The TERT/p53 Signaling Pathway Regulates Axon Regeneration

ObjectiveAxon regeneration depends on intrinsic capacity and external environment.Intrinsic axon regenerative mechanisms are tightly regulated by various genes after nerve injury.The telomerase reverse transcriptase(TERT)acts as an important catalytic subunit of telomerase and the p53 gene is an important transcription factor.They can regulate various physiological activities such as cell proliferation,cell growth and apoptosis.In addition,studies have shown that there is a functional link between TERT and p53.Thus,this study is to explore the role of the TERT/p53 signaling pathway in axon regeneration after nerve injury.Methods1.In this study,the mouse sciatic nerve injury model was used.After 1,3 and 7 days of sciatic nerve injury,qPCR and western blot were applied to detect the mRNA and protein expression level of TERT and p53 in dorsal root ganglion(DRG)neurons.2.DRG neurons were cultured in vitro with chemical inhibitors or small interfering RNA(siRNA)to inhibit the expression of TERT and p53.After 3 days of culture,axons were labeled with neuron marker by immunofluorescence staining.The length was measured to determine the effect of TERT and p53 on axon regeneration.3.DRG neurons were cultured in vitro with specific activators to enhance the expression level of TERT and p53.After 2 days of culture,the neuron marker was used to specifically label the axons by immunofluorescence staining.The length of axons was measured to determine the effect of TERT and p53 on axon regeneration.4.Embryonic day 18(E18)hippocampal neurons or E15 cortical neurons were treated with either the p53 inhibitor PFTa or activator Tenovin-6.After 3 days of culture,the axons were specifically labeled with immunofluorescence staining and the length was measured to determine the effect of p5 3 on axon regeneration in hippocampal neurons and cortical neurons.5.A mixture of TERT-specific siRNA or pEX-3-p53 plasmid together with green fluorescent protein(GFP)plasmid was transfected into the fourth and fifth lumbar vertebrae(Lumbar4-5,L4-5)DRG neurons that connected to the sciatic nerve in vivo,and performed sciatic nerve crush injury two days later.Three days after injury,sensory axon regeneration was analyzed.6.Using the optic nerve injury model,adeno-associated viral vector encoding p53(AAV2-p53)was microinjected into the vitreous body.The AAV2-GFP viral vector was used as the control.Optic nerve crush injury was performed two weeks after microinjection.After twelve more days,Alexa 488-CTB(Alexa Fluor 488-conjugated cholera toxin B subunit)was microinjected into the vitreous body to label regenerating axons.After 2 days,the optic nerve was fixed in 4%paraformaldehyde,followed by gradient dehydration with 10%,20%and 30%(w/v)sucrose solution.After frozen sectioning,the axon regeneration of the optic never was observed under a fluorescence microscope.Results1.TERT regulates axon regeneration of sensory neurons.a.After sciatic nerve injury,the mRNA and protein expression of TERT in DRG neurons was significantly increased,and it was the most obvious on the third day,showing a trend of increasing first and then decreasing;b.Inhibition of TERT with a specific pharmacological inhibitor BIBR 1532 impaired regenerative axon growth of cultured DRG neurons in vitro:c.Importantly,inhibiting TERT activity with a specific siRNA effectively blocked axon regeneration of DRG neurons in vitro and sciatic nerve in vivo;d.In contrast,activation of TERT with specific activator,CAG,axon regeneration of DRG neurons cultured in vitro was significantly promoted.2.p53 regulates axon regeneration of sensory neurons and central neurons.a.The protein expression of p53 in DRG neurons was up regulated following sciaticnerve injury;b.After attenuating p53 activity using the specific membrane permeable inhibitorPFT?,axon regeneration of DRG neurons cultured in vitro was markedly inhibited.Axon regeneration in hippocampal neurons and cortical neurons showed a consistent trend with DRG neurons;c.Conversely,the specific p53 activator,Tenovin-6,significantly promoted axonregeneration in cultured DRG neurons in vitro.Similar results were also observed with hippocampal neuron and cortical neurons axon regeneration assay;d.Moreover,overexpression of p53 was able to enhance sensory axon regeneration in vivo;e.Intravitreal microinjection of AAV2-p53 viral vector significantly promoted axon regeneration after optic nerve injury.3.TERT regulates axon regeneration of sensory neurons via p53.a.In DRG neurons cultured in vitro,the specific TERT inhibitor BIBR 1532,which has been shown to reduce the protein expression level of TERT,also led to reduced p53 protein level;b.In DRG neurons cultured in vitro,the p53 activator Tenovin-6 counteracts the inhibition of sensory axon regeneration caused by knocking down TERT via siRNA.ConclusionAxon regeneration after nerve injury is a complex biological process that is strictly regulated by a variety of genes.In general,the relevant transcription factors are activated after nerve injury,ensuring the protein synthesis required during axon growth.This study shows that telomerase reverse transcriptase and transcription factor p53 play an important role in regulating the process of axon regeneration after nerve injury.In conclusion,this study not only demonstrates for the first time that TERT can regulate axon regeneration of sensory neurons,but also reveals that TERT regulates axon regeneration through p53.More importantly,p53 overexpression can significantly promote the axon regeneration after optic nerve injury.