Mechanism Of Müller Cells' Regulation Of Retinal Remodeling By MGluR5/8 During Retinal Degeneration

Retinitis pigmentosa(RP)is an irreversible and serious blindnees-caused eye disease,which is characterized by the progressive apoptosis of photoreceptors.Over200 genes have been identified associated with RP,which affected around 1:4000individuals.Remodeling consists of three major phases.Early stage is characterized primarily by early markers of photoreceptor stress.Middle phase is the period of photoreceptor loss accompanied by glial remodeling of the outer nuclear layer.Last stage is characterized by loss of all photoreceptors,remodeling of the inner neurons and glial scar formed.And the glial scar isolated the remnat neurons from the retinal pigment epithelium(RPE)and choroids and could potentially act as a barrier for bionic,cell transplantation and gene therapy aimed at restoring vision.Therefore,the mechanism of Müller cells remodeling during retinal degeneration have to be settled urgently.The gliosis of Müller cells is characterized by the upregulated of the glial fibrillary acidic protein(GFAP).However,which factor initates the activation and gliosis of Müller cells is unclear.And we want to explore that how to reduce the expression level of gliosis,how to inhibit the apoptosis of neurons and whether the loss of reintal neural function could be delayed by downreglution of Müller cells'gliosis.Glutamate is the only neurotransmitter relased by photoreceptors and is released by all neurons in the retina.The released glutamate by photoreceptors in darkness is detected by second-order neurons via ionotropic glutamate receptors(iGlu Rs)and metabotropic glutamate receptors(mGluRs).Meanwhile,reliable synaptic glutamate relies not only on the release machinery of photoreceptors and the response mechanism of postsynaptic neurons but also on removal of glutamate from the synaptic cleft for avoiding the excitotoxicity of neurons and maintaining high signal to noise ratio.The‘glutamate–glutamine cycle'of Müller cells is important to the recycling of glutamate in glio-neuronal interactions.Glutamate is taken up by the glutamate/aspartate transporter(GLAST)expressed on Müller cells,and then it is intracellularly converted into glutamine by glutamine synthetase(GS),a glia-specific enzyme.Although it is reported that the bulk of glutamate from photoreceptor terminals was suggested to be removed by presynaptic EAAT5 and the glutamate in the inner retinal is regulated by Müller cells,it is reported that selective ablation of Müller cells resulted not only in disrupting the morphology of photoreceptors.Meanwhile,the glutamate metabolism of Müller cells is abnormal and the mGluR6 expressed on the bipolar cells is first affected at the early stage of retinal remodeling.Therefore,we hypothesize that the abnormal of glutamate release and the metabotropic glutamate receptor expression is initial factor of Müller cells remodeling during the retinal degeneration.Our main results are showed as following:First,we found that Müller cells regulated the a-wave of flash electroretinogram(FERG).More specifically,we found that the FERG a-wave which reflected the function of photoreceptors induced by light reduced after subretinal injection of GS inhibitor,DL-AAA or MSO,to the normal rats.Meanwhile,the metabotropic glutamate receptor 5(mGlu R5)was activated detected by immunohistochemistry(IHC),western blotting(WB)and quantitative real-time polymerase chain reaction(PCR).Moreover,selective antagonism of mGlu R5 by 2-methyl-6-(phenylethynyl)-pyridine increased the FERG a-wave amplitude and also increased rhodopsin expression.Conversely,activation of mGluR5 by the agonist,(R,S)-2-chloro-5-hydroxyphenylglycine,decreased both the a-wave amplitude and the expression level of rhodopsin,which was emolyed to aborbed photons by photoreceptors.Then we demonstated that overexpression of mGluR5 reduced the inward-rectifying potassium ion channel(K_ir)current by patch clamp and decreased the expression of K_ir4.1 and aquaporin-4(AQP4)by PCR and WB.Further experiments indicated that mGluR5 formed a macromolecular complex with these two membrane channels by co-immunoprecipitation.At last,the G?q-Ca²⁺pathway was proved to be involved in the regulation of mGluR5 to the Müller cells.Second,we found that the function of the inner retina cells,which refleted by the oscillatory(OPs)of FERG,could be modulated by Müller via subretinal delivery of GS inhibitor at early stage.We first illustrated that mGluR8 expressed on the Müller cells by immunohistochemistry in vitro and in vivo.Then the reduced FERG amplitude caused by inhibiting GS could be resotred after inhibiting the expression of mGlu R8 by subretinal injection of MSOP.Moreover,activation of mGlu R8 after subretinal delivery of DCPG reduced the amplitude of FERG b-wave and OPs.Meanwhile,we found that the expression level of GS was downregulated but the expression level of GFAP was upregulated by activating mGluR8.In addition,the origin of OPs was proved from the feedback circuit of GABA receptors and rod bipolar cells by intravitreal injection of different drugs to different retinal neurons and receptors.At last,we proved that the regulation of Müller cells to the inner retinal neurons after DCPG was activating GABAa by GABA released by Müller cells.Third,we investigated that the expression level of glutamate and mGlu R5/8during the retinal degeneration.At first,we found that the dysfunction of retinal photoreceptors started at the early stage of RCS by the decreased FERG amplitude,which had an exponential relationship with the ages of RCS rats.We then illustrated that GS changed at P20d of RCS rats by IHC and PCR.And the concentration of glutamate in the retina decreased first and then increased by HPLC.And the exrepssion level of mGluR5 and mGluR8 increased dramatically at middle and late stage of RCS rats by IHC and PCR.Based on these observations,we proposed the‘three stage of RP'on the viewed of Müller cells:early stage characterized by the gradually decreased GS expression and mGlu R5/8,middle stage characterized by graducally increased GS exression and mGlu R5/8,later stage characterized by dramatically decreased GS expression by increased mGluR5/8.Last,we illustrated that increased FERG amplitude and decreased GFAP expression of Müller cells were resulted from selectively inhibition of the overactivated mGluR5 or mGluR8 in the RCS rats.After subretinal delivery of MSOP to the RCS rats at P40d,the FERG amplitude was increased significantly at 2 and 4 days after injection,which continuted to the 28 days after delivery.Then the decreased expression of GFAP and increased expression of rhodopsin,GS,Kir4.1 and AQP4 were tested by IHC and PCR.Same expression changes of the same protein and increased FERG amplitude could also be found in the rats treated with MSOP,except the increased OPs amplitude in the latter.Based on the results,we concluded that:Müller cells regulated the outer retinal function via mGlu R5 which formed a macromolecular complex together with Kir4.1 and AQP4.Müller cells regulated the inner retinal function by mGluR8 via GABA receptor activities.Glutamate expression level and dysfunction of gluatamte recycling were the initated factor of the Müller cells activation during the retinal degeneration.mGluR5 and mGluR8 may be worth considering as a potential therapeutic target in RP.