Study On The Expression Changes Of DDC In The Midbrain Substantia Nigra In MPTP/probenecid-induced Progressive Parkinson’s Disease Mice

Background Parkinson’s disease(PD)is the second-largest neurodegenerative disease in the world characterized by the progressive loss of dopaminergic neurons in the substantia nigra compacta(SNc).The apoptosis of dopaminergic neurons will lead to a large reduction in the synthesis of dopamine in the brain,resulting in a series of movement and non-motor disorders in the body,which has a serious effect on patients’ lives.As an important rate-limiting enzyme in the synthesis of dopamine,DDC plays a particularly important role in the pathogenesis and treatment of PD.Objective This article was to explore and validate the role of DDC in PD mice,by establishing a mouse model of progressive Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine/probenecid(MPTP/p).The expression changes in the SNc of the midbrain provide a more comprehensive understanding of the pathogenesis of PD and a more efficient method for clinical diagnosis and treatment for PD.Methods1.Twelve-week-old C57BL/6J male mice were randomly divided into control group and MPTP/p model group.The MPTP/p model group mice were injected intraperitoneally with probenecid solution at a dose of 100 mg/kg,and half an hour later MPTP was injected at a dose of 25 mg/kg,once every 3.5 days,for 10 consecutive injections.The control group nuderwent the equal volume of normal saline.Those in the control group underwent the same way of injection with physiological saline simultaneously.At the specific time points,before modeling and after the 3rd,6th,and 10th administration,we conducted behavioral tests on olfactory test and beam traversal test evaluate their motor and non-motor function.Western blotting analysis was used to detect the changes in the expression of TH protein in the SNc.Experiments above were to evaluate whether the animal models were successfully established.2.According to the high-throughput transcriptome sequencing technology carried out in the early stage of the experiment,a transcriptome library of the substantia nigra of the MPTP/p model group and the control group was constructed.And databases such as GO and KEGG were used to screen out the differential gene DDC and carried out the follow-up verification.Through further bioinformatics analysis of differential genes,the key role of DDC gene in the pathway of dopamine synthesis and transport was screened.Combined with the expression changes of differential genes in the upstream and downstream,the main pathway of the role of DDC in the PD model was revealed by go analysis,and string was used(https://www.string-db.org/).Construction of protein network interaction,KEGG(https://www.kegg/).As an important rate limiting enzyme in dopamine synthesis,DDC plays a key role in the process of neurodegeneration in PD mice.3.Four time points,before modeling and the 3rd,6th,and 10th times of modeling,were selected to obtain the midbrain substantia nigra tissues of respectively.After that,we use qRT-PCR technology to detect DDC mRNA expression changes in the substantia nigra at different time points.Double immunofluorescence was used to detect the localization and expression of TH and DDC in substantia nigra at different time points,and immunohistochemical staining was used to detect the dynamic expression of DDC in striatum at different time points.Results1.The behavioral results showed that as the number of modeling times increases,the sense of smell and balance and coordination ability of mice gradually decreased.And the behavioral indicators of PD mice began to decrease significantly after the 3rd time injection(p<0.05).2.The results of Western Blotting were basically consistent with the results of behavioral test,showing that as the times of modeling increases,the expression of TH protein showed a gradual downward trend.There was a significant difference after 10 modeling(p<0.05).The above results are all shows that the model was stable and successful.3.According to the high-throughput transcriptome sequencing,we found DDC which was one of the DEGs relating to dopaminergic neurogenesis.According to the previous bioinformatics analysis of different genes in substantia nigra of mice,as an important rate limiting enzyme in the process of dopamine synthesis.DDC is consistent with other upstream and downstream differential genes involved in dopamine synthesis,transport and transport,such as Th,Dat and Vmat2,and cooperatively causes the neurodegenerative process of PD model mice.4.Through qRT-PCR detection,it was found that DDC in the substantia nigra of PD mice showed a significant decrease after 6 and 10 times of modeling compared with those in the normal group.The results were basically consistent with the previous sequencing results.The results of immunofluorescence chemical staining showed that in the substantia nigra,DDC and TH are mainly expressed in the cytoplasm and processes of neurons,and there is co-localization between the two.During the modeling process,the number of DDC and TH positive cells gradually decreased,And there is a significant difference in comparison with the control mice after 6 and 10 times of modeling.5.The results of immunohistochemical staining showed that the positive signal of DDC in striatum gradually decreased during the modeling process,and there was significant difference after 6 and 10 times of modeling.Conclusions1.A reliable chronic PD mice model induced by MPTP/p was successfully established.2.The expression of DDC and TH in substantia nigra of PD model mice was verified by QRT PCR and immunofluorescence staining,which was consistent with the change trend of sequencing.Immunofluorescence staining and single-cell data show that DDC is mainly distributed in dopamine neurons in the substantia nigra of the mouse midbrain.3.Immunohistochemistry further verified that the expression of DDC in striatum gradually decreased with the increase of modeling times.It is the key factor of PD like symptoms through nigrostriatal pathway and dopamine synthesis and transportation,which leads to motor dysfunction.4.DDC affects the synthesis,transport and metabolism of dopamine in midbrain,and cooperate with TH,DAT and VMAT2 to cause neurodegeneration in PD model mice.