The Role And Mechanism Of Glucose Regulated Protein75in The Mitochondrial Injury Induced By A53T Mutant α-synuclein Overexpression

a-synuclein, a main component of Lewy Body, is pivotally implicated in the pathogenesis of Parkinson’s disease (PD). Recently, the dysfunction of mitochondria and abnormal mitochondrial dynamics are reported to be involved in the a-synuclein-induced cell injury due to the abnormal aggregation,Glucose regulated protein75(GRP75) is a member of Heat Shock Protein70(HSP70) mainly locating in mitochondria. It was found to be associated with both a-synuclein and DJ-1, but the exact mechanism is still elusive. Here, we overexpressed the A53T mutant a-synuclein in SH-SY5Y cells and modulated the GRP75expression, to explore the role of GRP75in alleviating mitochondrial injury induced by A53T mutant a-synuclein and its underlying mechanism.PART I Effect of A53T Mutant a-synuclein on SH-SY5Y CellsObjective:To detect the effect of overexpressing A53T mutant a-synuclein in SH-SY5Y cells.Methods:The SH-SY5Y cells stably overexpressing A53T mutant a-synuclein were constructed by transfecting A53T mutant a-synuclein plasmid via Lipofectamine2000. To test its sensitivity to rotenone, an inhibitor of mitochondria complex I, the CCK-8test was used to assess the cell viability, and the LDH release was tested to show the cell injury. The GRP75expression level affected by A53T mutant a-synuclein overexpression was detected by Western Blot. The mitochondrial function involving the Mitochondria Membrane Potential (MMP), the expression ratio of Bax to Bc12, and Cytochrome C release into cytoplasma was tested to quantify mitochondrial dysfunction induced by the overexpression of A53T mutant a-synuclein. The expression of Drp1and Mfn2in mitochondria was analyzed to explain the mechanism involved in the mitochondrial dynamics.Results:A53T mutant a-synuclein could increase the cell sensitivity to rotenone, decreasing the cell viability (P<0.01) and increasing the LDH release (p<0.01). The expression level of GRP75decreased significantly when A53T mutant a-synuclein was overexpressed. The A53T mutant a-synuclein could also decrease MMP, increase Bax/Bcl2expression ratio, but not impact the Cytochrome C release in cytoplasma. The Drpl and Mfn2were not involved in abnormal mitochondrial dynamics induced by the overexpression of A53T mutant a-synuclein.Conclusion:The A53T mutant a-synuclein overexpression could cause the injury of SH-SY5Y cells, in which mitochondrial dysfunction was involved.PART Ⅱ Modulation of GRP75Expression in SH-SY5Y cells.Objective:To modulate the GRP75expression in the dopaminergic cell line.Methods:The ShRNA sequence was designed according to the GRP75sequence, and then transferred to the lentivirus plasmid. The lentivirus plasmid for overexpressing GRP75was also constructed. The lentiviruses were then packaged using the lentivirus system. After the centrifugation, the lentiviruses were collected to infect cells. The immunoflourence and Western Blot were used to assess the GRP75expression.Results:The ShRNA sequence we designed could significantly decrease the GRP75to less than50%of control. The lentiviruses we collected were then used to infect cells, and finally we selected the stable cell clones with GRP75modulation.Conclusion:Cells with upregulation and downregulation of GRP75were successfully constructed for the future research.PART Ⅲ Effect of GRP75on Mitochondrial Injury Induced by Overepression of A53T Mutant a-synucleinObject:To detect the role of GRP75in mitochondrial injury induced by A53T mutant a-synuclein and its underlying mechanism.Method:The GRP75expression was upregulated and downregulated in SH-SY5Y cells overexpressing A53T mutant a-synuclein, and the cells with stable GRP75expression were selected. The cell viability and injury induced by rotenone treatment were assessed to represent the cell sensitivity. The MMP, Bax/Bcl2expression ratio and cytochrome C release were also tested to assess the mitochondrial function. The mechanism of GRP75on mitochondria dynamics was explored by assaying Drpl and Mfn2expression in mitochondria.Results:No significant change of LDH release was detected after the modulation of GRP75, however, the downregulation of GRP75could enhance the cell resistance against rotenone, improve MMP, but not impact Bax/Bcl2expression ratio or cytochrome C release. In the mitochondria, overexpression of GRP75reduced Mfn2level, and downregulation of GRP75reduced Drp75expression.Conclusion:The downregulation of GRP75expression could alleviate the mitochondrial injury induced by overexpression of A53T mutant α-synuclein, a process in which fission and fusion of mitochondrial might be implicated.