The Effects Of Netrin-1 And Its Receptor DCC On The Death Of Dopaminergic Neurons In Parkinson’s Disease

Parkinson’s disease(PD)is a neurodegenerative disease with a very high incidence,and the affected population tends to be younger.The main clinical symptoms of PD are resting tremor,paralysis,and retardation of movement.In addition to these motor symptoms,there are also non-motor symptoms such as sleep,emotional,and cognitive impairment.When motor symptoms appear,the disease has already worsened.The pathological features of PD are the progressive loss of substantia nigra dopaminergic neurons and the deposition of Lewy bodies(LBs)and Lewy neuritis(LNs).The projection of midbrain SNpc DA neurons to the striatum requires a large number of axon branches and the establishment of hundreds of thousands of synapses,which are several orders of magnitude more than other neurons.These neurons need to maintain the cellular structure and basic homeostasis.The corresponding function requires a large amount of energy supply.When the energy supply is normal,the neurons behave well.When there is any interference,DA neurons will be more severely affected than other neurons.Therefore,midbrain DA neurons are lost first and more severely in PD than other neurons.The genome-wide association analysis study(GWAS)reported that DCC single nucleotide polymorphisms are associated with multiple aspects of PD,such as the age of onset,severity,and progression of the disease.DCC is the receptor of the axon guiding factor Netrin-1.DCC is expressed in the ventral midbrain of adults and can be co-localized with SNpc DA neurons.Most importantly,the ventral DA neurons of SNpc are the most prone to loss of DA neurons in PD,and they are also the subgroup of midbrain DA neurons with the highest expression of DCC.Consistent with DCC,Netrin-1 is also expressed in midbrain DA neurons.Studies have found that the expression level of Netrin-1 in the midbrain of PD patients decreases.Netrin-1 and DCC play an important role in the nervous system,such as promoting the development and maturation of synapses,enhancing the plasticity of synapses,and maintaining the integrity of the blood-brain barrier.During the development of the nervous system,it can induce midbrain DA neurons to project to the striatum.DCC is also a dependent receptor.In the absence of Netrin-1,cells expressing DCC will trigger programmed cell death signaling,leading to cell death.Therefore,Netrin-1 and DCC may be intrinsically related to the function or maintenance of SNpc DA neurons.The current studies of Netrin-1 and DCC in PD were based on the autopsy results of PD patients,and the changes of DA neurons after injury were also observed.This research intends to explore whether the early stage of nerve cell injury in the classic MPTP animal model and MPP~+cell model of PD is related to the changes of Netrin-1 and DCC,which provides new ideas for exploring the effective diagnosis and treatment of PD.Objective:To study the correlation between the changes of Netrin-1 and DCC and nerve damage in PDMethods:ELISA method was used to detect the content of Netrin-1 in the plasma of PD patients.The MPTP subacute PD mouse model was constructed,and the materials were collected and tested on the 1,3,and 7 days after the last administration.Using immunohistochemical methods,TH immunostaining was performed on the striatum and midbrain to detect the loss of midbrain DA neurons and striatal DA neurons caused by MPTP.By immunofluorescence method,TH was co-labeled with Netrin-1 and DCC to detect the expression of Netrin-1 and DCC in the midbrain.Through the Western blot(WB)method,the changes in the expression levels of Netrin-1 and DCC in the striatum and midbrain were studied.We use SH-SY5Y cells to give MPP~+stimulation to construct a cell injury model.We set different MPP~+concentrations(0,20,50,100,200,500μM)and 100μM MPP~+for different times(0,6,12,24,48,72h),using MTT to detect the viability of the cells after the administration,and using the WB method to detect the changes of Netrin-1 and DCC.WB detects the changes of Bax,Bcl-2,Caspase 3,and Cleaved Caspase 3 to determine the status of cell apoptosis.Immunofluorescence was used to stain DCC and Hoechst33342 to stain the nucleus to detect the relationship between apoptosis and DCC expression.WB was used to detect the changes in the downstream pathways FAK and Src,p FAK and p Src of Netrin-1 and DCC.Results:(1)First,we used ELISA to detect the level of Netrin-1 in the plasma of PD patients,and found that the level of plasma Netrin-1 in patients with early PD decreased.We guessed that the changes in Netrin-1 might be related to PD.(2)Immunofluorescence,the midbrain TH was co-labeled with Netrin-1 and DCC,which indicated that Netrin-1 and DCC were expressed on the ventral side of the midbrain SNpc DA neurons.(3)The TH immunostaining result of the striatum was given at the last time.At 1,3,and 7 days after MPTP,the expression of TH in the striatum gradually decreased,indicating that MPTP induced the gradual loss of the axon ends of DA neurons in the striatum over time.WB results showed that with the gradual decrease of TH,the expression of MAP2 decreased,and the expression of Netrin-1 gradually decreased,while the expression of Netrin-1 receptor DCC gradually increased,and when the neurons were not significantly lost,Netrin-1 There have been obvious changes in the midbrain and DCC.(4)According to the results of TH immunostaining in the midbrain,the expression of TH in the midbrain gradually decreased after 1,3,and 7 days after the last dose of MPTP,and the axon ends of DA neurons followed Gradually lost over time.The WB results of the midbrain showed that with the gradual decrease of TH,the expression of Netrin-1 gradually decreased,and the expression of Netrin-1 receptor DCC gradually increased.It shows that Netrin-1 and DCC are related to the loss of DA neurons.(5)After stimulating SH-SY5Y cells with different concentrations of MPP~+,the results of MTT showed that with the increase of drug concentration,cell viability gradually decreased.WB results showed that the expression of Netrin-1 decreased with the decrease of cell viability,and the expression of DCC increased with the decrease of cell viability.When the cell viability had not decreased significantly,Netrin-1 and DCC had already changed significantly,indicating that the changes of Netrin-1 and DCC are related to cell damage induced by MPP~+.(6)MPP~+induces an increase in Bax expression,a decrease in Bcl-2 expression,and an increase in Cleaved Caspase 3 expression,indicating that MPP~+induces cell apoptosis.(7)MPP~+induces increased staining of Hoechst33342 cells,showing bright blue,indicating that the cells have undergone apoptosis.In apoptotic cells,we found that the fluorescence intensity of DCC increased,which also indicates that DCC is related to cell damage.(8)With As the time of MPP~+stimulation is prolonged,cell viability gradually decreases.WB results showed that the expression of Netrin-1decreased with the decrease of cell viability,and the expression of DCC increased with the decrease of cell viability.(9)After MPP~+stimulated SH-SY5Y cells,FAK and p FAK,Src and p Src were detected by WB.The phosphorylations of FAK and Src are reduced,indicating that the binding of Netrin-1 and DCC is decreased.Conclusion:Netrin-1 and its receptor DCC are related to DA nerve injury induced by MPTP.And before the nerve damage is not obvious,Netrin-1 and DCC have changed,but the reason for the decrease of Netrin-1 in PD,and whether the changes of Netrin-1and DCC are the initiating events of DA neuron degeneration are still unclear.Exploring the pathophysiological role of Netrin-1 and DCC signals in PD may provide new ideas for disease treatment and diagnosis.Innovation:This study demonstrated that Netrin-1 and its receptor DCC are associated with early neurodegeneration in PD patients,MPTP subacute mouse model,and MPP~+cell model,providing a new target for the early diagnosis and treatment of PD.