Roles Of Microglia In Remodeling Of Outer Retina During Rat Retina Degeneration And Its Mechanisms

Background:Retinal degeneration(RD)represents a family of blinding retina disorders which are typically attributed to hereditary factors,light damage or aging.RD is characterized by a common pathological process—the progressive deterioration and loss of photoreceptors.The decline of visual function induced by photoreceptor degeneration leads to changes in the structure and function of neurons and glia cells in the retina,which further trigger retina remodeling of the outer and the inner layers.Retinal remodeling,especially for the inner retinal remodeling,are important reasons of limited effects in therapy.Inner retinal remodeling results from the loss of input from outer retina,regardless of the initiating insult.Therefore,it is important to maintain outer retina function by regulating outer retina remodeling,prior to the deleterious remodeling.The outer retina consists of photoreceptor layer and outer plexiform layer(OPL).The rods and cones in the photoreceptor layer sense light stimulus,then transmit the potentials to the inner retina through the synapse in the OPL.During the retinal degeneration,photoreceptor degeneration plays a crucial role in remodeling of outer retina.In most RD models,rod-specific damages,such as genetic mutation,lead to primary cell death of rods,resulting in night blindness,followed by a secondary degeneration of normal cones,which are essential for high acuity and color vision.Photoreceptor degeneration and loss are the most important pathological changes in the outer retinal remodeling.At the early stage of RD,photoreceptor degeneration causes OPL synaptic remodeling,characterized by the loss of synaptic proteins,ectopic neuritogenesis of bipolar cells and synaptic formation with survival photoreceptors.Therefore,synaptic remodeling in the OPL plays an important role in restoring the input activity of the inner retina.Microglia,the only resident immune cell in the central nervous system(CNS),plays crucial roles in the process of development and disease.During the development of RD,microglia,migrating from the inner retina to the photoreceptor layer,is activated with a less-ramified,more-amoeboid morphology.However,it remains controversial that microglia exerting positive or negative effect on photoreceptor degeneration,which is due to the limited understanding of the multiple phenotypes at different stages of microglia activation.In a variety of neurodegenerative diseases,activated microglia share the similar alteration of transcriptional profile,morphology and function,which has recently been identified as a novel microglia type of degeneration-associated microglia(DAM).DAM activation induces genes upregulation of lipid metabolism and phagocytic pathways,concomitantly with downregulation of microglia homeostatic genes.Developmental apoptosis of photoreceptors also promotes DAM activation to remove dead cells and maintain a normal developmental process.However,the mechanisms of DAM activation in the retina and its effects on photoreceptor degeneration remain unclarified.Here,we speculate that apoptotic photoreceptors in the RD may promote the activation of the microglia to DAM in the photoreceptor layer,which in turn regulates photoreceptor degeneration.Synaptic pruning is one of important functions of microglia during CNS development and injury,which maintains the normal transmission of neuronal circuits.Microglia protrudes processes to identify and engulf synaptic components in a complement-dependent manner,to remove abnormal synapses.The complement-dependent pathway is involved in the activation of microglia and promotes early synaptic loss in Alzheimer disease(AD)models,resulting in cognitive impairments.RD and AD are both neurodegenerative diseases,sharing the common pathological characters of early synaptic loss and cell degeneration.Thus,we speculate that microglia regulate synaptic remodeling in the OPL by synaptic pruning at the early stage of RD.However,the ways and mechanisms of synaptic pruning between photoreceptors and bipolar cells by microglia are not completely understood.Hypothesis:During the retinal degeneration of Royal College of Surgeons(RCS)rats,microglia in the photoreceptor layer is activated to DAM and regulates the photoreceptor degeneration,and microglia in the OPL regulates synaptic remodeling between photoreceptors and bipolar cells by synaptic pruning,which plays a key role in the remodeling of outer retina.Methods and Results:Part One:Characters of the outer retina remodeling during retinal degeneration in RCS rats1.The features of photoreceptor degeneration in RCS rats.The results from TUNEL staining showed that apoptotic photoreceptors could be found in the retina of RCS rats at P20,only remaining 35.26%photoreceptors compared with control group at P50.The structural damage and decline in quantity of cones mainly occurred after P40.Thus,photoreceptor degeneration,apoptosis and loss appeared in the outer retina of RCS rats at P15-P50,and cones degenerated after the loss of rods.2.The character of synaptic remodeling in the OPL of RCS rats.Using immunohistochemistry,it showed that the number of CtBP2-positive,mGluR6-positive and co-localized puncta decreased markedly at P20 in the retina of RCS rats compared with that of controls,the peak of the decrease appeared at P30 and 80%-90%reduction were observed in quantity of puncta at P50.However,the changes of cone synapses occurred after P40 in RCS rats.However,no significant decrease in the CtBP2 and mGluR6mRNA level were observed in the retinas of RCS rat.Electron microscopy(ECM)analyses further confirmed the disintegration of rod synapse in RCS rats after P15.It suggests that synapse loss and disintegration were the main changes during the synaptic remodeling in the OPL of RCS rats.Part two:Transformation of microglia in the outer retina during retinal degeneration in RCS rats1.Changes in the number and morphology of microglia in the outer retina of RCS rats.Immunohistochemical staining of IBA1,a cell marker of microglia,showed that the changes in the number and morphology of IBA1-positive microglia in RCS rats were mainly observed in the outer retina at P15-P50.After P20,microglia migrated to the photoreceptor layer and the number of microglia increased significantly as the development of retina degeneration.At P50,the number of microglia in photoreceptor layer increased to 74.21%of the total number of microglia in the retina.The number of OPL microglia in RCS rats increased from P15,reached a peak at P30,then gradually decreased.2.Characteristics of microglia in the photoreceptor layer of RCS rats.Retinal transcriptome sequencing analysis demonstrated that the expressions of DAM-related genes were significantly up-regulated in the retina of RCS rats compared to that of control rats.Using Axl staining to label different types of microglia,we observed that DAM activation mainly occurred in the photoreceptor layer of RCS rats.With CD45 staining,only a few microglia-like cells were observed in the subretinal space(SRS)of RCS rats near the RPE cells,which were probably derived from monocytes These results indicate that retinal degeneration promoted microglial activation to DAM in the photoreceptor layer of RCS rats.3.Characteristics of microglia in the OPL of RCS rats.Through confocal high-resolution imaging and morphological scoring,we found that OPL microglia in degenerative retinas maintained the homeostatic morphological scores.However,89.43%of microglial in the OPL of RCS rats protruded and directly contacted with synaptic proteins.CD68-labeled lysosome increased gradually in the OPL microglia of RCS rats.It suggests that microglia in the OPL of RCS rats touched directly to synapses with phagocytic enhancement.Part three:Effects of microglia on outer retinal remodeling in RCS rats1.Elimination of microglia with Colony-Stimulating Factor 1 Receptor(CSF1R)inhibitor in the retina of RCS rats.The rats were continuously fed with CSF1R blocker PLX3397 from P15 to analyze the effects on elimination of retinal microglia.It showed that PLX3397 at the dosage of 300 ppm(containing 300 mg of drug per 1 kg of feed)depleted 54.04%of retinal microglia in RCS rats,and PLX3397 at the dosage of 600ppm depleted 70.18%of retinal microglia,whereas the dosage of 1200ppm did not improve the clearance effect of microglia.Control rats treated with PLX3397 at the dosage of 600 ppm from P15 to P30 completely eliminated retinal microglia.63.22%of retinal microglia in RCS rats were depleted when treated with PLX3397at the dosage of 600 ppm from P15 to P50,and 80%of the survival microglia were mainly distributed in the photoreceptor layer.Therefore,the PLX3397 dosage of 600 ppm was selected to remove retinal microglial in subsequent experiments.2.Effects of microglial elimination on retinal function in RCS ratsScotopic Flash Electroretinogram(FERG)and grating optokinetic responses were used to evaluate the effect of microglial clearance on retinal function.Depleting microglia by PLX3397 in normal rats did not affect the retina function.In RCS rats,microglia elimination from P15 to P20 or P30 did not affect the retinal function;however,the microglia depletion from P15 to P40 mainly resulted in the decline of the b-wave amplitudes and b/a ratio from FERG;eliminating microglia from P15 to P50 resulted in a decrease of the a-and b-wave amplitudes,and impairments of visual acuity.It suggests that long-term continuous elimination of retina microglia in RCS rats by CSF1R inhibitor aggravated the function of photoreceptors and bipolar cells,and inhibited the transmission of information between photoreceptors and bipolar cells.3.Effects of microglia elimination on photoreceptor degeneration in RCS rats.TUNEL staining showed that the number of apoptotic nuclei in the photoreceptor layer increased markedly when the microglia were depleted by PLX3397 in the retina of RCS rats,while the number of survival photoreceptors decreased only at P50.Arrestin staining and photopic FERG showed that depleting microglia from P15 to P50 exacerbated secondary degeneration of cones.Retinal transcriptome analysis was used to further verify the inhibition of the functional and structural related genes in photoreceptors after the microglia elimination.It suggests that microglia elimination in the retina of RCS rats mainly exacerbated the secondary degeneration of photoreceptors,especially for cones.4.Effects of microglia elimination on synaptic remodeling in the OPL of RCS rats.It showed that removing microglia from P15 to P30 or P40 mainly increased the number of unpaired mGluR6 in the OPL of RCS rats.Depleting microglia from P15 to P30 had no effect on the expression of CtBP2 and mGluR6 genes.Thus,microglia elimination suppressed the loss of free post-synaptic proteins in rod bipolar cells.5.Effects of microglia elimination on ectopic neuritogenesis of rod bipolar cells.Using PKCαstaining,it showed that synapses and rod bipolar cells presented normal morphology in control rats with microglia elimination.In RCS rats,clearing microglia increased the number and length of ectopic dendrites of rod bipolar cells,without changes in the positive rate of mGluR6.The results suggest that microglia elimination promoted the development of ectopic neuritogenesis of rod bipolar cells.Part four:Mechanisms of microglia regulating the remodeling in the outer retina of RCS rats1.Mechanisms of microglial regulation of photoreceptor degeneration in RCS rats.Immunofluorescence staining and flow cytometry analysis revealed that depleting microglia from P15 to P50 mainly cleared DAM in the photoreceptor layer of RCS rats,without effects on the number of intermediate microglia.The activation of DAM mainly phagocytosed apoptotic cells and their fragments,no directly contacting stressed cones.Flow cytometry results showed that the number of neutrophils,Natural Killer(NK)cells,and CD4~+T cells in the retina of P50 RCS rats increased significantly,however microglia elimination only further aggravated neutrophil infiltration.Infiltrating neutrophils were mainly distributed in the photoreceptor layer,which produced a lot of harmful substances.Microglia suppressed neutrophil accumulation in the RCS rats by phagocytosing neutrophils and inhibiting the secretion of CXCL1 chemokines by Müller cells.It suggests that DAM activation in the photoreceptor layer of RCS rats improved the microenvironment by removing apoptotic photoreceptors and inhibiting the accumulation of neutrophils,which contributed to delay the secondary degeneration of photoreceptors,especially for cones.2.Mechanisms of microglial regulation of synaptic remodeling in the OPL of RCS rats.Immunofluorescence staining and three-dimensional reconstruction of the image showed that microglial in the OPL of RCS rats mainly engulfed unpaired mGluR6 on the rod bipolar dendrites.By analysing the expression of complement molecules in the OPL,it was found that complement molecules C1q and C3 were deposited in OPL of RCS rats and mainly bound to unpaired mGluR6.These results indicate that microglia in the OPL of RCS rats recognizeed and engulfed the free post-synaptic structure of rod bipolar cells through the complement pathway to regulate synaptic remodeling.Conclusion:1.Our study demonstrated for the first time that retinal degeneration in RCS rats can promote microglia conversion to intermediate types,and then activation to DAM in the photoreceptor layer.We also found that intermediate microglia survives and proliferates independent of CSF1R signaling.2.DAM activation in the photoreceptor layer of RCS rats contributes to suppress the secondary degeneration of photoreceptors,especially for cones.DAM is able to improve the microenvironment by removing accumulated apoptotic cells and inhibiting the infiltration of neutrophils,which maybe the main mechanism of suppressing the secondary degeneration of photoreceptors.3.Microglia in the OPL of RCS rats recognizes and engulfs unpaired post-synaptic structures on the dendrites of rod bipolar cells through the complement pathway,which is helpful to maintain the function of rod bipolar cells and inhibit ectopic neuritogenesis of rod bipolar cells.4.This study revealed that microglia plays a key role in the remodeling of outer retina in RCS rats.Innovative use of CSF1R blocker to eliminate retinal microglia of RCS rats,clarifies the role of microglia on photoreceptor degeneration and OPL synaptic remodeling and their mechanisms;which provide novel treatment strategies targeting microglia to delay photoreceptor degeneration,promote photoreceptor survival,inhibit retinal pathological remodeling,and improve retinal function.