Dissertation
Dissertation > Medicine, health > Neurology and psychiatry > Neurology > Neuromuscular disease > Muscular atrophy

The Protective Effect of EGCG on Cellular Model of TDP-43Related to Amyotrophic Lateral Sclerosis

Author LiWenJu
Tutor WangHuiJuan
School Hebei Medical University
Course Neurology
Keywords ALS Motor neuron disease Autophagy EGCG TDP-43
CLC R746.4
Type Master's thesis
Year 2014
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Objective: Amyotrophic lateral sclerosis (ALS) is aprogressive neurodegenerative disease, which selectively assault the brain andspinal cord, upper and lower motor neurons and are mainly for generalprogressive muscle weakness and muscle atrophy. Although in recentyears there are many studies and have been deeply about the ALS, but theexact etiology and pathogenesis is still unknown.Amyotrophic lateralsclerosis can cause deaths and long-term disabilities. Amyotrophic lateralsclerosis caused serious economic burden to the family and society, so amedical worker has undeniable responsibility to curb the occurrence andresearch the pathogenesis.Recently, the abnormal ubiquitin protein aggregates are found in thenucleus and cytoplasm of motor neurons and glial cells in ALS patients. Theirmain component is TAR DNA binding protein (TDP-43) of43kDa.TAR DNA binding protein (TDP-43) encoded by TARDBP gene, is akind of protein, with414amino acids and the molecular weight of43000. Itcontains two RNA recognition regions and a glycine end, so that it can becombined with single-stranded DNA. Its function is transcription andsplicing regulation. Other studies have found that there is mRNA reduced,splicing errors and some noncoding RNA in mice of TDP-43deletion. Thedistribution of physiological TDP-43protein are mainly in the nucleus, a fewappeared in the cytoplasm around nuclear. The expression of TDP-43protein is regulated by many signals such as nuclear localization and transfersignals, and is easy to be affected by the various physiological statuses. Inthe the condition of disease and stress, TDP-43protein can be expressed as aseries of pathological changes, such as nuclear inclusions, cytoplasmicaggregates.In the modified process,such as, mutations in the TARDBP gene, phosphorylation,ubiquitination and nterminal truncated can lead to theappearance of pathological TDP-43protein. Pathological TDP-43proteinstatus is not stable and more prone to aggregation, comparing with thephysiological structure.TDP-43which plays an important role in thepathological process of ALS, but the specific mechanism is still not clear.As everyone knows, epigallocatechin gallate (EGCG) is the highest,accounting for about25%-40%of the Green Tea polyphenols, and isconsidered to be the main source of Green Tea polyphenols biological activity.The Molecular mechanism of pharmacological activity of Green Tea poly-phenols is relating to metal chelating agent, free radical scavenger, affectingcell survival and the over expression of apoptosis gene and cell signaltransduction pathway, mitochondrial function and the regulation of ubiquitinproteasome system. Through these mechanisms, GTP had protective effects onneurodegenerative diseases like Parkinson’s disease (PD) and Alzheimer’sdisease (AD). Autophagy is a degradation mechanism of a long-lived proteinsand organelles recirculation and update. Many neurodegenerativediseases (Alzheimer’s disease, Parkinson’sdisease, poly glutamine disease,ALS and so on) are characterized by misfolded protein aggregation. In thecentral nervous system, autophagy has become an important way toremove misfolded proteins, and is necessary to maintain the normalfunction of neurons. In contrast, down-regulation or partial inhibition ofautophagy may stimulate the level of nerve degeneration.Therefore, we study the effect of EGCG on autophagy in amyotrophiclateral sclerosis model of TDP-43transfected NSC-34cells to investigatewhether EGCG has a protective effect on the TDP-43related cell modelof ALS.Methods:The NSC-34cells which were transfected by TDP-43are usedin this study as amyotrophic lateral sclerosis cell model. After treatment withdifferent concentration and acting time of EGCG in NSC34cells, its effect onthe cell viability was measured by the CCK-8assay. After NSC34cells wereexposed to appropriate concentrations and times of EGCG, we examined LC3-II protein levels by Western blot. Cells expressingpEGFP-LC3were treated with EGCG for different concentrations, andaccumulations of pEGFP-LC3were analyzed by fluorescence microscopy. Wealso determined which means EGCG caused the level of LC3-II protein usingthe autophagy inhibitor Baf A1. After the NSC-34cells which was transfectedby TDP-43, amyotrophic lateral sclerosis cell model, was exposed to theappropriate concentration of EGCG, we examined exogenous TDP-43proteinlevels by Western blot.Results:(1)There was no marked difference in the cell viability y from0to20μM, but there was significantly decreased in cells toxicity from20to80μM in NSC34cells after the NSC-34cells was exposed to differentconcentrations from0to100μM of EGCG. There was no marked decrease inthe cell viability from0to36hours after NSC-34cells were exposed to theconcentration of20μM.(2)The studies that the NSC-34cells were exposed to differentconcentrations from0to20μM of EGCG suggest the level ofLC3-II increased gradually by Western blot. We obtained the result that thelevel of LC3-II was highest in24h after NSC-34cells were exposed todifferent times from0to36hours of EGCG in the concentration of20μM byWestern blot. We also found that numbers of punctate pEGFP-LC3dots in24hof EGCG generally increase according to the dose-dependent manner by theImmunofluorescence technique.(3)The studies suggested that the level of LC3-II increased significantlyIn the presence of autophagy inhibitor Baf A1and20μM of EGCG whichwere exposed to24h by Western blot. We concluded that EGCG promotedthe protein degradation by autophagy pathway. NSC34cells were transientlytransfected with different human TDP-43plasmids, including WT, Q331K andM337V TDP-43.levels of Exogenous TDP-43are significantly reduced aftertreatment with20μM EGCG for24h by Western blot.Conclusion:We concluded that low doses of EGCG could raise the levelof autophagy t, and promote the degradation of exogenous TDP-43in TDP-43 related cell model of amyotrophic lateral sclerosis, which exerts aneuroprotective effect.

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