| Parkinson's disease(PD)is a chronic neurological disease that is prone to occur in people with increasing age.It is the second largest neurodegenerative disease in the world.Its main features are: myotonia,static tremor,unstable walking etc.The pathological features are mainly expressed in the accumulation of ?-synuclein in the substantia nigra of the brain,and it is easy to aggregate to form inclusion bodies-lewy bodies.The ?-synuclein mutation(A53T,A30 P,etc.)induces PD,and thus ?-synuclein A53 T transgenic mice are widely used as PD model mice.?-synuclein is a homologue of ?-synuclein.In vitro cell experiments have shown that wild-type ?-synuclein(?-syn WT)can inhibit the aggregation of ?-synuclein,but it has rarely been reported in vivo.Aim of this study was to investigate whether the overexpression of ?-synuclein WT can ameliorate the neurotoxicity caused by ?-syn A53 T mutation or not.Firstly,the ?-syn A53 T transgenic mice were genetically tested by PCR.The positive mice were selected as the experimental group,and the negative mice were used as the control group.The body weight,tandem gait test,balance beam,forced swimming test,tail suspension and elevated cross maze behavior test were applied.Biochemistry(Western blot)and immunohistochemistry analysis were used to detect the expression of ?-synuclein aggregation,tyrosine hydroxylase(TH),apoptosis-related protein,autophagy and related proteins in mitochondrial autophagy pathway.Then,the adenovirus with FLAG-tagged was injected into the substantia nigra of the ?-syn A53 T transgenic mice.After the virus was expressed for one month,all above mentioned markers were detected in the same way for behavioral learning and pathophysiological testing as it was done for A53 T transgenic mice.The results as followed:Behavior tests results indicate that ?-syn A53 T transgenic mice has less body weight along with reduce ability of physical activites especially elevated cross maze and forced swimming test indicate their less activity as compared to healthy mice and these physical inactivities are also associated with depression.The both western blot and immunohistochemical analysis for ?-synuclein,showed that the expression of ?-synuclein in the brain of ?-syn A53 T transgenic mice especially in the substantia nigra increased.TH is a key enzyme for the synthesis of dopamine.Western blot results showed that TH expression is decreased in the brain of ?-syn A53 T transgenic mice,indicating a decrease in dopaminergic neurons.Apoptosis related markers AIF,Bcl-2 and BAX were examined by western blot and results showed that ?-syn A53 T transgenic mice brain has increased expression of AIF and BAX and decreased expression of Bcl2.Another important marker cleaved-Caspase3 was also used to observe the apoptotic behavior in ?-syn A53 T transgenic mice in WB and results indicated that the expression of cleaved-Caspase3 is increased.These results indicated that A53 T mutation induce apoptosis.Autophagy is another mode of programmed cell death.To observe this phenomenon autophagy associated markers LC3-I and LC3-II were observed and our results showed that there was an increase in the ratio of LC3-II/LC3-I and in the expression of autophagy-related proteins Atg5,Atg4 D and p62 in transgenic mice.Mitochondrial autophagy is an important type of autophagy which is affected by multiple pathways.Western blot results showed that the expression of PINK1,Parkin and Beclin1 in the brain of ?-syn A53 T transgenic mice was increased,indicating that A53 T mutation leads to increased mitochondrial autophagy.The ?-syn-WT virus was injected into the substantia nigra of the ?-syn A53 T transgenic mice.After one month of injection,western blot and immunofluorescence staning were used to detect the successful expression of ?-syn-WT virus in the brain of mice,followed by behavioral tests.It was found that the exercise capacity of the mice has improved.Western blot results showed an increase in ?-synuclein expression,a decrease in ?-synuclein expression,and a decrease in ?-synuclein aggregates by immunohistochemistry.Increased expression of TH indicates that overexpression of ?-syn-WT can protect dopaminergic neurons.Overexpression of ?-syn-WT decreases the expression of apoptosis-related proteins p53,BAX and cleaved-Caspase3,and up-regulates Bcl-2 expression,indicating that ?-syn-WT can reverse A53 T mutation effects.The expressions of autophagy-related proteins p62,Atg5 and Atg4 D were decreased,and the ratio of LC3-II/LC3-I was also decreased,indicating that ?-syn-WT can reduce autophagy and reduce autophagic apoptosis.The expression of mitochondrial autophagy-related proteins Beclin1,Parkin and PINK1 also decreased,indicating that ?-syn-WT can attenuate mitochondrial autophagy induced by A53 T mutation along with a reduction in mitochondrial damage and play a role in cell protection.It is concluded that the ?-syn A53 T mutation can induce ?-synuclein aggregation,damage dopaminergic neurons and induce autophagy apoptosis,leading to motor dysfunction.Overexpression of ?-syn-WT can inhibit A53 T mutation-induced ?-Synuclein aggregation,reduce the damage of A53 T mutation on dopaminergic neurons,regulate autophagy pathway to reduce autophagic death,play a certain protective effect on cells,improve motor dysfunction induced by A53 T mutation,and can be the future Parkinson's syndrome therapeutic aspects and might be provide new therapeutic perspectives and targets. |
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