The Role Of Oxidative Modifications Of Mitochondrial MEF2D In The Pathogenesis Of PD

With the growing of population, Parkinson’s disease(PD) becomes a can not to be ignored problem to everyone. PD, as a neurodegenerative disorder which mainly involves movement function of the patient. But what PD’s pathogenesis remains unknown. The pathology demonstrates that the region of DA neurons degeneration and mutant, which can reduce the dopamine release. Previous literature demonstrates that 5-10% of PDs have a close relative of genetic. However, some PDs are accidental, which means that their precise causes are remain unclear. Epidemiological studies have identified the link between the exposure to certain environmental factors including toxicants and the incidence of PD, which show that neurotoxicants may paly an indespensable role in the pathology progress of PD. One widely accepted model is that genetic and environmental factors converge on cellular targets to interrupt the homeostasis and cause oxidative stress. One such key target is mitochondria. In support of this, accumulating experimental evidence indicates that several genes mutated in familial PD and toxins may exert pathogenic effects via modulating mitochondrial function. However there is less evidence to prove that mitochondrial function disorder, oxidative stress, protein aggregation and the key molecules and signaling pathways may induce neuronal death, which brings us difficulties in PD’s diagnosis and treatment, especially in beginnig of PD. Therefore, finding a reliable and sensitive biomarker of PD becomes a key problem that need to be solved immediately.MEF2 as a nuclear factor has some important function in the muscle cell differentiation progress. Although the cellular function for MEF2 s was first studied in muscle cells, the roles of MEF2 s have now been described in increasing numbers of cellular systems. Therefore, the most important task is to understand the role of MEF2 s in neurons. Different cell stress can regulate MEF2 D function through the corresponding signaling pathway inoder to change the rest of neurons. MEF2 D in DA neurons has effection on the activity of respiratory chain complex I. MEF2 D has ability to regulate ND6 gene transcription in mitochondria. ND6 is the unique light chain proteins encoded which has great effection on the chain complex I assembly process. The different expression level of MEF2 D can affect function of chain complex I, which will result intracellular peroxide levels increasing and neuronal death. Upregulated the expression of mitochondriatargeting MEF2 D will protect DA neurons from death caused by toxins.This research uses the MPTP-PD mice model. On the period of model construction, we explore the level of expression and oxidative modification of mitochondrial MEF2 D in DA neurons. We study the relationship between the level change of MEF2 D and ND6 and mitochondrial function and the death of DA neurons. Make a preliminary analysis on the feasibility of mitochondrial MEF2 D as biomarker of PD.Experiment IMethod(1) Divided animals into 4 different groups: Con(control) group, D1(the 1st day of injection) group, D3(the 3rd day of injection) group, D5(the 5th day of injection) group. On the experimental groups, MPTP were injected at 9 a.m. each day in a dose of 30 mg/kg in 5 days. Control group injected 0.9% Na Cl in the same dose and at the same time.(2) Sacrifice D1 group at 3 p.m. in the 1st day of injection, D3 group in the 3rd day, D5 and Con groups in the 5th day.(3) Immunofluorescence staining. Midbrain sections of each group were used for immunofluorescence staining of TH to detect the level change of DA neurons.(4) Isolate nuclei, cytoplasm and mitochondria. Using immunobloting to test the efficiency of the isolation.(5) Immunobloting was used to track the level of MEF2D(6) Isolate cerebellum, cortex, striatum and SN. Using immunobloting to detect the expression of MEF2 D in each region.Result(1) Immunofluorescence staining showed TH positive neurons significantly decreased,which prove that the model established successfully.(2) Western Blot results showed the separation were successful, achieved experimental requirement.(3) The expression of nuclei MEF2 D was not changed in midbrain DA neurons. The levels of mitochondrial MEF2 D were reduced at the first day of injection(P<0.05) and significantly reduced at the third and fifth day(P<0.01).(4) There were no change of mitochondrial MEF2 D in cerebellum, cortex and striatum.Conclusion(1) The expression of mitochondrial MEF2 D in midbrain DA neurons was changed fast than the histological in the period of modeling.(2) The level change of mitochondrial MEF2 D was not nonspecific change during the modeling.Experiment IIMethod(1) Divided animals into 4 different groups: Con group, D1 group, D3 group and D5 group.(2) Western Blot detecting the levels of Nurr1 and TFAM in DA neurons during the modeling.Result(1) The level of Nurr1 in midbrain DA neurons showed no change during the modeling of MPTP-PD mice model. The level of TFAM was significantly reduced at the fifth day of injection(P<0.05), but there is no change happens in the 1st and the 3rd day.Conclusion(1) Compared with Nurr1 and TFAM, mitochondrial MEF2 D was more sensitive to the death of DA neurons during the modeling of MPTP-PD mice model.Experiment IIIMethod(1) Prepare the MPTP-PD mice model. Divided Animals into 4 groups: Con group, D1 group, D3 group and D5 group.(2) Using Oxy Blot to test oxidative modification expression of mitochondrial MEF2 D.(3) Using q RT-PCR to identify the expression of ND6 m RNA.(4) Using 450 nm to test the oxidation rate of NADH which can demonstrate the change of complex I activity.Result(1) The level of oxidative modification of mitochondrial MEF2 D in midbrain DA neurons was significantly increased from the third day of injection during the modeling of MPTP-PD mice model.(2) The level of ND6 m RNA in DA neurons was significantly reduced from the third day of injection(P<0.05).(3) The activity of Complex I was significantly decreased at the 3rd and the 5th day of injection(P<0.01).Conclusion(1) During the modeling, the expression of oxidative modifications of mitochondrial MEF2 D in DA neurons was significantly increased from the 3rd day of injection, associated with the significantly reduce of the level of ND6 and complex I activity.