A Research Of Neurodegenerative Disease Mouse Which Induced By β-syn V70M And The Role Of Parkin In This Model

Parkinson’s Disease(PD)is a common neurodegenerative disorder.In Parkinson’s Disease(PD)patients the expression of α-syn in the neurons of substantia nigra accomulates significantly,and α-syn aggregates to form Lewy bodies.It has been demonstrated in vitro experiments that β-syn can inhibit the aggregation of α-syn,while β-syn mutations can promote α-syn aggregation and induce neuronal death.Since mutations of the gene which encoding β-syn protein have been found in some PD patients,there is a possibility that mutations in β-syn could be a reason to develop PD.What effects do the over expression of β-syn V70 M have on mice to develop PD,presently,there is no report on the β-syn V70 M mouse model.Parkin is an E3 ubiquitin ligase and plays a protective role in PD.Although there are many reports on parkin-mediated cytoprotection at home and abroad,but the role of parkin in the occurrence of neurodegenerative diseases that induced by β-syn V70 M mouse model is not very clear.In this study,transgenic mouse model was build to investigate the effects of β-syn V70 M mutation in mouse.Parkin virus was used to investigate the role of Parkin in neurodegenerative diseases induced by β-syn V70 M.It may provide new theoretical basis for the treatment and prevention of Parkinson’s disease.Firstly,a transgenic mouse model was designed contain β-syn V70 M and Flag tag causing overexpression of β-syn V70 M in the whole brain neurons.The β-syn V70 M transgenic mice was used to measure the Rot-Rod,tail suspension and other behavioral tests.A significant reduction was observed in these transgenic mice in ability to perform after reaching a certain age,and a particular degree of depression.Electrophysiological detection indicated that the frequency of neurons in the substantia nigra of the transgenic mice was significantly lower than the control group,and the activity of neurons in the substantia nigra of transgenic mice was also impaired as compared to control group.Immunoblotting and immunohistochemistry results revealed that the expression of α-syn and α-syn aggregation were significantly increased in transgenic mice as compared to control group.It was also found that neurons in the substantia nigra of the transgenic mice were significantly reduced.Moreover,the autophagy-associated proteins such as ATG4 D,apoptosis-associated proteins like p53 and BAX,and mitophagy-associated proteins like Parkin and PINK1,in β-syn V70 M transgenic mice were significantly higher than the control group.It concluded that β-syn V70 M can induce neuronal autophagy,apoptosis,and mitophagy,and results in neuron loss,eventually leading to motor dysfunction and depression in transgenic mice.Secondly,pCEP4 plasmids with wild-type Parkin and T240 R mutant Parkin with HA tag were successfully constructed,and parkin was detected express in B103 cells.Immunofluorescence experiments demonstrated that Parkin WT was uniformly distributed within the cells while Parkin T240 R accumulated within the cells.SH-SY5 Y cells which can stably express β-syn V70 M were transfected by PCEP4,Parkin WT and Parkin T240 R plasmids.Immunofluorescence was done by using three groups: dual-labeled β-syn and autophagy markers ATG4 D,β-syn and mitochondria,β-syn and lysozyme.The results showed that β-syn V70 M can be directly degraded by autophagy or indirectly through mitophagy into the autophagy pathway.Parkin WT can promote the degradation of β-syn V70 M while Parkin T240 R does not have this function.Parkin T240 R can inhibits the degradation of β-syn V70 M by the mitophagy pathway.Finally,Parkin WT and Parkin T240 R plasmids were packaged into a virus.Then,this virus were injected into the substantia nigra of mice together with AAV-hSyn-Cre virus,and the mice contain the β-syn gene but don’t expression of β-syn protein.Immunofluorescence and Western blotting detected the successful expression of mCherry,β-syn V70 M and Parkin in virus-injected mice.Behavioral test of virusinjected mice showed that Parkin WT can improved behavioral impairment caused by the β-syn V70 M,while Parkin T240 R could exacerbate mice’s behavioral impairment.The expression levels of α-syn,LC3,and other autophagy-related protein and apoptosis-associated proteins such as BAX and p53 were significantly increased in injected with Cre+Saline virus mice.The expression level of the protein α-Syn,ATG4 D,LC3,and other related protein were significantly decreased in injected with Cre+Parkin WT virus mice compared with Cre+Saline virus-injected mice.In contrast,the expression of these proteins in the Cre+Parkin T240 R group was significantly higher than that in the Cre+Saline group.The β-syn V70 M increased the expression of α-syn in the substantia nigra of mice and increased the expression of autophagy and apoptosis-related proteins.Parkin WT can alleviate this phenomenon and Parkin T240 R can aggravate this phenomenon.Observed the projection of neurons from the substantia nigra to other brain regions.It was not observed in the nerve fibers of substantia nigra in injected with Cre+Saline virus mice.Parkin WT can promote the projection of neurons from substantia nigra to other brain regions.Whereas Parkin T240 R inhibits the projection from substantia nigra to other brain regions.Taking together,β-syn V70 M can induce apoptosis,autophagy and mitophagy,induce neuronal death and motor dysfunction.Parkin WT can reverse this phenomenon while Parkin T240 R can exacerbate this phenomenon.