The Study Of The Roles Of Microglia In The Pre-clinical Mouse Models Of Retinitis Pigmentosa

BackgroundMicroglial cells（MGCs）,involved in maintaining tissue stability,play a key role in scavenging pathogens and phagocytosing cellular debris in the central nervous system and retina degeneration.The latest research results demonstrated that activated microglia contribute to the progression of multiple degenerative diseases,such as glaucoma,diabetic retinopathy and age-related macular degeneration.Given the potential damage created by MGCs,the role of the MGCs in the context of retinitis pigmentosa（RP）,however,is still unknown.In this study,we will investigate changes in MGCs over the course of RP disease progression and suggest new strategies for prevention and treatment of this clinically relevant disease.Objective1.Characterization the density,distribution,and activation of MGCs in the context of RP caused by various mutations in Pde6.2.Explore the changes of the density,distribution,and activation of MGCs throughout RP disease progression.3.Assess the effects of gene therapy on the course of RP disease using the Cre-LoxP-STOP system in the early,middle and late stages of RP,as well as on the changes in the density,distribution and activation of MGCs.Methods and resultsIn the first chapter of this study,we used two RP mouse models(Pde6α^D670G/D670G670G/D670G and Pde6β^H620Q/H620Q)each harboring a different mutation in the Pde6 gene and found that the resulting amino acid changes in PDE6 protein structure occur in the catalytic domain.Using the protein blast to compare the alignment between human and mouse,we found that both of these mutations in the human and mouse affect the catalytic domain in theαandβsubunit in PDE6.We thus hypothesized that the molecular mechanism of disease for both Pde6 mutations must be characterized by the loss of function or low function of PDE6,resulting in a low rate of hydrolysis of cGMP,and the accumulation of cGMP in the cell leading to cell death.In order to better visualize the MGCs in the retina,we crossed our RP mouse models with Cx3cr1^GFP/+mouse to generate Cx3cr1^GFP/+Pde6α^D670G/D670G670G/D670G and Cx3cr1^GFP/+Pde6β^H620Q/H620Q620Q/H620Q mice with GFP-labeled MGCs.In these new models,the thickness of the photoreceptor cells were decreased at 4 weeks,and the density of MGCs were increased.The MGCs were also activated,exhibiting a balloon-shaped cell body.Lately,we found that MGCs migrated beyond the layers where they are typically found to the inner and outer segments of the retina,where degeneration was ongoing.In the second chapter of this study,tamoxifen-inducible CreER was introduced to create a disease model(Cx3cr1^GFP/+Pde6γ^Cre/+Pde6β^H620Q/STOP)that could be used to explore changes in MGCs when RP progression halted with gene therapy.In this mouse model,we found that both the thickness of the photoreceptor layer and the ERG response of the photoreceptor cells decreased as disease progression.Moreover,the MGCs became activated,again exhibiting the morphology of a balloon-shaped cell body.In the middle stage of the disease,the density of the MGCs remained stable at a high level before decreaseing in the very late stage,in which all the photoreceptors have died.The MGCs were distributed across all layers in the retina,especially in the outer nuclear layer and the inner/outer segment layer where photoreceptor death was occurring.The second chapter of this stufy provided a basis for the following chapter,in which we explored changes in MGCs after RP progression is halted with gene therapy.Here,we found that the disease progression halted regardless when the treatment is provided,although earlier intervention resulted in better outcomes.In addition,no difference in MGC change was found regardless of whether the treatment was given at early,middle,or late disease stage.Disease progression was halted at the second week after treatment,while the density of the MGCswas found to be decreased at the second week until the sixth week,when the density returned to normal.Moreover,the distribution of the MGCs was limited to the inner and outer segment layers where they are typically found,and their morphology was highly branched after the disease progression was halted.Conclusion1.MGCs become activated in the form of a balloon-shaped cell body in Pde6-related RP.MGCs also migrate beyond the layers where they are typically found to the inner and outer segments,where degeneration was ongoing.2.In the middle stage of the disease progression,the density of the MGCs remains stable at a high level before decreasing in the very late stage,in which all photoreceptors have degenerated.3.Regardless of when the treatment is provided,RP disease progression halted at the second week after treatment.In addition,the density of the MGCs is found to be decreased by the second week,until the sixth week when the density returns to normal.The distribution of MGCs is also limited to the inner and outer segments layers where they are typically found,and the morphology of the MGCs becomes highly branched after the disease progression is halted.4.Although RP disease progression halts regardless of the time of treatment,earlier treatment results in better outcomes.MGC changes also occur independent of whether the treatment is administrated at early,middle,or late stage of disease.