The Role of the Microglial Cx3cr1 Pathway in the Postnatal Maturation of Retinal Photoreceptors.

Microglia are the resident immune cells of the CNS, and their response to infection, injury and disease is well documented. More recently, microglia have been shown to play a role in normal CNS development, with the fractalkine-Cx3cr1 signaling pathway of particular importance. This work describes the interaction between the light-sensitive photoreceptors and microglia during eye opening, a time of postnatal photoreceptor maturation. Genetic removal of Cx3cr1 (Cx3cr1GFP/GFP ) led to an early retinal dysfunction soon after eye opening [postnatal day 17 (P17)] and cone photoreceptor loss (P30 onward) in mice of either sex. This dysfunction occurred at a time when fractalkine expression was predominantly outer retinal, when there was an increased microglial presence near the photoreceptor layer and increased microglial-cone photoreceptor contacts. Photoreceptor maturation and outer segment elongation was coincident with increased opsin photopigment expression in wild-type retina, while this was aberrant in the Cx3cr1GFP/GFP retina and outer segment length was reduced. A beadchip array highlighted Cx3cr1 regulation of genes involved in the photoreceptor cilium, a key structure that is important for outer segment elongation. This was confirmed with quantitative PCR with specific cilium-related genes, Rpgr and Rpgrip1, downregulated at eye opening (P14). While the overall cilium structure was unaffected, expression of Rpgr, Rpgrip1, and centrin were restricted to more proximal regions of the transitional zone. This study highlighted a novel role for microglia in postnatal neuronal development within the retina, with loss of fractalkine-Cx3cr1 signaling leading to an altered distribution of cilium proteins, failure of outer segment elongation and ultimately cone photoreceptor loss.SIGNIFICANCE STATEMENT Microglia are involved in CNS development and disease. This work highlights the role of microglia in postnatal development of the light-detecting photoreceptor neurons within the mouse retina. Loss of the microglial Cx3cr1 signaling pathway resulted in specific alterations in the cilium, a key structure in photoreceptor outer segment elongation. The distribution of key components of the cilium transitional zone, Rpgr, Rpgrip1, and centrin, were altered in retinae lacking Cx3cr1 with reduced outer segment length and cone photoreceptor death observed at later postnatal ages. This work identifies a novel role for microglia in the postnatal maturation of retinal photoreceptors.

Knockdown of poc1b causes abnormal photoreceptor sensory cilium and vision impairment in zebrafish.

Proteomic analysis of the mouse photoreceptor sensory cilium identified a set of cilia proteins, including Poc1 centriolar protein b (Poc1b). Previous functional studies in human cells and zebrafish embryos implicated that Poc1b plays important roles in centriole duplication and length control, as well as ciliogenesis. To study the function of Poc1b in photoreceptor sensory cilia and other primary cilia, we expressed a tagged recombinant Poc1b protein in cultured renal epithelial cells and rat retina. Poc1b was localized to the centrioles and spindle bundles during cell cycle progression, and to the basal body of photoreceptor sensory cilia. A morpholino knockdown and complementation assay of poc1b in zebrafish showed that loss of poc1b led to a range of morphological anomalies of cilia commonly associated with human ciliopathies. In the retina, the development of retinal laminae was significantly delayed and the length of photoreceptor outer segments was shortened. Visual behavior studies revealed impaired visual function in the poc1b morphants. In addition, ciliopathy-associated developmental defects, such as small eyes, curved body axis, heart defects, and shortened cilia in Kupffer's vesicle, were observed as well. These data suggest that poc1b is required for normal development and ciliogenesis of retinal photoreceptor sensory cilia and other cilia. Furthermore, this conclusion is supported by recent findings that mutations in POC1B gene have been identified in patients with inherited retinal dystrophy and syndromic retinal ciliopathy.

RPGR: Its role in photoreceptor physiology, human disease, and future therapies.

Mammalian photoreceptors contain specialised connecting cilia that connect the inner (IS) to the outer segments (OS). Dysfunction of the connecting cilia due to mutations in ciliary proteins are a common cause of the inherited retinal dystrophy retinitis pigmentosa (RP). Mutations affecting the Retinitis Pigmentosa GTPase Regulator (RPGR) protein is one such cause, affecting 10-20% of all people with RP and the majority of those with X-linked RP. RPGR is located in photoreceptor connecting cilia. It interacts with a wide variety of ciliary proteins, but its exact function is unknown. Recently, there have been important advances both in our understanding of RPGR function and towards the development of a therapy. This review summarises the existing literature on human RPGR function and dysfunction, and suggests that RPGR plays a role in the function of the ciliary gate, which controls access of both membrane and soluble proteins to the photoreceptor outer segment. We discuss key models used to investigate and treat RPGR disease and suggest that gene augmentation therapy offers a realistic therapeutic approach, although important questions still remain to be answered, while cell replacement therapy based on retinal progenitor cells represents a more distant prospect.

Molecular complexes that direct rhodopsin transport to primary cilia.

Rhodopsin is a key molecular constituent of photoreceptor cells, yet understanding of how it regulates photoreceptor membrane trafficking and biogenesis of light-sensing organelles, the rod outer segments (ROS) is only beginning to emerge. Recently identified sequence of well-orchestrated molecular interactions of rhodopsin with the functional networks of Arf and Rab GTPases at multiple stages of intracellular targeting fits well into the complex framework of the biogenesis and maintenance of primary cilia, of which the ROS is one example. This review will discuss the latest progress in dissecting the molecular complexes that coordinate rhodopsin incorporation into ciliary-targeted carriers with the recruitment and activation of membrane tethering complexes and regulators of fusion with the periciliary plasma membrane. In addition to revealing the fundamental principals of ciliary membrane renewal, recent advances also provide molecular insight into the ways by which disruptions of the exquisitely orchestrated interactions lead to cilia dysfunction and result in human retinal dystrophies and syndromic diseases that affect multiple organs, including the eyes.

The Arf GAP ASAP1 provides a platform to regulate Arf4- and Rab11-Rab8-mediated ciliary receptor targeting.

Dysfunctional trafficking to primary cilia is a frequent cause of human diseases known as ciliopathies, yet molecular mechanisms for specific targeting of sensory receptors to cilia are largely unknown. Here, we show that the targeting of ciliary cargo, represented by rhodopsin, is mediated by a specialized system, the principal component of which is the Arf GAP ASAP1. Ablation of ASAP1 abolishes ciliary targeting and causes formation of actin-rich periciliary membrane projections that accumulate mislocalized rhodopsin. We find that ASAP1 serves as a scaffold that brings together the proteins necessary for transport to the cilia including the GTP-binding protein Arf4 and the two G proteins of the Rab family--Rab11 and Rab8--linked by the Rab8 guanine nucleotide exchange factor Rabin8. ASAP1 recognizes the FR ciliary targeting signal of rhodopsin. Rhodopsin FR-AA mutant, defective in ASAP1 binding, fails to interact with Rab8 and translocate across the periciliary diffusion barrier. Our study implies that other rhodopsin-like sensory receptors may interact with this conserved system and reach the cilia using the same platform.

Molecular assemblies that control rhodopsin transport to the cilia.

This review will focus on the conserved molecular mechanisms for the specific targeting of rhodopsin and rhodopsin-like sensory receptors to the primary cilia. We will discuss the molecular assemblies that control the movement of rhodopsin from the central sorting station of the cell, the trans-Golgi network (TGN), into membrane-enclosed rhodopsin transport carriers (RTCs), and their delivery to the primary cilia and the cilia-derived sensory organelle, the rod outer segment (ROS). Recent studies reveal that these processes are initiated by the synergistic interaction of rhodopsin with the active form of the G-protein Arf4 and the Arf GTPase activating protein (GAP) ASAP1. During rhodopsin progression, ASAP1 serves as an activation platform that brings together the proteins necessary for transport to the cilia, including the Rab11a-Rabin8-Rab8 complex involved in ciliogenesis. These specialized molecular assemblies, through successive action of discrete modules, cooperatively determine how rhodopsin and other rhodopsin-like signaling receptors gain access to primary cilia.

Ciliary signaling cascades in photoreceptors.

For being a polarized neuron and having a sensory cilium, photoreceptors attract remarkable attention. This is due their highly polarized structure and active visual signal transduction cascades and for the enrichment of complex networks of proteins in the cilium. Structural and functional maintenance of the photoreceptor sensory cilium, also called outer segment, ensures that light signal is received and relayed appropriately to the brain. Any perturbations in the protein content of the outer segment result in photoreceptor dysfunction, degeneration and eventually, blindness. This review focuses on the importance of photoreceptor sensory cilium to carry out signal transduction cascade for vision.

Disruption of intraflagellar protein transport in photoreceptor cilia causes Leber congenital amaurosis in humans and mice.

The mutations that cause Leber congenital amaurosis (LCA) lead to photoreceptor cell death at an early age, causing childhood blindness. To unravel the molecular basis of LCA, we analyzed how mutations in LCA5 affect the connectivity of the encoded protein lebercilin at the interactome level. In photoreceptors, lebercilin is uniquely localized at the cilium that bridges the inner and outer segments. Using a generally applicable affinity proteomics approach, we showed that lebercilin specifically interacted with the intraflagellar transport (IFT) machinery in HEK293T cells. This interaction disappeared when 2 human LCA-associated lebercilin mutations were introduced, implicating a specific disruption of IFT-dependent protein transport, an evolutionarily conserved basic mechanism found in all cilia. Lca5 inactivation in mice led to partial displacement of opsins and light-induced translocation of arrestin from photoreceptor outer segments. This was consistent with a defect in IFT at the connecting cilium, leading to failure of proper outer segment formation and subsequent photoreceptor degeneration. These data suggest that lebercilin functions as an integral element of selective protein transport through photoreceptor cilia and provide a molecular demonstration that disrupted IFT can lead to LCA.

Spotlight on childhood blindness.

Leber congenital amaurosis (LCA) is a rare disease that severely affects vision in early life. It is characterized by genetic and clinical heterogeneity due to complex and not fully understood pathogenetic mechanisms. It is also now widely known as a disease model for gene therapy. In this issue of the JCI, two independent research groups report valuable new data on LCA. Specifically, they provide important insights into the pathophysiological mechanisms of LCA and offer strong hope that the outcome of gene therapy for retinal degenerative diseases will be successful.