The Mechanism Of Amino-Nogo Inhibit Optic Nerve Regeneration

Eyes are organs detecting light and converting it into electro chemical impulses inretina. Retinal ganglion cells (RGCs) axons form the optic nerve (ON), injury of which hasbeen one of the causes of visual loss. Although ON injury accounts for only smallproportion of visual loss, the impaired vision is in general not recoverable. After injury, ONis unable to regenerate, which mainly because that RGCs retrogradely degenerate followinginjury and lack the ability of regenerating axons. Studies showed that the failure of axonsregeneration in adult mammalian central nervous system was partly due to the presence ofendogenous inhibitors of axon regeneration, which include Nogo A, myelin associatedglycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMGP). Among thoseendogenous inhibitors, Nogo A has received much attention. Nogo A is a myelin associatedinhibitor, the function of which depends on its two inhibitory domains:66amino aciddomain (Nogo66) and N terminal domain (Amino Nogo). The Nogo66domain bothinhibits axonal regeneration and regulates axonal growth, guidance and CNS plasticityneurogenesis. Conversely, Amino Nogo only exerts an inhibitory effect. However, the roleand the mechanism of Amino Nogo in the optic nerve have not been fully elucidated.Integrins are heterodimeric cell surface glycoproteins composed of α and β subunitsthat are non covalently connected. Integrins bind ligands in the extracellular matrix andform adhesion complexes that couple to the actin cytoskeleton. These complexes arenecessary for cellular expansion and axonal growth. Amino Nogo inhibits cell adhesion andaxonal outgrowth by inhibiting integrins, and the effect of Amino Nogo is related todifferent integrin subunits in axonal outgrowth. Integrins αv and α5, are widely expressedin the CNS, however, which integrin subunit is the main player in the Amino Nogo–integrinsignaling pathway operating in axonal outgrowth remains unknown.The study is divided into three parts:1、Investigating the proteins of Amino Nogosignalling pathway expression in the optic nerve system.2、Investigating the inhibitoryeffect of Amino Nogo on optic nerve regeneration.3、Investigating the Amino Nogo inhibiting RGC axonal outgrowth primarily through the integrin αv signaling pathway, butnot integrin α5signaling pathway.Aim: To regulat the Amino Nogo to confirm that the Amino Nogo inhibits nerveregeneration. To investigate the mechanism of Amino Nogo inhibiting optic nerveregeneration. To provide a new strategy for the treatment of optic nerve damage, as well asto provide reference for central nervous system injury repair research.Methods: In this study we detected the expression of integrin αv, integrin α5andfocal adhesion kinase (FAK) by immunohistochemistry. Screening for siRNA sequencetargeting rat Nogo A mRNA in vitro. Production and purification of RecombinantAdenoassociated VirusVectors (rAAV). rAAV2/8NC siRNA, rAAV2/8Nogo A siRNA, apeptide antagonist of Nogo66of Nogo A (Nep140), and a recombinant rat Nogo A Fcchimeric protein (△20) were added in the primary RGCs. The axonal length of RGCs wasassayed by Thy1immunofluorescence stained. We injected PBS, rAAV2/8NC siRNA,rAAV2/8Nogo A siRNA, Nep140and△20in vitreous cavity of the SD rats following opticnerve crush injury and observed the RGCs survival rate by retrograde fluorogold labeling,the number of regenerating axons by GAP43immunofluorescence stained and the opticnerve function recovery by F VEP recording. Western Blot assayed the expression changesof integrin αv, integrin α5and FAK. RhoA activition was measured by RhoA G LISA.The main results and conclusions are as follows:1、Integrin αv, integrin α5and focal adhesion kinase (FAK) were widely expressed inthe visual cortex, retina and optic nerve by immunohistochemical staining. TheAmino Nogo–integrin signaling pathway related key proteins expressed in the visualsystem indicating that Amino Nogo plays a role may through the integrin signaling pathway.This study establishes the foundation for further experiments.2、Our test showed the siRNA targeting to Nogo A could efficiently down regulatedNogo A expression in RGCs, but the control siRNA had no influence on the Nogo A level,which successfully screened effective RNAi sequence and laid the foundation forconstructing the recombinant virus vector. The recombinant virus vector could efficientlyinhibit the Nogo A protein of primary RGCs in vitro. In vivo, the inhibition has beenexisted4weeks after virus injection and maintained till8weeks in retinal ganglion celllayer, which confirmed it had been integrated into RGCs genome and might have longer inhibition3、In vitro test showed Nogo66antagonist peptide (Nep1–40) promoted RGCsoutgrowth, which indicated Nogo66functional domains can inhibit the outgrowth of RGCs.In addition, Nogo siRNA also promoted axonal outgrowth of RGCs and the length of RGCsaxons were longer than Nep140group, which suggested that except Nogo66functionaldomains can inhibit axonal growth, its other functional domain, Amino Nogo domain, canalso inhibit the outgrowth of RGCs. The recombinant rat Nogo A Fc chimeric protein (△20)can significantly inhibit the RGCs outgrowth, further indicated that Amino Nogo played aninhibitory effect.4、In vivo test showed Amino Nogo inhibited the survival rate of RGCs by fluoroGoldretrogradely labeled RGC neurons by injecting, and Amino Nogo inhibited nerveregeneration following the optic nerve injury by growth associatted protein43(GAP43)immunofluorescence staining. The F VEP recording also demonstrated that Amino Nogowas harmful to the recovery of optic nerve function. All of the above results suggest thatAmino Nogo inhibits optic nerve regeneration and functional recovery.5、In vitro and in vivo experiments showed Amino Nogo inhibited optic nerveregeneration through the integrin αv signaling pathway by observing the expressionchanges of integrin αv, integrin α5, FAK and RhoA, which were Amino Nogo signalingpathway downstream proteins.