The Changs Of Mitochondrial Dynamics In Lumbar Spinal Cord Of SOD1-G93A Transgenic Mice

Objective: Amyotrophic lateral sclerosis(ALS)is a neurodegenerativedisease that selectively affects upper and lower motor neurons (MNs). Clinicalmanifestation of ALS is muscle weakness, amyotrophy. The funtion of sensoryand sphincter is not affected. Its pathogenesis is still unclear up to now. Inrecent years, the majority of views on the pathogenesis of ALS have beenbased on the studies of mutant SOD1. Studies have found that ALS haverelated with oxidative stress, mitochondrial injury, glutamate excitotoxicity,inflammation, apoptosis. With the development of research, people found thatmitochondrial dysfunction plays an important role in ALS. It became thepathophysiologic changes of intersection in ALS.Mitochondria are highly dynamic organ. Fusion and fission are the twoimportant mechanisms for maintaining their shape, distribution ofmitochondria. In mammalian cells, fusion process including outer and innermembranes fusion, by Mfn1, Mfn2and Opa1protein regulation. Mitochondriaare connected to each other through the fusion process. Mitochondrial fusioncan make the exchange of material between neighboring mitochondria andinduce mitochondrial movement. Mitochondrial fission mechanism includeDrp1and Fis1. The loss of Drp1can inhibit mitochondrial division and lead tohighly tubular of inter-connected mitochondria. Mitochondrial fissionparticipates in mitochondrial update and distribution. Mitochondrial fusionand fission maintain the normal morphology and function of mitochondria. Itpromotes the mixed of mitochondrial DNA and the contents and determinesthe mitochondrial shape, quantity, and function. The disorders of fusion andfission process will lead to mitochondrial extension, fragmentation, todecrease mitochondrial biological activity and reduce cell energy supply.Eventually, the unbalance between fusion and fission will lead to cell damage. Studies have shown that the mutation of Mfn2cause Charcot-Marie-Toothtype2(CMT2A), the mutation of Opa1lead to optic nerve atrophy,autosomal dominant optic atrophy (ADOA). Fusion and fission protein havebeen studied deeply in Parkinson disease, Alzheimer's disease, Huntington'sdisease and other related neurodegenerative diseases. According to the report,In ALS patients and the various animal models of ALS have been found theabnormalities of mitochondrial morphology and function. It have shown thatthe changes of mitochondrial cristae, vacuolization and the decline ofrespiratory chain activity. In the pathogenesis of ALS disease, whether themitochondrial homeostasis is effected by mitochondrial dynamics, leading toabnormal accumulation of mitochondria, ultimately result in death of neuronalcell, it is not clear. In order to seek the possibilities for reasons ofabnormalities of mitochondrial morphology and function, In transgenichSOD1-G93A mice, we used a variety of methods to determine the expressionof mitochondrial dynamic proteins. Our aim is to observe the changes ofmitochondrial dynamics in lumbar spinal cord of ALS mouse and discusse theroles of mitochondrial dynamics in the occurrence and development of ALS.Methods: At present, the SOD1-G93A transgenic mice are one of themost ideal ALS models. Female SOD1G93A transgenic mice were used as theexperimental animals. The90days' female littermate controls served ascontrol group. There were four groups: control group,60-day-old group, onsetstage group and ending stage group. Each group included nine mice. After10%hydration aldehydes (350mg/kg body weight) intraperitoneal injectionanesthesia, decapitated, extracted the lumbar spinal cord of mice immediately,and they were frozen in liquid nitrogen and stored at-80, isolated spinalmitochondria and cytoplasm without mitochondria using Percoll densitygradient centrifugation, fixated tissues via heart perfusion by4%paraformaldehyde, dissected lumbar spinal cord of mice and fixated them in4%paraformaldehyde or2.5%glutaraldehyde. Using Electron microscopy,Western blot, Confocal microscopy and immunocytochemistry to detect themorphology, the protein expression of Opa1, Fis1and Mfn, p-Drp1in mitochondria and cytoplasm without mitochondria.Results:1. The changes of mitochondrial morphology and structure in the lumbarspinal cord of SOD1-G93A transgenic mouse. we observed that at60d of ALSmice, morphologic abnormalities of mitochondria were obvious, such asvacuolization, swelling and disappeared cristae. At onset and ending stage ofdisease, mitochondrial morphologic has undergone tremendous changes.2. The protein levels of Mfn1, OPA1, p-Drp1and Fis1in the lumbarspinal cord of SOD1-G93A transgenic mouse.(1) The protein levels of Mfn1, OPA1, p-Drp1and Fis1in the lumbarspinal cord of ALS mice：we find the protein levels of Mfn1and OPA1weregradually down-regulated at the onset stage and ending stage compared withWT mice, the difference was statistically significant; the protein levels ofMfn1were down-regulated at60d compared with WT mice, the protein levelsof OPA1were enhanced at60d compared with WT mice, the difference wasnot statistically significant. we find the protein levels of p-Drp1and Fis1weregradually enhanced at the onset stage and ending stage compared with WTmice, the difference was statistically significant; the protein levels of p-Drp1and Fis1were enhanced at60d compared with WT mice, but the differencewas not statistically significant.(2) The protein levels of Mfn1and p-Drp1in isolated mitochondria byWestern blot: we find the protein levels of Mfn1were decreased at the onsetstage and ending stage compared with WT mice, the difference wasstatistically significant; the protein levels of Mfn1were increased at60dcompared with WT mice, the difference was not statistically significant. Theprotein levels of p-Drp1were increased at the onset stage and ending stagecompared with WT mice, the difference was statistically significant; theprotein levels of p-Drp1were increased at60d compared with WT mice, butthe difference was not statistically significant.3. Double labelings of Mfn1, OPA1, p-Drp1, Fis1and SMI-32in WT andALS mice at different stages. Confocal microscopy shows that OPA1and Mfn1in SMI-32labeled MNs revealed lower immunoreactivity of OPA1andMfn1at the onset and ending stage of disease compared with WT mice.Thereis no obvious difference between the60-day-old group and control group.p-Drp1and Fis1in SMI-32labeled MNs revealed higher immunoreactivity ofp-Drp1and Fis1at the onset and ending stage of disease compared with WTmice.There is no obvious difference between the60-day-old group and controlgroup. IHC for OPA1and Mfn1in the lumbar spinal cord of ALS mice atdifferent stages of the disease revealed lower immunoreactivity of OPA1andMfn1in MNs at the onset and ending stage of disease.There is no obviousdifference between the60-day-old group and control group.4. IF for Mfn1, OPA1, p-Drp1, Fis1expression in GFAP labeled glialcells in the lumbar spinal cord of ALS mice at different stages of the diseaseDouble staining of OPA1and glial cell marker GFAP showed that the OPA1immunopositive cell co-localized with GFAP positive cell in the lumbar spinalcord of ALS mice at onset and ending stage of disease.There is no obviousdifference between the60-day-old group and control group. Double stainingof Mfn1, p-Drp1, Fis1and glial cell marker GFAP showed that there is noobvious difference in each group. IHC for OPA1in glial cells at differentstages of the disease revealed lower immunoreactivity of OPA1in MNs at theonset and ending stage of disease.There is no obvious difference between the60-day-old group and control group. IHC for Mfn1, p-Drp1, Fis1in glial cellsshowed that there is no obvious difference in each group.Conclusions: With the progress of ALS, the protein levels of Mfn1,Opa1in lumbar spinal cord induce; the protein levels of p-Drp1and Fis1inlumbar spinal cord increase. This indicates that mitochondrial dynamics inSOD1transgenic mice is impaired, it suggested that dysfunctionalmitochondria is closely related to the mechanism of mitochondrial dynamics.Instead, the protein levels of Opa1in glial cells in the lumbar spinal cord ofALS mice increase. it revealed mitochondrial fusion increase that is closelyrelated to mitochondrial damage.