Induction Of Mitophagy And Mitophagy Related Proteins BNIP3 Expression In The Brain Of Manganese Induced Parkinsonism Rats

Objectives:To determine the activity of mitophagy and mitophagy related proteins BNIP3、LC3、Drp1、FOXO3、HIF-1expression in substantia nigra（SN）in manganese induced parkinsonism rats model,which will provide a new insights into the pathogenesis of induced neurotoxicity.Method:64 healthy Sprague Dawley rats（male）,were assigned at random to sham control group and 3 dosages groups:low-dose group,middle-dose group,high-dose group.The rats in control group were given intraperitoneal injection（i.p）of normal saline while the rats in other three of experimental groups were injected（i.p）with Mn Cl2·4H₂O solution（5,15,20mg/kg of body weight,respectively）for 5 days once a week and lasting for 16 weeks.Motor coordination and balance is evaluated by Balance-beam test,catalepsy is also evaluated by Vertical-grid test.The content of manganese in the substantia nigra is detected by Inductively Coupled Plasma-Atomic Emission Spectrometry （ICP-AES）.Meanwhile,the tyrosine hydroxylase was tested by Immunofluorescence staining.Then,the transmission electron microscopy（TEM）was used to study the ultrastructure of the substantia nigra.The change of BNIP3、FOXO3、HIF-1 m RNA was measured by q RT-PCR.The expression of BNIP3、LC3、Drp1、FOXO3、HIF-1 protein were detected by Western blot.To analysis of the subcellular distribution of mitophagy related protein,it is executed with measuring the different expression of BNIP3、LC3 between the mitochondrial fractions and cytosolic fractions.Results:The results of behavioral test showed that the average time of manganese poisoning rats to cross the narrow beam and the latencies of manganese poisoning rats to move on the vertical grid were significantly increased compared to both the control group and the same group before exposure（P<0.05）.The results of immunofluorescence staining showed that after manganese administration,there was a marked loss of TH-immunoreactive neurons in the substantia nigra compared with the control group and showed a dose response related pattern.The TEM analysis demonstrated that the cells in the sham group possessed bright normal nuclei,endoplasmic reticulum,and mitochondria,which did not seem to be damaged,and the formation of autophagic vacuoles（AVs）was rare to be seen.In contrast,the damaged cells in the manganese poisoning rats displayed mitochondrial with the abnormal appearance such as swelling,distortion,fusion and vanish of the crista,numbers of AVs（including autophagosomes、mitophagosomes or autolysosomes）enhanced along with the increase of the dose,the differences achieved statistical significance（P<0.05）.Densitometric quantification of the total protein band densities demonstrated a significant increase（P<0.05）in the level of BNIP3 and LC3-II/LC3-I in all manganese poisoning rats vs.sham.Drp1 expression was also increased after intoxication,but only the middle and high dose group achieved statistical significance（P<0.05）.The expression of HIF-1 in manganese poisoning rats vs.sham was no difference（P>0.05）.Mitochondrial fractions and cytosolic fractions were separated and purified,the result shows that in control rats BNIP3、LC3-IIlevels were observed in mitochondrial fraction with a very low level of expression,whereas in manganese poisoning rats BNIP3、LC3-II were markedly increased.q RT-PCR demonstrated a significant increase（P<0.05）in the level of BNIP3 and FOXO3m RNA in all manganese poisoning rats vs.sham,and showed dose dependent manner.Conclusion:Sixteen weeks after Mn Cl2·4H2O administration,the rats developed behavior and typical pathological changes,which can serve as a reliable model.The biochemical mitophagy markers BNIP3、Drp1 expression and mitophagic activities were enhanced in the substantia nigra of manganese induced Parkinsonism rats.Simultaneous mitochondrial translocation of BNIP3 and LC3suggested BNIP3 may interacting with LC3 to trigger mitophagy.Transcription factor FOXO3 may regulate BNIP3.