Knocking Out Lca5 Gene Leads To Zebrafish Cone-rod Dystrophy

Leber congenital amaurosis?LCA?is a kind of hereditary retinal disease.The prevalence is about 1/30000 to 1/80000 in the general population.LCA usually develops in the first few months after birth,causing severe visual impairment and blindness.LCA is characterized by immense genetic and clinical heterogeneity.Clinically,the disease is defined by the following four characteristics:severe and early visual loss,sensory nystagmus,amaurosis pupils and absent signals on electroretinogram.Different patients may be accompanied by different clinical symptoms such as keratoconus,cataracts,refractive errors,photophobia,night blindness,mental retardation,autism and olfactory dysfunction.Meanwhile,the clinical heterogeneity of LCA is also reflected in the diversity of the patients'retinal phenotype.In genetics,twenty-six genes have been reported responsible for LCA,and these LCA genes encode proteins with a wide variety of retinal functions,such as photoreceptor morphogenesis,phototransduction,visual cycle,guanine synthesis,outer segment phagocytosis,and intra-photoreceptor ciliary transport processes.The LCA5 gene was located in chromosome 6q14.1 and cloned by den Hollander et al.in 2007.The mutations of LCA5 could lead to autosomal recessive leber congenital amaurosis.So far,36 pathogenic mutations of LCA5 gene have been reported,29 of these mutations are nonsense,frameshift or splice site mutations,which may lead to the loss of LCA5 protein function.LCA5 protein is mainly located in the region of photoreceptor connecting cilia and may be responsible for the ciliary transport of photoreceptor outer segment proteins.Inactivation of Lca5 in mouse leads to progressive photoreceptor degeneration.The rod and red/green cone opsins are retained in the outer nuclear layer,transducin and arrestin also exhibit ciliary transport defects.Although LCA5 is involved in ciliary transport processes of photoreceptor,the specific pathological mechanism of LCA caused by its mutations remains unclear.To further explore the pathogenic mechanism of LCA5,we obtained homozygous lca5 gene knockout zebrafish line through CRISPR/Cas9 gene editing technology.The visual function of lca5^-/-zebrafish was detected by ERG,and it was found that the scotopic b-wave amplitudes of 7 dpf lca5 knockout zebrafish was decreased by 50%when compared with wild type controls,indicating visual functional defects.The structure of zebrafish retina was analyzed by hematoxylin-eosin staining and immunofluorescence.The results showed that the deletion of zebrafish lca5 leads to progressive photoreceptor degeneration of both rods and cones with cones affected more severely and earlier than rods.In 21 dpf lca5^-/-zebrafish retina,the cone membrane discs were broken into segments and rod membrane discs showed disordered structure,and the cones outer segments almost completely disappeared in 7 mpf lca5^-/-zebrafish,showing a cone-rod dystrophy phenotype.This is different from the phenotype of severe degeneration of rod and cone in Lca5^-/-mouse at early stage.In lca5^-/-zebrafish,red-cone opsin was mislocalized,and its obvious localization signals were observed in inner segments,synaptic terminals and outer nuclear layer.This mislocalization is initiated in 15-day-old lca5 knockout zebrafish.However,the rod opsin and the other three types of cone opsins were located normally.By detecting the localization of cone transducin??c-T??,rod transducin??r-T??,G-protein-dependent receptor kinase 1?GRK1?and G-protein beta subunit 3?GNB3?in the retina of lca5^-/-zebrafish,we found that the c-T?mislocated to inner segment and synaptic terminal in lca5 knockout zebrafish,while the other three outer segment proteins were not affected.These results suggested that zebrafish lca5 gene may selectively participates in the trafficking of photoreceptor outer segment proteins.To explore the causes of ciliary transport defects in lca5 gene knockout zebrafish,we labeled the ciliary protein acetylated?-tubulin to detect the ciliary structure of photoreceptor.By measuring the length of photoreceptor cilia,we found that ciliary formation was not affected in lca5 knockout zebrafish.However,in the lca5 mutant,Ift88,the core component of IFT complex B,is abnormally distributed throughout the photoreceptor cilia,and this abnormal distribution has already occurred in the 5 dpf zebrafish retina.It is speculated that the loss of zebrafish lca5 gene function results in the failure of Ift88 to return to the cilia base,and thus causes the defect of photoreceptor ciliary transport.In conclusion,this study constructed the model of lca5 gene knockout zebrafish and found that the mutant resulted in early-onset visual defect and progressive photoreceptor cells degeneration with cones affected more severely and earlier than rods.In lca5^-/-zebrafish retina,red-cone opsin and c-T?were selectively mislocalized to inner segment,outer nuclear layer and synaptic terminal.Our zebrafish model well mimics the phenotype of leber congenital amaurosis phenotype caused by human LCA5 gene mutations,laying a solid foundation for further research on the function mechanism of LCA5 and prevention and treatment of LCA.