MTOR Signaling Pathway Promotes Neurite Outgrowth Of The Injuried Neuron-like Cells Through CD44

After central nervous system injury, because of ischemia, oxygen deficit and inflammation, it’s inevitable to cause degeneration and necrosis of neuron and myelin sheath. Due to the low regeneration capacity of neurons, axon is difficult to regenerate once injured. For decades, scientists are looking for the obstacle factors of axon regeneration after central nervous system injury, and the way to promote axon regeneration. More and more evidences proved that neuron’s regeneration capacity is an indispensable key factor of regeneration. Several studies have shown that the activity of mTOR signaling pathway is a key factor of axon growth ability, and that the lack of mTOR activity is the main reason for the barrier to axon regeneration, in injured CNS neurons. mTOR pathway is an important signaling pathway which regulates nerve regeneration, however, its mechanism remains elusive.To explore this mechanism, we induced the differentiation of human neuroblastoma cells（SH-SY5Y） into cell with the characteristics of mature neurons in vitro.SH-SY5 Y is similar to normal neural cells in cell morphology, physiology and biochemical functions. SH-SY5 Y also has obvious axons. Then we established neuron like cell damage model by H₂O₂ induction. mTOR patyway was activated by silenting PTEN gene using siRNA. After that, by applying morphology and molecular biology techniques, we elucidate that mTOR signaling pathway enhances neuron’s inherent regeneration capacity by CD44, participating in the outgrowth of axonal growth cone after central nervous system injury and thus promoting the regeneration of the damaged axons.Methods: SH-SY5 Y cells is cultivated in vitro and induced to differentiation with10 μM ATRA into neuron like cells. The concentrations of H₂O₂ were characterized by MTT method to establish oxidative damage model. On the basis of oxidative damage,we used PTEN specific siRNA transfected into cells to silence with PTEN. There are four groups: the control group, the H₂O₂ group, the H₂O₂+NC group and the H₂O₂+PTEN-RNAi group. To deternine the transfection efficiency of each group, the cells were exposed to fluorescence microscopy to detect the fluorescence intensity.Using RT-PCR, we detect the transcription of CD44 mRNA and PTEN mRNA. The expression level of CD44 protein, mammalian target of rapamycin（mTOR） as well asthe phosphorylated ribosomal（p S6k） protein were assayed by western blot. The F-actin was labeled by Phalloidin to observe the growth cone and the changes of neurites.Results: SH-SY5 Y induced by 10 μM ATRA for 72 hours can lead to neurite growth and enhanced differentiation compared with the non-induced cells. When the concentration of H₂O₂ was 50 μM and 100 μM induced cell for 24h, the cell morphology and survival rate were almost unchanged compared with that of control group. 200 μM H₂O₂ could change the cell morphology and survival rate. Compared to the control group, 200 μM H₂O₂ leaded to cell shrinkage, cytoplasmic refractive change, fuzzy boundary, shortened neurites and cell death. At the same time, MTT tests displayed that the survival rate was 74%. Therefore, we successfully established the oxidative stress injury cell model using 200 μM H₂O₂. On the basis of the model,we specific silence the gene of PTEN. The results showed that, compared with the control group, the H₂O₂ group and the H₂O₂+NC group, PTEN mRNA expression reduced significantly（P<0.01）in the PTEN interference group. On the other side,mTOR and p S6 k protein expression level were increased significantly（P<0.05）. The CD44 mRNA transcription level and CD44 protein expression level in the PTEN interference group increased significantly compared to the noninterference group（P<0.05）. The number of the filopodia of every growth cone was count and the length of cell neurite in each group was measured, and then statistical analysis were performed.The results showed that the control group had more neurites, and the growth cones were patchy and filamentous. The growth cones of the H₂O₂ damage group was shrunk into a spherical shape at the end of the cell neurite. The numbers of filopodia decreased significantly（P < 0.05）, and the neurite lengths were significantly shorter than the control group. The growth cones showed no obvious change between the H₂O₂+NC group and the H₂O₂ damage group, almostly spherical or rod-shape. While compared to the control group, the numbers of the filopodia reduced significantly（P <0.05）. Neurites in the H₂O₂+PTEN-RNAi group were longer and almost all growth cones were branched. The globular cones have filopodia and the numbers of filopodia increased significantly（P < 0.05） compared to the H₂O₂ group and the H₂O₂+NC group.When the expression of PTEN and CD44 were silenced at the same time, the results showed that mTOR and p S6 k protein expression level increased significantly,and that CD44 mRNA transcription and protein expression of CD44 was significantlyreduced（P<0.05）. The results also showed that the growth cone had no significant difference compared to the H₂O₂ group. The number of the filopodia was significantly less than that of the PTEN interference alone group（P < 0.05）.Conclusion: After the oxidative stress injury of neuron-like cells, activation of mTOR signaling pathway can up-regulated expression of CD44 to promote the growth of the growth cone and then to promote neurite outgrowth.