驱动蛋白家族成员5A介导的溶酶体功能在镉神经毒性中的作用

Effect of KIF5A mediated lysosomal functions in cadmium exposure-induced neurotoxicity in vitro

目的探讨驱动蛋白家族成员5A(kinesin family member 5A,KIF5A)介导的溶酶体功能损伤在镉神经毒性中的作用。方法以原代培养的SPF级C57BL/6J小鼠皮层神经元为模型,分为对照组和镉暴露组。检测氯化镉暴露24 h后皮层神经元的半数抑制浓度(IC_(50))。进一步用1、2、3μmol/L氯化镉处理神经元24 h,检测细胞存活率、神经元分化和溶酶体功能。RT-qPCR和Western blot检测KIF5A mRNA和蛋白的表达。腺病毒过表达KIF5A检测镉暴露后神经元的溶酶体功能、分化及细胞存活率。结果神经元暴露氯化镉24 h的半数抑制浓度IC_(50)为2.9μmol/L。与对照组相比,2、3μmol/L镉暴露24 h后神经元细胞活力受到抑制(P<0.01),突起生长的总长度和分枝点数目显著减少(P<0.01)。此外,与对照组相比,2、3μmol/L氯化镉处理后显著抑制神经元溶酶体组织蛋白酶B(cathepsin B,CTSB)的活性和改变其酸性环境(P<0.01),但是对组织蛋白酶D(cathepsin D,CTSD)活性没有影响。镉暴露后KIF5A的mRNA及蛋白表达显著降低(P<0.01)。过表达KIF5A可以显著改善镉暴露对神经元组织蛋白酶活性CTSB的抑制、稳定镉暴露造成的溶酶体pH值改变,拮抗镉暴露对神经元细胞活力及突起生长的抑制作用(P<0.01)。结论镉暴露显著减少神经元中KIF5A蛋白的表达,从而损伤溶酶体功能,抑制神经元细胞活力及突起生长。

Objective To investigate the effect of kinesin family member 5 A(KIF5 A) mediated lysosomal functions in cadmium(Cd) exposure-induced neurotoxicity. Methods Primary cortical neurons isolated and primarily cultured from specific pathogen free(SPF) C57 BL/6 J mice were used and divided into control and Cd-exposed groups. The 50% inhibitory concentration(IC_(50)) of cortical neurons after 24 h of Cd exposure was measured. Furthermore, the neurons were treated with 1, 2 and 3 μmol/L cadmium chloride(CdCl;) for 24 h, and then cell viability, neuronal differentiation and lysosomal function were measured. RT-qPCR and Western blotting were used to detect the changes of KIF5 A expression at mRNA and protein levels. The effects of overexpression of KIF5 A by adenoviral vector on cell viability, neuronal differentiation and lysosomal function were also investigated in the Cd-exposed cortical neurons. Results The IC_(50)value in neurons exposure to CdCl;for 24 h was determined to be 2.9 μmol/L. Compared with the control group, the cell viability was inhibited(P<0.01) and the total length of protrusion growth and the number of branching points were obviously reduced(P<0.01) after 2 and 3 μmol/L CdCl;exposure for 24 h. In addition, 2 and 3 μmol/L CdCl;treatment also resulted in dramatically decreased lysosomal activity of cathepsin B(CTSB) and altered acidic environment when compared with the control group(P<0.01). However, the activity cathepsin D(CTSD) was not significantly changed in CdCl;-exposed neuronal cells. More importantly, the mRNA and protein levels of KIF5 A were remarkably decreased after Cd exposure(P<0.01). Overexpression of KIF5 A effectively increased the CTSB activity, stabilized the lysosomal pH value, and antagonized the inhibitory effect on cell viability and protrusion growth induced by Cd exposure(P<0.01). Conclusion Cd exposure significantly reduces KIF5 A protein expression in neurons, and thereby impairs lysosomal function and inhibits neuronal cell viability and protrusion growth.