The Role And Mechanism Of CD82 In Regulating Glaucomatous Axonal Transport Deficits Of The Optic Nerve

PartⅠ:The role of CD82 in regulating axonal transport deficits of the optic nerve in acute ocular hypertension mouse modelPurpose:At present,there are limited treatments for glaucomatous optic nerve damage,and its specific mechanism is still unclear.Axonal transport deficit is the early pathological change of the optic nerve injury in glaucoma.Timely intervention of the optic nerve axonal transport disorder caused by intraocular hypertension could effectively alleviate the outcome of the optic nerve axonal injury and RGCs death.CD82 is an important member of the tetraspanin superfamily,which is involved in the regulation of the cytoskeleton and interacts with multiple signaling pathways in cells.It participates in the regulation of various physiological functions in cells.Currently,the role of CD82 in the nervous system is not clear.The purpose of this study is to observe the characteristics of optic nerve axonal transport deficts and the expression pattern of CD82 in a mouse model of acute ocular hypertension（AOHT）,and to explore whether CD82 overexpression could protect against the optic nerve axonal transport deficits as well as the outcome of axonal degeneration and ganglion cell death in the AOHT model.Methods:Acute ocular hypertension was induced in C57BL/6 mice by anterior chamber puncture and infusion of saline with high hydrostatic pressure.Mice were sacrificed at 8h,1d,2d,3d and 7d after modeling.Axonal transport function of the optic nerve was evaluated by intravitreal injection of fluorescently labeled cholera toxin B（CTB）as well as immunofluorescence staining and western-blot analysis of Synaptophysin in the optic nerve head.The expression level of CD82 was detected by immunofluorescence staining of tissue sections and western-blot analysis of tissue lysate.CD82 overexpression in eyeball tissues was achieved by intravitreal injection of recombinant adeno-associated virus overexpressing CD82（AAV-CD82）,and the axonal transport of CTB and Synaptophysin in the optic nerve at each time point after AOHT model was detected after virus injection.The axonal degeneration of the optic nerve was assessed by transmission electron microscopy imaging and Aβimmunohistochemical staining.The number of retinal nerve fibers and RGCs was assessed according to the immunofluorescence staining of Tuj1 and Rbpms in the retinal flat-mount.Retinoic acid-induced differentiated SH-SY5Y cell line and the primary hippocampal neurons were used for in vitro cell experiments with application of H₂O₂ to induce neurite damage.CD82 overexpression was achieved by plasmid transfection to verify its protective effect on neurite growth.Results:AOHT mouse model was successfully constructed,and the axonal transport deficits manifested as shortening of CTB transport distance in the optic nerve and accumulation of Synaptophysin in the optic nerve head.Axonal transport impairments started to appear at 8h after AOHT model,were most obvious at 2d,and recovered at 7d.The expression level of CD82 gradually decreased from 8h to 2d after AOHT model,and mildly recovered at 3d and later.After AAV-CD82 injection,the axonal transport disorders of CTB and Synaptophysin were significantly alleviated at all time points after AOHT modeling.At 7d after AOHT modeling,there were obvious destruction of axonal ultrastructure under transmission electron microscope and increased positive immunohistochemical staining of Aβin the optic nerve as well as significant reduction in nerve fiber and RGC counts in retinal flat-mount.After CD82 overexpression,the above axonal degeneration and RGC loss were significantly alleviated.In vitro experiments showed that the H₂O₂ injury model induced shortened neurite length in SH-SY5Y cells and primary hippocampal neurons,while CD82 overexpression had a protective effect on such injury.Conclusion:The optic nerve axonal transport deficits appeared at the very early stage of the AOHT model,and became gradually worsen with time extension,and then slightly recovered spontaneously.The expression level of CD82 was first down-regulated after AOHT model and then mildly increased over time,which was correlated with the spatiotemporal characteristics of the axonal transport disorders.Overexpression of CD82displayed protective effect on optic nerve axonal transport disorders and the subsequent axonal degeneration and loss of RGCs induced by AOHT model.Part Ⅱ:CD82 protected against axonal transport deficits via mTORC1 activation in AOHT mouse modelPurpose: The mTORC1 pathway is crucial for maintaining the normal physiological function of nerve cells.The mTORC1 pathway is closely related to optic nerve injury,and the activation of which can promote the survival of RGCs and the regeneration of axons under injury conditions.Some studies have suggested that there was an association between tetraspanins and mTORC1 pathway,but the interaction between the two in optic neuropathy remains unclear.The purpose of this study is to clarify the molecular mechanism of the neuroprotective effect of CD82,and to explore whether it exerts neuroprotective effect by activating the mTORC1 pathway.Methods: HEK-293 T cell line was used as a tool for molecular mechanism research.Cell damage was induced by H2O2 application and CD82 overexpression was achieved by plasmid transfection.Western-blot analysis was performed to detect the phosphorylation level of p70S6 K,a downstream substrate of mTORC1 pathway.The cells were treated with rapamycin,a specific mTORC1 pathway inhibitor,and then the effect of CD82 overexpression on the phosphorylation level of p70S6 K was detected by western-blot analysis.Acute ocular hypertension（AOHT）mouse model was established,and AOHT 2d was selected as the observation point.AAV-CD82 was injected into the vitreous cavity to induce CD82 overexpression.Immunofluorescence staining of p-S6 was performed to detect the activation of the mTORC1 pathway.Two methods were used to intervene mTORC1 pathway in vivo: intraperitoneal injection of the inhibitor rapamycin and intravitreal injection of AAV-hsyn-Cre in the Rptor fl/fl transgenic mice for ganglion cellspecific knockdown of Raptor.After the mTORC1 pathway inhibition,p-S6 and Tuj1 immunofluorescence co-staining was performed to indicate the activation of the mTORC1 pathway in RGCs.Intravitreal injection of CTB and the immunofluorescence staining of Synaptophysin were used to evaluate the optic nerve axonal transport function after mTORC1 pathway intervention.Results: In HEK-293 T cells,western-blot analysis showed that H2O2 stimulation induced a decrease in the phosphorylation level of p70S6 K,and overexpression of CD82 could reverse such decline.After rapamycin treatment in cells,the up-regulation effect of CD82 on p70S6K phosphorylation was blocked.In the AOHT mouse model,the immunofluorescence staining of p-S6 in Tuj1-positive RGCs was significantly reduced compared with that in the sham group.The p-S6 positive staining was recovered after CD82 overexpression,while the activation effect of CD82 on p-S6 disappeared after the application of rapamycin.Under mTORC1 pathway inhibition in Raptor knockdown mice,the protective effect of CD82 overexpression on CTB and Synaptophysin transport disorders was significantly diminished.Conclusion: In both in vitro and in vivo experiments,the mTORC1 pathway was inactivated under injury state.CD82 overexpression could reverse the mTORC1 pathway inactivation caused by injury.CD82 overexpression protected against optic nerve axonal transport deficits via mTORC1 activation.Part Ⅲ:CD82 exerted neuroprotective effects by upregulating TRAF2 thus activating the mTORC1 pathwayPurpose: CD82 is a transmembrane protein,and mTORC1 is an important intracellular signaling hub.There is no direct binding between CD82 and mTORC1.Previous studies have confirmed that CD82 overexpression activated the mTORC1 pathway,but the way how it induces the mTORC1 pathway activation remains to be further explored.The purpose of this study is to clarify the molecular mechanism of CD82 activating mTORC1 pathway: to find the intermediate molecules between CD82 and mTORC1 and to verify that CD82 mediates the activation of mTORC1 pathway through the intermediate molecule TRAF2 as well as to explore the specific mechanism of TRAF2 regulating mTORC1 pathway activity.Methods: Bioinformatics technology and the protein interaction network database were used to search for the relationship between CD82 and mTORC1.In HEK-293 T cells,H2O2 was used to induce cell damage and CD82 overexpression plasmid was transfected.RTPCR and western-blot analysis were used to detect the expression level of TRAF2.As for the animal experiments,retinal sections from AOHT 2d group were used for immunofluorescence staining of TRAF2.In HEK-293 T cells,small interfering RNA（si RNA）was used to inhibit the expression of TRAF2,western-blot experiments were performed to detect the phosphorylation level of p70S6 K.The combination of TRAF2 and Raptor was verified by co-immunoprecipitation.After interference with TRAF2,the K63 ubiquitination level of Raptor was detected by immunoprecipitation and western-blot analysis.The immunofluorescence co-staining of Raptor with the lysosomal marker Lamp1 was performed in cells to detect its lysosomal localization.The neurite outgrowth assay was performed in differentiated SH-SY5 Y cells and the primary hippocampal neurons under H2O2 injury with TRAF2 interference and CD82 overexpression.Results: The protein interaction network database suggested that TRAF2 had the strongest comprehensive association with CD82 and mTORC1.In HEK-293 T cells,RT-PCR and western-blot results showed that the m RNA and protein levels of TRAF2 were downregulated under H2O2 stimulation,and overexpression of CD82 could reverse such decrease.In AOHT mouse model,the immunofluorescence staining intensity of TRAF2 in the retinal ganglion cell layer was significantly reduced compared with that of the sham group.After overexpression of CD82,the down-regulation of TRAF2 induced by modeling was restored.After interfering with TRAF2,the CD82-mediated up-regulation of p70S6 K phosphorylation level was eliminated.Co-immunoprecipitation experiments showed that TRAF2 and Raptor were combined,and after interfering with TRAF2,the K63 ubiquitination modification of Raptor as well as its immunofluorescence colocalization with Lamp1 were reduced.The neurite outgrowth experiments in SH-SY5 Y cells and the primary hippocampal neurons showed that the promotion of neurite outgrowth by CD82 overexpression was inhibited by interfering with TRAF2.Conclusion: CD82 overexpression could up-regulate TRAF2 expression level.TRAF2 mediated the activation of the mTORC1 pathway by catalyzing Raptor for K63 ubiquitination and increasing its lysosomal localization.CD82 exerted neuroprotective effects through upregulation of TRAF2 which mediated the activation of mTORC1 pathway.