Src Stabilizes Golgi Apparatus Through ANLN-1 To Promote Neurite Formation Of Injured Neurons

The regenerative capacity of Central Nervous System?CNS?is much lower than that of PNS,because the axon extension capacity gradually shrinks during the maturation of neurons.An urgent problem is how to restore the regenerative capacity of injured mature CNS neurons.The phosphorylation of Src in injured neurons is inhibited,which affects the formation and growth of neurites.Activated Src can regulates the structure and subcellular location of the Golgi apparatus through F-actin assembly to avoid the dispersion of the Golgi apparatus.But Src mechanism is unclear.Activated Src family related protein YAP can promote the expression of ANLN and stabilize F-actin.ANLN can maintain the structural integrity of the Golgi apparatus through Septin.Knockout of ANLN disrupts outgrowth,guidance,and branching of neurite,indicating that ANLN regulates neuronal proliferation and neurite outgrowth.Our previous work showed that after 30 minutes of SCI,the Golgi apparatus dispersed and fragmented.We speculate that Src may stabilize the Golgi apparatus through ANLN and promote the neurite formation of damaged neurons.In the study,primary cortical neurons were injured by H₂O₂.To identify whether ANLN is a downstream molecule of Src that promotes neurite formation,Src activator was used to activate Src.ANLN RNAi was designed to interfere with the expression of neuronal ANLN.Using morphological and molecular biology techniques to clarify whether Src stabilizes Golgi apparatus through ANLN-1 and promotes the neurite formation.Methods:The MTT assay was used to determine the concentration of hydrogen peroxide.RT-PCR technology was used to detect changes in ANLN m RNA.An effective stimulator of Src activity was added after oxidative stress.Constructed RNAi technology was used to transfect cortical neurons.Immunohistochemical staining was used to observe the distribution of p Src and Golgi apparatus.Phalloidin was used to label F-actin to observe neuronal processes after ANLN interference.p Src and ANLN protein levels were evaluated by western blot analysis..Results:1.The establishment of oxidative stress injury model of neurons The viability of neuron was inhibited obviously by 100?m H₂O₂.The number of primary processes and branches complexity significantly decreased,increased after treated with Src activator.2.Effects of Src activation on ANLN-1,p Src and Golgi apparatus in H₂O₂ injured neurons Immunofluorescence staining of GM130 showed that Golgi apparatus dispersed,the length of Golgi apparatus was significantly longer in H₂O₂ group than that of the control group.The number of primary processes and branches increased significantly in H₂O₂+SA group.Western blot analysis showed that the level of p Src and ANLN in H₂O₂ group were significantly reduced.The expressions of p Src and ANLN increased significantly in H₂O₂+SA group.3.Src regulates the neurite formation of injured neurons by stabilizing the Golgi apparatus through ANLN-1Under oxidative stress,activation of Src,silencing ANLN,it was found that the expression of ANLN in the nucleus of H₂O₂+ANIi group and H₂O₂+ANIi+SA group was significantly reduced.Immunocytochemical staining showed that the p Src in the neurite end of the H₂O₂ group was significantly lower than that in the control group.thep Src expression in the neurite end of the H₂O₂+SA group was significantly higher than that in the H₂O₂ group;the p Src in the neurite end of the H₂O₂+SA+ANIi group was similar to that of H₂O₂+SA group,but it was significantly higher than that in the H₂O₂ group and the H₂O₂+ANIi group,suggesting that interference of ANLN didn't affect the activation of Src by the Src activator.Under oxidative stress,activation of Src,silencing ANLN,immunofluorescence results showed that GM130 was distributed in the nucleus of the control group.GM130 was dispersed,distributed in the cell body and processes in H₂O₂ group.GM130 was distributed in the nucleus of H₂O₂+SA group.Dispersion and disintegration of Golgi apparatus structure were in H₂O₂+ANIi group and H₂O₂+SA+ANIi group.Statistics on the length of the Golgi apparatus showed that the length in the H₂O₂ group was significantly higher than that in the control group;the length in the H₂O₂+SA group was significantly lower than that in the H₂O₂ group.That suggested that activation of Src could inhibit the dispersion of Golgi apparatus caused by oxidative damage.There was no significant difference in the length of Golgi apparatus between the SA+ANIi group and H₂O₂ group,but it was longer than H₂O₂+SA group,indicating that the interference of ANLN hindered the stabilization of Src on the Golgi apparatus.Statistic of the number of primary processes and branch complexity showed that the number of primary processes and branches in H₂O₂+SA group were significantly more complicated than those in H₂O₂ group.The number of primary processes and the complexity of branches in the H₂O₂+ANIi group and the H₂O₂+SA+ANIi group were significantly lower than those in the H₂O₂+SA group,suggesting that Src activation significantly promoted the formation of neurite and branch of injured neuron,and the interference of ANLN prevented Src from forming damaged nerve neuron protrusions.Conclusion:Under oxidative stress,Src mediates F-actin assembly through promoting the expression of ANLN-1 to stabilize the structure of Golgi apparatus and promote the formation of injured neuronal processes.