The Mechanistic Study Of Retinitis Pigmentosa Caused By Rpgra Knockout In Zebrafish

Retinitis pigmentosa（RP）is a kind of disease with progressive death of photoreceptor cells in the retina.It has high genetic and clinical heterogeneity.According to its genetic characteristics,it is mainly inherited as an autosomal dominant,autosomal recessive,X-linked,mitochondrial and oligogenic trait.X-linked RP accounts for 5%-15%of all RP cases and is recognized as one of the most common and severe forms of inherited retinal dystrophy.It is mainly manifested by degeneration of rod and cone photoreceptors in childhood,leading to narrowing vision field and early severe vision loss.Mutations in the RPGR（retinitis pigmentosa GTPase regulator）gene contribute to 70-90%of all XLRP cases,rod-cone dystrophy,cone and cone-rod dystrophy,atrophic macular degeneration,including syndromal RP with respiratory infection,hearing loss and primary ciliary dyskinesia.Multiple transcripts of RPGR gene can be produced by alternative splicing,among which the most important ones include RPGR^ORF15 and RPGR^EX1-19 isoform.As far as we know,most of the disease-causing variants are present in exons of the RPGR^ORF15 isoform,with only little present in exons 15-19,indicating the importance of the RPGR^ORF15isoform in the retina.Rpgra in zebrafish is homologous to the RPGR^ORF15 isoform in human.In this paper,a homozygous rpgra knockout zebrafish with c.1675＿1678delins21mutant was successfully constructed by Golden-gate Talen technique.The retina of 5 dpf rpgra^-/-zebrafish detected by HE staining did not show significant developmental defects,while ERG result showed a significant decreasing trend of b-wave in rpgra^-/-zebrafish,which confirmed visual dysfunction in rpgra^-/-zebrafish.HE staining of zebrafish at different stages showed that the outer segment of rpgra^-/-zenrafish showed a tendency of degeneration from 3 mpf,while the outer retinal nuclear layer becomes gradually thinner from 5 mpf.By 18 mpf,the photoreceptor cells were significantly reduced,especially in UV cones,suggesting that the loss of rpgra may cause retinal degeneration in zebrafish.Different opsin proteins were further detected by immunofluorescence that found that rod outer segment degenerated at 3 mpf while red cone outer segment degenerated at 6 mpf with mislocating in the outer plexus layer and the cell body region of the nucleus.Blue,green and UV cone were shorter,but their location did not change.It is indicated that knockout of rpgra causes progressive rod-cone degeneration in zebrafish.In order to further determine the physiological mechanism of retinitis pigmentosa caused by RPGR mutation,we detected the factors related to the light transduction pathway in rpgra^-/-zebrafish,and found that the expression of Gn AT1,GRK1,Rhodopsin and SAG was significantly down-regulated in 4 mpf rpgra^-/-zebrafish.In 6 mpf rpgra^-/-zebrafish,the phototransduction related factors of rods and cones were significantly down-regulated except for gnb1a and gnb1b,which further confirmed the retinal degeneration phenotype of rpgra^-/-zebrafish.Previous studies have shown that RPGR is involved in the formation of cilia and the regulation of actin stress filaments,and it can directly interact with a variety of transport-related proteins to form transport-complex involved in material transport.On this basis,we detected the localization of Rab8a and Gnb3 respectively,and found that both of them had mislocalization,and the expression of Rab8a was significantly down-regulated.Transmission electron microscopy（TEM）was used to detect the cilium of photoreceptor cells.A large number of abnormal vesicles were found in the cilium region of rpgra^-/-zebrafish.Transmission electron microscopy results also noticed the abnormal accumulation of lipid droplets in the RPE layer of knockout zebrafish,and further staining with Nile Red confirmed the presence of lipid droplets in rpgra^-/-zebrafish.In 24 mpf rpgra^-/-zebrafish RPE tile immunofluorescence staining,the morphological structure of RPE has changed,which is larger and disordered.Then,through transcriptome sequencing and enrichment,the signal pathways related to lipid metabolism were obtained,which is convenient for us to explore how rpgra knockout causes the accumulation of lipid droplets in the RPE layer.In summary,we had successfully first builded a rpgra^-/-zebrafish model using TALEN,which showed human XLRP phenotype.The absence of RPGR in zebrafish leads to the dysfunction of ciliary transport and the inability to properly transport the proteins associated with rods and cones,resulting in defective photoreceptor membrane disks and apoptosis of photoreceptor cells.Abnormal accumulation of lipid droplets may changed the morphology of RPE cells in knockout zebrafish.Using this model,we can try drug therapy research according to the molecular mechanism of retinal degeneration,and explore effective prevention and treatment methods for retinal degeneration.