Regulation Of Survival Factor MEF2D MRNA By Neurotoxin In Models Of Parkinson’s Disease

Background and objective:Parkinson’s disease (PD) is a neurodegenerative disorder severely affecting movementfunction. The incidence of this disease among old people above65years old is1%, whichaffects millions of people around the world. The mainly clinical symptoms of PD are statictremor, increased muscle tone and bradykinesia, accompanied by the symptoms ofautonomic nervous system and the change of emotion. Serious PD patient even can’t liveby himself. Thus, this disease adds a heavy economic and mental burden to the family andthe society. Current treatments mainly aim at improving symptoms, but all fail toencumber the pace of the progressive development of the disease. To find out the cause ofPD and to explore the pathogenesis of it are of great significance for the family and thesociety. Though the precise cause of PD is still not very clear, the generally acceptedtheory is the combination of genetic factors and environmental factors leading to the deathof dopaminergic neurons in the substantia nigra area of the midbrain. A growing numberof studies confirm the important role of neurotoxin in the pathogenesis of PD, for example,MPTP,6-OHDA, rotenone and paraquat, etc. As to the molecular mechanism underliePD, a large number of studies have been conducted. Studies have confirmed that autophagy is closely related to PD. The neurotoxic agents associated with PD caninfluence the process of autophagy. For example, rotenone, a mitochondrial complex Ⅰinhibitor, can inhibit autophagy. Also,6-OHDA can obviously increase the LC-3proteinlevel in the substantia nigra neurons, which will promote the autophagy. Other studiesshow that paraquat can lead to the accumulation of autophagy vesicles in the neurons.Myocyte enhancer factor2D (MEF2D) is a transcription factor. And MEF2D is one ofthe substrates of autophagy. Studies have showed that MEF2D is closely related to theonset of PD and the survival of dopaminergic neurons. Previous studies have found thatneurotoxins can promote the phosphorylation of MEF2D, which will in turn to accelerateits degradation. However, previous studies mainly focused on the post-translationalmodification of MEF2D when exposed to neurotoxins. Whether there is another pathwayby which neurotoxin modulate MEF2D is still unknown. Whether neurotoxin can affectthe level of MEF2D mRNA? Therefore, we designed the experiments to answer thesequestions.Methods:First, we exposed SN4741cells to MPP+, and then detected the change in the level ofMEF2D mRNA by real-time quantitative PCR. Second, in MPTP mouse PD model, weused laser capture micro dissection to obtain TH positive neurons selectively, and thenexamined the change in the level of MEF2D mRNA by real-time quantitative PCR.Further, by actinomycin D experiments, we explored how neurotoxins impact MEF2DmRNA level. Then, through lentivirus infection, we knocked down the MEF2D mRNAlevel in neurons. And then we estimated the effects of down-regulated MEF2D mRNAlevel on the survival of neurons by flow cytometry and western blot for cleaved caspase3.Results:Part one: the effects of neurotoxins on MEF2D mRNAlevel(1) SN4741cells were exposed to MPP+for12hours,24hours and36hoursrespectively, and then we collected the cells for detection respectively.Immunofluorescence staining and western blot showed that, compared with the controlgroup, MEF2D protein levels in MPP+treated group decreased obviously. Real-time quantitative PCR showed that, compared with the control group, MEF2D mRNA level inMPP+treated group also decreased significantly.(2) In MPTP mouse model, immunofluorescence staining showed that, compared withthe control group, the number of TH positive neurons in MPTP treated group decreasedobviously, which suggests that the model we use is a successful model. Then we use lasercapture micro dissection to obtain TH positive neurons selectively for real timequantitative PCR. The results showed that compared with the control group, MEF2DmRNA in the MPTP group dropped significantly (Р <0.01).Part two: the mechanism through which neurotoxins impact MEF2D mRNA leveland the effects of the decreased MEF2D mRNA level on neuronal survival(1) Actinomycin D is one of the most frequently used transcription inhibitors and it canprovide an efficient and rapid way to block gene transcription. We treated SN4741cellswith transcription inhibitor actinomycin D with or without co-treatment with MPP+andthen measured the levels of MEF2D mRNA over time by real-time quantitative PCR.Actinomycin D treatment alone indicated that MEF2D mRNA decays with a half-life ofabout1.5h. Addition of actinmoycin D and MPP+simultaneously to SN4741cellsresulted in significantly accelerated decrease in MEF2D mRNA level. The estimatedhalf-life of MEF2D mRNA was shortened to0.6h, suggesting that MPP+destabilizesMEF2D mRNAin SN4741cells.(2) Our above results showed that neurotoxin MPTP/MPP+reduced the stability ofMEF2D mRNA level in PD models. To test whether down-regulation of MEF2D mRNAaffects neuronal viability, we infected SN4741cells with lentivurs expressing shRNA toMEF2D for4days. Infection efficiency for SN4741cells was approximately80%, andshMEF2D significantly reduced the level of MEF2D mRNA. Analysis of cell viability byflow cytometry showed that decrease in MEF2D mRNA level caused a marked increase incell death (14%) compared to the control groups (0.5%). These data suggestdown-regulated MEF2D mRNAlevel is sufficient to reduce neuronal viability.(3) To test the effects of down-regulation of MEF2D mRNA level on the cell sensitivityto neurotoxin, we preinfected SN4741cells with lentivirus shRNA-MEF2D for4days and then treated cells with MPP+. Cell viability was determined by western blot for cleavedcaspase3. Analysis of the level of cleaved caspase3indicated that interfering MEF2DmRNA alone was sufficient to activate caspases3. Pre-inhibition of MEF2D mRNAfunction also markedly increased the level of activated caspases3following MPP+treatment. These findings showed that down-regulated MEF2D mRNA sensitized SN4741cells to MPP+-induced toxicity.Conclusion:The main innovations of this experiment lie in that we found a new way by whichneurotoxin regulates MEF2D. In addition, we used laser capture micro dissection toensure a much more precise and reliable quantification of the changes of MEF2D mRNAlevels in TH positive neurons in the SNc.(1) The level of MEF2D mRNA was significantly reduced by neurotoxins both in vitroand in vivo.(2) Neurotoxin MPP+destabilized MEF2D mRNAin dopaminergic neurons.(3) Down-regulation of MEF2D mRNAaffects the survival of dopaminergic neurons.(4) Down-regulation of MEF2D mRNAenhances MPP+-induced toxicity.