Modulation Of The Rheb/mTOR Pathway Promotes Neuroprotection And Axon Regeneration After Optic Nerve Injury

Purpose:The mammalian central nervous system lacks the ability to regenerate spontaneously,and damage to the optic nerve leads to irreversible death of retinal ganglion cells(RGCs)and axon atrophy,for which there is currently no cure.Rheb(Ras homolog enriched in brain)is a small GTPase that activates m TORC1(mammalian target of rapamycin complex 1).Dysregulation of Rheb was found in several neurodegenerative diseases and Rheb gene therapy showed protective effect in Alzheimer’s and Parkinson’s disease models.The aim of this study was to investigate the neuroprotective effect of Rheb gene therapy after optic nerve injury.Method:1.Adeno-Associated Virus Epitope 2(AAV2)was used to deliver constitutively active(constitutively active)Rheb gene to mice RGC by intravitreal injection,and its effects on RGC survival,axon regeneration and visual function were observed by retinal flat-mount,optic nerve section,flash visual evoked potential(FVEP)at different time points after optic nerve injury.Statistical data were compared between two groups using unpaired t-test,and multiple groups were compared using one-way or two-way ANOVA.2.The mode of action of Rheb-enhanced treatment was determined by inhibition of m TORC1 activity using rapamycin.The effect of Rheb overexpression on downstream molecules was determined by Western Blotting.3.Loss-of-function and gain-of-function assays were performed on4E-BP1(eukaryotic translation initiation factor 4E binding protein 1)and S6K1(ribosomal protein S6 kinase 1)downstream of the Rheb/m TOR pathway to elucidate the roles played by both in RGC injury and regeneration,respectively.Result:1.It was found that Rheb augmentation treatment activate the m TOR pathway in RGC and increased RGC survival(especially non-α-RGC subtypes),produced strong optic nerve regeneration and improved P-wave amplitude of FVEP at different time points after injury.Also,the results showed that conditional knockout of Rheb gene in mouse RGC significantly exacerbated RGC cytosolic death and axonal atrophy after optic nerve injury.2.The neuroprotective and pro-regenerative effects of Rhebenhanced treatment were m TOR-dependent.Rheb-enhanced treatment promoted 4E-BP1 and S6K1 phosphorylation downstream of m TORC1.3.Numbers of regenerating axons were significantly reduced by coexpression of Rheb with either the dominant negative S6K1 mutant(DNS6K1)or the constitutively activated 4E-BP1 mutant(4E-BP1-4A).This suggested that m TORC1-mediated S6K1 activation and 4E-BP1 inhibition are essential for Rheb induced axon regeneration.However,while the constitutively active S6K1 mutation(ca S6K1)was able to induce axon regeneration alone,we observe no regeneration by 4E-BP1 knockdown.Furthermore,S6K1 activation promoted RGCs survival after injury,whereas knockdown of 4E-BP1 unexpectedly reduced RGC survival,and overexpression of constitutively active 4E-BP1 promoted RGCs survival.These results suggested that the two downstream effectors play different roles in regulating the survival and regeneration of RGCs.Conclusion: In this study,we found that Rheb gene therapy displayed RGC protective effect and significantly promoted axon regeneration after optic nerve injury by manipulating downstream effectors S6K1 and 4E-BP1.