Involvement Of TFEB In The Lead Exposure-induced Neuronal Impairment And Its Mechanism

Objective:The purpose of the present study was to investigate the lead-induced neuronal impairment by employing SD rats and Neuro-2a(N2a)cells,and to clarify the involvement of abnormal autophagy in the lead-induced neuronal apoptosis.The role of TFEB in the autophagy and apoptosis following the lead exposure will also be evaluated.Methods:1.Animal model of lead exposure.Rats in the lead exposure group were given 300 ppm lead acetate in drinking water for 5 weeks,and the control group was given deionized water.At the end of the lead exposure,the blood lead level was measured by atomic absorption spectrophotometry.Water maze test was performed to investigate the learning and memory ability of the rats in each group.The protein level of Caspase-3 were determined by Western blot.2.In vitro model of lead exposure.The N2a cells were treated by 50?mol/L lead acetate.CCK-8 method was used to detect the cell viability at different time points following the lead exposure.The protein levels of apoptosis related proteins were determined by Western blot.3.Western blot was carried out to evaluate the protein expression of autophagy-related proteins.Immunofluorescence was applied to observe autophagy of N2a cells after lead exposure.Following the treatment of 3-MA,an autophagy inhibitor,the effect of inhibiting autophagy on lead-induced apoptosis was observed.4.Western blot was employed to detect the effect of lead exposure on TFEB expression level in rat hippocampus and N2a cells.Following the knocking down of TFEB expression by siRNA transfection,the protein levels of autophagy and apoptosis related proteins were analyzed by Western blot.Results:1.Effect of lead exposure on apoptosis of neuronal cellsCompared with the controls,the levels of blood lead and brain lead in lead-exposed rats increased significantly(P<0.05).The results from the water maze test showed decreased spatial learning and memory ability in the lead-exposed rats.The results of Western blot showed that the cleavage of Caspase-3 significantly increased following the lead exposure.The results of in vitro studies also confirmed that the viability of N2a cells decreased in a time-dependent manner after lead exposure.TUNEL staining showed that lead exposure resulted in a significantly increased number of apoptotic cells,accompanying with elevated levels of cleaved Caspase-3 proteins.These results suggest that lead exposure could result in the apoptosis in neuronal cells.2.The role of autophagy abnormality in lead exposure-induced neuronal damageThe results of Western blot assay revealed the increase in Atg5 protein level and LC3?/LC3 ? ratio,and the results from immunofluorescence assays showed that LC3-positive autophagic vacuole in N2a cells significantly increased after the lead exposure.As shown by TUNEL staining and Western blot,the treatment with 3-MA reduced the lead-induced cell apoptosis and the expression of cleaved Caspase-3.These results suggested that inhibition of autophagy may alleviate lead-induced neuronal apoptosis.3.The role of TFEB on autophagy and apoptosis of neurons induced by lead exposureThe results of Western blot assay showed that the protein level of TFEB in both of the rat hippocampus and N2a cells significantly elevated following the lead exposure.TFEB siRNA transfection resulted in increased protein levels of LC3,Atg5,and the cleaved Caspase-3 in N2a cells,suggesting that TFEB may be involved in the regulation of autophagy and apoptosis after lead exposure.Conclusions:1.Lead exposure activate apoptosis and autophagy in rat hippocampus and N2a cells,and inhibition of autophagy can ameliorate neuronal apoptosis induced by lead exposure,suggesting that lead-induced autophagy plays a regulating role in neuronal apoptosis.2.Lead can induce increased expression of TFEB,and inhibiting TFEB expression can increase the autophagy and apoptosis induced by lead exposure,suggesting that TFEB regulates autophagy and apoptosis induced by lead exposure.