Autophagic Pathway And Probable Mechanism In The Degradation Of α-synuclein

Part I Autophagic pathway and probable mechanism in the degradation of mutantα-synuclein in PC12 cellsOBJECTIVE: The accumulation of mutantα-synuclein plays a major role and morphologic evidence of autophagy has been reported in Parkinson disease. We aim to observe the effect of mutantα-synuclein (A30P) in autophagic programmed cell death by transfected PC12 cells and explored its probable role and pathway in PD.METHODS: We constructed definite PC12 cells which were transfected mutantα-synuclein (A30P) at first and administrate MPP+, Rapamycin and Wortmanin to transfected PC12 cells with mutantα-synuclein. We not only detected proliferative activity of cells with MTT method but also observed the ultrastructure changes of cells and expression ofα-synuclein in different circumstance by Transmission electron microscopy (TEM), Western Blotting and and the level of SOD.RESULTS: Mutantα-synuclein (A30P) leading to PC12 cells death involvesα-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutantα-synuclein (A30P) mediates the toxicity of MPP+. Rapamycin, an inducer of autophagy, reduces the aggregation ofα-synuclein in transfected cells. Meanwhile, Wortmanin, an inhibitor of autophagy, promotes the aggregation ofα-synuclein in transfected cells and induces cells to die.CONCLUSION: The abnormal aggregation ofα-synuclein induces autophagic programmed cell death in PC12 cells and mutantα-synuclein (A30P) mediates the toxicity of MPP~+. Meanwhile, Rapamycin may reduce the aggregation ofα-synuclein in transfected cells by activation of autophagic pathway. Part II Mutant -synuclein (A30P)are degraded by autophagic pathway and mechanism under oxidative stressObjective The accumulation of mutantα-synuclein plays a major role and morphologic evidence of autophagy has been reported in Parkinson disease. We aim to observe the effect of MPP~+ to mutantα-synuclein (A30P) in autophagic programmed cell death by transfected PC12 cells and explored its probable role and degradative pathway in PD. Method We constructed definite PC12 cells which were transfected mutantα-synuclein (A30P) at first and administrate MPP~+ to establish the the model of PD with cells under oxidative stress. Rapamycin and Wortmanin were administered to transfected PC12 cells with mutantα-synuclein respectively . We not only detected proliferative activity of cells with MTT method but also observed the ultrastructure changes of cells by Transmission electron microscopy (TEM). Meanwhile expression ofα-synuclein and LC3 in different circumstance was examined by Western Blotting. Moreover, the levels of CAT and SOD in cell cultures were also compared in order to discuss the relation between oxidative stress and autophagy. Results MPP~+ to mutantα-synuclein (A30P) leads to PC12 cells death by means of autophagy involvesα-synuclein accumulation, membrane lipid oxidation, and loss of plasma membrane integrity. Mutantα-synuclein (A30P) mediates the toxicity and enhances the sensitivity of MPP~+ . Rapamycin, an inducer of autophagy, reduces the aggregation ofα-synuclein in transfected cells. Meanwhile, Wortmanin, an inhibitor of autophagy, promotes the aggregation ofα-synuclein in transfected cells and induces cells to die. Conclusion Mutantα-synuclein (A30P) mediates the toxicity of MPP~+. Meanwhile, inducer of autophagy-Rapamycin may reduce the aggregation ofα-synuclein in transfected cells by activation of autophagic pathway. Part III Therapeutic effect and probable mechanisim of Rapamycin on MPTP-induced parkinsonism in miceOBJECTIVE: To investigate the effects of inducer of autophagy-Rapamycin on the functional and morphological outcome in a mice model of Parkinson s disease (PD) rendered by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP).BACKGROUND: In neurodegenerative disorders such as PD, autophagy participates in the cell death process of dopaminergic neuron.α-Synuclein is one of the major components of Lewy bodies and Lewy neuritis and mutation ofα-synuclein such as a-synuclein mutants (A53T and A30P) may be involved in the development of familial PD. Our previous study demonstrated that Rapamycin may induce autophagy and decrease aggregation ofα-synuclein and cell death.METHODS: Male C57BL mice were treated with Rapamycin(3mg/kg) by i.v for 7 days after MPTP administration(30mg/kg.14d), and were compared with saline-treated PD mice and normal control group. Immunohistochemistry and Western blot were used to detect the alterations of PD biomarker including tyrosine hydroxylase (TH), and the level of autophagy was investigated by microtubule-associated protein light chain 3(LC3) andα-synuclein cleavage. In addition, monoamine neurotransmitters in the striatum of mice were measured by the high performance liquid chromatography (HPLC).RESULTS: TH immunohistochemistry indicated that the number of dopaminergic neurons in the substantia nigra was increased in Rapamycin-treated mice compared with saline-treated mice (p=0.003). Western blot demonstrated the similar TH protein expression in striatum as the number of dopaminergic neurons.α-Synuclein was markedly decreased in Rapamycin-treated mice compared with the NS-treated mice(p=0.004) and LC3 was markedly increased in Rapamycin-treated mice compared with the NS-treated mice(p=0.003). Meanwhile, DAT and VMAT-2 were markedly decreased in Rapamycin-treated and NS-treated mice compared with control group. Although the degree was different, DAT and VMAT-2 were markedly increased in Rapamycin-treated compared with NS-treated mice. The concentrations of striatal dopamine and its metabolite DOPAC compared to the normal mice were decreased significantly in other groups. Rapamycin-treated mice minimized the reduction of DOPAC compared with the NS-treated mice (p=0.001).CONCLUSIONS: These results showed that Rapamycin is able to rescue the dopaminergic neurons in different doses and reverses the loss of DOPAC through activation of autophagy-lysosome pathway, and it may be a promising therapeutic agent in PD.