Mitophagy In SOD1Transgenic Mice

Objective: Amyotrophic lateral sclerosis (ALS) is a progressiveneurodegenerative disease, affecting upper and lower motor neurons (MNs),characterized by progressive limb weakness and muscle atrophy, whilesensory systems are generally spared. Lack of effective treatment and most ofthe patients die of respiratory failure3-5years later. ALS can be divided intofamilial amyotrophic lateral sclerosis (fALS) accounting for about5%-10%and sporadic amyotrophic lateral sclerosis (sALS) accounting for about90%-95%according to its speciality of episode and heredity. Approximately20%of fALS patients are caused by mutations of copper-zinc superoxidedismutase (SOD1) gene. Until now, the aetiology and pathogenesis of ALSremain largely unknown. Oxidative stress, glutamate excitotoxicity,mitochondrial dysfunction, abnormal protein aggregation, axonal transportbarriers, and apoptosis have been proved to participate in its pathogenesis.Recent years the dysfunction of mitochondria in ALS attracts more and moreattentions. Mitochondria are one of the most important organelles, providingenergy for cell survival, participating in information transmission, mediatingapoptosis, regulating calcium concentration and so on. Normal mitochondrialfunction is crucial for motor neurons.In physiological conditions, the organism can remove aberrant proteinsand damaged organelles by autophagy pathway, such as mitochondria,endoplasmic reticulum, peroxisomes, to update cell, maintain cellularhomeostasis and keep its physiological state. Impaired autophagy can lead tomany neurodegenerative diseases, and too much or too little autophagy isharmful. Mitochondria can be selectively cleared by autophagy, termedmitophagy. Mitophagy plays an important role to maintain mitochondrialnormal morphology, number and function. LC3II (Microtubule-associatedprotein1light chain3-II, LC3II) is specifically bound to autophagosome membranes and serves as an autophagic marker protein. P62servers as abridge between LC3II and the ubiquitin-conjugated cargo, which is degradedin the process of autophagy, so people often take p62as a marker ofautophagic flux. Research reported that skeletal muscle in Atg7(autophagyrelated gene7, Atg7) deficient mice showed mitochondria swollen, cristaebroken, respiratory chain activity declined and so on. In ALS, people have alsofound mitochondria cristae fragmentation, vacuolization and the respiratorychain activity decreased. Whether mitophagy in ALS is impaired? Are thereany relation between mitophagy and the accumulation of mutant SOD1? Itsrelevant reports are still poor, and need further study.At present, the SOD1-G93A transgenic mice are one of the most idealALS models. Here we focus on mitophagy in SOD1-G93A transgenic mice, tostudy its role in the pathogenesis of ALS.Methods: Female SOD1-G93A transgenic mice were used as theexperimental animals. The90days' female wild type controls served ascontrol group. There were four groups: control group,60-day-old group, onsetstage group and ending stage group. Each group included twelve 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 spinalcord mitochondria 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 and confocal microscopy to detect the morphology; the proteinexpression of LC3II and p62in mitochondria and cytoplasm withoutmitochondria.Results:1. The protein levels of LC3II in the lumbar spinal cord of ALS mice:using western blot, we find the protein levels of LC3II increase at the onsetstage and ending stage compared with WT mice. There is no obvious difference between the60-day-old group and control group. Confocalmicroscopy shows that LC3II immunofluorescence turns into a population ofpuncta in SMI32labeled MNs at the onset and ending stages of ALS, whileLC3II immunofluorescence lightly distributed homogeneously in thecytoplasm of MNs in WT mice and60d ALS mice. The morphologicalchanges in the lumbar spinal cord MNs of SOD1-G93A transgenic mice: usingelectron microscopy, we find there are a lot of autophagosomes in the MNs atthe onset and ending stage of ALS, while no accumulation of autophagosomesat the control group and60-day-old group.2. The protein levels of LC3II in isolated mitochondria and cytoplasmwithout mitochondria by western blot: the protein levels of LC3II inmitochondria increase at the onset and ending stages of ALS mice, while itdecreased in cytoplasm without mitochondria. By double stainings of LC3IIand mitochondria marker VDAC, we find VDAC immunopositive clustersco-localized with LC3II-positive puncta in MNs of ALS at the onset andending stages of ALS.3. The protein levels of p62in isolated mitochondria by western blot: theprotein levels of p62combined with mitochondria increase at the onset andending stages of ALS mice; there is no significant difference of the proteinlevels of p62combined with mitochondria between WT mice and60d groupof ALS mice.Conclusions: By morphological observation, protein quantitation andco-localization observation, we find: with the progress of ALS, the proteinlevels of LC3II in lumbar spinal cord increase; the protein levels of LC3II andp62in mitochondria component increase; the protein levels of LC3II incytoplasm without mitochondria decrease. This indicates that mitophagy inSOD1transgenic mice is impaired: excessive mitochondria stay at the earlystage of mitophagy, which may be the initiating or deteriorative factor of ALS.