MTOR Activates Src Through CD44 To Promote The Formation Of Growth Cones And The Neurite Growth Of Injuried Neurons

After central nervous system injury,how to improve the ability of neuronal regeneration,promote axonal regeneration,and restore neural function is a major medical problem in the world.With the development of science,the PI3K/PTEN/Akt/m TOR signaling pathway has been found to have an important role for regeneration.But its mechanism is not clear.In this study,we are committed to finding downstream targets related to axonal regeneration.,Our previous study found that CD44 is a key downstream target for m TOR in regulation of axonal regeneration and growth cone formation.In the literature,we found that CD44 has a relation with Src.CD44 silencing can inhibit Src,and changes in dendritic morphology resulting from CD44 silencing can be restored by Src activation.Src is a downstream molecule that regulates dendritic morphology by CD44.The phosphorylated tyrosine protein(p-Src)at the apex of filopodia promotes the formation and elongation of filopodia.No relevant literature reported that m TOR regulates Src activity through CD44 and then regulates the formation of growth cones and the growth of neurites in injured neurons.To identify whether Src is a downstream molecule that promotes axonal regeneration in m TOR,SD rat neonatal cortical neurons were used as in vitro neuronal models.PTEN sh RNA,CD44 sh RNA,and PTEN+CD44 sh RNA silencing plasmids were designed to interfere with the expression of neuronal PTEN and CD44.The morphological and molecular biology techniques were used to elucidate that m TOR activates Src through CD44 and regulates the formation of growth cones and the growth of damaged neurons.Methods:To determine whether m TOR in cultured cortical neurons modulate Src activity through CD44,PTEN sh RNA,CD44 sh RNA,and PTEN+CD44 sh RNA interference plasmids were used to transfect cortical neurons to observe the changes of PTEN,CD44,Src m RNA and PTEN,m TOR,CD44,and Src protein levels.To further investigate whether Src is a downstream target of neuronal projections regulated bym TOR/CD44,an effective inhibitor of Src activity(PP2)was added after PTENi was silenced,then changes in p Src protein levels was observed.At the same time,in order to explore whether m TOR activates Src through CD44 on cortical neurons cultured in vitro,.Expression of p Src in injured neurons of PTENi group,PTENi+PP2 group and PTENi+CD44i group was observed by immunofluorescence staining with p Src.We used F-actin fluorescent specific marker phalloidin(F-actin)to observe the effect of Src on growth cone formation and neurite outgrowth of injured neurons,and to analyze the complexity of neuron projection differentiation.RESULTS:In vitro,the cultured cortical neurons were transfected with PTEN sh RNA,CD44 sh RNA,and PTEN+CD44 sh RNA gene interfering plasmids for 48 hours.The effective interfering sequences were screened and named PTENi,CD44 i,and PTENi+CD44i for follow-up experiments.Western blot technique showed that when PTEN was silenced,the expression of CD44 and Src increased(P<0.001);when CD44 was silenced,the expression of CD44 and Src decreased(P<0.001);when PTENi+CD44i double interference were used,the expression levels of PTEN,CD44,and Src decreased,but the expression of m TOR increased(P<0.001).Src inhibitor PP2 can inhibit PTENi silencing and induced increase in p Src activity(P <0.001).Statistical analysis of growth cones and processes with F-actin markers showed that activation of Src in silenced PTENi group promoted growth cone formation and neurite outgrowth in injured neurons but could be blocked by Src inhibitors or CD44 i silencing(P < 0.01).At the same time,Sholl analysis showed that silence of PTENi significantly increased the complexity neurite of damaged neuronal processes(P<0.001).Conclusion:Src is an important target to promote growth cone formation and neurite outgrowth.m TOR activates Src through CD44 to promote growth cone formation and neurite outgrowth.