Studying The Molecular Pathogenesis Of SDCCAG8-Associated Retinal Ciliopathy

Retinal ciliopathies are a group of inherited retinal degenerative diseases caused by mutations in genes encoding ciliary proteins essential for photoreceptor morphology and function,presenting as syndromic or non-syndromic retinal degeneration.Over 100genes encoding retinal ciliopathy proteins have been associated with retinal ciliopathies to date.As a pathogenic gene of retinal ciliopathies,mutations in SDCCAG8,a serologically defined colon cancer autoantigen gene,have been first reported to cause two autosomal recessive ciliary disorders a decade ago,Senior L?ken syndrome（SLS）and Bardet-Biedl syndrome（BBS）.Affected individuals all developed progressive retinal degeneration in early childhood,presenting as Leber congenital amaurosis（LCA）or retinitis pigmentosa（RP）.There are no effective therapies available now.To date,19retinal ciliopathy-causative SDCCAG8 mutations have been identified,and all SDCCAG8 mutations are localized in C-terminal coiled-coil domains,and all of which are deletion or nonsense mutations.SDCCAG8 protein was found to co-localize and/or to interact with several ciliary proteins at the photoreceptor transition zone.Howerever,the function of SDCCAG8 is unkown.Therefore,this dissertation explored the function of SDCCAG8 and the molecular pathogenesis of SDCCAG8-associated retinal ciliopathy,the main research contents are as follows:1.To investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies in vivo,we selected the nonsense mutation（c.708C>G p.Y236X）in exon 7 of Sdccag8and a frameshift mutation（c.1352-1353ins G p.E451Gfs X467）in exon 11,and we employed Clustered Regularly Interspaced Short Palindromic Repeats（CRISPR）/Cas9-mediated homology-directed recombination（HDR）to generate two knock-in mouse models,Sdccag8^Y236X/Y236X and Sdccag8^{E451Gfs X467/E451Gfs X467} that carried these two truncating mutations of mouse Sdccag8.These knock-in mouse lines are the BBS mouse models,first to present with polydactyly phenotype.The results showed that mutations in SDCCAG8 resulted in truncated proteins in knock-in mouse retinas,and the expression of truncated proteins were significantly decreased,indicating instability of the truncated proteins.The Sdccag8 protein was localized around the photoreceptor inner segment（IS）and connecting cilium（CC）in wild-type mouse retinas,while the truncated Sdccag8 proteins were expressed at the same location in the mutant photoreceptors but with significantly deceased fluorescent signals.The results showed that these two mutant Sdccag8 knock-in mice faithfully recapitulated human SDCCAG8-associated BBS phenotypes of retinal degeneration,characterized by the progressive degeneration of rod and cone photoreceptors,cystic renal disorder,polydactyly,infertility,and growth retardation.The varied phenotypic onset of age and severity were directly proportional to the hypomorphic strength of the Sdccag8mutations.In addition,some of these two knock-in mice showed serious embryonic and early postnatal lethality.They also showed mislocalization outside the OS of major phototransduction proteins,including rhodopsin、GRK1、PDE6b、S-opsin、cone arrestin,mislocalizing after initiation of photoreceptor degeneration.2.To further investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies,we first examined the ciliary development of the mutant photoreceptors,renal epithelium cells,as well as the mouse embryonic fibroblasts（MEFs）derived from the knock-in mouse embryos.The results showed these cells formed abnormal cilia with decreased number and length.We then generated p EGFP-Sdccag8 plasmids expressing the recombinant mouse wild-type Sdccag8 protein in vitro,and two ciliopathy causative mutants Sdccag8（Y236X）and Sdccag8（E451Gfs X467）fused with a fluorescent protein tag,EGFP.The results indicated that their expression level of were consistent with the Sdccag8 mutants expressed in knock-in mouse retinas at reduced levels.And the truncated proteins caused the decrased number and length of cilia,demonstrating that SDCCAG8 was essential for cilia formation.It suggest the pathogenesis of SDCCAG8-associated retinal ciliopathy are cilium defects.3.To further investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies,we performed for RNA-sequencing after serum-starvation for the Sdccag8^Y236X/Y236XMEFs,as they had more severe phenotypes relative to the mutant Sdccag8 MEF cell line.The results showed the expression of many genes related to the formation of cilia and microtubules,as well as the development of kidney and retina were significantly reduced.Then,several representative genes with significant differences（Lama5,BMP4,Aqp1,Cfh,Stmn2,Sdccag8 and Hhip）were selected to verify their expression levels by real-time quantitative PCR,and the results showed that the expression of these genes decreased 79%,69%,45%,26%,26%,39%and 25%,respectively.Conclusively,our study demonstrates that SDCCAG8 is a ciliary protein involved in retinal ciliopathies and plays a role in cilium assembly and/or maintenance,and cilium defects are the primary driving force of SDCCAG8-associated retinal ciliopathies.The results preliminarily revealed the pathogenesis of SDCCAG8-associated retinal ciliopathies,and the results obtained from this study provide important mouse models for the effective treatment of such congenital retinal degeneration diseases.