The Mechanism Of GDF15 On Regulating Mitochondrial Function In The Progression Of Parkinson’s Disease

Background:Parkinson ’s disease(PD)is a common neurodegenerative disease in middle-aged and elderly people,characterized by bradykinesia,resting tremor and myotonia.The main pathological changes are gradual degeneration and injury of dopaminergic neurons in substantia nigra,locus coeruleus and raphe nucleus.Growth differentiation factor-15(GDF-15)is a member of the transforming growth factor-β superfamily,and is a ubiquitous pleiotropic cytokine.It has been suggested that GDF15 is an important factor in regulating mitochondrial function,and mitochondrial dysfunction is closely related to PD.In our previous clinical study,we found that the expression level of GDF15 in the serum of PD patients was significantly higher than that of normal people,and was related to the severity of PD.Therefore,the molecular mechanism of GDF15 in PD needs to be further studied.Part Ⅰ:The effect of GDF15 on rotenone-induced PD animal modelObjective:To investigate whether GDF15 elevation has a protective effect on dopaminergic neurons in a PD model.Methods:1.The cell and animal models of PD were established by inducing SH-SY5Y cells and C57BL/6J mice with rotenone.The skin tissues of PD patients were obtained by skin biopsy device and cultured into primary skin fibroblasts.The content of GDF15 in cell culture medium supernatant and mouse serum was detected by ELISA.The content of GDF15 protein in PD cell model and PD patient fibroblasts was detected by Western blotting(WB).2.Exogenous rhGDF15 protein was added into PD cell model,and the change of cell survival rate was detected by CCK8 assay.3.A GDF15 systemic knockout mouse model was constructed to monitor the effects on growth and development(length and weight)of WT,GDF15+/-and GDF15-/-mice within 16 weeks after birth.PD model was established in C57BL/6J mice induced by rotenone.The body weight of WT,rotenone,GDF15-/-and GDF15-/-+ rotenone groups were measured during 10 weeks.4.The effects of GDF15 on motor and cognitive function of PD model mice were evaluated by open field test,pole test,beam walking test,T maze,Barnes maze and novel object recognition.5.The effects of GDF15 on emotion and executive function of PD model mice were evaluated by elevated plus maze,tail suspension test,sugar preference test and nesting test.6.The effect of GDF15 on dopaminergic neurons in PD mice was determined by TH immunohistochemical staining and WB.Results:1.The level of GDF15 in PD patient fibroblasts and PD animal and cell models was significantly increased.2.The survival rate of PD cell model was positively correlated with rhGDF15 protein concentration.3.GDF15 knockout can aggravate the motor,cognitive and emotional damage of PD model mice.4.In a mouse model,the depletion of GDF15 will exacerbate the demise of dopaminergic neurons that are brought on by rotenone.Conclusion:The expression level of GDF15 was significantly increased in PD,and the addition of rhGDF15 protein increased the survival rate of PD cell models.Behavioral experiments showed that GDF 15 knockout could aggravate the motor,cognitive and emotional impairment of PD model mice.GDF 15 knockout aggravates dopaminergic neuron death in rotenone-induced animal models and GDF 15 can protect dopaminergic neurons.Part Ⅱ:The mechanism of GDF15 protecting rotenone-induced Parkinson’s disease model by inhibiting mitochondrial apoptosisObjectives:1.To determine whether GDF15 can protect dopaminergic neurons by regulating mitochondrial function and energy metabolism and inhibiting apoptosis in PD.2.The mechanism of GDF 15 protecting dopaminergic neurons by regulating mitochondrial function and inhibiting apoptosis of PD.Methods:1.Three mice in each of WT group,rotenone group,GDF 15-/-group and GDF 15-/-+rotenone group were dissected for brain slices and amygdala.The collected tissue samples were subjected to RNA-seq sequencing.2.MitoSox and DCFH-DA were used to detect the level of reactive oxygen species in mitochondria and cytoplasm,respectively.JC-10 probe was used to detect the change of mitochondrial membrane potential.Seahorse Mito Stress Test was used to measure mitochondrial function.Apoptosis was detected by flow cytometry and TUNEL staining.3.The expression of PGC-1 a and apoptosis related factors(p53,bcl-2/bax)were detected by WB and qPCR.4.The protein phosphorylation levels of Akt/mTOR pathway in brain tissue of PD model and PD cell model were detected by WB and qPCR.LY194002,an inhibitor of PI3K/Akt/mTOR pathway,was added to SH-SY5 Y cells to observe the changes of downstream factors.Results:1.Transcriptome sequencing showed that the pathogenesis of PD induced by rotenone exposure may be closely related to mitochondrial damage,oxidative stress,apoptosis and other biological processes.In the GDF15 knockout samples,the mechanisms leading to the aggravation of PD pathology mainly involve biological processes including mitochondrial dysfunction,inflammatory immunity,calcium ion channels and synaptic plasticity.2.GDF 15 can inhibit apoptosis and protect mitochondrial function of cells by reducing the expression of p53,increasing the expression of bcl-2/bax and increasing the expression level of PGC-1α,thus protecting dopaminergic neuron cells.3.The protective effect of GDF15 on Parkinson’s disease was found to be dependent on phosphorylation of the Akt/mTOR pathway by WB.Conclusion:GDF15 can inhibit apoptosis,protect mitochondrial function and protect dopaminergic neurons by decreasing the expression of p53,increasing the expression of bcl-2/bax and PGC1α.The protective effect of GDF15 on PD depends on phosphorylation of Akt/mTOR pathway.Part III:Explore the mechanism of GDF15 in regulating autophagy and apoptosis rebalance and protecting mitochondrial function in Parkinson diseaseObjectives:To explore whether GDF15 can protect mitochondrial function by regulating autophagy and apoptosis rebalance,reduce mitochondrial damage,and further alleviate the occurrence and development of Parkinson’s disease.Methods:1.The expression of P62 and LC3 protein in the substantia nigra were detected by immunohistochemistry and immunofluorescence,respectively.The protein levels of P62 and LC3 in PD cell model induced by rotenone were detected by WB.2.The GEO database and The Human Autophagy Database were used to analyze autophagy-related PD differential genes.3.The expression of autophagy-related PD differential genes in PD patients was verified byqRT-PCR.4.The differential expression of autophagy-related PD genes in rotenone-induced PD cell model with GDF15 overexpression was verified by qRT-PCR.Results:1.Overexpression of GDF-15 could down-regulate autophagy-related marker P62 and upregulate the expression of LC-3Ⅱ/Ⅰ,and P62 was up-regulated and LC3 down-regulated in GDF15-/-PD model mice.2.A total of 42 PD autophagy-related genes were identified from the public data set by bioinformatics analysis.By GO and KEGG enrichment analysis of differentially expressed autophagy-related genes,we found that these genes are involved in autophagy and other biological processes.3.The expression of BCL2,RAB 11 A,FAS,RELA and BCL-XL in PD patients was different from that in healthy controls.4.QRT-PCR showed that BCL2,RAB11A,FAS and RELA were differentially expressed in rotenone-induced PD cell model with GDF15 overexpression.Conclusion:Through bioinformatics analysis,42 differentially expressed genes of autophagy-related PD were identified from public databases.PPI analysis,GO and KEGG enrichment analysis confirmed that these genes are involved in multiple biological processes such as autophagy and mitosis.QRT-PCR was further used to verify that the expression levels of BCL2,RAB11 A,FAS,RELA,and BCL-XL were different between PD patients and healthy subjects.Overexpression GDF15 in PD cell models can affect the expressions of BCL2,RAB11A,FAS and RELA,and play a role in protecting mitochondrial function and reducing α-synuclein deposition.However,whether GDF15 affects the expression of the above differential factors by affecting the phosphorylation of Akt/mTOR pathway,increases the level of autophagy,and removes the accumulation of damaged mitochondria and α-synuclein in vivo needs to be further verified in PD cells and animal models.