The Role And Mechanism Of Dynactin In The Degeneration Of Dopamine Neurons

Objectives:Dynactin is one of the members of synapse-cytoplasmic retrograde transport complex,which plays an important role in the transport of synapses to cytoplasm.The expression of dynactin in the brain of normal people increased with age,while the expression of dynactin in the brain of patients with Parkinson’s disease decreased in the time axis,suggesting that the change of dynactin may be related to the pathogenesis of Parkinson’s disease.Objective to find out the reasons for the degeneration of dopamine neurons in the early stage of Parkinson’s disease,and to provide basis for early diagnosis of Parkinson’s disease.Revolves around dynactin in this experiment,whether its dysfunction can cause symptoms of Parkinson’s disease,whether can create dopamine neurons degenerative diseases,if dopamine neurons caused by degenerative diseases,so what are the mechanism,whether can cause synuclein abnormal protein aggregation and autophagy body such as increased.If caused,its mechanism is related to the function of dynactin retrograde axoplasmic transport.Methods:This experimental method is divided into two parts: vivo and in vitro.(1)in vivo experiment,dynactin was used to inhibit Tg(vmat2-gfp)zebrafish dynactin expression to construct zebrafish model,and the statistical survival rate was calculated.Observe the appearance change and behavior change of zebrafish;Comparing with the MPTP Parkinson’s disease zebrafish model,the systematic evaluation was different from that of Parkinson’s disease.Using live cell workstations,laser co-focusing and other devices to observe the changes of dopamine neurons in the brain in real time;The aggregation of synuclein was observed by the whole embryo immunofluorescence method.(2)in vitro experiments,dynactin-sirna knocked down sh-sy5 y cell lines.Fluorescence immunological observation of synuclein expression in the cell,transfection of mtagrfp-mwasabi-lc3 in sh-sy5 y cell lines and the expression and distribution of Wasabi and TagRFP,using WB to determine lc3-ii,and P62 expressionlevel to determine the autophagy flow pathway.The axonal transport state of sh-sy5 y cell lines of synuclein-GFP plasmid and mtagrfp-mwasabi-LC3 plasmid was observed by using live cell workstation and laser co-focusing device.Immunofluorescence bistandard dynactin and dynein determine the obstruction of retrograde axoplasmic transport.Results:(1)Inhibition of zebrafish dynactin expression,zebrafish embryo,the larval survival rate and hatching efficiency drops,embryo incubation delay,tail bending deformity,rachis rigidity,balance gaffes,tremors,pericardial edema.The movement ability of zebrafish decreased,and the movement rate slowed down after the free movement rate and mechanical stimulation.Under the living status of dopamine neurons,by observing the living Tg(Vmat2 GFP transgenic zebrafish diencephalon dopamine neurons,found that lower dynactin express a number of dopamine neurons decreased significantly.(2)The expression of dynactin was reduced in sh-sy5 y cells,resulting in the changes in gene expression of Parkinson’s disease,and the large aggregation and distribution of synuclein protein,mainly in axon and cytoplasmic near axons.In the dynactin-sirna knockdown group,the autophagy vesicles marked by LC3 were clustered around the cytoplasm near the axon and the membrane,which is different from the typical induced autophagy pathway,and the autophagy is not dominated by the nuclear cycle.Combined with WB results,it is proved that the decrease of dynactin can result in the change of autophagy position,which may be one of the reasons for the eventual aggregation of synuclein in the above position.(3)Real-time observation of sh-sy5 y cells showed that there were a large number of nerve beads in the cells,and the axoplasmic retrograde transport of LC3 was obstructed.The dynein-dynactin complex was reduced and distributed in the reverse axoplasm transport,and the distribution of dynein-dynactin complex was changed into the distribution of the proximal axons and synapses of the cytoplasm.Dynactin decreased the transport of retrograde axoplasm,resulting in degeneration of dopamine neurons.Conclusions:This experiment established MB concentration is 33.4 ~ 50.3 mu g/L range of zebrafish model is set up,on the behavior and morphology and traditional MPTP to establish the animal model of Parkinson’s disease was similar,explain dynactin reduce symptoms of Parkinson’s disease,mainly for sports ability is abate,abnormal posture,etc.Specific knockdown and inhibition of dynactin and the decrease of dopaminergic neurons in zebra fish showed that inhibiting dynactin caused the degeneration of dopamine neurodegenerative disease.A large aggregation of synuclein in zebrafish and sh-sy5 y cells was consistent with the formation of the synaptic and the synapses in the small body of lewis,suggesting that dynactin dysfunction could lead to pathological changes in Parkinson’s disease.LC3 near the axon and membrane around the large accumulation characteristics may and synuclein near axons in the cytoplasm and synaptic costimulation,and confirmed by WB autophagy flow induced by way of increased speculation may be retrograde axoplasmic transport caused obstacles.The living cells observed intracellular material transport,and synuclein,LC3 transport,dynein and dynactin distribution anomalies all indicated the intracellular retrograde axoplasmic transport barrier.Above all,a reduction in retrograde axoplasmic transport dynactin blocked,causing the synuclein protein in axons and cytoplasmic gathered near the axon,metabolism,autophagy increases,eventually lead to dopamine neurons regression lesions.