Mitochondrial Fusion And Fission Of Pc12 Cells In Response To Manganese

【Background】Manganese (Mn) is considered an essential nutrient and is crucial for maintaining the proper function and regulation of many biological processes such as producing ATP and blood clotting. On the other hand, after chronic exposure of Mn is known to result in neurotoxicity. Manganese can selectively destroy DA neurons and induce clinical symptoms similar to those of PD in humans and animals.Mitochondria play essential and diverse roles in the metabolism and physiology of eukaryotic cells. Mitochondrial defect relates with a variety of disease. Recently there has been increasing evidence that manganese could have a special affinity for mitochondria, and its neurotoxicity is closely associated with mitochondrial dysfunction, mitochondrial function would play a key role in Mn neurotoxicity. Studies have demonstrated that Mitochondria dysfunction is associated with the pathogenesis of several neurodegenerative disorders.The mitochondrial population and morphological are in fact dynamic, this plasticity is based on the ability of mitochondria to undergo both organellar fusion and fission, but their physiological means remains unclear. Mitochondrial dynamics involves the two opposing processes of fusion and fission, and the balance of these opposing processes regulates mitochondrial morphology. Unbalance of fusion and fission is always accompanied by mitochondrial dysfunction.Inhibition of mitochondrial fusion results in mitochondrial fragmentation by progression of mitochondrial division and the mitochondria function impairment is observed; whereas an arrest of mitochondrial division causes the formation of highly connected and elongated mitochondria, which may play a protective role in cell activity. At the cellular level, Mn preferentially accumulates in mitochondria, where it disrupts mitochondria function, suggesting that mitochondrial fusion and fission may be involved in manganese neurotoxicity.The mitochondrial fusion and fission are controlled by the particular molecules. Mfn1 and Mfn2 are each essential for embryonic mitochondrial fusion, and OPA1 play important roles in mitochondrial fusion. Mitochondria depleted of either Mfn protein fail to fuse after the cell fusion reaction, disruption of both Mfn1 and Mfn2 leads to severe mitochondrial fragmentation, retained low levels of mitochondrial fusion and escaped major cellular dysfunction. There is not the formation of highly connected and elongated mitochondria, mitochondrial is in dysfunction. OPA1 is required for developmentally regulated fusion of mitochondrial inner membrane. The loss of OPA1 protein leads to dispersal of mitochondrial fragments throughout the cytosol and results cell apoptosis. The fission of the outer mitochondrial membrane is regulated by Drp1and Fis1 for complete fission of the mitochondria. Drp1 which is essential for mitochondrial fission, Fis1 is directly involved in membrane distribution and function of Drp1-containing complexes during fission.【Objective】To investigate the neurotoxicity and the molecular mechanism of manganese to rat pheochromocytoma cells (PC12) through mitochondrial fusion and fission sight. Our work may provide evidence for mechanism of manganese neurotoxicity, and this may give some help to prevent and treat manganese induced neurotoxicity.【Methods】1) We used rat pheochromocytoma cells (PC12) as the cell model; 2) the cell viability was detected by MTT assay and cell count, and the morphology was detected by microscope and electron microscope; 3) Commercial reagent to analyze the changes of mitochondrial function; 4) Mito green Tracker was used to track mitochondrial morphology; 5) Western blot analysis to measure the expression of mitochondrial-shaping protein; 6) Disruption of Drp1 by RNAi.【Results】1) Manganese induced PC12 cell injuryMTT assay and cell count showed that the cell viability is reduced when PC12 cell exposed to manganese, and this has concentration-time affection; cell morphology shows shrinkage, neurites shortened and floatation by microscope; the cell has a bad liveness by electron microscope.2) The mitochondrial fusion and fission play a role in manganese induced cell injury Manganese induced a time-dependent reduction in ATP and the inner mitochondrial membrane potential (?Ψm). And the activity of mitochondrial complexⅡandⅤdecreased, the activity of complexⅠ,ⅢandⅤchanges was not observed. At the same time, the mitochondrial morphology was analyzed by fluorescence microscopy, in control group without manganese showing that the normal morphology such as elongation or shortening or swelling, and when treatment with manganese, the elongated mitochondria decreased and the mitochondrial fragmentation is observed. Cell shrinkaged, retraction, cell bodies enlarged while neurites shortened or disappeared. The expression level of Drp1 increased gradually when mfn2 decreased. All this indicate mitochondrial fission take advantage in manganese induced PC12 cell injury.3) The important function of Drp1 in manganese neurotoxicityManganese has a significant effect on cell proliferation and mitochondrial fragmentation, which may has a relation with mitochondrial fission molecule Drp1. Disruption of Drp1 by RNAi, MTT assay show a marked increase in cell viability and cell proliferation decreased. The morphology of mitochondrial changed from fragmentation to normal, elongation is observed. It shows Drp1 play an important role in manganese induced cell injury.【Conclusions】1) Manganese induced cell injury and had a significant effect on cell proliferation. This may relate with the dysfunction of mitochondrial because mitochondrial dysfunction always associates with cell injury. Mitochondrial dysfunction display as ATP and the inner mitochondrial membrane potential reduced, the activity of mitochondrial complexⅡandⅤdecreased. 2) The mitochondrial fusion and fission play a role in manganese induced cell injury. When PC12 cell exposed to manganese, increased mitochondrial fission hastens mitochondrial fragmentation and cell proliferation.3) Drp1 may play an important role in manganese induced cell injury.In conclusion, manganese can selectively destroy DA neurons. In this progress, mitochondrial morphology changed and mitochondrial had dysfunction. And all this are related with mitochondrial fusion and fission, we can infer mitochondrial fusion and fission play an important role. We provide some evidence for manganese neurotoxicity investigation.