Effects Of Resveratrol On LC3B In Lumbar Spinal Cord And Motor Cortex Of SOD1-G93A Mice

Objective: Amyotrophic lateral sclerosis (ALS) is a progressive fatalneurodegenerative disease, having the feature of affecting upper and lowermotor neurons. Its clinical presentation is muscle weakness, muscle atrophy,breathing myoparalysis, and average survival period is3to5years. A varietyof mechanisms involve in ALS, including misfolded protein aggregation,oxidative stress, calcium overload, glutamate excitotoxicity, the lack ofnutrition factor, mitochondrial dysfunction and so on. These pathologicalmechanisms are still unable to explain the selective motor neuron injury. Inour previous tests, we found in ALS, there is obstacle about the fission andfusion of mitochondrion, also exists obstacle in mitochondrial autophagy.Autophagy is a protective mechanism to remove harmful material ineukaryotic cells. It can sequestrate and degrade the excess injury or abnormalcytoplasmic components that provides a stable environment for cell normalsurvival and update. Meanwhile, it protects the high-efficiency of cellmetabolism and thus realizes the the body’s balance and stability. Autophagyincludes three major types: macroautophagy, microautophagy, andchaperone-mediated autophagy (CMA). Macroautophagy, presently, is a hotpoint and also is a direction in our research. Microtubule-associated protein1light chain3(LC3) is a homolog of yeast autophagy related gene Atg8inmammalian, LC3has three variants, named LC3A,LC3B,LC3C. LC3has twoforms--LC3I and LC3II. LC3I presents in the cytoplasm, LC3II presents onthe cell membrane. LC3II is the composition of autophagosome and serves asan autophagic marker protein. When macroautophagy enhancing, more LC3Iconverts to LC3II. In many neurodegenerative diseases, abnormal proteinaggregates in cytoplasm, the nucleus or extracellular matrix, which has thetoxic effects on cells, the induction of apoptosis, and leads to the occurrence of the disease. Macroautophagy, while, can remove these harmful substances andprotect nerve cells. Therefore, Macroautophagy becomes a target for thetreatment of neurodegenerative diseases.Resveratrol is a naturally derived polyphenols. There is a report that in theneurodegenerative disease, such as Alzheimer’s disease and Parkinson’sdisease, resveratrol can activate silent mating type information regulation2homolog1(Sirt1) or AMP-activated protein kinase (AMPK) to inducemacroautophagy, affect LC3and regulate the macroautophagy. Resveratrol hasthe function of protecting neurone. Then in ALS whether the Resveratrol alsoaffects LC3levels and has the same function? In this test, we try to observethe changes of LC3B in different period of SOD1-G93A mice lumbar spinalcord and motor cortex. We also want to observe the influence of resveratrol tothe LC3B, further understand the pathogenesis of ALS disease and provide adirection for the diseases treatment.Methods: SOD1-G93A mice were used as the experimental animals,and the90-day-old wild type mice served as the control group. There werefour groups: control group, presymptom group (60-day-old group), onset stagegroup, and ending stage group. Every group has6mice. Starting from the70days, we gave SOD1-G93A mice three different treatments. Resveratrolgroup mice were infused resveratrol solvent (30mg/kg/d), vehicle group micewere infused equal volume of alcohol solvent, and blank group mice were nottreated until onset stage or end-stage. Every group has6mice. After theintraperitoneal injection of10%hydration aldehydes (350mg/kg body weight),we decapitated the mice and extracted the lumbar spinal cord and motor corteximmediately, then frozen them in liquid nitrogen and stored in freezer at-80℃.What is more, the mice hearts were perfused4%paraformaldehyde, andthe lumbar spinal cord and motor cortex also were dissected and stored by4%paraformaldehyde. According to the technology of electron microscopy,western blot and confocal microscopy, we can detect the different changes ofLC3B in different conditions, thus analyzing the data by the softwarespss13.0. Results:1The different LC3B contents of the lumbar spinal cord and motor cortex indifferent stages: by western blot, we found that compared with the WT mice,the LC3BII contents in the lumbar spinal cord and motor cortex ofSOD1-G93A mice increased at the onset stage and ending stage and there wasno obvious difference between the presymptom group and control group. Onthe other side, immunohistochemistry showed that at the onset stage andending stage, motor neurons of ALS mice became less and LC3B increased inthe lumbar spinal cord and motor cortex. There is no obvious differencebetween the presymptom group and control group. Confocal microscopy,moreover, showed the colocalization condition of LC3B and SMI32.Compared with WT mice, the LC3B contents of ALS mice in motor neuronincreased at the onset stage and ending stage.There is no obvious differencebetween the presymptom group and control group.2Effects of resveratrol on LC3B content in the lumbar spinal cord and motorcortex about SOD1-G93A mice:2.1Onset stage: by western blot, compared with blank group ALS mice, theLC3BII protein levels of ALS mice in the lumbar spinal cord and motor cortexincreased in resveratrol group and vehicle group. Immunohistochemistryshowed the protein levels of LC3BII in the lumbar spinal cord and motorcortex also increased in resveratrol group and vehicle group. Confocalmicroscopy showed the colocalization of LC3B and SMI32and found theprotein levels of LC3B in the lumbar spinal cord still increased in resveratrolgroup and vehicle group, compared with blank group ALS mice.2.2Ending stage: by western blot and immunohistochemistry, for ALS micewe found there was no obvious difference between resveratrol group, vehiclegroup and blank group in the lumbar spinal cord and motor cortex. Confocalmicroscopy showed the colocalization of LC3B and SMI32and also had noobvious difference between resveratrol group, vehicle group and blank groupin the lumbar spinal cord. Conclusion:1With the progress of the ALS, the level of LC3B gradually increases inthe lumbar spinal cord and motor cortex, which indicates the tendencyenhancing of macroautophagy.2The impact of resveratrol on the expression of LC3B is limited in thelumbar spinal cord and motor cortex about ALS mice. Whether resveratrol canprotect neurons by influencing the macroautophagy deserves further study.