CERKL Gene Knockout Disturbs Photoreceptor Outer Segment Phagocytosis And Causes Rod-cone Dystrophy In Zebrafish

Retinitis pigmentosa(RP)is one of the major causes that results in hereditary visual impairment and blindness charaterized by the progressive degeneration of photoreceptors.Patients with RP typically appear loss of night vision in adolescence,gradually aggravating the form of tubular vision,and can further cause blindness because of the serious degeneration of rod and cone photoreceptor cells.CERKL is an important RP/CRD pathogenic gene cloned in 2004.Until now twelve mutants have been reported worldwidely.In human beings,the process of CERKL mutations causing the retinal degeneration includes early dysfunction and loss of rod and cone photoreceptors in the outer retina and,progressively,death of cells in the inner retina.Human CERKL contains 13 exons that encode a polypeptide of 532 amino acids.This protein is a homolog of ceramide kinase(CERK),containing a Pleckstrin homology(PH)domain and an evolutionarily conserved diacylglycerol kinase(DAGK)domain.Despite intensive efforts,both its physiological and pathological roles of CERXL remain obscure.Studies in animal models have been especially important to clarify the function of CERKL.Zebrafish is an ideal animal model to study retina disease.The development of the eye,anatomical structure and the formation of photoreceptor cells are very similar to human beings.CERKL is highly conserved over the course of evolution.The zebrafish CERKL shares 65%amino acid identity with human.To clarify the disease mechanism of CERKL mutations,we generated a CERKL knockout zebrafish model by TALEN technology.5-to 7-day old CERKL-/-zebrafish examined by ERG exhibited a significantly reduced light response.The b wave amplitude in CERKL-/-zebrafish was reduced by 30%compared with that of control,indicating that deletion of CERKL affected visual function at an early stage.Histological examination of the retina of CERKL-/-zebrafish was carried out at different ages.At 2 months of age,the outer segments(OSs)of photoreceptors arrangement showed early signs of disorganization.At 3-12 months old,the OSs of photoreceptors showed gradually degeneration,appearing shorter than normal.At 12 months of age,the CERKL-/-zebrafish showed significant decreases in both the length of the OSs and the thickness of the outer nuclera layer(ONL),indicating the loss of rod and cone photoreceptors.We extended the morphological studies for rod and cone photoreceptors at different stages and found that the rod degeneration in CERKL-/-zebrafish occurred earlier(3 months old)than that in cone cells(7 months old),indicating a phenotype of rod-cone dystrophy.To further investigate the physiological function of CERKL in retina development,proteins required for phototransduction that expressed specifically in rod or cone cells were examined at different ages.Results from Western-blotting assays showed that in CERKL-/-zebrafish the expression of rhodopsin,GRK1,GNAT1 and GNB1 in rod rapidly decreased from the age of 1-2 months.By contrast,the cone proteins GNB3 and GCAP1 showed no significant changes in CERKL-/-zebrafish,further confirming that rod cells showed earlier degeneration than cone cells in CERKL-/-zebrafish.Transmission electron microscopic(TEM)analysis of CERKL-/-zebrafish retinas at ages of 3 and 5 months showed that the OSs of most rod photoreceptors were detached.A massive accumulation of vesicular membrane structures in the interphotoreceptor spaces were observed in CERKL-/-zebrafish.These results suggested an important role of CERKL in OS phagocytosis by the RPE and indicate that the CERKL mutations may lead to defects in photoreceptor outer segment phagocytosis,and eventually result in photoreceptor degeneration.The components of phagocytosis were categorized by their involvement in photoreceptor recognition,engulfment,and degradation in different stages of the renewal process.To explore the underlying mechanism of dysfunction of RPE phagocytosis,the mRNA levels of phagocytosis-associated genes in WT and CERKL-/-,zebrafish of 1 month old were measured by qRT-PCR.As a result,we found a significantly decreased mRNA level of the phagocytosis-associated gene MERTK in CERKL-/-zebrafish.Additionally,Western-blot assays showed that,compared to WT,the protein expression levels of MERTK were reduced by 23%and 36%in retina of the CERKL-/-zebrafish at the age of 1 and 3 months,respectively.Likewise,in ARPE-19 cell lines,knockdown of CERKL also decreased the mRNA and protein level of MERTK,as well as the ox-POS phagocytosis.In conclusion,we had successfully established a CERKL deficiency zebrafish model,in which we observed a phenotype of rod-cone dystrophy,but not the cone-rod dystrophy.Knockout of CERKL in zebrafish caused earliest damage at the phototransduction by disturbing the expression of the phototransduction-associated proteins,while the CERKL deficiency-mediated down-regulation of the MERTK expression impaired the phagocytosis function of RPE.Our result will not only uncover the important roles of CERKL in retina physiology and new mechanism of how CERKL mutations cause retinitis pigmentosa,but also provide evidences and support in developing effective strategy for RP/CRD prevention and treatment.