The Effects Of Aging And Prior Exercise Training On MPTP-Induced Parkinson's Disease In Mice: Mechanism Research Of Mitochondria And Autophagy Pathway

Objective:Parkinson's disease (PD) is a neurodegenerative disease which is a serious threat tothe elderly group. Recent studies have shown that Autophagy play a key role in theremoval of neurodegenerative disease-related organelles. But its mechanism is notclear yet. The purpose of this study was to investigate the relationship betweenAutophagy, mitochondrial function and mitochondrial dynamics during aging processof the PD model mice established by injection of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine). To investigate the effects on the above indicators in young PDmodel mice, after prior forced chronic treadmill running. To study the role ofautophagy and mitochondrial fusion/fission in vitro PD model on PC12cells inducedby MPP~+with different autophagy level.The research could further reveal thepathogenesis of Parkinson's disease and provide an effective non-pharmacologicalprevention and treatment method for related neurodegenerative disease.Methods:1,90male C57BL/6mice were divided into six groups: four month age controlgroup(4MC), four month age PD group(4MP), eight month age control group(8MC),eight month age PD group(8MP), fifteen month age control group(15MC) and fifteenmonth age PD group(15MP). The PD group were administered moderate dose ofMPTP (30mg/kg×2, ip,16hr apart). The behavioral test (pole test and hanging test)were conducted in succession1day before MPTP injection, and2,5,8day afterMPTP. Then all animal was sacrificed.the changes of the midbrain substantia nigracells were observed by the method of immunohistochemical staining for TyrosineHydroxylase(TH) positive cell. Nerve ultrastructure were observed in midbrain underTEM. Mitochondria state3respiration, state4respiration, respiratory controlratio(RCR), and ATP synthesis activity and ROS generation rate in isolated midbrainand striatum mitochondria were detected. Mitochondrial dynamic-related protein(Mfn2, Fis1, Drp1) and Autophagy-related protein(Beclin1, LC3-II) expression were measured by Western-blot.2,60Male C57BL/6(6-8weeks) mice were randomly divided into sedentary orexercise group(12m/min,20minutes/day, and training on treadmill for6weeks,6days/week). Each group were divided in to two groups again, and were injected withmoderate dose of MPTP or saline after training. Finally, mice were divided in to thefour groups: saline injected(N); exercise and saline injected(EN); MPTP injected(P);pre-exercise and MPTP injected(EP),(n=15). Mfn2, Fis1, Drp1, Beclin1and LC3gene expression were measured by Real-time PCR. The other indexes were same asthe former part. GDNF content in blood, midbrain and striatum were measured byELISA3,PC12cell were randomly divided into six group: control group(C),~+rapamycingroup(R),~+wortmannin group(W), MPP~+induced group(P), MPP~+~+rapamycingroup(PR), MPP~+~+wortmannin group(PW). Cell survival rate and MDA content wasmeasured. Cell ultrastructure were observed under TEM. The happening of autophagyafter MPP~+induced with GFP-LC3plasmid transfected into PC12cells was detect byusing laser confocal microscopy. The mPTP and ΔΨm changes in each group weredetect by using laser confocal microscopy. The Beclin1, LC3-II, OPA1, Fis1andDrp1protein expression in each group were measured by Western-blot.4,SPSS13.0was used for statistical analysis. Behavioral test scores among thegroups was analysised by repeated measures analysis of variance.The difference wasanalysised by the two way analysis of variance (ANOVA).Results:1,Compare with4MC group,the body weight, Fis1and LC3-II protein in8MC groupwere significantly higher. Compare with4MC group,TH positive cell, mitochondriastate3respiration,RCR, ATP synthesis activity, Mfn2, Beclin1and LC3-II proteinwere significantly lower in15MC with a significantly higher body weight, ROSgeneration rate, Fis1and Drp1protein expression. After building the PD model withMPTP, compare with control group, the score of behavioral test, TH positive neurons,mitochondria respiration capacity and Mfn2protein expression were significantlylower in PD group, whose ROS generation rate, Fis1, Drp1, Beclin1and LC3-IIprotein expression were significantly higher. The changes in15MP were moresignificantly compare with4MP and8MP group. There was a significant interactionof 'aging' and 'MPTP' in related indicators of mice with statistical analysis.Accompanied with aging process, the sensitive of mice to MPTP was gradually increased. Effects of MPTP were the most obvious in old mice.2,Compare with N group, the score of behavioral test, TH positive neurons,mitochondria state3respiration, RCR, ATP synthesis activity Mfn2gene and proteinexpression were significantly lower in P group whose ROS generation rate, Fis1,Drp1, Beclin1and LC3-II gene and protein expression were significantly higher.Compare with P group, the score of behavioral test, TH positive neurons,mitochondria respiration capacity, Mfn2, Fis1, Beclin1and LC3-II gene and proteinexpression were significantly higher in EP group whose ROS generation rate wassignificantly lower. Compare with N group, neurons ultrastructure damage is obviousin P group which could be improved by pre-trainning. There was a significantinteraction of 'exercise' and 'MPTP' in the related indicators (behavioral test, THpositive neurons, mitochondria state3respiration, RCR, LC3-II protein) of mice withstatistical analysis. Compare with N group, GDNF in blood was significantly higherin EP group and GDNF in brain was significantly higher in P and EP group. Comparewith P group, GDNF in blood and brain was significantly higher in EP group.3,Compare with C group, cell survival rate, ΔΨm, OPA1protein expression weresignificantly lower in P group whose MDA, MPT, Beclin1, LC3-II, Fis1, Drp1proteinexpression were significantly higher. Compare with P group, MDA, MPT weresignificantly lower in PR group whose cell survival rate, ΔΨm, Beclin1, LC3-II, Fis1,Drp1protein expression were significantly higher. Compare with P group, the changesin PW group were opposite as that in the PR group. Compare with C group, cellultrastructure was impaired and autophagic vacuoles were increased in P group.Compare with P group, autophagic vacuoles increased significantly, in which thenumber of mitochondria was increased, in PR group. Compare with P group, cellultrastructure was impaired significantly in PW group.Under laser confocalmicroscopy it was observed that the cell autophagy levels increased after MPP~+treatment, and the occurrence of mitophagy was increased. There was a significantinteraction of 'MPP~+' and 'Drug Intervention' in the related indicators (cell survivalrate, MDA, MPT, ΔΨm, Beclin1, LC3-II, Fis1protein) of mice with statisticalanalysis.Conclusion:1, Accompanied with the aging process, ROS generation gradually increased;mitochondrial respiratory function decreased; the mitochondria tent to split;autophagic level was lower. A large number of abnormal mitochondria could not be cleared, which resulted in the substantia nigra damage significantly.2,Moderate dose of MPTP could damage the mitochondrial function which lead toMitochondrial split and increase autophagy levels which might be a compensatoryreaction. With the aging process, the sensitivity to MPTP in mice midbrain andstriatum was gradually increased. Effects of MPTP were the most obvious in old mice.Compensatory ability of autophagy was insufficient in old mice. When old micesubjected to the same external damage, abnormal mitochondria could not beeffectively removed, which result in the increased ROS production, a further declinein mitochondrial function, and the most severe neuronal damage.3,Prior exercise training did initially start autophagy in mice midbrain and striatum,which enhanced autophagy levels induced by mitochondrial fission when MPTPdamage occurs. As a result,mitochondrial respiratory function was improved andROS was reduced, which played a neuroprotective role.4,The raise of autophagy level was the self-protection mechanism for PC12cellsresistant to the damage of MPP~+. The upward or downward regulation of theautophagy level could improve or aggravate cell damage correspondingly. Mitophagymay play an important role in this process.