Role And Mechanism Of PHB In Regulating The Occurrence And Progression In Parkinson's Disease

Parkinson's disease(PD),also named Paralysis agitans,is a common neurodegenerative disorder.The main clinical features of PD include resting tremor,bradykinesia,rigidity and postural instability.In the late stage,it may be accompanied by depression,dementia,sleep disorders and other non-motor symptoms.The main pathological features of PD include the loss of dopaminergic(DA)neurons,degeneration of residual neurons and Lewy bodies in cytoplasm.Autopsy showed that the main component of Lewy body was ?-synuclein,and the mutation of ?-synclein gene could lead to familial PD.This indicates that ?-synuclein played a crucial role in the development of PD.Prohibitin(PHB)is a kind of tumor inhibiting protein,whose structure is highly conserved and widely distributed in organisms.PHB protein,locating on the inner membrane of mitochondria,forms a circular complex and plays the role of molecular chaperone to maintain the integrity of mitochondrial structure and function.It also involves in autophagy and aging,which may participate in the evolution of neurodegenerative diseases.In the substantia nigra of Parkinson's disease patients,the level of PHB protein decreased significantly.Based on the above,we raise the following questions:Can PHB regulate the damage of nigra-striatal pathway in PD?Can PHB regulate the toxic effect induced by a-synclein abnormal aggregation in PD?In current study,the SH-SY5Y cells were incubated with the MPP+to establish the PD in vitro model,PHB is over-expressed or knocked down,cell viability,apoptosis and autophagy in different group were detected.To detect whether the regulatory effect of PHB by clearing the a-synclein,a-synclein expression is inhibited by shRNA.We also applied the A53T PD mice to detect the role of PHB in PD model.Part ?:Regulatory effect of PHB on the dopaminergic neuron injury in PD models Objective:To investigate the regulatory effect of PHB on the nigra-striatal pathway injury in PDMethod:(1)The SH-SH5Y cells were incubated the with MPP+to establish the PD in vitro model,PHBwere over-expressed or inhibited by the retrovirus technique in the SH-SY5Y cells.Cells were divided into normal control,PD,PD+PHB+/+ and PD+PHB-/-group.The cell viability and apoptosis were detected by CCK-8 assay and ICC assay.(2)The A53T transgenic PD mice model was established,the PHB and TH expression was detected by the immunofluorence assay.(3)To collect the blood of 27 patients with Parkinson's disease and 21 normal persons as control from the department of Neurology,Subei People's Hospital,Yangzhou City.The expression of PHB in PD patients and normal person was detected by the qRT-PCR assay.Result:(1)The expression of PHB in SH-SY5Y is increased nearly 350 times after infected with retrovirus.The level of PHB in SH-SY5Y cells decreased significantly after shPHB.(2)After incubated with MPP+,the cell viability decreased and apoptosis increased significantly;Over-expression of PHB could inhibit the apoptosis of PD cells and increased the cell viabiltiy.Surppressing the PHB level promoted the apoptosis and decreased the viability of PD cells.(3)Western blotting results showed that the level of PHB increased while TH decreased with the age of PD mice.The immunological analysis showed that PHB and TH co-expressed in the PD mice.(4)The mRNA level of PHB in the blood of PD patients was obviously lower than that of normal people.Conclusion:PHB may play a potential protective effect on the nigra-striatal pathway injury in PD.Part ?:Regulation of PHB on abnormal aggregation of a-synclein protein in PDObjective:To investigate the regulatory effect of PHB on neurotoxicity induced by abnormal aggregation of a-synclein.Method:(1)The SH-SH5Y cells were incubated the with MPP+to establish the PD in vitro model,the PHB and a-synclein protein levels were analysis by the Western Blotting assay.The Pearson correlation test was used for analysing the correlation of that two protein levels.(2)The PHB protein was over-expressed or suppressed by the retrovirus technique in the SH-SY5Y cells.The flow assay and flow assay was used to observe cell activity and apoptosis.(3)The expression of apoptosis related protein(Bcl-2,Bad,cleaved-caspase-3),related protein(LC3?/??Beclin)of different groups were detected by Western Blotting assay.(4)The expression of PHB and a-synclein were detected by Western Blotting assay in the A53T PD mice.The Pearson correlation test was used for analysing the correlation of that two protein levels.Result:(1)PHB expression revealed a constant increase,in a time and concentration dependent manner.Moreover,a-synclein protein expression pattern was consistent with that of PHB.The Pearson correlation test showed a significant positive correlation between the two protien levels.(2)Western blotting analysis showed that MPP+ incubation increased the level of Bad,cleaved-caspase-3,LC3 ?/? and Beclinl while downregulated the level of Bcl-2.PHB overexpression could partially alleviate autophagic stress by inhibiting the levels of LC3 ?/? and Beclinl,induced by MPP+.(3)Kockdown of PHB excerbated the apoptosis in PD cells,envidenced by increasing the level of apoptosis level.(4)Western blotting results showed that the levels of PHB and a-synclein increased with the age of PD mice.The Pearson correlation test showed significant positive correlation between the PHB and a-synclein.Conclusion:Over-expression of PHB could increase cell viability,inhibit the autophagy stress and the apoptosis in dopaminergic neurons in PD models.Knockdowon of PHB excerbated the neurodegeneration happened in PD models.PHB may play its potential protective role by inhibiting the toxicity of abnormal aggregation of ?-synuclein in PD models.