| Background:Primary glaucoma has became the second leading cause of blindness worldwide,which characterized by loss of vision,optic nerve damage and loss of visual field.Once RGCs degenerate,it is difficult to reverse.How to prevent and reverse the degeneration of RGCs has been a difficult problem in glaucoma treatment.Currently,the exact pathogenesis of glaucoma is not completely understood.Elevated intraocular pressure(IOP)has been considered as a major risk factor for glaucoma and is the only factor that can be controlled in clinical practice.But elevated intraocular pressure is neither a necessary factor nor a sufficient factor for glaucoma.However,vision and the visual field gradually deteriorate even in the presence of stable IOP in some patients.RGCs degeneration in glaucoma is regarded as a result of degeneration and apoptotic mechanisms triggered by multiple factors,including elevated IOP,glutamate excitotoxicity,ischemia,oxidative stress,hypoxia,and deficiency of neurotrophic factors and so on.RGCs degeneration induced by glutamate excitotoxicity is more representative of the pathological mechanism of glaucoma.Vacuolar protein sorting 35(Vps35)has been regarded as the core component of the Retromer complex,which is responsible for sorting and recognizing membrane protein and transporting them from the endosome to trans-Golgi network(TGN)or to plasma membrane to avoid degradation within lysosome.Researches about Vps35 have focused on degenerative diseases of the central nervous system,such as Alzheimer's disease(AD)and Parkinson's disease(PD).Tau protein is a microtubule-associated protein expressed in the axons of neuron.Hyperphosphorylation of tau protein(p-tau)can decrease binding with tubulin,lead to unstability of microtubules,decrease axonal transportation.Hyperphosphorylation of tau protein(p-tau)has been confirmed to be associated with a variety of neurodegenerative diseases,such as AD,familial frontotemporal dementia.Tau protein is widely expressed in normal retina,while p-tau is hardly expressed in normal retina.Degeneration of RGCs is similar to those of central degenerative diseases such as AD,both are increase with age,characterized by neurodegeneration,and increase of p-tau in both brain and retina.Previous studies have shown that Vps35 was mainly expressed in RGCs,deficiency of Vps35 resulted in increased of p-tau in the retina,increased apoptosis of RGCs,disordered arrangement,reduced axons and dendrites,thinning of nerve fiber layer.We refer that the degeneration of RGCs caused by Vps35 reduction is related to the increase of p-tau.Cyclin-dependent protein kinase 5(CDK5),as an important regulator of neuronal postmitotic activity,plays an important role in cell architecture,cytoskeleton,axonal guidance,migration and differentiation of neurons,membrane transport and synaptic plasticity of the central nervous system,and regulates apoptosis signal transduction pathways.CDK5 mediated hyperphosphorylation of tau protein leads to abnormal microtubule structure and cytoskeleton collapse is considered to be the main pathological mechanism of neurodegenerative diseases such as AD and PD.And studies have shown that increased CDK5/p35 activity lead to apoptosis of RGCs.Therefore,we hypothesize that Vps35 deficiency may increase Cdk5/p35 kinase activity and lead to increase of p-tau of retina.Object:Previous studies have found that Vps35 deficiency lead to increase of p-tau in retinal ganglion cells.The purpose of this study is to explore the mechanism of Vps35 in retinal neurodegeneration;to confirm that Vps35 decrease the activity of CDK5/p35 kinase by regulating the degradation of CDK5/p35,and then reduce the production of p-tau to prevent degeneration of RGCs.Methods:1.The concentration of glutamate in the vitreous humor was detected on the 1st,3rd,7th and 14 th day after intravitreal injection of glutamate 20 nmoles,50 nmoles and NMDA 40 nmoles.The expression of cleaved-caspase-3 and phosphorylated tau s396 and taus404 were detected in fresh retina at different time points.2.The expression and interaction of Vps35,CDK5/p35,tau s396,LAMP1 and EEA1 in the glutamate excitotoxic model was studied by immunofluorescence,immunoblotting,Q-PCR and immunoprecipitation.3.Primary retinal ganglion cells transfected with siRNA or overexpression plasmid were detected with immunofluorescence,immunoblotting,co-immunoprecipitation and Q-PCR to explore the effect of Vps35 reduction on the expression of p-tau by affecting the degradation of CDK5/p35 lysosome pathway.Results:1.Intravitreal injection of 50 nmoles of glutamate resulted in decrease of retinal thickness,increase of apoptosis of RGCs,cleaved-caspase-3 and phosphorylated p-tau s396.2.Down-regulation of Vps35 led to increase activity of CDK5/p35 and the expression of p-tau s396 and s404.Vps35 interacted with p35,CDK5 interacted with p35.Inhibition of Vps35 decreased the expression of LAMP1 and EEA1,LAMP1 interacted with p35,Vps35 interacted with LAMP1;indicating that dificiency of Vps35 decrease the lysosomal degradation of CDK5/p35,increase intracellular CDK5/p35 activity,which leads to increase of p-tau.Conclusion:We investigated the effects of Vps35 on the activity of CDK5/p35 in neurodegeneration of RGCs by immunoblotting,co-immunoprecipitation,immunohistochemistry,immunofluorescence,TUNEL assay,Q-PCR,primary cell culture and cell transfection,combined with a glutamate-induced retinal neurodegeneration rat model.The study confirmed that inhibition of Vps35 could increase the activity of CDK5/p35 kinase by affecting the lysosomal degradation pathway of p35.and lead to the degeneration of RGCs.This study revealed the possibility of CDK5/p35 acts as "cargo" of Vps35,and provided new insights into the pathogenesis of RGCs degeneration caused by overphosphorylated tau protein activated by protein kinase.Vps35 is a potential target for basic research and clinical treatment of RGCs degeneration in many ocular diseases such as glaucoma. |
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