The Impact Of Rapamycin On The Mitochondrial Quality Control In SOD1-G93A Mice

Objective: Amyotrophic lateral sclerosis (ALS)is a progressivelydegenerative disease, causing selective loss of MNs from spinal cord,brainstem, and cortex, and results in paralysis and death within3–5years fromdiagnosis. The pathogenesis of ALS involves multiple mechanisms, includingglutamate excitotoxicity, oxidative stress, mitochondrial dysfunction, proteinaggregation and so on, but there is not a reasonable hypothesis that canexplain the real reason of MNs degeneration and there is not any effectivetreatmen until now.As the develepment of the researches, people found that mitochondrialdysfunction played an important role in the process of ALS; it was not only aconverging point of multiple pathological pathways in ALS, but also theinitiating factor of ALS. However, the real reason of mitochondrialdysfunction is still unclear. In physiological conditions, the cell can repairdamaged mitochondria, remove aging mitochondria, promote mitochondrialrenewal, and maintain the morphology and function of mitochondria in thehelp of mitochondrial quality control mechanism. Mitochondrial qualitycontrol includes mitochondrial fission, fusion and mitophagy, two processesthat regulate mitochondria morphology, distribution, and function. Theimbalance of mitochondrial quality control would lead to mitochondrialultrastructural changes, which are extremely similar to the changes ofmitochondria in ALS. In addition to that, some researches have provend thatthere were amounts of accumulation of mitochondria and abnormal protein inMNs, indicating that the clearance of mitochondria and protein was impaired.Our initial experiments have confirmed that the mitochondrial quality controlmechanisms were really impaired in the lumbar spinal cord of SOD1G93Amice.(including impaired mitochondrial fission, fusion mechanism and mitiphagy). In mammals, autophagy contributes to remove abnormal proteins anddamaged organelles such as mitochondria, endoplasmic reticulum, etc, and itwas the key factor that maintains normal physiological function of cells.Mitochondrial fission, fusion and mitophagy are two processes interactingwith each other. Mitochondrial fission can trigger mitophagy, and mitophagycan regulate mitochondrial fission. Rapamycin firstly used as immunosupp-ressants, which belongs to a classic autophagy activation agent by inhibitionof mTOR pathway, is closely related to cell growth, differentiation and energyutilization Because of the relation of mitochondrial fission, fusion andmitophagy, there may be some relations between impaired mitochondrialfission, fusion and impaired mitophagy. Therefore, we hypothesized thatapplication of autophagy activation agent may improve mitochondrial fissionand fusion.Originally, it was believed that autophagy was a non-selective process,randomly engulfing cytosolic components and organelles (29-30), but recentstudies had found the time when the cargos were surrounded by AVs wasdifferent, indicating that the choice of "cargos" was procedural. In the cellsdeprived of nutrient, mitophagy accounted for80%of all autophagy flow,while the autophagy of remaining components accounted for only20%,indicating that there is certain priority choice for mitochondria instarvation-induced autophagy.Our previous study found, as the progression of the disease, the distributionof autophagic vacuoles changes (accumulation of mitochondrially-targetedautophagosomes), there may be some relations between the abnormaldistribution of autophagy flow and the accumulation of inclusions in ALS.In our research, through short-term given rapamycin treatment, weobserve the protein level of dynamics associated protein1(p-Drp1), opticatrophy protein1(OPA1) and Mfn1in the lumbar spinal cord of SOD1-G93Atransgenic mice. At the same time, observe the distribution of LC3II and P62after activation of autophagy. Make sure the ralations of impairedmitochondrial fission, fusion and impaired mitophagy, understand deeply of selectivity characteristics of autophagy, explor the ralations of abnormaldistribution of autophagy flow and the accumulation of proteins, and assessthe treatment feasibility of autophagy activation agent on ALSMethods: Rapamycin was dissolved in dimethyl sulfoxide (DMSO)(25mg/ml) and further diluted with1:200v/v PBS before intraperitoneally(i.p)injection. ALS transgenic mice in90ds were randomized divided into2groups:rapamycin treatment at the dose of2mg/kg body weight/day in ALS mice(ALS-Rapa mice, n=3) and vehicle treatment in ALS mice (ALS-Vehiclemice, n=3). Continuous intraperitoneally injection for8days. Mice weresacrificed and seperation of the spinal cord for total protein extraction,isolation of mitochondria, extraction the protein of mitochondrial andcytosolic (not including mitochondrial protein,). Finally, observation theprotein level of p-Drp1OPA1and Mfn1using Western blot method.Simultaneous observe the distribution of LC3II and P62in mitochondrial andcytosolic. The data were analyzed with SPSS13.0.Results:1The protein level of OPA1, Mfn1and p-Drp1in the lumbar spinal cordof SOD1-G93A transgenic mouse after given rapamycin intervention.(1) The protein levels of p-Drp1were gradually down-regulated in thelumbar spinal cord of SOD1-G93A transgenic mouse after given rapamycinintervention, compared to vehicle treatment group.(P <0.05)(2) The protein levels of Opa1and Mfn1were gradually enhanced in thelumbar spinal cord of SOD1-G93A transgenic mouse after given rapamycinintervention, compared to vehicle treatment group.(P <0.05)2The distribution of LC3II and P62in mitochondrial and cytosolicfractions after given rapamycin intervention.(1) The protein levels of LC3II in mitochondria were gradually enhancedin the lumbar spinal cord of SOD1-G93A transgenic mouse after givenrapamycin intervention, but protein levels of LC3II in cytosolic fractions weredecreasing, compared to the vehicle treatment group (P <0.05).(2) The protein levels of P62in mitochondria were gradually enhanced in the lumbar spinal cord of SOD1-G93A transgenic mouse after givenrapamycin intervention, the difference was statistically significant (P <0.05).Conclusions: In this experiment, we found that Rapamycin couldsignificantly improve excessive and unbalanced fission of mitochondria,indicating that excessive and unbalanced fission of mitochondria were closelyrelated impaired mitophagy; we found the protein level of P62inmitochondrial are increased, because P62act as a marker of autophagic flux,further illustrates the impaired mitophagy; we also observed the distribution ofLC3II in mitochondria and cytosolic fractions, the MNs are rich inmitochondrially-targeted autophagosomes, further illustrates the impairedmitophagy; in addition, we also found activation of autophagy, the number ofautophagic vacuoles in the cytoplasm did not increase with the activation ofautophagy, howere, because of the accumulation of autophagic vacuoles in themitochondrial fractions, the content in the cytoplasm is further reduced.Excessive and unbalanced fission of mitochondria were caused by impairedmitophagy, and further leading to disorders of mitochondrial quality controlmechanisms, that may be the fundamental reason of abnormal morphologyand function of mitochondria in ALS. At the same time, the abnormaldistribution of autophagy flow may become an important breakthrough pointin exploring the real reason of deposition of abnormal proteins in ALS;application of autophagy activation agent may not increase the degradation ofabnormal protein in cytosol, and have not benifical effect on the treatmen toALS.