The Role Of Autophagy Change In Manganese-induced Cytotoxicity Of PC12 Cells

【Background】Manganese is an element widely distributed in nature, and it's also a necessary trace element for human beings. However, excess exposure to manganese can cause several neural disorders and it is a well-recognized environmental health risk factor. It can cause certain extracorticospinal tract symptoms similar to those of Parkinson's disease. So the pollution of manganese and the health problem caused by it have been regarded as an important public health problem.Autophagy-lysosome pathway (ALP), which is responsible for the degradation of dysfunctional proteins and organelles, plays a critical role in the maintenance of internal environment homeostasis together with ubiquitin- proteasome pathway (UPP). Accumulated evidence in the past few years has shown that the disfunction of ALP plays a key role in the pathology of several neurodegenerative diseases. The inhibition of ALP can lead to the accumulation of several toxic proteins in neurons, which further leads to the exacerbation of pathological changes. On the contrary, over-activated ALP can also lead to the degeneration of neurons, especially in certain neurotoxins-induced toxicity.【Aims】In the present study, we have investigated the effect of manganese on autophagy and the effect of autophagy change in manganese-induced neurotoxicity. Our data may shed new light for the mechanisms of manganese-induced neurotoxicity and pave a new way for the prevention of neurotoxicity induced by manganese.【Methods】(1) An in vitro model of manganese-induced neurotoxicity was set by using rat pheochromocytoma PC12 cell line. (2) MTT assay, LDH activity analysis, and flow cytometry were used to determine the viability and cytotoxicity. (3)The change of autophagy was detected by electron microscopy, immunofluorescence, and Western Blot. (4) Western Blot was used to determine the expression of autophagy-targeted proteinα-synuclein.【Results】1. The in vitro model of manganese-induced cytotoxicity. The results of MTT showed that manganese inhibited the cell viability of PC12 cells in a concentration-dependent manner. The result of LDH indicated that the injuries of PC12 cell induced by manganese were enhanced with the increase of manganese concentration. Furthermore, as detected by flow cytometry, the death rate of PC12 cells was enhanced with the increment of manganese concentration.2. Manganese can induce the change of autophagy in PC12 cells.In our present study, manganese induced the change of autophagy. As shown by electron microscopy results, manganese of 300μmol/L induced typical ultrastructural characteristics after 24 h treatment. The results of MDC staining and EGFP-LC3 transfection also showed typical morphological changes of autophagy. Moreover, the ratio of LC3-II/LC3-I, a microtubule-associated protein, and the expression of Beclin 1, an autophagy-related protein, were enhanced at both 12h and 24h. Furthermore, we also found the enhanced autophagy was due to the increased autophagic vacuole generation rather than the repression of lysosomal function. The generation of autophagic vacuoles was inhibited by 3-MA, an inhibitor of autophagy. On the other hand, the addition of lysosomal inhibitor Leu led to the increase of autophagic vacuoles.3. The role of autophagy in manganese-induced cytotoxicity in PC12 cellsThe MTT, LDH, and flow cytometry results showed that the enhancement of autophagy alleviated manganese-induced viability inhibition, LDH release, and all death while the inhibition of autophagy intensified manganese-induced cytotoxicity. Moreover, the treatment of manganese also led to the overexpression ofα-synuclein, which was enhanced by the inhibition of autophagy and alleviated by the enhancement of autophagy. 【Conclusions】1. While leading to the inhibition of cytotoxicity, manganese can induce the change of autophagy.2. The enhanced autophagy was mainly due to the increased autophagic vacuole generation rather than the repression of lysosomal degradative function.3. Manganese-induced autophagy can be a protective mechanism against its toxicity. The enhancement of autophay can inhibit manganese-induced neurotoxicity while the inhibition of autophagy can promote such neurotoxicity. The regulation of autophagy may be a potential strategy for the prevention and treatment of manganese-induced neurotoxicity.4. The inhibition of autophagy can promote the cytotoxicity through its promotive effect on the accumulation ofα-synuclein while the enhanced autophagy can exert its protective effect through the promoted degradation ofα-synuclein.Taken together, our research has been focused on the change of autophagy in PC12 cells treated by manganese. We have also determined the role of such change in manganese-induced neurotoxicity. Our data may further our understanding of manganese-induced neurotoxicity and pave a new way for the prevention of it.