Zinc Dyshomeostasis-induced Golgi Fragmentation Is Involved In The Damage Of HT22 Cells

[Background and Objective]Intracellular zinc homeostasis is maintained by a dynamic process of zinc influx,efflux,and reservation.Zinc dyshomeostasis,including abnormally increase or decrease of intracellular zinc,leads to neuronal damage,which in turn causes neurodegenerative diseases?NDDs?.The integrity of the Golgi structure is necessary for the normal function of cells.Golgi fragmentation is commonly observed in NDDs.Zinc directly participated in the interaction between GRASP55 and Golgin45,which is indispensable for Golgi cisternae stacking.In this study,we used mouse hippocampal neuron HT22 cells to explore whether zinc dyshomeostasis induces nerve cells damage through breaking the Golgi apparatus via disrupting the formation of the GRASP55-Golgin45 complex.[Methods]1.The cell viability of HT22 cells was detected by CCK8.2.The relative fluorescence intensity of Zn²⁺in HT22 cells was evaluated by Zinpyr-1 fluorescence dye.3.The colocalization of GRASP55 and Golgin45 and the interaction between GRASP55 and Golgin45 in HT22 cells were detected by immunofluorescence and co-immunoprecipitation.4.Golgi fragmentation was observed using Golgi-Tracker Red and GRASP55 fluorescence.5.The expression of GRASP55 and Golgin45 was detected by western blotting.[Results]1.Zinc deficiency promotes Golgi fragmentation by inhibiting the formation of GRASP55-Golgin45 complex and induces HT22 cells damage1.1.After treatment of HT22 cells with TPEN?2,4,8?M?for 10 h,the relative fluorescence intensity of Zn²⁺was significantly reduced.1.2.After treatment of HT22 cells with TPEN?2,4,8?M?for 10 h,the cell viability of HT22 cells was decreased.1.3.The co-localization and the interaction between GRASP55 and Golgin45 in HT22 cells were significantly decreased by treatment with TPEN?4?M,for 10 h?.However,TPEN did not affect the expression of GRASP55 and Golgin45 proteins in HT22 cells.1.4.After treatment of HT22 cells with TPEN?4?M?for 10 h,the Golgi apparatus was significantly fragmentated.2.Rotenone-induced HT22 cells damage is involved in increasing zinc concentration and Golgi fragmentation2.1.After treatment of HT22 cells with ROT?0.5,1,2?M?for 24 h,the cell viability of HT22 cells was significantly decreased.2.2.After treatment of HT22 cells with ROT?0.5,1,2?M?for 24 h,the relative fluorescence intensity of Zn²⁺was obviously increased.2.3.After treatment of HT22 cells with ROT?0.5,1,2?M?for 24 h,the formation of GRASP55-Golgin45 complex and the expressions of GRASP55 and Golgin45 were not changed.2.4.After treatment of HT22 cells with ROT?0.5,1,2?M?for 24 h,the Golgi apparatus were significantly fragmentated.[Conclusion]1.Zinc deficiency promotes Golgi fragmentation by inhibiting the formation of GRASP55-Golgin45 complex and induces HT22 cells damage2.Rotenone promotes Golgi fragmentation by increasing zinc concentration and induces HT22 cells damage.