Animal Models And Genetic Studies Of Retinal Degeneration Diseases

Purpose:The mammalian target of rapamycin(mTOR)is a conserved Ser/Thr kinase that regulates cell growth and survival.Previous studies indicate mTOR may play important roles in regulating the functions of retinal pigment epithelial(RPE).To investigate the consequences of genetically stimulating wTOR pathway in the RPE,we mutated tuberous sclerosis complex 1(Tscl),a critical negative regulator of mTOR,by using tissue-specific knockout mouse model.Mice received intraperitoneal injection of rapamycin daily to test whether it was efficient to prevent the pathology in retina.Methods:To inactivate Tscl in the RPE cells,we crossed Tscl conditional targeted mice(Tsclf/f)with a transgenic Bestl-Cre mouse line to generate a mouse model in which Tscl was specifically eliminated in RPE cells(Tscf/f Best1-Cre9 cKO).PCR was used for genotyping of Tscl gene and Cre transgene.The activation of mTOR pathway was examined by western blotting.Immunofluorescence on cryosection was used for detecting the distribution of Tscl?mTOR?s6?and p-s6 in patient's eyecups well as the distribution of Cre and p-s6 in our cKO mice.Contour length of outer segments and thickness of the RPE layers were measured by unbiased stereology.We also examined the morphological and eletrophysiological changes of cKO mice by funduscopy and electroretinogram respectively.Immunofluorescence on RPE flat mounts were performed to assess the cuboidal appearance and the junctional integrity of RPE cells.Electronmicrographs of cKO mice and control groups were used to reveal the abundant structures consistent with degraded mdanosomes.We treated cKO mice daily with rapamycin or vehicle when they were 4-week-old until sacrifice.Littermate controls also received rapamycin or vehicle.Western blotting and immunofluorescence were performed to determine whether rapamycin rescued the mTOR pathway and preserved RPE differentiation.Results:The highest expressing percentage of Cre transgene reached to 70%in RPE cells and displayed a patchy mosaic expression pattern,in the cKO mice.Within the eyes of cKO mice,only the RPE cells were immuno-reactive with an anti-Cre antibody.The level of Tscl expression was almost diminished and mTOR was activated.The cKO mice showed a gradual thinning of the outer nuclear layer and the redistribution of melanosomes in RPE cells.These morphological abnormalities indicated the process of RPE dedifferentiation.B-wave of electroretinographic responses was reduced compared to the control group.In the Tscl deficiency RPE cells,the distributions of ?-catenin were increased in cytoplasm and decreased on cell boundary,and the phosphorylation of Y489 in ?-catenin increased with age.Furthermore,rapamycin treatment inhibited mTOR-mediated RPE dedifferentiation and attenuated loss of RPE-characteristic markers.Conclusion:The mTOR pathway was successfully activated after the deletion of Tscl in RPE cells and the cKO mice displayed some typical phenotypes of AMD disease.This mice model enabled us to investigate physical role of mTOR signaling in RPE cells and rapamycin treatment in the cKO mice.Furthermore,this mice model may provide new insights into the pathogenic mechanism of AMD and new therapeutic targets for thisdisease.Purpose:Inherited retinal dystrophies(IRDs)are a group of progressive and irreversible disorders characterized by progressive dysfunction of photoreceptors and vision loss.Despite its clinical and genetic heterogeneities,IRDs are mostly monogenic diseases.This study aims to determine the causative mutations of patients diagnosed as IRDs and to explore the genotypic-phenotypic correlations.Methods:Medical histories were carefully reviewed.All patients received comprehensive ophthalmic evaluations included best-corrected visual acuity(BCVA)assessment,slit-lamp examinations,dilated fundus examination,optical coherence tomography(OCT)examinations,electroretinogram(ERG)test,and visual field(VF)measurements.Next-generation sequencing(NGS)approach targeting a panel of 205 retinal diseases relevant genes and 15 candidate genes was selectively performed on probands from the nineteen recruited families for mutation detection.Online predictive software and crystal structure modeling were also applied to test the potential pathogenic effects of identified mutations.Results:We identified 3 reported mutations and 10 novel mutations including 2 putative pathogenic frameshifts and 8 missense mutations that are very likely pathogenic.For the first time we reported the correlation between EYS mutation and RPSP,we also for the first time reported the correlation between INPP5E mutation and CRD.Conclusion:Identification of the novel IRDs-causative mutations in our study using NGS approach not only provide a efficient and economical method for heterogeneous IRDs,but also provide a better understanding of genotype-phenotype relations in IRDs of Chinese patients.