Primary cilia in retinal pigment epithelium development and diseases

Abstract Retinal pigment epithelium (RPE) is a highly polarized epithelial monolayer lying between the photoreceptor layer and the Bruch membrane. It is essential for vision through participating in many critical activities, including phagocytosis of photoreceptor outer segments, recycling the visual cycle‐related compounds, forming a barrier to control the transport of nutrients, ions, and water, and the removal of waste. Primary cilia are conservatively present in almost all the vertebrate cells and acts as a sensory organelle to control tissue development and homeostasis maintenance. Numerous studies reveal that abnormalities in RPE lead to various retinal diseases, such as age‐related macular degeneration and diabetic macular oedema, but the mechanism of primary cilia in these physiological and pathological activities remains to be elucidated. Herein, we summarize the functions of primary cilia in the RPE development and the mutations of ciliary genes identified in RPE‐related diseases. By highlighting the significance of primary cilia in regulating the physiological and pathological processes of RPE, we aim to provide novel insights for the treatment of RPE‐related retinal diseases.

retina. Interestingly, primary cilia are dynamically changed during the developmental and maturation process of RPE, and defective primary cilia of RPE layer are associated with retina-related diseases, inferring the indispensable role for primary cilia in RPE. 7 Herein, we review the alterations of primary cilia during the RPE development and the physiological and pathological roles of primary cilia in regulating RPE development and homeostasis, aiming to highlight the significance of primary cilia in RPE and provide therapeutic strategies for RPE-related diseases.

| PRIMARY CILIA AND RPE DE VELOPMENT
Primary ciliogenesis is a dynamic process oscillating during the cell cycle. Primary cilia are formed in G 0 phase and resorbed as cells reenter the cell cycle. Similarly, primary cilia are changed dynamically during RPE development. During mice embryonic development, primary cilia are detectable in RPE layer at E14.5, and reach the highest to 70% at E16.5, and resorb at E18.5 with a decreased ciliary length.
In the RPE layer of postnatal mouse, primary cilia can be observed only in a little of cells 8 (Figure 2). The narrow expression window of primary cilia during the development of the RPE suggests that primary cilia may play a crucial role in RPE and their activity must be tightly controlled.
A recent study revealed that RPE is crucial for photoreceptor development and function, and primary cilia is required for complete maturation of RPE probably through WNT pathway. 9 Defects in primary ciliogenesis lead to RPE immaturation and consequently photoreceptor degeneration. 9 Several ciliary genes in ciliopathies are found to be responsible for RPE development and retinal functions. For example, Bardet-Biedl syndrome 8(BBS8), an important member of BBSome complex, is critical for the biogenesis of the ciliary membrane and ciliary protein trafficking. BBS8 gene mutations are associated with retinitis pigmentosa and early vision loss through affecting the development of outer segments in photoreceptor neurons. 10 Centrosomal protein 290 (CEP290) is essential for the assembly of ciliary transition zone, and its mutations are frequently found in Leber congenital amaurosis (LCA), an autosomal recessive childhood blindness disorder. 11 Retinal pigment epithelium 65(RPE65), an isomerase in the visual cycle, is highly expressed in the RPE. Light that reaches the RPE activates the visual pigment and converts the all-trans retinol to alltrans retinol, which in turn is oxidized by RPE65 to 11-cis retinol. 12 Retinitis pigmentosa 1(RP1), a homolog of RPE65, is located at the ciliary axeneme and its mutation is associated with retinitis pigmentosa (RP). 13 It is worth exploring whether RPE65 is involved in primary ciliogenes is and RPE development. 14 In addition, primary cilia play an important role in the development of RPE; however, the ciliated cells remarkably decrease in mature RPE, it is thus interesting to investigate the physiological role of primary cilia in mature RPE.

| PRIMARY CILIA AND RPE-REL ATED DISE A SE S
Abnormal structure and function of RPE leads to impaired bloodretina barrier, which affects cell diffusion, active transport and intron mutation is the most common cause of LCA, which leads to splicing errors and premature termination. 18 This causes defects in primary ciliogenesis and RPE transport. 19 The frameshift mutation of pre-mRNA processing factor 31 (PRPF31) suppressed primary ciliogenesis, resulted in an incorrect connection between the RPE and photoreceptor and impaired barrier function 20,21 (Figure 3).
Although the association of ciliary gene mutations with ciliary defects and retinal diseases have been observed, the causal relationship and the underlying mechanisms of how primary cilia lead to RPE-related diseases remain to be characterized.

| TRE ATMENT OF RPE-REL ATED DISE A SE S
Gene therapy has been clinically used to correct the mutated genes for retinal diseases, 22 of which the most used delivery vector is recombinant adeno-associated virus (AAV). 23 AAV-based gene therapy targeting RPE cells has shown promising therapeutic prospects for RP patients with PRPF31 mutations. Inducing pluripotent stem cells (iPSCs) harbouring PRPF31 mutations differentiation into RPE cells is able to recapitulate PRPF31 mutation-caused pathological phenotypes. F I G U R E 3 Abnormal primary cilia lead to RPE dysfunction and related diseases. Primary cilia regulate RPE maturation and homeostasis through WNT pathways. Mutations of the ciliary genes, such as RP1, RPE65, CEP290, BBS8, and IFT88 are often found in retinal diseases. Lossof-function of these ciliary proteins may lead to defects in primary ciliogenesis, which in turn results in immature RPE, thereby contributing to retinal diseases, including diabetic retinopathy, diabetic macular edema, and age-related macular degeneration. The short red lines indicate primary cilia and the small blue dots represent the centrosomes AAV-based PRPF31 gene therapy can restore the defects in phagocytosis and ciliary formation of RPE, showing promising therapeutic prospect for RP patients. 24,25 By sub-retinal injection of AAV2-RPE65 into LCA patients for dual-allele replacement, visual impairment in RPE can be restored, and the clinical trials are currently ongoing. 12 The abnormal splicing of CEP290 can be effectively blocked by antisense oligonucleotides in LCA patients. 19 In addition, CRISPR/Cas9 technology has also been successfully used to correct CEP290 mutations in LCA patients. 26 In addition, stem-cell therapy has shown the potential for the treatment of retinal diseases. 27 RPE patch composed of RPE monolayers differentiated from human embryonic stem cells (hESCs) was implanted into the sub-retinal space of patients with AMD. After a period of time, the biological microscope and optical coherence tomography showed that the RPE patch could improve the vision of AMD patient. 28 Notably, the conditioned medium used for hESC induction contained PDGF-AA, TGFα and IGFBP-2, which is conducive to primary ciliogenesis and RPE functions. 29 Moreover, mature retinal organoid generated by co-culture of human epidermal keratinocytes and human pluripotent stem cells contains primary cilia and is able to replace defective RPE and photoreceptors for the treatment of AMD patients. 30

| CON CLUS I ON S AND PER S PEC TIVE S
Emerging studies reveal that functional RPE is essential for

This work was supported by grants from the Taishan Scholars
Program of Shandong Province (20161201) and Natural Science Foundation of Shandong Province (ZR2016CM28).

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.