energy supply of neurons.Studies have confirmed that mitochondria abnormal functions is one of the important mechanisms for the pathogenesis of AD.Mitochondrial function is regulated by various mechanisms,among which the continuous mitochondrial fission,fusion dynamics and transport are the keys to maintain mitochondrial function.Mitochondrial fission fusion is unbalanced and transport is abnormal,suggesting that abnormal mitochondrial dynamic changes may be one of the key causes of mitochondrial dysfunction to induce neurodegenerative neuropathy in AD brain.Reversion of the changes and the improvement of exercise in neurodegenerative diseases are closely related,so it is important to investigate the mechanisms of exercise in the prevention and mitigation of mitochondrial dynamic in AD.Research purposesThis study adopted 3-month-old APP/PS1 double transgenic mice with moderate intensity treadmill exercise intervention for 12 weeks to explore whether the effects of long-term moderate intensity aerobic exercise for APP/PS1 double transgenic mice spatial learning and memory ability,and the influence of the exploratory behavior is possible mechanisms of moderate intensity treadmill exercise as intervention for AD from the aspects of the hippocampus mitochondrial dynamics.Research methodsAt the age of 3 months,all male wild-type mice were randomly divided into wild-type sedentary control group(WSED)and wild-type exercise group(WEXE),as well as the same age APP/PS1 double Transgenic mice were randomly divided into two groups:transgenic sedentary control group(TSED)and transgenic exercise group(TEXE).Mice in the WEXE and TEXE groups were given 12 weeks of moderate-intensity treadmill exercise intervention,while mice in the WSED and TSED groups were fed statically.After the treadmill exercise intervention,the water maze experiment and the opening experiment were carried out to detect the spatial learning and memory ability and the autonomous exploration behavior of the experimental mice.After behavioral experiment,all the experimental mice fasted and gave them unlimited water for 12h,and then entered the tissue sampling stage the next day.Selected six mice in each group to perform heart perfusion fixation with paraformaldehyde and to rapidly strip bilateral hippocampal tissues in the brain.Three of the stripped hippocampal tissues were placed in ice packs,1mm~3of tissue blocks were taken out with sharp blades in hippocampal CA1 region,and placed in the fixed solution of electron microscope for preparation of electron microscope specimens and the morphological changes of mitochondria and synapse in hippocampal CA1 region of mice were observed.In addition,the whole brain was fixed with 4%paraformaldehyde,and paraffin sections were prepared for immunofluorescence and immunohistochemical experiments,so as to evaluate the senile plaque and nerve cell apoptosis in the hippocampal area of mice.Each group selected 12 mice randomly to pick the eye for blood,then sacrified and separated bilateral hippocampus.Selected six mice from each group extracted mitochondria immediately,six other just for a set of bilateral hippocampal tissue in liquid nitrogen preservation,then turn on-80?refrigerator,in case of late for enzyme-linked Immunosorbent(ELISA)experiments and Western Blot(WB)experiments.The specific research contents are as follows:(1)Behavioral experiment:Morris water maze experiment was used to detect the spatial learning and memory ability of the experimental mice,and the opening experiment was used to detect the autonomous exploration behavior of the experimental mice.(2)Detection of A?content and senile plaque deposition:the contents of soluble A?40 and A?42 in the hippocampus of mice were detected by ELISA,and the status of senile plaque deposition in the hippocampus of mice was detected by sulfuretin staining.(3)Hippocampal mitochondria related nerve damage index detection:using ELISA experiment to test mt COX?,mt ROS activity and ATP content of hippocampus.WB assay detected the expression levels of SYN,PSD95 and GAP43,and the contents of Cyp D,Cyt-C,AIF,Apaf-1,P53,Bcl-2/Bax and Caspase-3,which were related to hippocampal apoptosis.The electron microscopy was used to test mice hippocampus synaptic number and the length of PSD.TUNEL assay was used to detect the apoptosis of hippocampal cells in mice.(4)The hippocampus mitochondrial dynamics related indicators of detection:using electron microscopy to test mice hippocampus mitochondrial shape,number and length.Using WB experiment to test the protein levels of mitochondrial fission and fusion dynamics indicators:Drp1,Fis1,Mff,Mfn1,Mfn2 and Opa1 in the hippocampus.At the same time,testing the protein levels of mitochondrial transporters KIF5A,KIF5B,KIF5C,KLC1,KLC2,SYBU,DIC,DLC and DCTN1in the hippocampus respectively.Research results(1)In the Morris water maze experiment,escape latency and total swimming distance of six-month old APP/PS1 double transgenic mice increased significantly(P<0.05),and the times of cross the platform position,platform quadrant distance percentage and platform time percentage significantly decreased(P<0.05).In the opening experiment,the number of poking,percentage of central distance and central time significantly decreased(P<0.05).After 12 weeks of moderate-intensity treadmill exercise intervention,escape latency and total swimming distance of AD mice decreased significantly(P<0.05),times of cross the platform position,platform quadrant distance percentage and platform time percentage increased significantly(P<0.01)in Morris water maze experiment.In the opening experiment the number of poking,percentage of central distance and central time percentage increased significantly(P<0.05).The content of soluble A?40 and A?42 in the hippocampal of APP/PS1 transgenic mice at six months of age were significantly increased(P<0.001),and the level of senile plaque deposition was significantly increased(P<0.001).After12 weeks of moderate-intensity treadmill exercise intervention,hippocampal A?40,A?42 and senile plaque deposition in AD mice were significantly reduced(P<0.01).(2)As the mitochondrial energy index of six-month-old APP/PS1 double transgenic mice hippocampus,mt COX?,mt ROS activity and ATP levels were significantly reduced(P<0.001).The protein expression levels of involved in hippocampal synaptic SYN,PSD95 and GAP43 decreased significantly(P<0.05)and apoptosis of hippocampal cells related proteins Cyp D,Cyt-C,Apaf-1,AIF,P53 and Caspase-3were significantly increased(P<0.01).The ratio of Bcl-2/Bax was significantly decreased(P<0.01).The number of synapse was significantly decreased(P<0.01),and the synaptic length was significantly shortened(P<0.01),and the proportion of TUNEL positive cells in the hippocampus was significantly increased(P<0.001).After 12 weeks of moderate-intensity treadmill exercise intervention,AD mice hippocampus mt COX?,mt ROS activity and ATP levels were significantly increased(P<0.05).The number of synapse and synaptic length were significantly increased(P<0.05).The protein expression levels of SYN,PSD95 and GAP43 were significantly increased(P<0.05).The protein expression levels of Cyp D,Cyt-C,Apaf-1,AIF,P53 and Caspase-3 were significantly decreased(P<0.05),and the Bcl-2/Bax ratio were significantly increased(P<0.01).The proportion of TUNEL positive cells in the hippocampus was significantly decreased(P<0.001).(3)The mitochondrial morphology in the hippocampal CA1 region of the6-month-old APP/PS1 double transgenic mice was fragmented,a large number of mitochondria were vacuolated,and the mitochondrial cristae were fractured.The number of mitochondria was significantly increased(P<0.001),and the mitochondrial length was significantly shortened(P<0.001).The levels of mitochondrial fission proteins Drp1,Fis1,and Mff were significantly increased(P<0.01),while the fusion protein Mfn1/2 and Opa1 were significantly decreased(P<0.05).The levels of mitochondrial transporters KIF5A,KIF5B,KIF5C,KLC1,KLC2,SYBU,DIC,DLC and DCTN1 in the hippocampus were significantly decreased(P<0.05).After 12weeks of moderate-intensity treadmill exercise intervention,mitochondrial morphology in hippocampal CA1 region of AD mice was significantly improved,the number of mitochondria was significantly reduced(P<0.001),and the length of mitochondria was significantly increased(P<0.001).The levels of fission proteins Drp1,Fis1,and Mff were significantly decreased(P<0.05),and the fusion protein MFN1/2 and Opa1 expression levels were significantly increased(P<0.05).The expression levels of mitochondrial transporters KIF5A,KIF5B,KIF5C,KLC1,KLC2,SYBU,DIC and DCTN1 were significantly increased(P<0.05).Research conclusions(1)The levels A?and senile plaque deposition in six months old APP/PS1 double transgenic mice hippocampus increased.The spatial learning and memory ability,autonomous exploratory behavior decreased,and 12-week moderate-intensity treadmill exercise can reduce levels of A?and senile plaque deposition in the hippocampus of APP/PS1 mice,and improve the ability of learning and memory,as well as the independent exploratory behavior.(2)The decreased learning and memory ability of APP/PS1 mice induced by A?may be related to mitochondrial dysfunction,while 12-week moderate-intensity treadmill exercise can inhibit synaptic damage and neuronal apoptosis associated with mitochondrial dysfunction by improving hippocampal mitochondrial dysfunction.(3)Hippocampal mitochondrial dynamics and abnormal transport are important causes of mitochondrial dysfunction in six-month-old APP/PS1 mice.12-week moderate-intensity treadmill exercise can alleviate mitochondrial abnormalities,improve mitochondrial function and subsequently inhibite mitochondria-related synaptic damage and neuronal apoptosis,and ultimately improve the learning and memory ability of APP/PS1 mice by balancing the fission and fusion and transport.